بسم اهلل الرمحن الرحيم
قالوا سبحانك ال علم لنا إال ما علمتنا إنك أنت العليم احلكيم*
البقرة ()23
دراسة بعض املكونات الكيميائية النباتية لنوعني من ( )Pistacia sppمتأصل من قضاء ماوت – السليمانية - كردستان العراق
مقدمة اىل جملس فاكليت العلوم و الرتبية العلوم سكول العلوم يف جامعة السليمانية كجزء من متطلبات نيل شهادة
ماجستري علوم حياة /علم النبات
من قبل علي سعيد كريم بكالرويوس علوم حياة ( )8002جامعة السليمانية
املشرف أ.م.د نوري حسن غفور
صفر 6341
سةرماوةرز 8763
Appendix
Phenolic compounds
Rt.
Gallic acid
1.19
Cinnamic acid
1.76
Stilbene
2.32
Ellagic acid
2.80
Catechin
3.40
Tannin
4.50
Anthocyanin
4.92
Appendix 9: chromatogram of Rt. (min.) of different phenolic compounds in the leaves of P. eurycarpa
Phenolic compounds
Rt.
Gallic acid
1.22
Cinnamic acid
1.80
Stilbene
2.39
Ellagic acid
2.87
Catechin
3.47
Tannin
4.53
Anthocyanin
5.02
Appendix 10: chromatogram of Rt. (min.) of different phenolic compounds of P.eurycarpa from cluster without fruit
Appendix
Volatile oil
Rt.
Sabienene
1.18
Limonene
2.16
β- Pinene
3.23
β- Myrecene
4.29
α- Pinene
5.36
Phellandrine total
6.28
3-Carene
7.43
Aldehyde-citral
8.24
Myretenol
9.09
Carveol
10.04
Terpineolene
11.04
Appendix 16: chromatogram of Rt. (min.) of different volatile oil compounds from the leaves of P. eurycarpa
Volatile oil
Rt.
Sabienene
1.21
Limonene
2.13
β- Pinene
3.37
β- Myrecene
4.25
α- Pinene
5.42
Phellandrine total
5.94
3-Carene
7.43
Aldehyde-citral
8.15
Myretenol
9.12
Carveol
10.02
Terpineolene
10.96
Appendix 17: chromatogram of Rt. (min.) of different volatile oil compounds of P.eurycarpa from the cluster without fruits.
Appendix
Phenolic compounds
Rt.
Gallic acid
1.18
Cinnamic acid
1.76
Stilbene
2.34
Ellagic acid
2.78
Catechin
3.41
Tannin
4.49
Anthocyanin
4.93
Appendix 7: chromatogram of Rt. (min.) of different phenolic compounds of P.eurycarpa from the seed
Phenolic compounds
Rt.
Gallic acid
1.18
Cinnamic acid
1.75
Stilbene
2.33
Ellagic acid
2.80
Catechin
3.42
Tannin
4.49
Anthocyanin
4.96
Appendix 8: chromatogram of Rt. (min.) of different phenolic compounds of p. eurycarpa in the outer shell
Appendix
Phenolic compounds
Rt.
Gallic acid
1.18
Cinnamic acid
1.77
Stilbene
2.35
Ellagic acid
2.83
Catechin
3.42
Tannin
4.50
Anthocyanin
4.91
Appendix 5: chromatogram of Rt. (min.) of different phenolic compounds in the leaves of P. khinjuk
Phenolic compounds
Rt.
Gallic acid
1.15
Cinnamic acid
1.70
Stilbene
2.28
Ellagic acid
2.78
Catechin
3.36
Tannin
4.44
Anthocyanin
4.85
Appendix 6: chromatogram of Rt. (min.) of different phenolic compounds of P. khinjuk from the cluster without fruit
Appendix
Phenolic compounds
Rt.
Gallic acid
1.17
Cinnamic acid
1.75
Stilbene
2.34
Ellagic acid
2.83
Catechin
3.41
Tannin
4.49
Anthocyanin
4.90
Appendix 3: chromatogram of Rt. (min.) of different phenolic compounds in the seed of P. khinjuk
Phenolic compounds
Rt.
Gallic acid
1.18
Cinnamic acid
1.76
Stilbene
2.33
Ellagic acid
2.81
Catechin
3.41
Tannin
4.49
Anthocyanin
4.91
Appendix 4: chromatogram of Rt. (min.) of different phenolic compounds of P. khinjuk from the outer shell
Appendix
Volatile oil Sabienene Limonene β- Pinene β- Myrecene α- Pinene Phellandrine total 3-Carene Aldehyde-citral Myretenol Carveol Terpineolene
Rt. 1.20 2.10 3.35 4.30 5.35 6.20 7.42 8.18 9.19 10.08 10.99
Appendix 12: chromatogram of Rt. (min.) of different standards of volatile oils.
Volatile oil Sabienene Limonene β- Pinene β- Myrecene α- Pinene Phellandrine total 3-Carene Aldehyde-citral Myretenol Carveol Terpineolene
Appendix 13: chromatogram of Rt. (min.) of different volatile oil compounds of P.eurycarpa in the gum.
Rt. 1.19 2.12 3.34 4.40 5.47 6.34 7.49 8.25 9.15 10.07 11.01
Appendix Phenolic compounds
Rt.
Gallic acid
1.30
Cinnamic acid
1.75
Stilbene
2.43
Ellagic acid
2.93
Catechin
3.51
Tannin
4.59
Anthocynidin
5.01
Appendix 1: chromatogram of Rt. (min.) of different standards of phenolic compounds
Phenolic compounds
Rt.
Gallic acid
1.19
Cinnamic acid
1.77
Stilbene
2.33
Ellagic acid
2.41
Catechin
3.53
Tannin
4.53
Anthocynidin
4.9
Appendix 2: chromatogram of Rt. (min.) of different phenolic compounds of P. eurycarpa in the gum
List of abbreviations
List of abbreviations Abbreviations
Full name
AFLP
Amplified Fragment Length Polymorphism
BC
before Christ
DDH2O
Double demonized water
DPPH test
α, α Di-Phenyl-β-picrylhydrazyl test
DW
distil water
GC
Gas Chromatography
GC/MS
Gas Chromatography/ Mass Spectrophotometer
H2O2
Hydrogen peroxide
HPLC
High Performance Liquid Chromatography
K. bneshtatall
Kurdish bneshtatall (gum)
K. kojila
gum collector
K. tazbeh
Kurdish tazbeh (beads)
M.a.s.l
Meter above sea level
Mg
milligram
NADPH
Nicotinamide adenine dinucleotide phosphate hydrogenise
RAPD
Random Amplified Polymorphic DNA
RFLP
Restriction Fragment Length Polymorphism
Rpm
round per minutes
Rt.
Retention time
UV
ultra violet
XII
List of Appendix No
Title
1
Chromatogram of Rt. (min.) of different standards of phenolic compounds.
2
Chromatogram of Rt. (min.) of different phenolic compounds of P.eurycarpa in the gum
3
Chromatogram of Rt. (min.) of different phenolic compounds in the seed of P. khinjuk
4
Chromatogram of Rt. (min.) of different phenolic compounds of P. khinjuk from the outer shell
5
Chromatogram of Rt. (min.) of different phenolic compounds in the leaves of P. khinjuk
6
Chromatogram of Rt. (min.) of different phenolic compounds of P. khinjuk from the cluster without fruit
7
Chromatogram of Rt. (min.) of different phenolic compounds of P.eurycarpa from the seed
8
Chromatogram of Rt. (min.) of different phenolic compounds of P.eurycarpa in the outer shell
9
Chromatogram of Rt. (min.) of different phenolic compounds in the leaves of P.eurycarpa
10
Chromatogram of Rt. (min.) of different phenolic compounds of P.eurycarpa from cluster without fruit
11
Chromatogram of Rt. (min.) of different phenolic compounds in the gum of P. khinjuk
12
Chromatogram of Rt. (min.) of different standards of volatile oils
13
Chromatogram of Rt. (min.) of different volatile oil compounds of P.eurycarpa in the gum
14
Chromatogram of Rt. (min.) of different compounds of leaves in P. khinjuk
15
Chromatogram of Rt. (min.) of different volatile oil compounds of P. khinjuk from cluster without fruits
16
Chromatogram of Rt. (min.) of different volatile oil compounds from the leaves of P.eurycarpa
X
17
Chromatogram of Rt. (min.) of different volatile oil compounds of P.eurycarpa from the cluster without fruits
18
Chromatogram of Rt. (min.) of different compounds of the gum in P. khinjuk
19
chromatogram of Rt. (min.) of different compounds from the outer shell in P.eurycarpa
XI
List of figures List of Figures Figure No
Title
page No
1 A simplified view of the major pathways of secondary carbon -metabolite Biosynthesis in plant cells ........................................................................................... 10 2 Schematic of Anthocyanins Biosynthetic pathways ................................................. 11 3 Chemical Structure of Tannin ................................................................................... 14 4 Chemical Structure of ellagic acid ............................................................................ 14 5 Chemical Structure of Anthocyanin.......................................................................... 14 6 Chemical Structure of Gallic acid ............................................................................. 14 7 Chemical structure of Pinene ................................................................................... 27 8 Chemical structure of Limonene .............................................................................. 27 9 Chemical structure of Myrtenol ............................................................................... 27 10 Barbard-Mawat Location the Wild habitat of the P. eurycarpa and P. khinjuk where samples were collected ..................................................................................... 34 11 P. khinjuk shrubs .................................................................................................... 44 12 P.eurycarpa trees .................................................................................................... 44 13 Gum of P. khinjuk .................................................................................................. 44 14 Gum of P.eurycarpa ............................................................................................... 44 15 Leaves of P. khinjuk............................................................................................... 45 16 Leaves of P.eurycarpa ............................................................................................ 45 17 Clusters of fruits P. khinjuk ................................................................................... 45 18 Clusters of fruits P.eurycarpa................................................................................. 45 19 Immature fruit P. khinjuk ....................................................................................... 46 20 Immature fruit P.eurycarpa .................................................................................... 46 21 Mature fruit P. khinjuk (show mature seed) .......................................................... 46 22 Mature fruit P.eurycarpa (show mature seed) ........................................................ 46 23 Mature fruit P. khinjuk ........................................................................................... 46 24 Mature fruit P.eurycarpa ........................................................................................ 46 25 The percentage concentration of Tannin in different parts of the two Pistacia spp. ................................................................................................................ 58 26 The percentage concentration of Stilbene in different parts of the two Pistacia spp. ................................................................................................................ 59
VII
List of figures 27 The percentage concentration of Catechin in different parts of the two Pistacia spp ...................................................................................................................................... 60 28 The percentage concentration of Ellagic acid in different parts of the two Pistacia spp ................................................................................................................................ 61 29 The percentage concentration of Anthocyanin in different parts of the two Pistacia spp ................................................................................................................................ 62 30 The percentage concentration of Gallic acid in different parts of the two Pistacia spp ................................................................................................................................ 63 31 The percentage concentration of Cinnamic acid in different parts of the two Pistacia spp .................................................................................................................. 64 32 The percentage concentration of α – Pinenein different parts of the two Pistacia spp. at different growth stage ....................................................................................... 76 33 The percentage concentration of Phellandrine in different parts of the two Pistacia spp. at different growth stage ....................................................................................... 77 34 The percentage concentration of β - Pinenein different parts of the two Pistacia spp. at different growth stage .............................................................................................. 78 35 The percentage concentration of Sabienene in different parts of the two Pistacia spp. at different growth stage ....................................................................................... 79 36 The percentage concentration of Limonene in different parts of the two Pistacia spp. at different growth stage ....................................................................................... 80 37 The percentage concentration of Aldehyde-citral in different parts of the two Pistacia spp. at different growth stage ......................................................................... 81 38 The percentage concentration of 3 – Carenein different parts of the two Pistacia spp. at different growth stage. ...................................................................................... 82 39 The percentage concentration of Myrecene in different parts of the two Pistacia spp. at different growth stage ....................................................................................... 83 40 The percentage concentration of Terpinene in different parts of the two Pistacia spp. at different growth stage. ...................................................................................... 84 41 The percentage concentration of Carveol in different parts of the two Pistacia spp. at different growth stage .............................................................................................. 85 42 The percentage concentration of Myretenol in different parts of the two Pistacia spp. at different growth stage ....................................................................................... 86
VIII
List of tables List of tables
Table No.
Titles
Page No
1. Some morphological and physiological comparative among two Pistacia species growing in Iraqi – Kurdistan region............................................................................... 4
2. Scientific classifications of P. eurycarpa and P. khinjuk ........................................ 5
3. List of materials ....................................................................................................... 31
4. List of equipments.................................................................................................... 32
5. Preliminary Detection of some phytochemicals of the two Pistacia spp ............... 47
IX
Chapter one
Introduction
Introduction Plants are the major source of natural product of many useful applications such as spices, perfume basis, insecticide, growth hormone and pharmacological activity, whereas only a few plant-derived secondary metabolites have been directly used as drugs. Many pharmacologically active compounds have served as leading models for semi synthetic and synthetic drugs (Butler, 2005). Additionally, there is a growing interest in the application of standardized extracts, complex Phytochemical mixtures with a well-defined content of the bioactive constituents, emphasizing the therapeutic importance of natural compounds (Newmann and Cragg, 2007; Potterat and Hamburger, 2008). The genus Pistacia is a member of the Anacardiaceae family, and it consists of 11 species (Zohary, 1952). They are shrubs or trees. Three Pistacia species occur naturally in Iraq, including Pistacia vera, Pistacia khinjuk and Pistacia eurycarpa. P. vera is the only species in this genus that is successfully grown in orchards less than 900 m.a.s.l., P. vera is dioecious tree and deciduous plant, which produces edible seeds large enough to be commercially acceptable (Tous and Ferguson, 1996). But P. khinjuk and P. eurycarpa species are native to northern Iraq, and they are dioecious plants and deciduous plants are spread in places where the attitude is 700 - 2000 m.a.s.l., P. khinjuk is a shrub, but P .eurycarpa is a tree (Behboodi, 2003). Pistachios are utilized mostly in the unripe outer shell, for fresh consumption; processed uses include candy, baked goods, and ice cream. They also have folklore, medicinal and non- food uses such as toothache relief. And, fruits of both species are used as edible wild fruits are used as tasteful and flavor added to wheedle and vinegar. The resin is used as antibacterial in Europe and North America; gum (dried resin) is used as a bloodclotting agent in Europe and the Middle East (Hormaza et al., 1994, 1998). The essential oil of the oleo-gum resin of P. eurycarpa without treatment strongly inhibited microorganisms associated with common wound infections and skin diseases. Husks are used in India for dying, and tanning Pistachio wood is good for carving, cabinetry, and firewood as is that of other species of Pistacia (Mohannad et al., 2006). The stems exudate gum (K. bneshtatall) which is freshly collected and boiled with water, Upon cooling, the water soluble compounds are discarded and the water insoluble residue is used as a chewing gum, and the dry ripe fruits are bored two sides and converted into necklaces and worry beads (k. tazbeh).(Ahmad, 2013).
1
Chapter one
Introduction
Pistachios are adapted to a variety of soils and are probably more tolerant of alkaline and saline soil than most tree crops; Pistachios thrive in hot, dry desert-like conditions (Gale, 1975). Different parts of Pistacia species have been used in traditional medicine for various purposes like tonic, aphrodisiac, antiseptic, antihypertensive, management of dental, gastrointestinal, liver, urinary tract, and respiratory tract disorders. Scientific findings also revealed the wide pharmacological activities from various parts of these species, such as antioxidant, antimicrobial, antiviral, anticholinesterase, anti-inflammatory, antidiabetic, anti-tumor, antihyperlipidemic, antiatherosclerotic, and hepatoprotective activities (Villar et al., 1987and Kordali, et al., 2003). Various types of phytochemical constituents like terpenoids, phenolic compounds, fatty acids, and sterols have also been isolated and identified from different parts of Pistacia species (Villar et al., 1987; Kordali et al. 2003; Benhammou et al. 2008 and Mahbube et al., 2013). The total area of Great Kurdistan (Kurdistan of Iraq, Iran, Turkey and Syria) was estimated to be approximately 39.2 million hectare (392000 Km²), there is around 16 million hectare of total forests such as Oaks, pistacia, Firs and other conifers can be found in those forests (http://groups.myspace.com/Kurdistan1, (2003-2008). According to Talib (2004), the Iraqi Kurdistan Region area is about 7361800 hectare (73618 Km²). The total area of mountain forest land computes to 1777600 hectare, of which 36.2% falls in Duhok Governorate, 39% falls in Erbil Governorate, and 23.7% falls in Sulaimani Governorate and the remaining in Kirkuk and Diyala Governorate. The forest zone has annual rainfall of 700-1400 mm. The lower limit of the forest in Iraq is generally about 500 m, while the higher limit reaches 1750-1800 m. (Chapman, 1957).
The aims of the current study are 1. Preliminary tested for detection of some secondary metabolites from two Pistacia spp., such as Flavonoid, phenolic compounds, Tannin, Saponin, Terpenoids, Steroids, Anthocyanin, Resin, Glycosides, Fatty Acids, Leucoanthocyanidin, Alkaloid, Coumarin, and Emodin. 2. To extract the total phenolic compounds and essential oil (volatile oil) from leaves, seeds, outer shells, cluster without fruits and gum. 3. To quantify and qualify total phenolic compounds and essential oil (volatile oil) using High perphormance liquid chromatography (HPLC).
2
Chapter two
Literature review
Literature Review 2.1. Pistacia spp. The genus Pistacia is characterized by its dioeciously reproductive system and homeochlamydic perianth (or naked flowers), Pistacia, distinguished from other Anacardiaceae members by its reduced flower structure, plumose styles, and unusual Pollen (Pell, 2004; Tingshuang et al., 2008). The first classification of the genus Pistacia was done by Zohary (1952). In his monographic study, the genus was subdivided into four sections. The main diagnostic traits used to distinguish between the various species were, and remain, leaf characteristics and nut morphology. Zohary (1972) classified Pistacia species in Israel, and Yaltirik (1967a) classified Pistacia species in Turkey, identifying P. atlantica var. kurdica Zoh. As a different species, which he named P. eurycarpa (Al-Yafi, 1978) morphology described a few P. atlantica subspecies on the basis of their leaf morphology and retained P. eurycarpa as a variety of P. atlantica (Kafkas and Perl-Treves, 2001). On the basis of the morphology of leaves, leaflet, inflorescence, flowers, fruits, and the seedlings, Zohary (1952) divided Pistacia into four sections: Lentiscella Zoh., including P. Mexicana HBK, and P. texana Swingle, Eu Lentiscus Zoh., including P. lentiscus L., P. saportae Burnat., and P. weinmannifolia Poisson; Butmela Zoh., including P. atlantica Desf.(P. eurycarpa); and Eu Terebinthus Zoh., including P. chinensis Bge., P. khinjuk Stocks, P. palaestina Boiss., P. terebinthus L., and P. vera L. On the basis of plastid restriction site analysis and morphological characters, Parfitt and Badenes (1998) suggested the division of the genus into two sections: Lentiscus and Terebinthus. Section Lentiscus includes Zohary (1952) sects, Lentiscella and Eu Lentiscus and consists of the evergreen species with par pinnate leaves and smaller seeds. They also suggested that Zohary (1952) sects. Butmela and Eu Terebinthus are combined as sect. Terebinthus, which includes the deciduous species with impair pinnate leaves and large seeds. Section Terebinthus was supported by recent molecular studies on Mediterranean Pistacia species (Kafkas and Perl-Treves, 2001, 2002; Golan-Goldhirsh et al., 2004 and Kafkas, 2006) However the phylogenetic relationships among Pistacia species were also estimated by plastid DNA restriction site analysis and RFLP (Parfitt and Badenes, 1998),
3
Chapter two
Literature review
RAPD (Katsiotis et al., 2003), RAPD and AFLP (Katsiotis et al., 2003 and Tingshuang et al., 2008). Three Pistacia spp. can be seen in many places in the northern mountain forest of Iraqi- Kurdistan which lies between 900-2500 m.a.s.l, contours (flora of Iraq). The botanical features of the two Pistacia spp. in Kurdistan region are given below:
Table 1: Some morphological and physiological comparisons between two Pistacia species growing in Iraqi – Kurdistan region (Flora of Iraq, 1969) No
P. eurycarpa
P. khinjuk
1
Dioecious plant
Dioecious plant
2
Deciduous plant
Deciduous plant
3
Large tree 4-20m tall in figure 12
Small tree 3-7m tall in figure 11
4
Leaves imparipinnate up to 20cm long
Leaves imparipinnate 7 - 15cm long
5
3-7 ovate-lanceolate leaflets show in figure 16
3-9 oblong – lanceolate to ovate leaves show in figure 15
6
Male panicles 3-20 cm but female panicles 8-20 cm long
Male panicles 5-12 cm but female panicles 7-15 cm long
Indehiscent and paniculate Fruits contain 1 - seeded drupe in figures 22 and 24
Indehiscent and paniculate Fruits contain 1 - seeded drupe in figures 21 and 23
Fruits red - violet – green colour 5 8 mm long , 7 - 10 mm broad Show in figure 24
Fruits coppery – green colour 4 – 7 mm long , 4 - 5.5 mm broad shown in figure 23
7
8
9
Release high amount of colour less gum in figure 14
Release low amount of honey yellow colour gum in figure 13 4
Chapter two
Literature review
Table 2: Scientific classification of P. eurycarpa and P. khinjuk taken from (Nahida et al., 2012).
Kingdom: Plantae
Kingdom: Plantae
Division: Magnoliophyta
Division: Magnoliophyta
Order: Sapindales
Order: Sapindales
Family: Anacardiaceaae
Family: Anacardiaceaae
Genus: Pistacia
Genus: Pistacia
Species: Pistacia khinjuk
Species: Pistacia eurycarpa
Binomial name: Pistacia khinjuk Stocks
Binomial name: Pistacia eurycarpa Yalt
According to the World Health Organization (WHO), plants are a supplier of medicines for human consumption (Ghasemi, 2009; Ghasemi et al., 2009 and WHO 2001). Factors such as lack of access to the majority of chemical drugs and/or the cost of chemical drugs, as well as the adverse side effects of chemical drugs, lead to the advantages of using herbal or plant drugs instead of chemical medicines (Ghasemi, 2009 and Ghasemi et al., 2009). Herbal or plant remedies used for hundreds of years by stock raisers could be put to commercial use, but scientists are demanding that traditional knowledge should be validated so as to verify the safety and efficacy of the treatments (Mahmoud and Zohre, 2012).
2.2 Primary and secondary metabolites Plants synthesize a vast range of organic compounds that are traditionally classified as primary and secondary metabolites although the precise boundaries between the two 5
Chapter two
Literature review
groups can in some instances be somewhat blurred. Primary metabolites are compounds that have essential roles for the life of the plant associated with photosynthesis, respiration, growth and development. These include phytosterol, acyl lipids, nucleotides, simple sugar amino acids and organic acids. Other phytochemicals, many of which accumulate in surprisingly high concentrations in some species, are referred to as secondary metabolites. These are now known to be important for the survival and propagation of the plants that produce them and structurally diverse and many are distributed among a very limited number of species within the plant kingdom and so can be diagnostic in chemotaxonomic studies. Although ignored for long, their function in plants is now attracting attention as some appear to have a key role in protecting plants from herbivores and microbial infection, as attractants for pollinators and seed-dispersing animal, as allelopathic agents, protection from the sun (UV protectants) and chemical signals that enable the plant to respond to environmental changes . Secondary metabolites are also of interest because of their use as dyes, fibres, glues, oils, waxes, flavouring agents, drugs and perfumes, and they are viewed as potential sources of new natural drugs, antibiotics, insecticides and herbicides (Dewick, 2002 and Peter et al., 2013). Based on their biosynthetic origins, plant secondary metabolites can be divided into three major groups: (i) Phenolics (including Flavonoids, tannins, catechols, lignins and salicylic acid) (ii) Terpenoids (including essential oils, taxol, isoprene and (iii) Alkaloids and sulphur-containing compounds such as; morphine, cocaine, caffeine and nicotine (Peter et al., 2013). 2.2.1 Properties and classification of phenolic compounds Phenolic compounds are ubiquitously distributed throughout the plant kingdom. There is increasing evidence from epidemiological, in vivo, in vitro, and clinical trials clearly suggesting that the phenolic compounds present in fruits, vegetables, leaf, root and grains may reduce risk of chronic diseases such as cancer, anti-inflammatory, cardiovascular, and neurodegenerative diseases (Rice-Evans et al., 1996). Over 8000 phenolic compounds with diverse structural configurations (although each has at least one aromatic ring with one or more hydroxyl group) and polarities have been isolated and reported from plant sources (Robbins, 2003). Phenolic compounds can be chemically classified into three major categories: simple phenols, polyphenols and a miscellaneous group. Simple phenols primarily consist of phenolic acids (cinnamic acid and benzoic acid derivatives). 6
Chapter two
Literature review
However, polyphenols are further subdivided into two main classes: tannins (polymers of phenolic acids, catechins, or epicatechins) and flavonoids (flavones, isoflavones, anthocyanins, chalcone, flavonols, flavanones, etc.). The common structure of Flavonoids consists of two aromatic rings linked by three carbons that usually form an oxygenated heterocyclic. In plants, Flavonoids can be found as aglycones, although they are usually found as glycosides contributing to the color (blue, scarlet, orange) of leaves, flowers, and fruits (Wu and Prior, 2005; Escarpa, and Gonzalez, 2008). The third, the miscellaneous group consists of other phenolic compounds such as Coumarins, Stilbenes, and Lignins (Luthria, 2006). Today's society is characterized by having many unhealthy dietary habits. Not only snacking but also the inadequate intake of healthy foods triggers a major dietary imbalance, this being a major cause of chronic diseases such as obesity, diabetes mellitus, cardiovascular disease, hypertension, stroke, and several types of cancer. Therefore, it is vital to ascertain the composition and nutritional value of these products. To prevent the above-mentioned diseases, epidemiological studies recommend the consumption of whole fruits and vegetables that contain high amount of phenolic compounds (Espinosa et al., 2006 and Mertz et al., 2009). Polyphenols, widely distributed in plants, contribute to fruit organoleptic and nutritive quality in terms of color, taste, aroma, and flavor (Serrano et al., 2010)., also being involved in astringent and bitter tastes. It is known that, amongst other factors, such as maturity stage or light exposure, phenolic composition varies with the cultivar. In addition, the phenolic profile has already been revealed to be a useful parameter for the discrimination of the different fruit parts (Ferreres et al., 2009). The intake of these compounds is an important health-protecting factor. These bioactive compounds retard or inhibit lipid autoxidation by acting as radical scavengers and, consequently, are essential antioxidants that protect against the propagation of the oxidative chain (Navarro et al., 2006). Evidence for their role in the prevention of degenerative diseases is emerging. Experimental studies on animal and human cell lines have demonstrated that polyphenols can play a role in preventing cancer and cardiovascular diseases, when taken daily in adequate amounts (Wijngaard, et al., 2009). The determination of phenolic compounds in plant parts and other foods has been of increasing interest in recent years (Palma et al., 2002). Therefore, the objective of the 7
Chapter two
Literature review
present review is to show the classification of the polyphenolic compounds, taking into account different aspects related to these compounds. Moreover, our aim is to examine the various methods used for preparing and/or treating samples to determine the phenolic content in plants including the different factors that affect the content in plant bioactive compounds, such as light, temperature, mineral nutrition, pathogens, mechanical damage, plant-growth regulators, and other factors (Dinelli et al., 2006). Polyphenols have been a feature of plants since their early appearance. These compounds, also called secondary metabolites, are indeed crucial for many important functional aspects of plant life, including structural roles in different supportive or protective tissues, involvement in defense strategies, and signaling properties, particularly in the interactions between plants and their environment. Collectively, higher plants synthesise several thousand different known phenolic compounds, and the number of these which have been fully characterized is continually increasing (Boudet, 2007). According to the epidemiological studies, the intake of phenolic compounds is inversely correlated with the risk of coronary heart disease (Aparicio-Fernandez et al., 2005 and Fang et al, 2005). In the human body, these phytochemical compounds are thought to provide health benefits by several mechanisms, including: (1) free-radical scavenging (2) Protection and regeneration of other dietary antioxidants (i.e. vitamin E) (3) Chelating of pro-oxidant metal ions The qualitative and quantitative aspects of phenolic compounds vary dramatically among plants, and their different structures or levels are likely to have different functional properties (Huang et al, 2007 and Magalhães et al., 2009). Besides the general properties of the compounds, a number of polyphenolic compounds, especially Catechin, have been found to be potent antioxidants and to be effective in preventing cancer (Costa et al., 2009). While tannins have been reported to exert other physiological effects; e.g. they can reduce blood pressure, accelerate blood clotting, lower serum-lipid levels, modulate immunoresponses and cause liver necrosis (Muchuweti et al., 2006). As mentioned above, it is impossible to separate the closely relationship between the structure and properties of polyphenolic. The structure of phenolic compounds is a key determinant of their radical scavenging and metal-chelating activity. For example, in the case of phenolic acids, the antioxidant activity depends on the numbers and positions of 8
Chapter two
Literature review
the hydroxyl groups in relation to the carboxyl functional group. Thus, the antioxidant activity of phenolic acids increases with higher degree of hydroxylation (Naczk and Shahidi, 2006). As a result, it is important to analyze the composition of phenolic compounds in all plants before their health-promoting properties can be adequately studied. The analysis of phenolic compounds in plant samples is difficult because of the great variety of their structure and the lack of appropriate standards (Huang et al., 2007 and Magalhães et al., 2009). In the secondary carbon metabolism malonic acid and Shikmic acid pathways are key biosynthetic intermediates for the formation of the secondary metabolite phenolic compounds (Mann et al., 1994 and Zhang et al., 2004). Acetyl co-enzyme A, mevalonic acid and methyl-erythritol-4-phospate (MEP) play a key role in the synthesis of various terpenoids (Ramawat, 2004). As shown in (figure 1). Figure (2) showed Schematic of Anthocyanins biosynthetic pathways, and produce some other phenolic compounds such as Stilbenes, Coumarin, Tannin and Flavonols and the key enzymes involved. Branched pathways leading to other metabolites are also indicated. Phenylalanine Ammonia Lyase (PAL); cinnamate-4-hydroxylase (C4H); 4coumarate-CoA ligase (4CL); acetyl-CoA synthetase (AS); acetyl-CoA carboxylase (AC); chalcone synthase (CHS); chalcone isomerase (CHI); flavanone 3β-hydroxylase (F3H); flavonoid 3′-hydroxylase (F3′H); dihydroflavonol 4-reductase (DFR); leucoanthocyanidin dioxygenase (LDOX (ANS)); UDP-glucose:cyanidin 3-O-glucosyltransferase (UFGT) (Zhang et al., 2004).
9
Chapter two
Literature review
Figure 1: a simplified view of the major pathways of secondary carbon-metabolite biosynthesis in plant cells taken from (Zhang et al., 2004).
01
Chapter two
Literature review
Figure 2: Schematic of Anthocyanins biosynthetic pathways taken from (Zhang et al., 2004).
2.2.1.1 Some type of active medicinal compounds in plant 2.2.1.2 Tannins Tannic acid is a polymer of gallic acid molecules and glucose as shown in (figure 3). There are 3 gallic acid molecules, but normally there are about 8. Since there are different molecular structures for tannic acid it would be better to discuses tannic acids in plural. Tannic acid hydrolyzes into glucose and Gallic or ellagic acid units. Tannic acid is odorless but has a very astringent taste. Pure tannic acid is a light yellowish and amorphous powder (Ramakrishnan and Krishnan, 1994). Oak tree is very rich in tannic acid. High levels of tannic acid are found in some plant galls. These are formed by plants when they are infected by certain insects. These insects pierce the plant leaves and when 00
Chapter two
Literature review
the egg hatches out into a larva, the plant produces a gall which surrounds the larva (Evans, 2002). Tannic acid has anti-bacterial, anti-enzymatic and astringent properties. Tannic acid has constringing action upon mucous tissues such as tongue and inside of mouth. The ingestion of tannic acid causes constipation and can be used to treat diarrhea (in the absence of fever or inflammation). The anti-oxidant and anti-mutagenic properties of tannic acid are beneficial; however, tannic acid should not be used continuously or in high quantities as it slows down the absorption of iron and possibly other trace minerals. A study by Afsana "Reducing effect of ingesting tannic acid on the absorption of iron, but not of zinc, copper and manganese by rats" published by Bioscience, Biotechnology, and Biochemistry in March 2004, concluded that the usual intake of polyphenols is relatively safe, but that a high intake by supplementation or by dietary habit of tannin affects only the iron level. Tannic acid can also reduce the effectiveness of digestive enzymes. Externally, tannic acid is used to treat ulcers, toothache and wounds. The best known is the tanning of leather. Tannic acid is sometimes used to clear wines. Tannic acid reacts with proteins in wine to form insoluble complexes and the sediment can be filtered (Chung et al., 1998). 2.2.1.3 Ellagic acid Ellagic acid is a fused four-ring polyphenol, displayed in (figure 4). Pure ellagic acid is a cream to light yellow crystalline solid. Ellagic acid (EA) is a naturally occurring plant polyphenol, present in high concentrations in various fruits and nuts regularly consumed by humans. EA has been shown to possess numerous anticarcinogenic and antimutagenic properties towards a variety of different carcinogens, including nitrosamines, azoxymethane, mycotoxins and polycyclic aromatic hydrocarbon. EA has also antiviral and antibacterial activities. In plants EA is present in the form of ellagitannin, which is ellagic acid bound to a sugar molecule (Glen and Halverson, 2001). EA has antioxidant, anti-mutagen and anti-cancer properties. Studies have shown the anti-cancer activity on cancer cells of the breast, oesophagus, skin, colon, prostate and pancreas. More specifically, EA prevents the destruction of P53 gene by cancer cells. EA can bind with cancer causing molecules, thereby making them inactive. EA causes a decrease in total hepatic mucosal cytochromes and an increase in some hepatic phase II enzyme activities, thereby enhancing the ability of the target tissues to detoxify the 01
Chapter two
Literature review
reactive intermediates (Ahn et al., 1996). EA showed also a chemo protective effect against various chemically induced cancers. A study by Thresiamma and Kuttan (1996)., Indian Journal Physiology and Pharmacology, indicate that oral administration of ellagic acid by rats can circumvent the carbon tetrachloride toxicity and subsequent fibrosis of the liver. 2.2.1.4 Anthocyanins Anthocyanins are water-soluble phytochemicals with a typical red to blue color. Anthocyanins belong to the group of Flavonoids compounds that are responsible for the colors of many flowers, vegetables, fruits and berries, in addition to their value as a source of natural food colorants (Pazmiño et al., 2001). Anthocyanins polyphenolic molecules containing 15 carbon atoms which can be visualized as two benzene rings joined together with a short three carbon chain see (figure 5). They can be found in tissues of plants, including leaves, stems, roots, flowers and fruits. Anthocyanins occur mainly as glycosides of Anthocynidins such as cyanidin, delphinidin, pelargonidin, and Malvidin. Anthocyanins can be found in numerous plants. Although anthocyanins are powerful antioxidants in vitro, their real biological activity will be low because of their low stability and poor absorption. Most studies on the potential health benefits of anthocyanins have been focused on its effect on cardiovascular health, its anti-cancer activity and antiinflammatory properties (Tamura and Yamaganci, 1994; Tsai et al., 2002). Moreover, the identification of anthocyanins may help to better understanding of environment-induced changes in plant secondary metabolism (Harborne, 1993). and of relation between the chemical composition of vegetable tissues and specific biological/pharmacological activities (Larson, 1995). Plants produce anthocyanins for many different reasons. Animals are attracted to the red and purple colours in fruits, whereas the bright Anthocyanin colors in flowers attract insects for pollination. Anthocyanins also protect the plant cells against damage caused by UV radiation (Luigia et al., 2007). 2.2.1.5 Gallic acid Pure Gallic acid is a colorless crystalline organic powder. Gallic acid occurs as a free molecule or as part of a tannin molecule explained in (figure 6). and is found in almost all plants. Plants known for their high gallic acid content include gallnuts, grapes and oak bark. Gallic acid seems to have anti-fungal and anti-viral properties. Gallic acid acts as antioxidant and helps to protect our cells against oxidative damage (Ow and Stupans, 03
Chapter two
Literature review
2003). Gallic acid was found to show cytotoxicity against cancer cells, without harming healthy cells. Gallic acid is used a remote astringent in cases of internal haemorrhage. Gallic acid is also used to treat albuminuria and diabetes. Some ointment to treat psoriasis and external haemorrhoids contains gallic acid. Gallic acid is also used for making dyes and inks. Gallic acid does not combine with protein and has therefore no astringent taste (Kumar et al., 2013).
Fig. 3: Chemical structure of Tannin
Fig. 5: Chemical structure of Anthocyanins
Fig. 4: Chemical structure of Ellagic Acid
Fig. 6: Chemical structure of Gallic acid
2.2.1.6 Four steps in analyzing all types of phenolic compounds The four common steps for any analytical method are sampling, sample preservation, sample preparation, and analysis (separation and detection) (Mitra and Brukh, 2003). Over 90% of the development made during the past few decades has focused on the final analysis step. Remarkable developments in instrumentation, spectroscopy, and chromatography have resulted in rapid advancement of methods for high-through 04
Chapter two
Literature review
separation and detection of complex multi-component mixtures containing trace quantities of the analytes of interest. However, there has been limited research in the area of sampling, sample preservation and sample preparation, which are the basic foundations for developing a quality and rugged analytical procedure. It is well documented in the literature that approximately 60% of analysis time is spent in sample preparation and around 30% of analytical error stems from the sample preparation step (Majors, 1999). 2.2.1.6.1 Sampling The initial step in any analysis is sampling, where a representative sample is collected from the entire sample matrix that needs to be analyzed. The sample is obtained in such a way that it truly represents the entire sample. It is often observed in published manuscripts that details about the samples are poorly documented. Authors often overlook and fail to provide information regarding what part of the fruit, vegetable or plant material was used during analysis; for example, whether peels or seeds of the fruits or vegetable were included as a part of sample during analysis or only the fresh or edible part was used (Bocco et al., 1998). 2.2.1.6.2 Sample preservation This is an important step as there is often some delay between sample collection and analysis. Proper sample preservation ensures that the sample retains its physical and chemical characteristics from the time it is collected to the time it is analyzed (Mitra and Brukh, 2003). the excellent example depicting the influence of sample preservation on assay of the phenolic compounds is the following Over 50% reduction in the levels of three components, namely total flavonoids, total caffeoyl-quinic and total sinapic and feruloyl derivatives, were obtained when samples were either stored at 1◦C for 7 days or at 15◦C for 3 days. Therefore it is essential to inactivate all enzymatic, metabolic, and chemical reactions during the sample preservation step to maintain accurate sample identity, and demonstrate the effectiveness of such procedures in any report. Thus researchers need to study in detail the influence of storage temperature on the analytes of interest and preserve samples under appropriate conditions (Vallejo et al., 2003). 2.2.1.6.3 Sample preparation This step may consist of multiple steps such as sample drying, homogenization, sieving, extraction, pre-concentration, derivatization, and hydrolysis. The motive behind sample preparation can be multi-fold: to increase the efficiency of an assay procedure, to 05
Chapter two
Literature review
eliminate or reduce potential interferences, to enhance the sensitivity of the analytical procedure by increasing the concentration of the analytic in the assay mixture, and sometimes to convert the analyte of interest to a more suitable form that can be easily separated, detected, and/or quantified. Phenolic compounds are known to exist as free glycons or as conjugates with sugars or esters, or as a polymer with multiple monomeric units (Naczk and Shahidi, 2004). It is also well documented that phenolic compounds are not uniformly distributed and may be associated with other cellular components such as cell walls, carbohydrates, or proteins. In addition, the stability of phenolic compounds varies significantly; some are relatively stable and others are thermally labile, unstable, and easily prone to oxidation (Antolovich et al., 2000). Therefore, it is practically impossible to develop an efficient and uniform method for extraction of all phenolic compounds with a single solvent system, as polarities of different phenolic compounds vary significantly due to their conjugation status and their association with the sample matrix. Thus, optimization of the sample preparation procedure is essential for the accurate extraction in any report. Thus, researchers need to study in detail the influence of storage temperature on the analytes of interest and preserve samples under appropriate conditions (Mukhopadhyay et al., 2006). The initial step is to check for the existence of multiple forms of the analyte of interest. It may not always be possible to extract multiple forms of an analyte of interest with a single extraction solvent or solvent mixture. One may require multiple solvent mixtures to extract different forms of varying polarities of conjugated mixtures. The second step is to select an extraction technique that will enable the researcher to efficiently extract the analyte of interest from the sample matrix. The third step will be evaluation of extraction solvents and/or solvent mixtures, as polarity matching between the analyte of interest and extraction solvent is critical for optimum extraction. The fourth logical step is optimization of extraction conditions (extraction temperature, number of extraction cycles, matrix particle size and solid-to-solvent ratio, flush volume, pressure, and static time) which are directly dependent on the availability of the extraction technology. Optimization of extraction parameters not only increases extraction efficiency of the analyte of interest but also reduces the solvent consumed and the waste generated during an extraction process (Devanand, 2006).
06
Chapter two
Literature review
2.2.1.6.4 Extraction systems for phenolic compounds Extraction is one of the most important steps in sample pretreatment. Generally, it is a separation process where the distribution of analyses (in this case, a phenolic compound) between two immiscible phases is made in order to arrive at the appropriate distribution coefficient, the extraction procedure is sequential and systematically carried out using an aqueous organic solvent to extract phenolic compounds in plant samples. This traditional method is called liquid-liquid extraction (LLE) and different extraction solvents have been mentioned in the literature such as ethanol, acetone or methanol, or a mixture with water (Ross et al., 2009). Soxhlet system is used to extract the lipidic fraction from food and other solid samples, using suitable solvents. Although it is not specific for phenolic compounds extraction, usually the extraction yields are compared to those obtained with another type of polyphenol extraction systems (Arias et al., 2009). The ultimate goal of sample preparation is to eliminate or reduce potential matrix interferences (Luthria, 2008). The extraction must be performed with the most adequate solvent and under ideally predetermined analytical conditions of temperature and pH. Moreover, it is essential to take account the polyphenolic structure because these compounds may have multiple hydroxyl groups that can be conjugated to sugars, acids or alkyl groups. Thus, the polarities of phenolic compounds vary significantly and it is difficult to develop a single method for optimum extraction of all phenolic compounds. Hence, the optimization of the extraction procedure is essential for an accurate assay of phenolic compounds from different food matrices. In the end, the effort amounts to lowering costs and reducing sampling time during the abovementioned conventional extraction. In any case, the extraction stage is extremely important, as its outcome will determine the release of analytes from the Plant part matrix into the medium, and this in turn will allow the quantitative determination of the extract (Escarpa and Gonzalez, 2008). For this reason, modern extraction and isolation techniques will be described as alternative techniques to considerably reduce solvent consumption and accelerate the extraction process. These modern techniques include: Supercritical Fluid Extraction (SFE), Pressurized Liquid Extraction (PLE), Microwave-Assisted Extraction (MAE), and Accelerate Solvent Extraction, Solid-phase extraction (SPE), Accelerate Solvent Extractor (ASE), and Ultrasound-Assisted Extraction (UAE) (Klejdusa et al., 2009).
07
Chapter two
Literature review
2.2.1.6.5 Liquid-liquid extraction (LLE) Solubility of phenolics is governed by their chemical nature in the plant, which may vary from simple to very highly polymerize. Plant materials may contain varying quantities of phenolic acids, phenylpropanoids, anthocyanins, and tannins, among others. There is a possibility of interaction of phenolics with other plant components such as carbohydrates and proteins that may lead to the formation of complexes that may be quite insoluble. Likewise, the solubility of phenolics is affected by the polarity of solvent(s) used. Therefore, it is very difficult to develop an extraction procedure suitable for the extraction of all plant phenolics. The phenolic extracts from plant materials are always a diversified mixture of plant phenolics soluble in the solvent system used. Additional steps may be required to remove the unwanted phenolics and non-phenolic substances such as waxes, terpenes, fats, and chlorophylls by another solvents; n-Hexan, dichloromethane, chloroform (Gómez et al., 2005; Naczk, and Shahidi, 2006). The extraction methods for simple phenolic compounds (benzoic acids, benzoic aldehydes, cinnamic acids, and catechin) from solid or semi-solid materials have been focused on maceration using organic solvents. The current official analytical method for extracting phenolic compounds is liquid-liquid extraction (LLE) for liquid samples. This method requires expensive and hazardous organic solvents, which are undesirable for health and disposal reasons, and they require a long time per analysis, giving rise to possible degradations. The process of degradation can be triggered both by external and internal factors. Light, together with air and temperature, are the most important factors that facilitate degradation reactions. The extraction temperature usually needs to be high in order to minimize the duration of the process. For these reasons, these traditional extraction sample methods have been replaced by other methodologies which are more sensitive, selective, fast, and environmentally friendly (Liazid et al., 2007). In any case, LLE is still used as the standard pre-concentration step for phenol determination in water because it is a cheap and easy method. Solvents, such as methanol, ethanol, propanol, acetone, ethyl acetate, and their combinations have also been used for the extraction of phenolics, often with different proportions of water. For example, phenolic compounds can be efficiently extracted from plant samples using an methanol/water, ethanol/water and acetone/water (70:30 v: v) system (Prati et al., 2007).
08
Chapter two
Literature review
Generally, LLE is used at room temperature to avoid the degradation of phenolic compounds, but there are many studies such as Costa et al., (2009), Aparicio-Fernández et al.(2005) and Magalhães et al.(2009) using temperatures around 20 to 40 ºC or less, when hydrolysis of phenolic compounds is carried out, the temperature is usually 80–95 ºC for acid hydrolysis or 45 ºC for basic hydrolysis (Nuutila et al., 2003). Otherwise, extraction times depend on several factors such as maceration time, centrifugation time or the time spent on the evaporation of solvents. Anthocyanins are usually extracted from plant material with an acidified organic solvent, most commonly methanol, This solvent system destroys the cell membranes, simultaneously dissolves the anthocyanins, and stabilizes them. However, the acid may bring about changes in the native form of anthocyanins by breaking down their complexes with metals and co-pigments (Naczk and Shahidi, 2006). 2.2.1.7 Purification technique Multilayer silica gel column clean-up: after concentration of the extracts, the clean-up of samples was accomplished using a multilayer silica gel column, and eluate concentration was accomplished using a rotary evaporator. Final concentration was accomplished using the nitrogen evaporation technique. Extract clean-up, using a multilayer silica gel column, was performed after sonication, (ASE), and Soxhlet extraction. This was not performed for SFE (Jeong et al., 1999).
2.2.1.8. Determination of phenolic compounds by different methods 2.2.1.8.1 Colorimetric reactions or Folin-Ciocalteu method Colorimetric reactions are widely used in the UV/VIS spectro-photometric method, which is easy to perform, rapid and applicable in routine laboratory use, and is low cost (Pelozo et al., 2008). However, it is important that colorimetric assay need to use a reference substance, and then this method measures the total concentration of phenolic hydroxyl groups in the plant extract. Polyphenols in plant extracts react with specific redox reagents (Folin-Ciocalteu reagent) to form a blue complex that can be quantified by visible-light spectrophotometry. The Folin-Ciocalteu method is described in several pharmacopoeias. The reaction forms a blue chromophore constituted by a phosphor tungstic - phospho molybdenum complex (Milan and Jana 2007).where the maximum absorption of the chromospheres depends on the alkaline solution and the concentration of phenolic compounds. However, this reagent rapidly decomposes in alkaline solutions, 09
Chapter two
Literature review
which makes it necessary to use an enormous excess of the reagent to obtain a complete reaction. This excess can result in precipitates and high turbidity, making spectrophotometry analysis impossible. To solve this problem, Folin and Ciocalteu included lithium salts in the reagent, which prevented the turbidity. The reaction generally provides accurate and specific data for several groups of phenolic compounds, because many compounds change color differently due to differences in unit mass and reaction kinetics (Andressa et al., 2013). 2.2.1.8.2 High performance liquid chromatography High-performance liquid chromatography (HPLC; formerly referred to as highpressure liquid chromatography), is a technique in analytic chemistry used to separate the components in a mixture, to identify each component, and to quantify each component. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column (Snyder et al., 2009). Quantification of phenolic compounds was achieved by the absorbance recorded in the chromatograms relative to external standards. The concentration of extracts was calculated from peak area according to calibration curves. The amount of each phenolic acid was expressed as microgram per gram of dry weight (μg /g DW) (Suarez et al., 2005 and Ya-Qin et al., 2009).
2.2.2 Volatile oils Essential oils are defined as volatile, natural with a wide range of bioactive complex compounds characterized by a strong odour, formed by plants as aromatic secondary metabolites (Ramanadhan, 2005 and Amar et al., 2012). Obtained from plant materials (flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits and roots). They can be obtained by expression, fermentation, effleurage or extraction; Steam or hydro-distillation is the most commonly used method for the production of Essential oils, first developed in the middle ages. Essential oils are highly concentrated and must be diluted with carrier oil before applying to the skin. At present, approximately 3000 Essential oils are known, 300 of which are commercially important especially for the pharmaceutical, agronomic, food, sanitary, cosmetic and perfume industries (Abi-Ayada et al., 2011). Essential oils are
11
Chapter two
Literature review
complex mixtures comprising of many single compounds (prabuseenivasan et al., 2006). Analysis showed that many of them contain more than 100 individual compounds (pauli, 2006). Chemically they are derived from terpenes and their oxygenated compounds (prabuseenivasan et al., 2006). Oxygenated compounds derived from these hydrocarbons include alcohols, Aldehyde, esters, ethers, ketones, phenols and oxides (Svoboda and Hampson, 1999). Essential oils are used in the cosmetics industry (Chowdhury and Nishteswar, 2013) food industry (Bozin, et al., 2007) aromatherapy (Lis-Balchin, 2009) and as antimicrobial activity (Cardile et al., 2009) the chemical composition of the Essential oils depends on climatic, seasonal, geographic and soil condition; harvest period; and distillation technique (Kan et al., 2000). In pharmacopoeias of European countries, Essential oils are accepted and recommended, e.g. for the treatment of catarrhal in children (1991). In each Indian state about one–third of the governmental medical posts were occupied by physicians who belonged to the traditional systems (pauli, 2006). Various industries are now looking into sources of alternative, more natural and environmentally friendly antimicrobials, antibiotics, antioxidants and crop protection agents. The possibility of utilizing volatile oils is now being investigated. Although their biological activity has been known for centuries, their mode of action was not fully understood. It has been suggested that volatile oils are either inhaled or absorbed by the skin. The most important suggested areas of Essential oils use are in urology, dermatology, sleep and nervous disorders, laxatives, erosive gastritis, cardiac and vascular systems, immune modulating drugs, colds and coughs. Various plant species are being investigated in detail and are being thoroughly tested for their pharmacological properties (Svoboda and Hampson, 1999). These conditions include stress-related illness, formation of stomach ulcers and tumour growth. Their effects on blood circulation, nerve growth, nucleic acid, liver, protein, lipid, and carbohydrate and cholesterol metabolisms are also monitored, as well as any effects on the activity of the adrenal gland and the body's immune system (Svoboda and Hampson, 1999). In the last few years, the search continues for safe and effective antimicrobial agents with which a wide variety of bacterial infections can be treated. This need has been heightened recently by the emergence of many antimicrobial-resistant organisms. The use 10
Chapter two
Literature review
of natural substances such as essential oils for the control of postharvest diseases and their effective usage as antimicrobial agents, as functional ingredients in foods, drinks, toiletries and cosmetics is gaining momentum, both for the growing interest of consumers in ingredients from natural sources and also because of increasing concern about potentially harmful synthetic additives (Abi-Ayada et al., 2011). The antimicrobial properties of essential oils derived from many plants have been empirically recognized for centuries, but is scientifically confirmed only recently (Deans and Ritchie, 1987). Practical uses of these activities have long been suggested in humans and animals, but only in the last few years it has been reported that some essential oils are capable of inhibiting food borne bacteria and extending the shelf-life of processed food (Smith-Palmer et al., 1998). The physical properties and hence the biological efficacy of essential oils vary. Accordingly, research about the properties of essential oils has focused on the active constituents of essential oils instead of focusing on the essential oils themselves. The antimicrobial property of essential oils is attributed to the monoterpenes and monoterpenoids present in these oils (Cardile et al., 2009). The site of action of terpenes and their derivatives is the cell membrane (Pasqua et al., 2007). Due to their lipophilicity, terpenes and their derivatives insert in the membrane, causing membrane expansion and increased membrane fluidity/ permeability that subsequently affects the transport processes (Sikkema et al., 1994). These actions contribute to the antimicrobial activity of terpenes. Studies on the effects of selected Essential oils components on outer membrane permeability in Gram-negative bacteria have shown that the monoterpene uptake is partly determined by the permeability of the outer envelope of the microorganism under investigation (Helander et al., 1998). 2.2.2.1 General remarks of essential oils uses Essential oils have become an integral part of everyday life. They are used in a great variety of ways: as food flavorings, as feed additives, as flavoring agents by the cigarette industry, and in the compounding of cosmetics and perfumes. Furthermore, they are used in air fresheners and deodorizers as well as in all branches of medicine such as in pharmacy, balneology, massage, and homeopathy (Vilaplana and Romaguera, 2002).
11
Chapter two
Literature review
2.2.2.2 Factors affecting the yield and quality of essential oils A number of important agronomic factors have to be considered before embarking on the production of essential oils, such as climate, soil type, influence of drought and water stress and stresses caused by pathogen, propagation (seed or clones), and cultivation practices. Other important factors include precise knowledge on which part of the biomass is to be used, location of the oil cells within the plant, timing of harvest, method of harvesting, storage, chemotaxonomy and preparation of the biomass prior to essential oil extraction (Kan et al., 2000). 2.2.2.2.1 Soil pH Soil pH affects significantly oil yield and oil quality. Figueiredo et al., (2005) found that the pH value "strongly influences the solubility of certain elements in the soil. Iron, zinc, copper and manganese are less soluble in alkaline than in acidic soils because they precipitate as hydroxides at high pH values" It is essential that farmers determine the limits of the elemental profile of the soil. Furthermore, the spacing of plantings should ensure adequate supply with essential trace elements and nutrients. Selection of the optimum site coupled with a suitable climate plays an important role as they will provide a guarantee for optimum crop and essential oil quality (Figueiredo et al., 2005). 2.2.2.2.2 Water stress and drought It is well known to every gardener that lack of water, as well as too much water, can influence the growth of plants and even kill them. The tolerance of the biomass to soil moisture should be determined in order to identify the most appropriate site for the growing of the desired plant. Since fungal growth is caused by excess water, most plants require well-drained soils to prevent their roots from rotting and the plant from being damaged, thus adversely affecting essential oil production. Lack of water, for example, dryness, exerts a similar deleterious influence. Flowers are smaller than normal and yields drop. Extreme drought can kill the whole plant as its foliage dries closing down its entire metabolism (Yanive and Palevitch, 1982). 2.2.2.2.3 Insect stress and micro-organisms Plants are living organisms capable of interacting with neighbor plants and warning them of any incipient danger from insect attack or pathogen factors. These warning signals are the results of rapid changes occurring in their essential oil composition, which
13
Chapter two
Literature review
are then transferred to their neighbors in turn, transmit this information on to their neighbors forcing them to change their oil composition as well. In this way, the insect will come into contact with a chemically modified plant material, which may not suit its feeding habits thus obliging it to leave and look elsewhere. Microorganisms can also significantly change the essential oil composition as shown in the case of elderflower fragrance. Headspace gas chromatography coupled with mass spectroscopy (GC/MS) has shown that linalool, the main constituent of elderflowers, was transformed by a fungus present in the leaves, into linalool oxide (Erich, 2010). 2.2.2.2.4 Timing of the harvest The timing of the harvest of the plant or herbal crop is one of the most important factors affecting the quality of the essential oil. It is a well-documented fact that the chemical composition changes throughout the life of the plant. Occasionally, it can be a matter of days during which the quality of the essential oil reaches its optimum. Knowledge of the precise time of the onset of flowering often has a great influence on the composition of the oil. The chemical changes occurring during the entire life cycle of Vietnamese Artemisia vulgaris have shown that 1, 8-cineole and β-Pinene contents before flowering were below 10% and 1.2%, respectively, whereas at the end of flowering they reached values above 24% and 10.4% (Nguyen et al., 2004). 2.2.2.2.5 Location of oil cells As already mentioned, the cells containing essential oils can be situated in various parts of the plant. Two different types of essential oil cells are known, superficial cells, for example, glandular hairs located on the surface of the plant, common in many herbs such as oregano, mint, lavender, and so on, and cells embedded in plant tissue, occurring as isolated cells containing the secretions (as in citrus fruit and eucalyptus leaves), or as layers of cells surrounding intercellular space (canals or secretory cavities), for example, resin canals of pine. Professor Dr. Johannes Novak (Institute of Applied Botany, Veterinary University, Vienna) has shown impressive pictures and pointed out that the chemical composition of essential oils contained in neighboring cells (oil glands) could be variable but that the typical composition of a particular essential oil was largely due to the averaging of the enormous number of individual cells present in the plant (Novak, 2005). It has been noted in a publication entitled “Physiological Aspects of Essential Oil Production” that those individual oil glands do not always secrete the same type of 14
Chapter two
Literature review
compound and that the process of secretion can be different (Kamatou et al., 2006). Different approaches to distillation are dictated by the location of the oil glands. Preparation of the biomass to be distilled, temperature, and steam pressure affect the quality of the oil produced.
2.2.2.3 Phytochemical variation and Chemotaxonomy The ability to accumulate essential oils is not omnipresent in plants but scattered throughout the plant kingdom; in many cases, however, very frequent within—or a typical character of—certain plant families. From the taxonomical and systematic point of view, not the production of essential oils is the distinctive feature since this is a quite heterogeneous group of substances, but either the type of secretory containers (trichomes, oil glands, lysogenic cavities, or schizogenic oil ducts) or the biosynthetically specific group of substances, for example, mono- or sesquiterpenes, phenylpropenes, and so on. (Vetter et al, 1997). 2.2.2.3.1 α- Pinene One of the common antimicrobial monoterpenes found in several essential oils is αPinene shown in (figure 7). The strained four- member ring of α- Pinene is very reactive and makes it prone to opening and skeletal rearrangements (Kamath and Ojima, 2012). In addition to research about the antimicrobial activity of monoterpenes/monoterpenoids, there has been some recent work by Wilder man and colleagues that has shown that (+) αPinene is a potent P450 2B inhibitor (Wilderman et al., 2013). With microbes becoming increasingly resistant to known antibiotics, it is imperative to develop drugs that are active against the next generation of microbes. Both enantiomers of α-Pinene are readily available, and hence it is an excellent starting material for synthesizing a variety of functionally and skeletally altered α-Pinene derivatives. In this study, investigated the structure−antimicrobial activity relationship for both (+) and (−) α-Pinene and the corresponding
derivatives.
Antimicrobial
activity was
assessed
on
four
test
microorganisms (Staphylococcus aureus, Micrococcus luteus, Escherichia coli, and Candida albicans) using a bioautographic assay. This bioassay gave good reproducibility (Rios et al., 1988 and Brantner, 1997).
15
Chapter two
Literature review
2.2.2.3.2 Limonene Pure limonene is a clear liquid. Limonene is a monoterpene, made up of two isoprene units displayed in (figure 8). Limonene occurs in two optically active forms, l-limonene and d-limonene. Both isomers have different odours: l-limonene smells piney and turpentine like and d-limonene has a pleasing orange scent. One Phytochemical of particular importance in cancer prevention is d-limonene (also known as limonene). Limonene is a widely distributed, natural, nontoxic compound found in citrus fruits, spices, herbs, and some conifer essential oils. Squeezing orange and lemon rinds and collecting the oily residue concentrates limonene. In fact, the essential oils of grape fruit, tangerine, and orange contain over 90 percent limonene (Bonithon-Kopp et al., 2000). This simple monocyclic monoterpene compound has been found in nearly 100 studies in animals and in humans to prevent cancer, stop the progression of cancer, and destroy and dismantle cancer. Limonene has been shown to be active against several types of tumors, including mammary, skin, lung, liver an fore stomach in rodents and colon and breast cancer in humans. Incorporating limonene in your diet is a healthy lifestyle choice that helps promote normal cell life cycles. Studies have shown that limonene have anti- cancer effects. Limonene increase the levels of liver enzymes involved in detoxifying carcinogens. The Glutathione Stransferase (GST) is a system which eliminates carcinogens (toxogenices). Limonene seems to promote the GST system in the liver and small bowel, thereby decreasing the damaging effects of carcinogens. Animal studies demonstrated that dietary limonene reduced mammary tumor growth. Limonene is also used as a solvent and cleaner. It can replace white spirit and other solvents (Elegbede et al., 1984 and Bonithon-Kopp et al., 2000).
2.2.2.3.3 Myrtenol Medicinal plants have been used in many cultures for thousands of years and information on the use of natural resources has played a vital role in the discovery of novel products from plants as chemotherapeutic agents (Maria et al., 2012). Although a considerable number of analgesic and anti-inflammatory drugs are available for the treatment of pain and inflammation, there is a continuous search for new compounds as therapeutic alternatives, because these drugs exert a wide range of side effects and low efficacy, especially for chronic diseases (Maria et al., 2012). In this context, natural products have been one of the most successful sources for the discovery of new therapeutic agents to benefit those afflicted by inflammatory diseases (Alan, 2000). 16
Chapter two
Literature review
Essential oils are volatile compounds produced as secondary metabolites by medicinal plants, and have been reported to exhibit a variety of biological properties; the majority of these effects is attributed to monoterpenes, Myrtenol is a monoterpene alcohol and found in the essential oils of some aromatic plants represented in (figure 9), and can also be obtained through the oxidation of the α-Pinene ( Bell et al., 2003). Although it is used for its flavoring properties, several studies have shown that Myrtenol has sedative properties (DeSousa, 2007) and hypotensive (Márcio et al., 2011) and inhibitory effects against the growth of harmful intestinal bacteria (Haribalan et al., 2013).
Fig. 7: Chemical structure of Pinene
Fig. 8: Chemical structure of Limonene
Fig. 9: Chemical structure of Myrtenol
2.2.2.4 Commercial essential oil extraction methods There are three methods in use. Expression is probably the oldest of these and is used almost exclusively for the production of Citrus oils. The second method is hydro distillation or steam distillation while dry distillation is used only rarely in some very special cases.
17
Chapter two
Literature review
The most commonly used one of the three methods is hydro-distillation of the plant material performed in a Clevenger-type apparatus for 3 hour. The oil obtained was light yellow, liquid at room temperature and its odor was agreeable. After its isolation, the essential oil is collected and stored in steeled glass vials in refrigerator at 0 -15°C (AbiAyada et al., 2011).
2.2.2.5 Determination of volatile oils by different methods Preparation of volatile oils for detection (analysis) by HPLC is a technique in analytic chemistry used to separate the components in a mixture, to identify each component, and to quantify each component. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column (Snyder et al., 2009). GC (Nadhir et al., 2010) and GC-MS (Kafkas et al., 2007) are other standard techniques for analyzing the volatile fractions of essential oils.
2.2.3 Previously isolated compounds of the genus Pistacia Phenolic composition of the most Pistacia species consisting of gallic acid, Catechin, anthocyanins and Tannin extracted from pistacia areal parts (Romani et al., 2002; Mahbubeh et al., 2013). Essential oils of pistacia species contain high amount of some compounds (Pinene, limonene, Myrecene, phellandrene and Sabinene, etc. (Duru, et al., 2003), (Demirci et al. 2001) investigated the composition of micro-distilled and hydro-distilled essential oils of the mastic gum of P. eurycarpa by GC/MS. The same authors detected α- and β-Pinene as the major constituents, and results showed (α-Pinene 66 % - 75%, β-Pinene 4.9 % - 5.6%, Myrecene 0.1 % - 0.2%, limonene 1.3 % - 1.6%, phellandrene 0 – 0.1%, and Sabinene 0.4 % - 0.5%) respectively Mahbubeh et al., (2013) investigated both essential oils (twigs/leaves) of P. lentiscus contained, at different levels. These are the major components: α- Pinene (3.8 / 1.6%), Myrecene (34.1 / 25.3%), and limonene (9.6 / 15.7%). These components account for approximately 47.5% and 42.9% of the chemical composition of twigs and leaves
18
Chapter two
Literature review
essential oils respectively. In general, the two chemical compositions are quantitatively and qualitatively different. This seems obvious because the structure of the two plant parts is very different. A previous study has also shown that there is a difference between the chemical compositions of essential oils extracted from different plants’ parts. Others were (α-Pinene 20.6 %, Sabinene 1.9 %, β-Pinene 9.6 %, Myrecene 3.4 %, limonene 15.3 % and terpinene 8.2% are detected by Amri et al., (2012) from pistacia lentiscus L. Taran et al., (2010b) researches investigated that some major constituents of essential oil from the aerial parts of P. khinjuk are (α-Pinene 2.11%, Sabinene 1.67%, β-Pinene 1.49%, Myrecene 2.85%, 3-carene 0.29%, limonene 0.58% and terpinene 1.15% and according to Ghasemi and Alghaee (2011) P. khinjuk contain four main compounds of fruit essential oils: phellandrene 52.33%, α-Pinene 15.28 %, limonene 4.08 % and Sabinene 0.9 %.
2.2.3.1 Traditional uses of some Pistacia species In traditional uses, plant parts used and their pharmacological activities of P. khinjuk, P. eurycarpa, P. lentiscus, P. terebinthus, and P. vera from different regions. Different parts of Pistacia species including resin, leave, fruit and aerial part have been traditionally used for a wide range of purposes. Among them, P. lentiscus is the most commonly used in different regions and resin of that has been utilized for as long as 5000 years. Resin of P. lentiscus has been used for a variety of gastric ailments in the Mediterranean and Middle East countries for the last 3000 years (Dimas et al., 2009). Most of the traditional uses reports for resin of P. atlantica is from Middle East and have been used for the treatment of digestive, hepatic and kidney diseases (Avicenna, 2008). Fruit of P. vera (pistachio) is used all over the world. Records of the consumption of pistachio as a food date to 7000 BC (DerMarderosian, and Beutler, 2010). Pistachio is cultivated in the Middle East, United States and Mediterranean countries. Iranian Agricultures is one of the biggest producers and exporters of pistachio nuts (Kashaninejad et al., 2006). In Traditional Iranian Medicine (TIM), different parts of P. vera, P. eurycarpa, P. khinjuk P. terebinthus, and P. lentiscus have been used for a long time as useful remedies for different diseases. For example, the fruit kernel of P. vera as a cardiac, stomach, hepatic, and brain tonic; the fruits of P. atlantica, P. khinjuk and P. terebinthus for its aphrodisiac activity and treatment of liver, kidney, heart and respiratory system 19
Chapter two
Literature review
disorders, and the gum resin of P. lentiscus, P. atlantica, P. khinjuk and P. terebinthus for its wound healing activity, and treatment of brain and gastrointestinal disorders (Avicenna, 2008).
2.2.3.2 Pharmacological aspects of the genus Pistacia Different parts of P. eurycarpa and P. khinjuk have been used in traditional medicine for various purposes like tonic, aphrodisiac, antiseptic, antihypertensive and management of dental, gastrointestinal, liver, urinary tract, and respiratory tract disorders. Scientific findings also revealed the wide pharmacological activities from various parts of these species,
such
inflammatory,
as
antioxidant,
antimicrobial,
antinociceptive,
antidiabetic,
antiviral,
anticholinesterase,
anti-tumor,
anti-
antihyperlipidemic,
antiatherosclerotic, and hepatoprotective activities and also their beneficial effects in gastrointestinal disorders, various types of phytochemical constituents like terpenoids, phenolic compounds, fatty acids, and sterols have also been isolated and identified from different parts of Pistacia species (Mahbubeh et al., 2013).
31
Chapter three
Materials & Methods
Materials and Methods 3.1. Materials and Equipments Table 3: List of materials No.
Material
Source
1
Ferric Chloride (FeCl3)
Sigma, Germany
2
Ethanol (CH3CH2OH)
Scharlau , Spain
3
Methanol (CH3OH)
Scharlau, Spain
4
Acetone (CH3)2CO)
Scharlau, Spain
5
Chloroform (CHCl3)
Scharlau, Spain
6
n- hexane(C 6 H 14)
Scharlau, Spain
7
Sulfuric acid(H2SO4)
Himedia , India
8
Di-chloromethane (CH2Cl2)
Sigma, Germany
9
Sodium citrate (NaH2C6H5O7)
Fluka, Germany
10
Potassium hydroxide(KOH)
Fluka, Germany
11
Ammonium Molybdat [(NH4)6Mo7O24.4H2O]
Fluka, Germany
12
Calcium Carbonate (CaCO3)
BDH, England
13
Deionized Distilled water (DDH2O)
College of Agriculture
14
Hydrogen chloride (HCl")
Fluka, Germany
15
Bismuth sub nitrate
NFPA, United kingdom
16
Potassium iodide(KI)
Sigma, Germany
17
Mercuric chloride ( HgCl2)
Fluka, Germany
18
sodium hydroxide (NaOH)
MERCK, Germany
19
Rochelle's salt (KNaC4H4O6·4H2O)
NFPA, United kingdom
20
Iodine (I)
Sigma , Germany
21
Cupric sulphate (CuSO4)
MERCK, Germany
22
Lead acetate Pb(CH3COO)
MERCK, Germany
23 24
Acetic acid anhydride (CH3COOH) Sodium sulfide(Na2SO4)
Sigma, Germany MERCK, Germany
13
Chapter three
Materials & Methods
Table 4: List of equipments
No.
Equipments
Source
1
Water Deionizer
Human Power Integrate
2
Sensitive Balance
Mettler Toledo
3
Rotary Evaporator
Heidolph
4
Drying Oven
Fisher Scientific
5
Hot plate magnetic stirrer
Bibby sterilin
6
Water bath
BUCHI
7
Inductive Couple Plasma (ICP)
Perkin Elmer
8
Clevenger
Lab TECH
9
Thermo circulator (chiller )
Lab TECH
10
Water purification system
BUCHI
11
Dark glass
Biology department
12
Filter paper
Wattman
13
High Performance Liquid Chromatography (HPLC)
Shimadzu , koyota , Japan
13
Chapter three
Materials & Methods
3.2 Methods 3.2.1 Sample collection and identification Fresh samples of the two Pistacia species; p. eurycarpa and p. khinjuk were collected during premature samples in May and during mature samples in September in 2013 from Barbard– Mawat region. The studies site is located at benchmark of (34*54'22''N and 45*23'54''E) with altitude of 900 m.a.s.l with a slope of NE. Mawat site is located 55 Km NE of Sulaimani city. Photos presented in figure 10 show sample collection locations in Barbard mountain forests. Samples were identified at the Herbarium of the Department of Crops Science, College of Agriculture-University of Sulaimani by Dr. Saman A. Ahmad. Plant samples were identified in the location depending on the variation among the plant species description, variation in leaves ,seed and fruit , size , shape , stem , gum also the presence of the plant at different altitudes according to the key that is available in Flora of Iraq. (Figures 11-24) show the photo of the tree or shrub, leaves, gum with gum collector, and cluster of mature and immature fruit of both species.
3.2.2 Sample preparation After collection, samples were prepared for analysis and investigation in Botany Research Laboratory/ Biology Department/ College of Science/ University of Sulaimani. Leaf, seed, outer shell, cluster without fruit, were air dried at room temperature and ground to a fine powder by electric blender, then stored in plastic containers at 4Cº. Each plant parts (Leaf, seed, outer shell, cluster without fruit) separately were prepared. But gum accumulated in the gum collector (K. kojila) not needed drying and ground.
3.2.3 Preparation of plant extracts 3.2.3.1 Aqueous extracts preparation The aqueous extract of each sample was prepared by soaking 15 gm of the dry ground sample in 100 ml of DDH2O for 24 hour. The extract was centrifuged for 10 min. at 2500 rpm then filtered using Wattman filter paper No.42. All extracts were placed in Rotary evaporation under vacuum at 750C to obtain dried extract. One gm of dry powdered extract was dissolved in 5 ml DDH2O. A standard procedure for identification of the various classes of Phytochemical constituent was used (Abdul-Aziz, 2005).
11
Chapter three
Materials & Methods
Figure 10: Barbard-Mawat Location the Wild habitat of the P. eurycarpa and P. khinjuk where samples were collected
13
Chapter three
Materials & Methods
3.2.3.2 Preparation of alcoholic extracts Alcoholic extract of each sample was prepared by added 15 gm of the dry ground sample in 100 ml of 70% ethanol for 24 hour. The extract centrifuged for 10 min. at 2500 rpm. , then filtered using Wattman filter paper No.42. All extracts were placed in rotary evaporated under vacuum at 300C. One gm of dry powdered extract was dissolved in 5 ml of absolute ethanol. A standard procedure for identification of the various classes of Phytochemical constituents was used (Okogun, 2000).
3.2.4 Preparation of reagents 3.2.4.1 Fehling reagent preparation A Fehling A. quantity of 35 gm of cupric sulphate (CuSO4) was dissolved in 100 ml of DDH2O, and then the volume was completed to 500 ml of DDH2O. B. Fehling B. quantity of 7 gm of sodium hydroxide (NaOH) with 175 gm of Rochelle's salt was dissolved in 100 ml DDH2O, and the volume was completed to 500 ml of DDH2O. C. Equal volumes from (A) and (B) solutions were mixed together (vogel, 1974). 3.2.4.2 Wagner's reagent preparation A quantity of 2 gm of potassium iodide (KI) with 1.27 gm iodine (I2) was dissolved in 5 ml of DDH2O, the volume was completed to 100 ml with DDH2O (Smolensk et al., 1972) 3.2.4.3 Benedict's reagent preparation A. A quantity of 137 gm sodium citrate (NaH2C6H5O7) with 117 gram sodium carbonate anhydride (Na2CO3.H2O) was dissolved in 700 ml DDH2O. B. A quantity of 17.3 gm cupric salphate (CuSO4) was dissolved in 100 ml of DDH2O. C. (A) And (B) solutions were mixed gently; the volume was completed to 1000 ml of DDH2O (Vogle, 1974). 3.2.4.4 Mayer's reagent preparation A. quantity of 1.58 gm mercuric chloride (HgCl2) was dissolved in 60 ml of DDH2O B. 5 gram of potassium iodide (KI) was dissolved in 10 ml of DDH2O. C. (A) And (B) solutions were mixed, and the volume was completed to 100 ml with DDH2O (Smolensk et al., 1972). 3.2.4.5 Dragendroffs reagent preparation A. Aliquot of 2 ml of concentrated HCl was added to 0.6 gm of Bismuth sub nitrate and then 10 ml of DDH2O was added. B. 6 gm of potassium iodide (KI) was added to 10 ml of DDH2O. 13
Chapter three
Materials & Methods
C. Aliquot of 7 ml of concentrated HCl was added to solution (A) and (B) then mixed; the volume was completed to 400 ml DDH2O (Harborne, 1973). 3.2.4.6 Kidde reagent preparation A. A quantity of 0.5 gm of 3-5 Nitro benzoic was dissolved in 25 ml of methanol (95%). B. A quantity of 2.5 gm of sodium hydroxide (NaOH) 1N was dissolved in 25 ml of methanol (95%). C. Aliquot of 0.4 ml of solution (A) was mixed with 0.6 ml of solution (B) (Al-Shahat, 1986).
3.2.5 Phytochemical detection Preliminary qualitative phytochemical detection is carried out with the following tests. 3.2.5.1 Steroids One ml of the ethanolic extract is added in 10 ml of chloroform, and equal volume of concentrated sulphuric acid is added by sides of the test tube. If the upper layer turns red and sulphuric acid layer shows yellow with green fluorescence, this indicates the presence of steroids (Trease and Evan, 1989). 3.2.5.2 Terpenoids Two ml of ethanolic extract is added to 2 ml of each acetic anhydride and concentration of H2SO4. Formation of blue-green rings indicates the presence of terpenoids (Ayoola, 2008). 3.2.5.3 Fatty acids Amount of 0.5 ml of ethanolic extract is mixed with 5 ml of ether. These extracts were allowed to evaporate on filter paper and dried the filter paper. The appearance of transparence on filter paper indicates the presence of fatty acids (Ayoola, 2008). 3.2.5.4 Anthocyanins Two ml of aqueous extract is added to 2 ml of HCl 2N and ammonia. The appearance of pink-red turning blue-violet indicates the presence of anthocyanins (Paris, 1969). 3.2.5.5 Leucoanthocyanins Two ml of aqueous extract added to 5 ml of isoamyl alcohol. The appearance of red upper layer indicates the presence of Leucoanthocyanins (Paris, 1969). 3.2.5.6 Tannins Two ml of the ethanolic extract is added to few drops of 1% lead acetate. A yellowish precipitate indicates the presence of tannins (Trease and Evan, 1989).
13
Chapter three
Materials & Methods
3.2.5.7 Saponins Five ml of ethanolic extract was mixed with 20 ml of distilled water and then agitated in a graduated cylinder for 15 minutes. Formation of foam indicates the presence of saponins (Kumar, 2009). 3.2.5.8 Coumarins Three ml of 10% NaOH was added to 2 ml of aqueous extract. Formation of yellow color indicates the presence of Coumarins (Rizk, 1982). 3.2.5.9 Emodin Two ml of NH4OH and 3 ml of Benzene are added to the ethanolic extract. the appearance of red color layer indicates the presence of emodins (Rizk, 1982). 3.2.5.10 Alkaloids A quantity of 0.2 gm of the alcoholic extract is heated on a boiling water bath with 2N HCl (5ml). After cooling, the mixture is filtered and the filtrate is divided in to three equal portions. One portion was treated with few drops of Mayer’s reagent; the second portion was treated with equal amounts of Dragendroffs and added to the last portion three drops of Wagner's reagent. Turbidity of the resulting precipitate in all portion indicate the presence of alkaloids (Trease and Evans, 1987 and Sharma, 2010). 3.2.5.11 Flavonoids A.A sample 10 gm of dry powdered plant material is dissolved in 5 ml of ethanol (95%) then filtered. B. Aliquot of 10 ml of KOH (50%) was added to 10 ml ethanol (50%). C. Equal volume of (A) and (B) solutions were mixed; development of a yellow color indicates the presence of Flavonoids (Jaffer et al., 1983). 3.2.5.12 Phenolic acid (phenolic compounds) Five ml of the aqueous filtrate of each plant extract 1-2 drops of 1% of ferric chloride is added, a blue green coloration indicates the presence of phenolic compounds (Harborn, 1973). 3.2.5.13 Glycosides One ml of the aqueous filtrate of each plant extract is placed in a clean test tube, and treated with 2 ml Fehling reagent [1 ml Fehling (A) + 1 ml Fehling (B) ], then placed in water bath for 10 minutes till boiling, and cooled. The appearance of red - brown precipitation indicates the presence of saccharides, and 5 ml benedicts' reagent added to 1 ml of the aqueous plant extract to. Appearance of red precipitate indicates the presence of
13
Chapter three
Materials & Methods
saccharides. Addition of few drops of Kidde reagent if produce violet-blue coloration in each extract indicated the presence of glycosides since (Al-anzy, 2004). 3.2.5.14 Resins Ten ml of methanol (95%) is added to 1 gm dry powdered sample and placed in water bath for 2 minute and filtrated. Then 20 ml of DDH2O was added and mixed with 4% HCl, The appearance of turbidity indicates the presence of Resins (Al-anzy, 2004).
3.2.6. Extraction, preparation and Analysis of samples 3.2.6.1 Extraction of total phenolic compounds Five gm of each plant sample was purified with 100 ml of Dichloromethane for 1 hour to remove pigment, non pigment, lipid and non polar material
The dry samples then extracted (3 times) with 80 ml of 70% aqueous acetone for 2 hour
Discard solvent (dichloromethane) with solutes
Remove insoluble plant parts
Add 4gm of Na2SO4 to remove some water; the extracts were centrifuged for 20 minutes at 2000 rpm to precipitate all debris.
All extracts were pooled in Rotary evaporate under vacuum at 300C to remove acetone,
Then partitioned first with 80 ml of n-Hexane for de-fatting
Remove upper portion (n-Hexane with oil)
Then partitioned with 40 ml of dichloromethane to remove pigment, non pigment, lipid and non polar material
Remove deposit portion Dichloromethane
Upper portion pooled in rotary evaporated under vacuum 600C to remove water with other solvents
Keep remain gummy phenolic compound in dark glass at deep freezer ready for chromatographic study using HPLC. (Yu and Dahlgren, 2000 and Yu et al., 2005) 13
Chapter three
Materials & Methods
3.2.6.2 Preparation of phenolic acids for detection by (HPLC) Amount of 0.1g of dry sample was dissolved into 5 ml of ethanol – water (80:20, v/v) in glass tube. The suspension was subject to ultra-fornication (Branson sonifier, USA) at 60% duty cycles for 25 min at 25 0C followed by centrifugation at 7500 rpm for 15 min. The clear supernatant treatment of each sample was subjected to charcoal treatment to remove pigments prior to evaporation under vacuum (Buchi Rotavapor Re type). Dried samples were re-suspended in 1.0 ml HPLC grade methanol by vortexing, the mixture was passed through 2.5 nm disposable filters, and stores at 40C for analysis. Then μ ml sample was injected to HPLC system according the optimum condition (Suarez et al., 2005 and Mauricio et al, 2007).
3.2.6.3 Analyses of phenolic compounds in pistacia spp by (HPLC) The main compounds were separated on FLC (Fast Liquid Chromatographic) column under the optimum condition, of the following Column: phenomenex C-18, 3 μ m particle size (50×2.0mm I.D) column. Mobile phase: linear gradient of A 0.1% formic acid: solvent B was (6:3:1, v/v) of acetonitrile: methanol: 0.1% formic acid, gradient program from 0 % B to 100%for 10 minutes. Detection: UV at 270nm. Flow rate 1.2ml/min. The sequences of the eluted materials of the standard were as follow, each standard was 20 μ g / ml. All standards with retention times, area and concentration listed in appendix (1 -11) The separation occurred on liquid chromatography Shimadzu 10AV-LC equipped with binary delivery pump model LC-10A Shimadzu; the eluted peaks were monitored by UVVes 10A-SPD spectrophotometer, the data recorded on Shimadzu CR-8A Chromatopack integrator (Shimadzu, koyota, Japan) (Cu, 1986; Cu et al., 1990). Calculation equation Area of sample Conc. of sample μ g / ml (ppm) = .................................× conc. of standard × dilution Factor Area of standard To obtain percentage results conc. of each sample (μ g / ml) was divided by summation (aggregation) of all sample values.
13
Chapter three
Materials & Methods
3.2.6.4 Extraction of the essential oils The essential oils were extracted by hydro-distillation of plant material, 100g of each sample was added to 500 ml of distilled water in a Clevenger-type apparatus, and distillation process for 3 hour according to the standard procedure described in the European pharmacopoeia (2004). The oils were stored at low temperature -20 C° prior to analysis. The oils were dried using anhydrous sodium sulfate (according to sample size and water content) and stored in sealed glass vials in refrigerator before analysis.
3.2.6.5 Preparation of volatile oils for detection by (HPLC) Ten mg of the sample extract was dissolved in a mixture of 20 ml of mobile phase A: B 50:50 v/v and, 2 ml of 0.2 M acetonitrile and KOH added. The mixture was shaken for 2 min and allowed to stand for 10 min. The upper layer was removed and washed with water. Then, the mixture was passed through 205 um disposable filters, and then 20μm was injected onto HPLC column and analyzed by HPLC according to optimum separation condition. The separation occurred on liquid chromatography Shimadzu 10AV-LC equipped with binary delivery pump model LC-10A Shimadzu, the eluted peaks were monitored by UVVes 10A-SPD spectrophotometer, the data recorded on Shimadzu CR-8A Chromatopack data processor (Shimadzu, koyota, Japan).(Lawrence et al., 1986; Cu et al., 1990).
3.2.6.6 Analysis the main constitutes of volatile oils in tow pistacia spp. by (HPLC) The main compounds were separated on FLC (Fast Liquid Chromatographic 2.7 μ m with high surface area and short analysis time) column under the optimum condition. Column: nucleoshell PR 18r (50×4.0mm I.D) column., Mobile phase: THF (tetrahydrofuran) 0.1% formic acid (solvent A): acetonitrile HPLC grade (solvent B) using linear gradient program from 0%Bto 100%for 15 minutes. Detection: UV at 234 nm. Flow rate 1.2ml/min. (Shimadzu, koyota, Japan) The sequences of the eluted materials of the standard listed in appendix 12-19 which were purchased from Sigma Comp LTD.
34
Chapter three
Materials & Methods
Calculation equation Area of sample Conc. of sample μ g / ml (ppm) =................................. × conc. of standard × dilution Factor Area of standard To obtain percentage results conc. of each sample (μ g / ml) divided by summation (aggregation) of all sample values.
3.2.7 Statistical analysis Statistical analysis was performed using Minitab 13.1 for Windows. The results from the measurement of total phenolic compounds and volatile oils were analyzed by a twoway analysis of variance (ANOVA), the solvents and the plants being the three parameters and concentration of phenolic compounds and volatile oils (mean values) the response variable, with three repetitions. Also the results from the HPLC analysis were statistically analyzed by ANOVA, two species, two growth stage (ripe & unripe)and wounded to obtains gum from each plant being the three parameters and concentration of phenolic compounds the response variable, with three repetitions.
33
Conclusion and Recommendations
Conclusions and Recommendations Conclusions To summarise the experimental results, several main points need to be highlighted which are: 1. Tannin has the highest concentration of phenolic compounds for both Pistacia species, while cinnamic acid is the lowest, α- Pinene has the highest concentration and Myretenol has the lowest concentration in all plant parts of the volatile oil compounds. 2. Both Pistacia species are releasing considerable amount of gum in spring rather than summer, while the concentration of volatile oil in the gum in summer collection higher than the gum in spring collection. 3. Volatile oil is not produced in mature leaf, seed, and outer shell of both Pistacia species, but phenolic compounds are presented in all parts, while leaves of the two pistacia spp in spring contain large amount of phenolic compounds than other parts even in different growth stages (ripe and unripe). 4. P. khinjuk contains high amount of some phytochemicals in volatile oils such as: Phellandrene, β- Pinene, Limonene, Sabinene, Aldehyde – citrate and 3-Carene rather than P. eurycarpa. On the other hand, P. eurycarpa is richer than P. khinjuk from α- Pinene, Myrecene, Terpinene, Carveol and Myretenol. 5. Stem wound is a most important factor to induce the amount of some phytochemicals in both Pistacia species, and the important parts for extraction of all phytochemicals of both Pistacia species are cluster without fruit and gum in different growth stages.
78
Conclusion and Recommendations
Recommendations Further investigations following recommendations may be useful:
1. Extraction of fixed oil from the two Pistacia spp. during maturation and determination by HPLC, both plants contain considerable amount of fixed oil. 2. A survey and extraction of all elements such as: P, S, Cu, Mg, Ca, Fe, Ca, Mn, K from all plant parts of the two Pistacia spp. 3. Extraction of protein and carbohydrate of the two Pistacia spp at the different growth stage (ripe and unripe). 4. Extension biological studying and investigating the effect of phytochemical compounds isolated from two Pistacia spp in various fields of health science as a possible source of medicine such as; antimicrobial, antifungal and anti viral activity.
77
References
References Abdul-Aziz, J.M.R. (2005) ''the potential of aqueous and acetone extraction of galls of Quercus infectoria as anti-bacterial agent''. Indian j. of pharm. 37(1):26-29. Abi-Ayada, F., Abi-Ayada, M., Lazzounia, H.A., Rebiahib S.A. and Bessierec. (2011) ''Antibacterial activity of essential oil extracted from leaves of Tetraclinis articulata (Vahl) Masters from Algeria flora''. J. Microbiol. Biotech. Res. 1 (1): 1-6 Afsana, K. (2004) the tremendous cost of seeking hospital obstetric care in bangladesh, Reproductive Health Matters. 12(24):171- 180. Aggarwal, K. K. Khanuja, S. P. S. Ateeque Ahmad, Santha Kumar, T. R. Vivek K. Gupta and Sushil Kumar (2002) ''Antimicrobial activity profiles of the two enantiomers
of
limonene
and
carvone
isolated
from
the
oils
of Mentha
spicata and Anethum sowa'', Flavor and Fragrance Journal. 17 (1): 59–63, Aglika Edreva (2005) ''The importance of non-photosynthetic pigments and cinnamic acid derivatives in photo protection'', Agri. Ecosys. & Envir.. 106(2): 135–146. Ahmad S. A. (2013) ''Flora of Hawraman Mountain''. PhD. Thesis. Fac. Agriculture science, Sulaimani University. Iraq. Ahn, J.C., Hwang, B., Tada, H. (1996) ''Polyacetylenes in hairy roots of Platycodon grandiflorum''. Phytochemistry. 42: 69–72. Al – Shahat, N. Z. (1986) ''plant and Herbal medicine''. Dar- albehar publisher, Baurit. (in Arabic). Alan Harvey (2000) ''Strategies for discovering drugs from previously unexplored natural products'', Drug Discovery Today. 7 (1):294–300. Al-anzy, M.A.K. (2004) ''Effect of Eruca sativa M.extracts on the growth of some pathogenic microorganisms''. MSc. Thesis. Coll. Science. Baghdad Univ. Iraq. Ali Asghar Hatamnia, Nasser Abbaspour and Reza Darvishzadeh (2014) ''Antioxidant activity and phenolic profile of different parts of Bene (Pistacia atlantica subsp. kurdica) fruits'', Food Chemistry. 145:306–311. Alma, M.H., Nitz, S., Kollmannsberger, H., Digrak, M., Efe, F. T. and Yilmaz, N. (2004) “Chemical composition and antimicrobial activity of the essential oils from the gum of Turkish Pistachio (Pistacia vera L.)” J. of Agri. and F. Chem. 52(12): 3911– 3914.
98
References Al-Yafi J. (1978) ''New characters differentiating Pistacia atlantica subspecies''. Candollea. 33:201–208. Amar
Zellagui, Noueddine
Gherraf, and Salah
Rhouati
(2012)
''Chemical
composition and antibacterial activity of the essential oils of Ferula vesceritensis Coss et Dur. leaves, endemic in Algeria'', Org Med Chem. 46:446-475.. Amri, I., Homrouni, L., Hanana, M. and Jamoussi, B. (2012) ''Chemical composition and herbicidal effect of Pistacia lentiscus L essential oil against weeds'', Int. J. Med.Agrom. Plants. 2(4): 558-565. Andersen, O. M. and Markham, K. R. (2006) ''Flavonoids Chemistry, Biochemistry and Applications: Isoflavonoids and Human Health''. Taylor & Francis Group, New York. PP.450-471. Andressa Blainski, Gisely Cristiny Lopes and João Carlos Palazzo de Mello (2013) ''Application and Analysis of the Folin Ciocalteu Method for the Determination of the Total Phenolic Content from Limonium Brasiliense L''. Molecules. 18: 6852-6865.
Anne Johansson and Margot Lundborg (1997) ''Effects of low concentrations of 3carene on alveolar macrophages in vitro'', Toxicology. 120(2): 99–104.
Antolovich, M. Prenzler, P. Robards, K. and Ryan, D. (2000) ''Sample preparation in the determination of phenolic compounds in fruits''. Analyst. 125:989–1009. Aparicio-Fernández, X., Yousef, G.G., Loarca-Pina, G., de Mejia, E. and Lila, M.A. (2005) ''Characterization of polyphenolics in the seed coat of black jamapa bean (Phaseolus vulgaris L.)''. J. Agr. Food Chem. 53: 4615-4622. Arias, M., Penichet, I., Ysambertt, F., Bauzab, R., Zougaghc, M., Ríos, A. (2009) ''Fast supercritical fluid extraction of low- and high-density polyethylene additives: Comparison with conventional reflux and automatic Soxhlet extraction''. J. Supercrit. Fluid. 50: 22-28.
Avicenna (2008) the canon, translated by: A. Shrafkandi, Soroush Press, Tehran.
Ayoola, G.A., Coker, H.A.B. Adesegun, S.A. Adepoju-Bello, A. A. Obaweya K. Atangbayila, T.O.
(2008) ''Phytochemical screening and, E.C. Ezennia and
antioxidant activities of some selected medicinal plants used for malaria therapy in South Western Nigeria''. Trop. J. Pharm. Res. 7: 1019-1024.
Balachandran S., Kentish, S. E., Mawson R. and Ashokkumar, M. (2006) ''Ultrasonic Enhancement of the Supercritical Extraction from Ginger''. Ultrason. Sonochem. 13: 471-477. 89
References Behboodi B. Sh. (2003) ''Ecological distribution study of wild pistachios for selection of rootstock''. Options Mediterran. 63:61-6. Bell SG, Chen X, Sowden RJ, Xu F, Williams JN, Wong LL and Rao Z. (2003) ''Molecular recognition in (+)-alpha-pinene oxidation by cytochrome P450cam''. J. of the Amer.Chem. Society. 125(3):705-14.
Bénédicte Lepoittevin , Xiaolu Wang, Jean-Pierre Baltaze, Hefang Liu, JeanMarie Herry, Marie-Noëlle Bellon-Fontaine and Philippe Roger (2011) ''Radical polymerization and preliminary microbiological investigation of new polymer derived from Myrtenol'', Eur. Polymer J. 47(9): 1842–1851.
Berrin O¨zc-elik, Mustafa Aslan,, Ilkay Orhan, and Taner Karaoglu (2005) ''Antibacterial, antifungal, and antiviral activities of the lipophylic extracts of Pistacia vera'', Microbiolo. Res. 160: 159—164.
Bhatia S.P., McGinty D., Letizia C.S., Api A.M. (2008) ''Fragrance material review on Carveol'', F. and Chem. Toxi. 46 (11): 85–87.
Bocco A, Cuvelier ME, Richard H and Berset C, (1998) ''Antioxidant activity and phenolic composition of citrus peel and seed extracts''. J. of Agr. and F. Chem. 46:2123–2139. Bohn, A.A., Forsyth, C.S., Stoner, G.D., Reed, D.J. and Frank, A.A (1998) ''Effect of cocaine, 95% oxygen and ellagic acid on the development and antioxidant status of cultured rat embryos'', Toxicology Letters. 95(1): 15–21. Bonithon-Kopp, C., Kronborg, O., Giacosa, A., Rath, U., Faivre, J., (2000) ''Calcium and Fibre Supplementation in Prevention of Colorectal Adenoma Recurrence'': a Randomised Intervention Trial. European Cancer Prevention Organisation Study Group. Lancet. 356(9238):1300. Boudet, A.M. (2007) ''Evolution and current status of research in phenolic compounds''. Phytochemistry. 68: 2722-2735. Bozin, B.; Mimica-Dukic, N.; Samojlik, I. and Jovin, E. J. (2007) ''Antimicrobial and antioxidant properties of rosemary and sage (Rosmarinus of f icinalis L. and Salvia of f icinalis L., Lamiaceae) essential oils''. J. Agric. Food Chem. 55:7879−7885. Brantner, A. H. (1997) ''Influence of various parameters on the evaluation of antibacterial compounds by the bioautographic tlc assay''. Pharm. Pharmacol. Lett. 4:152−154.
89
References Butler,M.S. (2005) ''Natural products to drugs: natural product derived compounds in clinical trials''. Nat. Prod. Rep. 22: 162–95. Cardile, V., Russo, A., Formisano, C., Rigano, D., Senatore, F., Arnold, N. A. and Piozzi, F. (2009) ''Essential oils of Salvia bracteata and Salvia rubifolia from Lebanon: chemical composition, antimicrobial activity and inhibitory effect on human melanoma cells''. J. Ethnopharmacol. 126:265−272. Chapman, G.W. (1957) ''Forest and Forestry in Iraq''. Government of Iraq, Ministry of Agriculture, Directorate general of Forest, Printed Government press, Baghdad, Pp 37. Chowdhury, S. and Nishteswar, K. (2013) ''Role of volatile oil of natural source in the cosmetics and health care industries''. Pharma Sci. Monit. 4:3738−3750. Chung, King-Thom, Wong, Tit Yee, Wei, Cheng-I, Huang, Yao-Wen, and Lin, Yuan (1998) "Tannins and Human Health: A Review". Critical Rev. in F. Science and Nut. 38 (6): 421–64. Costa, R.M.; Magalhães, A.S.; Pereira, J.A.; Andrade, P.B.; Valentão, P.; Carvalho, M.; Silva, B.M. (2009) ''Evaluation of free radical-scavenging and antihemolytic activities of quince (Cydonia oblonga) leaf: a comparative study with green tea (Camellia sinensis)''. F. Chem. Toxicol. 47: 860-865. CU, J.Q. (1986) ''the kingdom of essential oil'', in: Lawrence. B.M., Mookherjee, B.D and Willis, B.J (eds) Flavour and Fragrances: A World perspective, proceedings of the 10th international congress of Essential Oils, Washington, DC 16-20 November. Elsevier, Amsterdam. Cu, J.Q., Perineau, F. And Goepfert, G. (1990) ''GC/MS analysis of star anise oil''. J.of Esse.Oil Res. 2: 91-92. De Sousa D.P., Raphael E., Brocksom U., and Brocksom TJ. (2007) ''Sedative effect of monoterpene alcohols in mice: a preliminary screening''. J. Z Naturforsch C. 62(78):563-576. Deans, S.G. and Ritchie, G.A. (1987) ''Antimicrobial properties of plant essential oils''. Int. J. of F. Microbio. 5:165–180. Deba, F., Xuan, T.D. Yasuda M. and Tawatu, S. (2008) ''Chemical composition and antioxidant, antibacterial and antifungal activities of the essential oils from Bidens pilosa Linn. Var. Radiata'', Food control.19: 346-352. Demirci F., Baser, K.H.C., Calis, I. and Gokhan, E. (2001) ''Essential oil and antimicrobial evaluation of the Pistacia eurycarpa'', Che. of Nat. Comp. 37(4): 332-335. 89
References
DerMarderosian, A. Beutler, J. A. (2010) ''The Review of Natural Products'', sixth ed., Wolters Kluwer Health, Missouri.
Devanand Luthria, (2006) ''Perspective Significance of sample preparation in developing analytical methodologies for accurate estimation of bioactive compounds in functional foods''. J. of the Sci. of F. and Agri. 86:2266–2272. Dewick, P.M. (2002) ''Medicinal Natural Products: A Biosynthetic Approach'', 2nd edn., JohnWiley and Sons, Chichester. 134-145.
Dimas, K., Hatziantoniou, S. Wyche, J.h. and Pantazis, P. (2009) ''a mastic gum extract induces suppression of growth of human colorectal tumor xenografts in Immunodeficient mice'', In vivo. 23: 63-68.
Dinelli, G., Bonetti, A., Minelli, M., Marotti, I., Catizone, P., Mazzanti, A. (2006) ''Content of flavonols in italian bean (Phaseolus vulgaris L.) ecotypes''. Food Chem. 99: 105-114. Duru ME, Cakir A, Kordali S, Zengin H, Harmandar M, Izumi S. (2003) ''Chemical composition and antifungal properties of essential oils of three Pistacia species''. Fitoterapia. 74: 170 – 176 Elegbede, J. A.,.Elson, C. E, Qureshi, A., Tanner, M. A. and Gould, M. N. (1984) ''Inhibition of DMBA induced mammary cancer by the monoterpene d-limonene''. Carcinogenesis. 5 (5):661-664. Erich Schmidt (2010) ''chapter 4: Production of Essential Oils', 'handbook of essential oil Science, Technology, and Applications, edited by K. Hüsnü Can Bas and Gerhard Buchbauer, Taylor & Francis Group, pp.90-91. Escarpa, A. and Gonzalez, M.C. (2008) ''An overview of analytical chemistry of phenolic compounds in foods''. Crit. Rev. Anal. Chem. 75: 57-139. Espinosa-Alonso, L.G.; Lygin, A.; Widholm, J.M.; Valverde, M.E.; Octavio Paredes-López, O.( 2006) ''Polyphenols in wild and weedy mexican common beans (Phaseolus vulgaris L.)''. J. Agric. Food Chem. 54: 4436- 4444. European pharmacopoeia, (2004) 5th edition; council of Europe: Strasbourg Cedex, France, 1: 217-218. Evans, W.C. (2002) ''Trease and Evans pharmacognosy''. 15th Ed. Elsevier science limited. London. UK. PP. 219 – 224. Fang, Z., Zhang, M., Wang, L. (2005) ''HPLC-DAD-ESI-MS analysis of phenolic compounds in bayberries (Myrica rubra Sieb. et Zucc.)''. Food Chem. X. 100: 845-852. 89
References Faraidoon Haghdoost, MohammadMehdi Baradaran Mahdavi, Alireza Zandifar, Mohammad Hossein Sanei, Behzad Zolfaghari, and Shaghayegh Haghjooy Javanmard (2013) ''Pistacia atlantica Resin Has a Dose-Dependent Effect on Angiogenesis and Skin Burn Wound Healing in Rat'', Evidence-Based Compl. and Alter. Med. 8:1155-1164. Ferreres, F., Gomes, D., Valentão, P., Gonçalves, R., Pio, R., Alves, E., Seabra, R.M., Andrade, P.B. (2009) ''Improved loquat (Eriobotrya japonica Lindl.) cultivars: variation of phenolics and antioxidative potential''. Food Chem. 114: 1019-1027. Figueiredo, A.C., J.G. Barroso, L.G. Pedro, and J.J.C. Scheffer, (2005) ''Physiological aspects of essential oil production''. Plant Sci. 169(6): 1112–1117. Flora of Iraq. (1969) ''Forest trees in Iraq''. vol. 4 Faculty of Agriculture and Veterinary science, Mosul University, pp 494 -499. Gale, J. (1975) ''Water balance and gas exchange of plants under saline conditions. In: Plant in Saline Environments'', Polijakoff-Maber, A. and Gale, J. (eds). Springer Verlage, Berlin, 168-185. Ghasemi Pirbalouti A (2009) ''the third list plants, traditional medicine and ethnoveterinary'', 1st Ed. Medicinal and aromatic plant. Shahrekord: Saman-Danesh Pub, 158–190 (Persian) Ghasemi Pirbalouti, A. Bahmani M. and Avijgan, M. (2009) ''Anti-Candida activity of some of the Iranian medicinal plants''. E. J. Bio. 85–88. Ghasemi Pirbalouti, A Aghaee, K. (2011) ''Chemical Composition of Essential Oil of Pistacia khinjuk Stocks Grown in Bakhtiari Zagross Mountains'', Iran. Elect.c J. of Bio. 7(4): 67-69. Glen, A. Halverson M.D. (2001) ''Chemo-protective properties of phytochemicals featuring ellagic insurance formula''. Published by: Weltek, Inc7925.A north oracle Road. Golan - Goldhirsh, A. , Barazani, O. Wang, Z . S. Khadka, D. K. Saunders, J. A. Kostiukovsky, V. and Rowland, L. J. (2004) ''Genetic relationships among Mediterranean Pistacia species evaluated by RAPD and AFLP markers''. Plant Syst. and Evol. 246: 9 – 18. Goli, A.H., Barzegar, M. and Sahari, M.A. (2005) ''Antioxidant activity and total phenolic compounds of pistachio (Pistachia vera) hull extracts'', F. Chem. 92: 521-525.
89
References Gómez, A.M. Carrasco, A. Cañabate, B. Segura, A. and Fernández, A. (2005) ''Electrophoretic identification and quantitation of compounds in the polyphenolic fraction of extra-virgin olive oil''. Electrophoresis. 26, 3538-3551. Gottlieb, O.R. and Borin, M.R. (2000) ''Medicinal products: Regulation of biosynthesis in space and time''. Memorias do Instituto Oswaldo Cruz j. 95: 115–120.
Haghdoost F., Baradaran Mahdavi MM., Zandifar A, Sanei MH., Zolfaghari B., and Javanmard SH.( 2013) ''Pistacia atlantica Resin Has a Dose-Dependent Effect on Angiogenesis and Skin Burn Wound Healing in Rat''. Evid Based Compl. Alter. Med. 13(4) 25-56.
Harbone, J. B. (1973) ''Phytochemical methods, A Guide to modern Techniques of Plant analysis''.2nd Ed, Chapman and hall Ltd. London. P. 37-89, 176-192.
Harborne, J. B. (1993) ''Introduction to ecological biochemistry''. London: Academic Press. Haribalan Perumalsamy, Moon Young Jung, Seung Min Hong and Young-Joon Ahn, (2013) ''Growth-Inhibiting and morphostructural effects of constituents identified in Asarum heterotropoides root on human intestinal bacteria'', Compl. and Alter. Med. 13:245. Helander, I. M., Alakomi, H.-L., Latva-Kala, K., Mattila- Sandholm, T., Pol, I.,Smid, E. J.; Gorris, L. G. M.; von Wright, A. (1998) ''Characterization of the action of selected essential oil components on Gram-negative bacteria''. J. Agric. Food Chem. 46: 3590−3595. Hormaza, J. I., Pinney, K. and Polito, V. S. (1998) ''Genetic diversity of Pistachio (Pistacia vera, Anacardiaceae) germplasm based on randomly amplified polymorphic DNA (RAPD) markers''. Economic Botany. 52: 78-87. Hormaza, J. I., Dollo, L. and Polito, V. S. (1994) ''Determination of relatedness and geographic movements of Pistacia vera (Pistachio; Anacardiaceae) germplasm by RAPD analysis''. Economic Botany. 48: 349-358.
http://groups.myspace.com/Kurdistan1, (2003-2008).
Huang, Z., Wang, B., Eaves, D.H., Shikany, J.M., Pace, R.D. (2007) ''Phenolic compound profile of selected vegetables frequently consumed by african americans in the southeast'' US F. Chem. 103: 1395-1402. Jaffer, H.J., Mahmod, M.J., Jawad, A.M., Naji,A. and Al-naib, A.(1983) ''Phytochemical and biological screening of some Iraqi Plant''. Fitoterapia, Lix. pp299. 89
References Jahangiri, Y., Ghahremani, H., Abedini Torghabeh, J. and Hassani, S. M. (2012) ''The effects of operational conditions on the total amount of anthocyanins extracted from Khorasan's native fig fruit “Ficus carica''. Schol. Res. Lib. 5:2181-2186. Jeong Soo Yang, Sung Kwang Lee, Young Hun Park, and Dai Woon Lee, (1999) ''Analytical Method for Dioxin and Organo-Chlorinated Compounds: (II) Comparison of Extraction Methods of Dioxins from XAD-2 Adsorbent'', Bulletin of the Korean Chemical Society. 20: (6) 689. Kafkas, E., Kurkcuoglu, M., Kafkas, S. and Baser, K. H. C. (2007) ''Analysis of the fatty oil of Pistacia eurycarpa nuts by Gas Chromatography /Mass Spectromery''. Chem. of Natu. Compo.. 43: 1-3. Kafkas, S. (2006) ''Phylogenetic analysis of the genus Pistacia by AFLP markers''. Plant Syst. and Evol. 262: 113 – 124. Kafkas, S. and R. Perl - Treves. (2001) ''Morphological and molecular phylogeny of Pistacia species in Turkey''. Theor. and Applied Gen.. 102: 908 – 915. Kafkas, S. and R. Perl - Treves. (2002) Inter-specific relationships in Pistacia based on RAPD fingerprinting. HortScience 37: 168 – 171. Kamath, A. and Ojima, I. (2012) ''Advances in the chemistry of β-lactam and its medicinal applications''. Tetrahedron. 68:10640−10664 Kamatou, G.P.P., R.L. van Zyl, S.F. van Vuuren, A.M. Viljoen, A.C. Figueiredo, J.G. Barroso, L.G. Pedro, and P.M. Tilney, (2006) ''Chemical composition, leaf trichome types and biological activities of the essential oils of four related salvia species indigenous to Southern Africa''. J. Essent. Oil Res. 18 (Special edition): 72–79. Kan, Y., Ucan, U., Kartal, M., Altun, M., Aslan, S., Sayar, E., Ceyhan, T. (2000) ''GC-MS Analysis and antibacterial activity of cultivated Satureja cuneifolia Ten. Essential oil''. Turk J. Chem. 30: 253 – 259. Karuo, D., Mamoudou, H. D., Jaques, S. and Alfred, S. T. (2005) ''Antioxidant and antibacterial activities of poly phenols from ethno-medicinal plants of Burkina Faso''. Afr. J. Biotechnol. 4(8): 823 – 828.
Kashaninejad, M., Mortazavi, A., Safekordi, A. and Tabil, L.G. (2006) ''some physical properties of Pistachio (Pistacia vera L.) nut and its kernel'', J. F. Eng.72:3038.
89
References
Katsiotis, A., Hagidimitriou, M. Drossou, A. Pontikis C. Loukas, M. (2003) ''Genetic relationships among species and cultivars of Pistacia using RAPDs and AFLPs''. Euphytica. 132: 279-286.
Kevin, T. Watts, Pyung C. Lee and Claudia Schmidt-Dannert (2006) ''Biosynthesis of plant-specific stilbene polyketides in metabolically engineered Escherichia coli'', BMC Biotechnology. 6:1-22. Klejdusa,
B.,
Kopecký,
J.,
Benesˇová,
L.,
Vaceka,
J.
(2009)
''Solid-
phase/supercritical-fluid extraction for liquid chromatography of phenolic compounds in freshwater microalgae and selected cyanobacterial species''. J. Chromatogr. A. 1216: 763-771. Kordali, S. Cakir, A. Zengin, H. and Duru, ME. ( 2003) ''Antifungal activities of the leaves of three Pistacia species grown in Turkey''. Fitoterapia. 74: 164 - 167. Kumar KJ, Vani MG, Wang SY, Liao JW, Hsu LS, Yang HL and Hseu YC. (2013) ''In vitro and in vivo studies disclosed the depigmenting effects of gallic acid: a novel skin lightening agent for hyperpigmentary skin diseases''. Biofact. 39(3):259- 270.
Kumar, A., Ilavarasn, R., Jayachandran, T., Decaraman, M., Aravindhan, P., Padmanaban, N. and Krishnan, M.R.V. (2009) ''phytochemical investigation on a tropical plant''. Pak. J. Nutri. 8: 83-85.
Larson, R. A. (1995) ''Anti-oxidant mechanisms of secondary natural products''. In S. Ahmad (Ed.), Oxidative Stress and Antioxidant Defense in Biology. New York: Chapman and Hall. pp.210−237
Lawrence, James F. Paula Michalik , Gerard Tam, Henry B. S. and Conacher (1986) ''Identification of arsenobetaine and arsenocholine in Canadian fish and shellfish by high-performance liquid chromatography with atomic absorption detection and confirmation by fast atom bombardment mass spectrometry'' J. Agric. F. Chem. 34 (2):315–319.
Liazid, A., Palma, M., Brigui, J. and Barroso, C.G. (2007) ''Investigation on phenolic compounds stability during microwave-assisted extraction''. J. Chrom. A. 1140: 29-34.
Lis-Balchin, M. (2009) ''Aromatherapy with essential oils. In Handbook of Essential Oils''; CRC Press: Boca Raton, FL, USA, pp 549−736.
89
References Liu AL, Shu SH, Qin HL, Lee SM, Wang Y.T., and Du GH. ( 2009) ''In vitro antiinfluenza viral activities of constituents from Caesalpinia sappan'', Planta Med. 75: 337-9. Luigia Longo, Anna Scardino and Giuseppe Vasapollo, (2007) ''Identification and quantification of anthocyanins in the berries of Pistacia lentiscus L., Phillyrea latifolia L. and Rubia peregrina L''. Inno. F. Sci. and Eme. Tech.. 8: 360–364. Luthria DL, (2006) ''Influence of sample preparation on assay of phytochemicals''. Amer. Lab. News 38:12–14. Luthria DL, Mukhopadhyay S and Kwansa AL, (2006) ''A systematic approach for extraction of phenolic compounds using parsley (Petroselinum crispum) flakes as a model substrate''. J of the sci. of F. and Agri. 86:1350–1358. Luthria, D.L. (2008) ''Influence of experimental conditions on the extraction of phenolic compounds from parsley (Petroselinum crispum) flakes using a pressurized liquid extractor''. F. Chem. 107: 745-752. Magalhães, A.S.; Silva, B.M.; Pereira, J.A.; Andrade, P.B. Valentão, P.; Carvalho, M. (2009) ''Protective effect of quince (Cydonia oblonga Miller) fruit against oxidative hemolysis of human erythrocytes''. F. Chem. Toxicol. 47: 1372-1377. Mahbubeh Bozorgi, ZahraMemariani, MasumehMobli, Mohammad Hossein, Salehi Surmaghi, Mohammad Reza, Shams-Ardekani, and Roja Rahimi (2013) ''Five Pistacia species (P. vera, P. atlantica, P. terebinthus, P. khinjuk, and P. lentiscus): A Review of Their Traditional Uses, Phytochemistry, and Pharmacology'', The Sci. world J. 33: 1155-1167. Mahmoud Bahmani and Zohre Eftekhari (2012) ''an ethno-veterinary study of medicinal plants in treatment of diseases and syndromes of herd dog in southern regions of Ilam province'', Iran. Comp Clin Pathol. 12 (11): 1527-1532. Majors RE, (1999) An overview of sample preparation methods for solids. LC-GC. 17:8–13. Mallikharjuna, P.B., L.N. Rajanna, Y.N. Seetharam and G.K. Sharanabasappa, (2007) ''Phytochemical studies of Strychonos potatorum L.F. Amedicinal plant''. E.J. Chem. 4: 510-518. Mann, j., Dazvidson, R.S., Hobbs, J.B., Banthorp, D.V., and Harborne, J.B. (1994) ''Natural product: their Chemistry and Biological Significance''. Longman, Harlow.
89
References Márcio R. V. Santos, Flávia, V., Moreira, Byanka P. Fraga ,Damião P. De Sousa, Leonardo R. Bonjardim, Lucindo J .And Quintans-Junior, (2011) ''Cardiovascular effects of monoterpenes''. Bra. J of Pharm. 21(4): 764-771. Maria I. G. Silva, Carla T. V. de Melo, Leonardo F. Vasconcelos, Alyne M. R. de Carvalho, Francisca C. F. Sousa, (2012) ''Bioactivity and potential therapeutic benefits of some medicinal plants from the Caatinga semi-arid) vegetation of Northeast Brazil''. Bra. J. of Pharm. 22(1): 193-207. Mecherara-Idjeri, S., Hassani, A., Castola, V., and Casanova J. (2008) ''Composition of leaf, fruit and gall essential oils of Algerian Pistacia atlantica Desf'', J. Essent. Oil Res. 20: 215-219. Mecherara-Idjeri, S., Hassani, A., Castola, V., and Casanova, J. (2008) ''Composition and chemical variability of the essential oil from Pistacia lentiscus L. growing wild in Algeria Part 1: Leaf oil'', J. Essent. Oil Res. 20: 32-38. Mehmet Öztürk (2012) ''Anticholinesterase and antioxidant activities of Savoury (Satureja thymbra L.) with identified major terpenes of the essential oil'', F. Che. 134(1):48–54.
Mélanie Hall, Bernhard Hauer, Rainer Stuermer, Wolfgang Kroutil, Kurt Faber (2006) ''Asymmetric whole-cell bioreduction of an α,β-unsaturated aldehyde (citral): competing prim-alcohol dehydrogenase and C–C lyase activities'', Tetrahedron Asymmetry. 17 (21): 3058–3062.
Mertz, C., Gancel, A-L., Punata, Z., Alter, P., Dhuique-Mayer, C., Vaillant, F.,Perez, A.M., Ruales, J., Prat, P.( 2009) ''Phenolic compounds, carotenoids and antioxidant activity of three tropical fruits''. J. F. Compos. Anal. 22: 381-387. Milan Suhaj and Jana Horvayhova (2007) ''Changes in antioxidant activity induced by irradiation of clove (Syzygium aromaticum) and ginger (Zingiber officinale('' J. of F. and Nut. Res. 46 (3)112-122.
Mitić-Ćulafić, D. Žegura, B. Nikolić, B. Vuković-Gačić, B., Knežević-Vukčević, J. Filipič, M. (2009) ''Protective effect of linalool, Myrecene and eucalyptol against tbutyl hydro peroxide induced genotoxicity in bacteria and cultured human cells''. F. and Chem. Toxi. 47(1): 260–266.
Mitra S and Brukh R. (2003) ''Sample preparation: an analytical perspective, in Sample Preparation Techniques in Analytical Chemistry'', ed. by Mitra S.Wiley, New York, pp.1–36. 88
References Mohammad Sharif Sharifi and Stuart Loyd Hazell (2011) ''GC-MS Analysis and Antimicrobial activity of the essential oil of the trunk exudates from Pistacia atlantica kurdica'', J. of Pharm. Sci. and Res. 3(8):1364-1367. Mohannad, G., Duncan, m. porter and Saghai-Maroof. (2006) ''Phylogenetic Analysis of the genus Pistacia (Anacardiaceae)''. University of virginia. p 4-5. Moller, C., Hansen, S.H., Cornett, C. (2009) ''Characterisation of tannin-containing herbal drugs by HPLC. Phytochemical''. Analysis journal. 20: 231–239. Muchuweti, M. Ndhlala, A.R. Kasiamhuru, A. (2006) ''Analysis of phenolic compounds including tannins'', gallotannins and flavanols of Uapaca kirkiana fruit. Food Chem. 94: 415-419. Mukhopadhyay, S. Luthria, D.L. and Robbins, R.J. (2006) ''Optimization of extraction process for phenolic acids from Black cohosh (Cimicifuga racemosa) by pressurized liquid extraction''. J. of the sci. of F. and Agr. 86:156–162. Naczk M and Shahidi F. (2004) ''Extraction and analysis of phenolics in food''. Journal of Chromatography A. 1054:95–111. Naczk, M. and Shahidi, F. (2006) ''Phenolics in cereals, fruits and vegetables: occurrence, extraction and analysis''. J. Pharm. Biomed. Anal. 41: 1523-1542. Nadhir Gourine, Mohamed Yousfi, Isabelle Bombarda, Boubakeur Nadjemi and Emile Gaydou(2010) ''Seasonal variation of chemical composition and antioxidant activity of essential oil from Pistacia atlantica Desf. Leaves''. J Am Oil Chem Soc. 87:157–166. Nahida, A, Ansari S.H. and Siddiqui A.N. (2012) ''Pistacia lentiscus L. A review on Phytochemistry and Pharmacological properties'', international journal of pharmacy and pharmaceutical sciences. 4(4): 975-1491. Navarro, J.M., Flores, P., Garrido, C., Martinez, V. ( 2006) ''Changes in the contents of antioxidant compounds in pepper fruits at different ripening stages, as affected by salinity''. Food Chem. 96: 66-73.
Navindra P. Seeram, Lynn S. Adams, Susanne M. Henning, Yantao Niu, Yanjun Zhang, Muraleedharan G. Nair and David Heber (2005) ''In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice'', The J. of Nutr. Bioch. 16 (6): 360–367
999
References Newmann, D.J. and Cragg, G.M. (2007) ''Natural products as sources of new drugs over the last 25 years''. J. Nat. Prod. 70: 461–77. Nguyen, T.P.T., Nguyen, T.T., Tran, M.H., Tran, H.T., Muselli, A., Bighelli, A., Castola, V., and Casanova, J., (2004) ''Artemisia vulgaris L. from Vietnam, chemical variability and composition of the oil along the vegetative life of the plant''. J. Essent. Oil Res. 16: 358–361. Novak, J., (2005) ''Lecture held on the 35th Int. Symp. on Essential Oils'', Giardini Naxos, Sicily.
Nurulain T. Zaveri (2006) ''Green tea and its polyphenolic Catechin: Medicinal uses in cancer and noncancer applications'', Life Sciences. 78(18): 2073–2080.
Nuutila, A.M., Puupponen-Pimia, R., Aarni, M., Oksman-Caldentey, K.M. (2003) ''Comparison of antioxidant activities of onion and garlic extracts by inhibition of lipid peroxidation and radical scavenging activity''. Food Chem. 81: 485-493. Okogun, J.I. (2000) ''Methods of Medicinal Plant extract. National Institute for Pharmaceutical Research and Development (NIPRD)''. Idu Abuja, Nigeria. Okuda, T.; Ito, H. (2011) ''Tannins of constant structure in medicinal and food plants hydrolysable tannins and polyphenols related to tannins''. Molecules. 16: 2191-2217. Okwu, D.E. and Josiah, C. (2006) ''Evaluation of the chemical composition of two Nigerian medicinal plants''. Afri. J. Biotech.5: 357-361. Ossipova, S., Ossipov, V., Haukioja, E., Loponen, J., Pihlaja, K. (2001) ''Proanthocyanidins of mountain birch leaves: Quantification and properties. Phytochemical''. Analysis journal.12:128–133. Ow Y.Y. and Stupans I. (2003) ''Gallic acid and gallic acid derivatives: effects on drug metabolizing enzymes''. Curr Drug Metab. 4(3):241-8. Palma, M.; Piñeiro, Z. and Barroso, C.G. (2002) ''In-line pressurized-fluid extractionsolid-phase extraction for determining phenolic compounds in grapes''. J. Chromatogr. A. 968: 1-6. Parfitt, D. E. and Badenes, M. L. (1998) ''Molecular phylogenetic analysis of the genus Pistac''. Acta Horticulturae. 470: 143 – 151.
Paris, R. and H. Moyse, (1969) ''Precis de matiere medicinale''. Paris: Masson.
Pasqua, R. D.; Betts, G.; Hoskins, N.; Edwards, M.; Ercolini, D.; Mauriello, G. (2007) ''Membrane toxicity of antimicrobial compounds from essential oils''. J. Agric. Food Chem. 55:4863−4870. 999
References Pauli, A. (2006) ''Anticandidal low molecular compounds from higher plants with special Reference to compounds from essential oils'', Med. Res. Rev. (26):223-268. Pazmiño-Duràn, A., Giusti, M., Wrolstad, R., & Glòria, M. (2001) ''Anthocyanins from Oxalis triangularis as potential food colorants'', Journal of Food Chemistry. 75: 211−216. Pell, S. K. (2004) ''Molecular systematic of the cashew family (Anacardiaceae)''. Ph.D. dissertation, Louisiana State University, Baton Rouge, Louisiana, USA. Pelozo, M.I.G., Cardoso, M.L.C., Mello, J.C.P. (2008) ''Spectrophotometric determination of tannins and caffeine in preparations from Paullinia. cupana var sorbilis''. Bra. Archi. of Bio. and Tech.. 51, 447–451. Peter H. Raven, Ray F. Evert and Susan E. Eichhorn (2013) ''Biology of plants'', New York W.H. Freeman publisher. 30-35. Potterat, O. and Hamburger, M. (2008) ''Drug Discovery and development with plant derived compounds''. Prog. Drug Res. 65: 47–118. Prabuseenivasan, S., Jayakumar M., and Ignacimuthu, S. (2006) ''In vitro antibacterial activity of some plant essential oils. BMC complement and alternate''. Medici. 6:39-42. Prati, S., Baravelli, V., Fabbri, D., Schwarzinger, C., Brandolini, V., Maietti, A., Tedeschi, P., Benvenuti, S., Macchia, M., Marotti, I., Bonetti, A., Catizone, P., Dinelli, G. (2007) ''Composition and content of seed flavonoids in forage and grain legume crops''. J. Sep. Sci. 30: 491-501. Rajaei, M. Barzegar,
Z. Hamidi1 and M. A. Sahari (2010) Optimization of
Extraction Conditions of Phenolic Compounds from Pistachio (Pistachia vera) Green Hull through Response Surface Method J. Agr. Sci. Tech. 12: 605-615. Ramakrishnan, K. and Krishnan, M.R.V. (1994) ''Tannin–classification, analysis and applications''. Ancient Science of Life. PP 232 – 238. Ramanadhan, B. (2005) ''Microwave extraction of essential oils (from black paper and coriander) AT 2.46 GHz'', Balasundaram ramanadhan, (Thesis). Ramawat, K. (2004) ''Plant biotechnology'', 1st edition ,Chand and Company ltd, New Delhi. pp 105 – 109. Rice-Evans CA, Miller NJ and Paganga G. (1996) ''Structure–antioxidant activity relationships of flavonoids and phenolic acids''. Free Radical Biology and Medicine. 20:933–956. 999
References Rios, J. L., Recio, M. C. and Villar, A. (1988) ''Screening methods for natural products with antimicrobial activity: a review of the literature''. J. Ethnopharmacol. 23:127−149.
Rivas da Silva AC, Lopes PM, Barros de Azevedo MM, Costa DC, Alviano CS, Alviano DS. (2012) ''Biological activities of α-pinene and β-pinene enantiomers''. Molecules. 17(6):6305-16.
Rizk, A.M. (1982) ''Constituents of plants growing in Qatar''. Fitoterapia. 52: 35-42.
Robbins R.J. (2003) ''Phenolic acids in foods: an overview of analytical methodology''. Journal of Agriculture and Food Chemistry. 51:2866–2887. Rodrigues, S. and Pinto, G. A. S. (2006) ''Ultrasound Extraction of Phenolic Compounds from Coconut (Cocos nucifera) Shell Powder''. J. Food Eng. 80: 869-872. Romani, A. Pinelli, P. Galardi, C. Mulinacci, N. and Tattini, M. (2002) ''Identification and Quantification of Galloyl Derivatives, Flavonoid Glycosides and Anthocyanins in Leaves of Pistacia lentiscus L''. Phytochem. Anal. 13: 79 - 86. Ross, K.A., Beta, T. and Arntfield, S.D.( 2009) ''A comparative study on the phenolic acids identified and quantified in dry beans using HPLC as affected by different extraction and hydrolysis methods''. Food Chem. 113, 336-344. Santhi, R., G. Lakshmi, A.M. Priyadharshini and Anandaraj, L. (2011) ''Phytochemical screening of Nerium oleander leaves and Momordica chrantia leaves''. Inter Res. J. Pharm. 2: 131-135. Serrano, M., Zapata, P.J., Castillo, S., Guillén, F., Martínez-Romero, D., Valero, D. (2010) ''Antioxidant and nutritive constituents during sweet pepper development and ripening are enhanced by nitrophenolate treatments''. Food Chem. 118: 497-503.
Sharma, M.C. and Sharma, S. (2010) ''Phytochemical screening of methanolic extract and antimicrobial activity of Eclipta alba and Morinda Citrifolia L''. MiddleEast J. Sci. Res. 6(5): 445-449.
Shi, J., K. Arunasalam, D. Yeung, Y. Kakuda, G. Mittal and Jiang, Y. (2004) ''Saponins from edible legumes: Chemistry, processing and health benefits'', J. Med. Food. 7: 67-78. Sikkema, J., de Bont, J. A. and Poolman, B. (1994) ''Interactions of cyclic hydrocarbons with biological membranes'', J. Biol. Chem. 269:8022−8028.
999
References Smith-Palmer, A., Stewart, J. and Fyfe, L. (1998) ''Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens''. Letters in Applied Microbiology. 26 (2): 118–122. Smolensk, S. J., Silnis, H. and Farnsworth, N. R. (1972) ''Alkaloid screening''. Liydia. 35(1): 31- 34.
Snyder, L.R, Kirkland, J.J and Dolan, J.W. (2009) ''Introduction to Modern Liquid Chromatography'', John Wiley & Sons, New York.
Suarez, B., Palacios, N., Fraga, N., and Rodriguez, R. (2005) ''Liquid chromatographic method for quantifying polyphenols in ciders by direct injection'' J. chromatography A. 1066:105-110. Svoboda, P. and Hampson, J. (1999) ''Bioactivity of essential oils of selected temperate aromatic plants: antibacterial, antioxidant, anti-inflammatory and other related pharmacological activities'', Plant Biology Department, SAC Auchincruive, Ayr, Scotland, UK. Talib, J.T. (2004) ''the political and geographical study of Iraqi Kurdistan region''. Ph.D. dissertation, Univ. of Sulaimani, Coll. of Hum. Tamura, H., and Yamaganci, A. (1994) ''Antioxidative activity of monoacylated anthocyanins isolated from Muscat Bailey a grape''. Journal of Agricultural and Food Chemistry. 42: 1612−1615. Taran, M., Mohebali, M. and Esmaeli, J. (2010) ''In vivo efficacy of gum obtained Pistacia atlantica in experimental treatment of cutaneous leishmaniasis'', Iran J Public Health. 39: 36-41. Thresiamma KC and Kuttan R., (1996) ''Oral administration of ellagic acid by rats can circumvent the carbon tetrachloride toxicity and subsequent fibrosis of the liver'', Ind. J. Phys. and Pharm.32:234 – 258. Tingshuang, Yi., Jun Wen, Avi Golan-Goldhirsh, Dan E. and Parfitt (2008) ''Phylogenetics and reticulate evolution in Pistacia (Anacardiaceae)'', American Journal of Botany. 95(2): 241–251. Tous, J. and Ferguson, L. (1996) “Mediterranean Fruits”. In: J. Janick, Ed., Progress in New Crops, ASHS Press, Ar-lington, pp. 416-430. Trease, GE. Evans, WC. (1989) ''Pharmacognosy''. 11th edn. Brailliar Tiridel Can. Macmillian publishers.
999
References Trease, GE. Evans, WC. (1987) ''Pharmacognosy''. Billiare Trindall.London 13th Edn. PP: 61-62. Tsai, P. J., McIntosh, J., Pearce, P., Camden, B., and Jordan, B. R. (2002) ''Anthocyanins and antioxidants capacity in Roselle (Hibiscus sabdariffa L.) extract''. F. Res. Int. 35: 351−356. Vaghasiya, Y., Dave R. and Chanda, S. (2011) ''Phytochemical analysis of some medicinal plants from Western region of India'', Res. J. Medic. Plant. 5: 567-576. Vallejo F., Toams-Berberan F. and Garcia-Viguera C. (2003) ''Health promoting compounds in broccoli as influenced by refrigerated transport and retail sale period''. J.of Agri. and F.Chem. 51:3029–3034. Vetter, S., C. Franz, S. Glasl, U. Kastner, J. Saukel, and J. Jurenitsch, (1997) ''Inheritance of sesquiterpene lactone types within the Achillea millefolium complex (Compositae). Plant Breeding. 116: 79–82. Vilaplana, J. and C. Romaguera (2002) ''Contact dermatitis from the essential oil of tangerine in fragrance''. Cont. Dermat. 46: 108. Villar A, Sanz MJ, Payo M. (1987) ''Hypotensive effect of Pistacia lentiscus L''. Int. J. Crude Drug Res. 25 (1): 1 - 3. Vogel, A. I. (1974) ''practical organic chemistry including qualitative organic analysis''. 3rd (ed.) Longman group limited. London. U.K. p.330-453. WHO. (2001) ''General guidelines for methodologies on research and evaluation of traditional medicine''. Geneva, Switzerland. Wijngaard, H.H., Rößle, C. and Brunton, N. (2009) ''A survey of Irish fruit and vegetable waste and by products as a source of polyphenolic antioxidants''. Food Chem. 116: 202-207. Wilder man, P. R., Shah, M. B., Jang, H.-H., Stout, C. D. and Halpert, J. R. (2013) ''Structural and thermodynamic basis of (+)-α-pinene binding to human cytochrome P450 2B6''. J. Am. Chem. Soc. 135:10433−10440. Wu, X. and Prior, R.L. (2005) ''Identification and characterization of anthocyanins by high-performance liquid chromatography-electro spray ionization-tandem mass spectrometry in common foods in the United States: vegetables, nuts, and grains''. J. Agr. Food Chem. 53: 3101-3113. Yaltirik F. (1967a) ''Anacardiaceae''. In: Davis PH (ed) Flora of Turkey, volume 2. Edinburgh University Press, pp 544–548. 999
References Yanive, Z., and Palevitch, D. (1982) ''Effect of drought on the secondary metabolites of medicinal and aromatic plants''. In: Cultivation and Utilization of Medicinal Plants, C.V. Atal and B.M. Kapur (ed). CSIR Jammu Tawi, India. Ya-Qin Ma, Jian-Chu Chen, Dong-Hong Liu and Xing-Qian Ye. (2009) ''Simultaneous extraction of phenolic compounds of citrus peel extracts: Effect of ultrasound'', Ultrason. Sonochem. 16: 57–62. Yu, J., Ahmedna M. and Goktepe, I. (2005) ''Effect of processing methods and extraction solvent on concentration and antioxidant activity of peanut skin phenolics''. F. Chem. 90:199-206.
Yu Z. and Dahlgren R.A. (2000) ''Evaluation of methods for measuring polyphenols in Conifer foliage'', J Chem Ecol. 26:2119-214.
Zeki Haznedaroglu M., Ulku Karabay N. and Ulvi Zeybek (2001) ''Antibacterial activity of Salvia tomentosa essential oil'', Fitoterapia. 72(7): 829–831
Zhang w., franco, C. Curtin, C. And Conn, S. (2004) ''to stretch the boundary of a case study'' J. of biomed. And biotech. 5:264-271. Zohary, M. (1972) ''Pistacia L. Flora Palestine''. Isr Acad Sci and Hum. 2:297–300. Zohary, M. (1952) ''a monographic study of the genus pistachio Palestine'' J. Bot., Jerusalem Series, 5(4): 187-228.
999
Results and Discussion
Chapter four
Results and Discussion 4.1 Distribution of pistacia eurycarpa and pistacia khinjuk Both Pistacia spp are widely spread through Zagros Mountains region, particularly in West and North Iran, East and North Iraq, South Turkey and North Syria so called great Kurdistan (Mohammad and Stuard, 2011). The distribution of the two Pistacia spp. in Iraq which has been mentioned in the flora of Iraq based on different altitudes p. khinjuk grows at an elevation varying from 450-1750 m.a.s.l in rocky places and P. eurycarpa grows at about 750-1800 m.a.s.l in rocky places with oak and pine forest on limestone. Altitudinal variation of climate, and environmental stress, caused many visible changes in the composition of vegetation and in the growth habit of individual plants. Figures (11 – 24) represent some parts of the two Pistacia spp. such as: (leaves, gum, fruit, cluster and tree or shrub)
4.2 Preliminary detection of phytochemical compounds in the two Pistacia spp Results revealed the presence or absence of some phytochemicals such as: Flavonoids, Phenolic acid, Tannin, Saponins, Terpenoids, Steroids, Anthocyanins, Resins, Glycosides, Alkaloid, Fatty acids, Leuco-anthocyanins, Coumarins and Emodins in all parts (Leaves, Seed, Cluster without fruits, outer shell and Gum,) of P. eurycarpa and P. khinjuk. Table (5) showed that Flavonoids, Phenolic acid, Tannin, Saponins, Terpenoids, Steroids and Anthocyanins present in all parts (Leaves, Seed, Cluster without fruits outer shell, and Gum) of P. eurycarpa and P. khinjuk. While other compounds such as alkaloid, Coumarins and emodin are absent in all parts in the two pistacia spp. on the other hand Glycosides, Resins, Fatty acids and Leuco-anthocyanins are present only in some parts. Terpenoids and tannins impart for analgesic and anti-inflammatory activities. Apart from this tannins contribute property of astringency i.e., enhances the healing of wounds and inflamed mucous membrane (Okwu and Josiah, 2006). Traditionally saponins have been extensively used as detergents, as pesticides and molluscicides, in addition to their industrial applications as foaming and surface active agents and also have beneficial health effects (Shi et al., 2004). It should be noted that steroidal compounds are of importance and of interest in health science due to their relationship with sex hormones (Santhi et al., 2011). Steroids and Terpenoids are found in most parts of the medicinal plants of the present study; the 24
Results and Discussion
Chapter four
presence of bioactive compounds indicates the medicinal value of the plants. Antioxidants and antimicrobial properties of various extracts from many plants have recently been of great interest in both research and the food industry, because their possible use as natural additives emerged from a growing tendency to replace synthetic antioxidants and antimicrobials with natural ones (Deba et al., 2008). Preliminary qualitative test according to Mallikharjuna et al., (2007) is useful in the detection of bioactive principles and subsequently may lead to drug discovery and development (Vaghasiya et al., 2011). Appendix (1-11) represent the chromatograms of all parts of the two Pistacia spp. of the phenolic compounds extracted according to seven standard reference compounds available seven peaks were detection including the following phenolic compounds (Tannin, Stilbene, Catechin, Ellagic acid, Anthocyanin, Gallic acid and Cinnamic acid) in addition some minor peaks which could not be identified were also shown in some chromatograms.
4 .3 Phenolic compounds Traditional medicines based mostly on medicinal plants have been used for the treatment of various diseases by mankind for centuries. Plants are also well-known to be the rich sources of biologically active compounds. Therefore, one approach that has been used for the discovery of antimicrobial agents from natural sources is based on the evaluation of traditional plant extracts (Berrin et al., 2005). Phenolic compounds are the most diverse or largest branch of the secondary metabolites that are produced by most type of plants. Phenolics include simple phenols, phenolic acids (benzoic and cinnamic acid derivatives), Coumarins, Flavonoids, stilbenes, hydrolysable and condensed tannins and lignins. These compounds are acting mainly as phytoalexins, attractants for pollinators, contributors to plant pigmentation, antioxidants, and protective agents against UV light, among others (Gottlieb and Borin, 2000). Among the many varieties of natural phenolic compounds, the procyanidins are an important subgroup. These substances are composed of oligomers and polymers that consist of Catechin and/or epicatechin units (Okuda and Ito, 2011). Although quantitative determination of polyphenols is hampered by their structural complexity and diversity, several methods have been used to determine polyphenols in plant extracts (Moller et al, 2009).
24
Results and Discussion
Chapter four
Fig 11: P.khinjuk shrub
Fig 12: P.eurycarpa tree
Fig 13: Gum of P.khinjuk
Fig14: Gum of P.eurycarpa
22
Results and Discussion
Chapter four
Fig 15: Leaves of P. khinjuk
Fig 16: Leaves of P. eurycapa
Fig 17: Cluster of fruits of P. khinjuk
Fig 18: Cluster of fruits of P. eurycapa
24
Results and Discussion
Chapter four
Fig19: Immature fruit of P.khinjuk
Fig 20: Immature fruit of P.eurycarpa
Fig 21: Mature fruit of P.khinjuk (show mature seed)
Fig 22: Mature fruit of P. eurycapa (show mature seed)
Fig 23: Mature fruit of P.khinjuk
Fig 24: Mature fruit of P. eurycarpa
24
Results and Discussion
Chapter four
Table 5: Preliminary quantitative detection of some phytochemicals of the two Pistacia spp
(+) Positive result
Phytochemical compounds
(-) Negative result
Species and parts P. khinjuk cluster seed shell
P. eurycarpa
leaf
gum
cluster seed
shell
leaf
Gum
Flavonoids
+
+
+
+
+
+
+
+
+
+
Phenolic compounds Tannin 1
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Tannin 2
+
+
+
+
+
+
+
+
+
+
Saponins
+
+
+
+
+
+
+
+
+
+
Terpenoids
+
+
+
+
+
+
+
+
+
+
Steroids
+
+
+
+
+
+
+
+
+
+
Anthocyanins
+
+
+
+
+
+
+
+
+
+
Resins
+
+
+
-
+
+
+
+
-
+
Glycosides 1
+
+
+
-
-
+
+
+
-
-
Glycosides 2
+
+
+
-
-
+
+
+
-
-
Glycosides 3
+
+
+
-
-
+
+
+
-
-
Fatty acids
-
+
+
+
-
-
+
+
+
-
Leucoanthocyanin
-
+
+
-
-
-
+
+
-
-
Alkaloid 1
-
-
-
-
-
-
-
-
-
-
Alkaloid 2
-
-
-
-
-
-
-
-
-
-
Alkaloid 3
-
-
-
-
-
-
-
-
-
-
Coumarins
-
-
-
-
-
-
-
-
-
-
Emodins
-
-
-
-
-
-
-
-
-
-
24
Results and Discussion
Chapter four
Figures (25 - 31) shows the total phenolic compounds in parts (leaves, outer shell, cluster without fruit, seed and gum) of the two Pistacia spp. samples were collected during different growth stage (ripe and unripe) in May and September with other parameters from the same parts such as wounded and unwounded stems. Identification and determination of phenolic compounds are performed by HPLC according to seven standards that are presented below:
4.3.1 Tannin Figure (25) shows the total tannin of the two pistacia spp. parts (leaves, outer shell, cluster without fruit, seeds and gum) the significant differences were observed between the highest levels of tannin are present in ripe outer shell of P. eurycarpa spring wounded (31.72%, T11) and the lowest level appears in the gum of P. eurycarpa in spring collection (3.59%, T8), while Significant differences present in the unripe outer shell of the P. khinjuk spring collected contain high amount of tannin (23.33%, T1), and the lowest level appear in the mature leaves of P. khinjuk spring wounded (9.39%, T13). Outer shell of the P. khinjuk from unripe, spring collection contain high amount of tannin (23.33%, T1), and have significant differences were observed in comparison the other plant parts, ripe outer shell spring wounded (16,785%, T15), gum in spring collection (16.58%, T4), ripe cluster without fruit spring wounded (16.280%, T14), unripe cluster without fruit in spring (16 %, T2), ripe outer shell summer wounded (15.4%, T19), ripe seeds spring wounded (15.07%, T16), immature leaves in spring (14.8%, T3), mature leaves summer wounded (14.18%, T18), ripe cluster without fruit summer wounded (12.555%, T20), ripe seeds summer wounded (10.987%, T17) and mature leaves spring wounded (9.39%,T13), But no significant differences were observed between gum of P. khinjuk in spring collection (16.58%, T4) with ripe outer shell of the P. khinjuk spring wounded (16,785%, T15). While tannin in the ripe outer shell of P. eurycarpa spring wounded (31.72%, T11), shows a significant difference were observed in comparison the other plant parts such as; ripe outer shell summer wounded (19.21%, T25), ripe seed spring wounded (15.665%, T12), unripe cluster without fruit in spring (15.662%, T6), ripe cluster without fruit spring wounded (15.180%, T10), mature leaves summer wounded (15.050%, T22) higher than other parts respectively than unripe leaves in spring (13.967%, T5), mature leaves spring wounded (13.510%, T9), ripe seeds summer wounded (13.330%, T24), gum in summer collection (12.155%, T26), ripe cluster without fruit summer wounded (11.603%, T23), unripe seeds in spring (10.1%, T27), unripe outer shell in spring (8.630%, T7) and gum in 24
Results and Discussion
Chapter four
spring collection (3.59%,T8), during unripe gum samples of P. eurycarpa contain more tannin than unripe gum of same species. Tannin has antioxidant, antibacterial, anti-inflammatory and antifungal activity. The mechanism of tannin toxicity against microbes may be related to inhibition of hydrolytic enzymes (proteases and carbohydrolases) or other interaction to inactivate microbial adhesions, cell envelope transport proteins, and non specific interactions with carbohydrates (Karuo et al., 2005).
4.3.2 Stilbene Figure (26) shows the percentage value of stilbene in all parts of the two pistacia spp. parts, the significant differences were observed appear between largest concentration from mature leaves of P. khinjuk spring wounded (31.525%, T13) and the lowest concentration present in the immature outer shell of P. khinjuk (12.47%, T1) while significant differences appear between highest concentrations presents in gum of P. eurycarpa in spring (29.74%, T8) spring collection with lowest concentration in the ripe leaves of P. eurycarpa (15.315%, T9) spring wounded. Mature leaves (31.525%, T13) and cluster without fruit (27.56%, T14) of P. khinjuk spring wounded in summer collection richer in Stilbene than other parts of the same species among (ripe and unripe) parts and significant differences were observed with outer shell (24.747%, T19) summer wounded, gum (24%, T21) in summer, cluster without fruit (23.78%, T20) summer wounded, mature leaves (23.675%, T3) in spring, ripe seeds (23.4%, T16) spring wounded, unripe cluster without fruit (22.645%, T2), ripe outer shell (20.803%, T15) spring wounded, gum (18.895%, T4) in spring and smallest percentage value of unripe outer shell of the same species (12.47%, T1). Otherwise no significant differences were observed between mature leaves (26.550%, T18) with seeds (26.403%, T17) of P. khinjuk summer wounded and ripe cluster without fruit (23.78%, T20) summer wounded with unripe leaves (23.675%, T3) in spring, Percentage value of Stilbene of the gum of P. eurycarpa in spring (29.74%, T8), and ripe seeds of (27.3%, T24) summer wounded, richer than all parts and significant differences were observed present with unripe outer shell (27.14%, T7), ripe outer shell (25.08%, T25) summer wounded, unripe seeds (24.035%, T27) in spring, ripe cluster without fruit (23.48%, T23) summer wounded, ripe seeds (23.455%, T12), gum (23.055%, T26) in summer collected, mature leaves (21.645%, T22) summer wounded, unripe cluster without fruit (20.635%, T6) in spring, ripe outer shell (20.230%, T11) spring wounded, mature leaves (18.703%, T5) in spring, ripe cluster without fruit (17.93%, T10) in spring 24
Results and Discussion
Chapter four
and mature leaves of P. eurycarpa spring wounded (15.315%, T9) and the aftermost is very poor in stilbene than all parts of the same species. Otherwise no significant differences were observed between (ripe cluster without fruit (23.48%, T23) summer wounded, ripe seeds (23.455%, T12) summer wounded, and gum (23.055%, T26) in summer collected, Stilbene is very important compounds as anti bacterial activity against gram negative and gram positive bacteria (Kevin et al., 2006).
4.3.3 Catechin Figure (27) reveals the percentage value of Catechin in all parts of the two pistacia spp. The significant differences were observed among two Pistacia spp with highest concentration values in the ripe seeds of P. eurycarpa summer wounded (26.4%, T24) and the lowest concentration present in the gum of P. khinjuk in spring collection (9.58%, T1). Significant differences are found present among P. eurycarpa between highest amount in ripe seeds (26.4% T24) in summer wounded with lowest amount in mature leaves of P. eurycarpa (13.735%, T9) spring wounded, while significant differences present between highest amount in ripe cluster without fruits of P. khinjuk (24.885%, T14) spring wounded and lowest amount in unripe outer shell of P. khinjuk (9.58%, T1). ripe seeds of P. eurycarpa (26.4% T24) in summer wounded and unripe (26.14%, T27) contain higher amount of Catechin than all parts from spring to summer collection and have significant differences compared with the following plant parts, gum (22.92%, T8) in spring, ripe outer shell (22.788%, T25) summer wounded, ripe seeds (22.425%, T12) spring wounded, gum (21.435%, T26) in summer collection, ripe cluster without fruit (21.305%, T23) summer wounded, ripe cluster without fruit (20.89%, T10) spring wounded, mature leaves (20.64%, T22) summer wounded, unripe outer shell (19.32%, T7) in spring, immature leaves (18.705, T5) in spring, unripe cluster without fruit (14.875%, T6) in spring, ripe outer shell (14.415%, T11) spring wounded and mature leaves of P. eurycarpa (13.735%, T9) spring wounded, and the aftermost contain low concentration of catechin than other parts during sampling from spring to summer. No significant differences are found between gum (22.92%, T8) in spring with ripe outer shell (22.788%, T25) summer wounded, gum (21.435%, T26) summer collection with cluster without fruits (21.305%, T23) of P. eurycarpa summer wounded and ripe cluster without fruit (20.89%, T10) spring wounded with mature leaves (20.64%, T22) summer wounded,
45
Results and Discussion
Chapter four
Ripe cluster without fruits of P. khinjuk (24.885%, T14) spring wounded is richer in Catechin than other parts (seeds, leaves, outer shell and cluster without fruit),and significant differences are found among some plant parts, but the concentration of Catechin arranged from largest to smallest of samples from the following in the mature leaves (21.51%, T13) in spring, ripe outer shell (20.485%, T15) spring wounded, ripe seeds (19.33%, T16) spring wounded, gum (19.32%, T4) in spring collected, gum (19.215%, T21) in summer collection, ripe cluster without fruits (19.155%, T20) summer wounded, mature leaves (18.625%, T18) summer wounded, ripe outer shell (17.07%, T19) summer wounded, ripe seeds (16.775%, T17) summer wounded, unripe cluster without fruits (13.235%, T2) in spring, immature leaves (11.94%, T3) and unripe outer shell of P. khinjuk (9.58%, T1). no significant differences were found between ripe outer shell (20.485%, T15) spring wounded, ripe seeds (19.33%, T16) spring wounded, gum (19.32%, T4) in spring collected, gum (19.215%, T21) in summer collection and ripe cluster without fruits (19.155%, T20) summer wounded, Catechin
is
very
important
phytochemicals
for
human
health
such
as:
anticancer, cardiovascular disease and neuroprotection activity (Nurulain, 2006).
4.3.4 Ellagic acid Figure (28) represents percentage concentration of Ellagic acid in two pistacia spp. The significant differences were observed among Pistacia spp between highest values from mature leaf of P. eurycarpa spring wounded (21.925%, T9) and the lowest concentration value of mature leaves P. khinjuk spring wounded (4.315%, T13) but ellagic acid is not detected in the immature outer shell of P. eurycarpa in spring (0.0%, T7) Significant differences were observed between immature clusters without fruit of P. khinjuk in spring (14.5%, T2) with mature leaves (4.315%, T13) spring wounded, while significant differences are found between large amount of ellagic acid present in mature leaves of the P. eurycarpa (21.925%, T9) spring wounded with ripe seeds (4.46%, T12) spring wounded. Ellagic acid are present in the highest to lowest level and significant differences were observed between them in immature cluster without fruit of P. khinjuk in spring (14.5%, T2), immature leaves (13.15%, T3) in spring, immature outer shell (13%, T1) in spring, mature cluster without fruit (11.765%, T20) summer wounded, ripe seeds (11.425%, T16) spring wounded, mature outer shell (10.965%, T15) spring wounded, mature outer shell (10.3%, T19) summer wounded, mature seeds (9.922%, T17) summer wounded, mature leaves (9.61%, T18) summer wounded, gum (9.51%, T21) in summer, gum (9.17%, T4) in 45
Results and Discussion
Chapter four
spring respectively of the same species, otherwise mature cluster without fruit (4.92%, T14) spring wounded and mature leaves (4.315%, T13) in summer (spring wounded) contain lowest level of Ellagic acid and no significant differences were observed between (unripe leaves (13.15%, T3) in spring, immature outer shell (13%, T1) in spring) and (mature cluster without fruit (4.92%, T14) spring wounded and mature leaves (4.315%, T13) in summer spring wounded). However a large amount of ellagic acid is present at the mature leaves of P. eurycarpa (21.925%, T9) in summer collected (spring wounded) than other parts. Percentage concentration level of ellagic acid in other parts are the following immature cluster without fruit (11.51%, T20) in spring, gum (11.2%, T26) in summer collected, unripe clusters without fruit (10.385%, T10) in spring, immature leaves (9.96%, T5) in spring, immature seeds (9.225%, T27) in spring, ripe outer shell (9.18%, T25) summer wounded, ripe clusters without fruit (7.87%, T23) summer wounded, gum (7.17, T8) in spring, mature seeds (5.73%, T24) summer wounded, mature leaves (5.085%, T22) summer wounded, mature outer shell (4.53%, T11) spring wounded, ripe seeds (4.46%, T12) spring wounded. In this study ellagic acid is absent only in the outer shell of unripe P. eurycarpa in spring collection (0.00%, T7), no significant differences were observed between immature leaves (9.96%, T5) in spring with immature seeds (9.225%, T27) in spring and mature outer shell (4.53%, T11) spring wounded with mature seeds (4.46%, T12) spring wounded. Ellagic acid is one type of Phenolic phytochemicals that are important components of fruits and vegetables and are partly responsible for their beneficial health effects against oxidation-linked chronic diseases such as cancer and cardiovascular diseases, and function either by countering the negative effects of oxidative stress by directly acting as an antioxidant or by activating/inducing cellular antioxidant enzyme systems (Navindra et al., 2005).
4.3.5 Anthocyanins Figure (29) displays the percentage concentration of Anthocyanin in all parts of the two Pistacia spp., the significant differences were observed between concentration value of highest level in the gum of P. khinjuk in summer collection (19.39%, T21) and the lowest concentration value of ripe seeds P. khinjuk spring wounded (8.7%, T16) while Outer shell of P. eurycarpa (17.350%, T7) in spring collection with ripe outer shell (10.18%, T25) summer wounded samples. Arrangement of Anthocyanin values from high to low and significant differences were observed from Gum of P. khinjuk in summer (19.39% T21), unripe seeds (17.37%, T4) in 44
Results and Discussion
Chapter four
spring, immature leaves (15.825%, T3) in spring, immature outer shell (15.67%, T1) in spring, unripe cluster without fruit (14.485%, T2) in spring, ripe outer shell (14.385%, T15) spring wounded, ripe cluster without fruit (12.605%, T20) summer wounded, ripe seeds (12.52%, T17) summer wounded, mature leaves (12.26%, T13) spring wounded, mature leaves (11.075%, T18) summer wounded, ripe cluster without fruit (10.67%, T14) spring wounded, ripe outer shell (10.605%, T19) summer wounded and while the lowest concentration present in seeds of P. khinjuk spring wounded (8.7%, T16), no significant differences were observed between (immature leaves (15.825%, T3) in spring with immature outer shell (15.67%, T1) in spring), (unripe cluster without fruit (14.485%, T2) in spring with ripe outer shell (14.385%, T15) spring wounded), (ripe cluster without fruit (12.605%, T20) summer wounded, ripe seeds (12.52%, T17) summer wounded and mature leaves (12.26%, T13) spring wounded) , (ripe leaves (11.075%, T18) summer wounded, ripe cluster without fruit (10.67%, T14) spring wounded and ripe outer shell (10.605%, T19) summer wounded. Outer shell of P. eurycarpa (17.350%, T7) in spring collection rich in Anthocyanin than other parts (leaves, seeds, cluster without fruits and gum), and significant differences were observed with other parts, but other value respectively arranged, ripe seeds (16.17%, T24) summer wounded, mature leaves (16.03%, T9) spring wounded, immature leaves (15.95%, T5) in spring, ripe seeds (15.94%, T12) summer wounded, unripe cluster without fruit (15.845%, T6) in spring, ripe cluster without fruit (15.475%, T23) summer wounded, ripe outer shell (15.160%, T11) spring wounded, gum (15.095%, T26) in summer collection, ripe cluster without fruit (13.99%, T10) spring wounded, mature leaves (13.915%, T22) summer wounded, gum (13.703%, T8) in spring, unripe seed (10.895%, T27) in spring and ripe outer shell (10.18%, T25) summer wounded, no significant differences were observed between ripe seeds (16.17%, T24) summer wounded), mature leaves (16.03%, T9) spring wounded, immature leaves (15.95%, T5) in spring, ripe seeds (15.94%, T12) summer wounded, unripe cluster without fruit (15.845%, T6) in spring, ripe cluster without fruit (15.475%, T23) summer wounded, ripe outer shell (15.160%, T11) spring wounded, gum (15.095%, T26) in summer collection), (ripe cluster without fruit (13.99%, T10) spring wounded, mature leaves (13.915%, T22) summer wounded, gum (13.703%, T8) in spring) and (immature seed (10.895%, T27) in spring with ripe outer shell (10.18%, T25) summer wounded). Anthocynidin help the human immune system to work more efficiently to protect against viral infections, specific types of Anthocynidins may have a direct effect in 44
Results and Discussion
Chapter four
decreasing influenza viruses infectivity by decreasing the ability of the virus itself to get into the human cell or to be related to infected cells or by having a viricide effect (Liu et al., 2009).
4.3.6 Gallic acid Figure (30) reveals that gallic acid is found in all parts of the two Pistacia spp., significant differences were observed between highest values from gum of P. eurycarpa spring collection (17.595%, T8) and the lowest concentration is present in the immature cluster without fruit of P. khinjuk spring collection (6.57%, T2). Significant differences were observed between highest values from gum of P. eurycarpa spring collected (17.595%, T8) with the lowest concentration present in the ripe outer shell (8.335%, T25) summer wounded while in P. khinjuk significant differences are found between highest level unripe outer shell (14.770%, T1) in spring with lowest level present in unripe cluster without fruit (6.57%, T2) Gallic aid found in all parts of both Pistacia species, from largest to smallest concentration value appear in gum of P. eurycarpa in spring collection (17.595%, T8) and have significant differences were observed with other parts, ripe cluster without fruit (15.695%, T23) summer wounded, mature leaves (14.87%, T22) summer wounded, immature outer shell (14.71%, T7) in spring, mature leaves (13.5%, T9) spring wounded, ripe cluster without fruit(13.49%, T10) spring wounded, mature seeds (11.55%, T12) spring wounded, unripe cluster without fruit(11.34%, T6) in spring, immature leaves (11.315%, T5) in spring, ripe seeds (9.08%, T24) summer wounded, immature seeds (8.99%, T27) in spring, gum (8.91%, T26) in summer collection, ripe outer shell (8.885%, T11) spring wounded and ripe outer shell (8.335%, T25) summer wounded of same species. But no significant differences were observed between P. eurycarpa parts (mature leaves (14.87%, T22) summer wounded, immature outer shell (14.71%, T7) in spring, mature leaves (13.5%, T9) spring wounded, ripe cluster without fruit (13.49%, T10) spring wounded, ripe seeds (11.55%, T12) spring wounded, unripe cluster without fruit(11.34%, T6) in spring, immature leaves (11.315%, T5) in spring, and unripe seeds (8.99%, T27) in spring, gum (8.91%, T26) in summer collection, ripe outer shell (8.885%, T11) spring wounded). The amount of gallic acid from unripe outer shell (14.770%, T1) of P. khinjuk in spring higher than all parts from spring to summer, and arranged from high to low and significant differences were observed with other parts such as; ripe seed (13.165%, T16) 42
Results and Discussion
Chapter four
spring wounded, ripe cluster without fruit (12.995%, T14) spring wounded, mature leaves (12.905%, T13) spring wounded, mature leaves (12.855%, T18) summer wounded, immature leaves (12.835%, T3) in spring, ripe seeds (11.95%, T17) summer wounded, ripe outer shell (10.36%, T15) spring wounded, ripe outer shell (10.055%, T19) summer wounded, ripe cluster without fruit (9.87%, T20) summer wounded, gum (7.1%, T4) in spring collection and lowest level present in unripe cluster without fruit (6.57%, T2) in spring. No significant differences were observed between some P. khinjuk parts from largest to smallest from mature seed (13.165%, T16) spring wounded, ripe cluster without fruit (12.995%, T14) spring wounded, mature leaves (12.905%, T13) spring wounded, mature leaves (12.855%, T18) summer wounded, immature leaves (12.835%, T3) in spring, ripe outer shell (10.36%, T15) spring wounded, ripe outer shell (10.055%, T19) summer wounded, ripe cluster without fruit (9.87%, T20) summer wounded) and (gum (7.1%, T4) in spring collected and lowest level present in unripe cluster without fruit (6.57%, T2) in spring. Gallic acid is an organic acid found in a variety of foods and herbs that are well known as powerful antioxidant, anti bacterial and anti-inflammatory effect (Bohn et al., 1998).
4.3.7 Cinnamic acid Figure (31) shows that cinnamic acid in all parts of the two pistacia spp. however significant differences were observed between concentration value of highest level in the unripe clusters without fruit of P. eurycarpa spring collection (13.875%, T6) and lowest concentration value present from seed of P. eurycarpa summer wounded (3.49%, T24) while in P. khinjuk significant differences were observed between largest amount immature cluster without fruit (11.84%, T2) in spring with smallest level mature cluster without fruit (5.195%, T14) spring wounded of the same species. The result of cinnamic acid revealed that the high concentration of cinnamic acid to low value appear in immature cluster without fruit (11.84%, T2) in spring, ripe outer shell (11.80%, T19) summer wounded, gum (11.535%, T4) in spring, mature seeds (11.385%, T17) summer wounded, gum (11.325%, T21) in summer, immature leaves (10.765%, T3) in spring, mature cluster without fruit (10.33%, T20) summer wounded, ripe leaves (10.075%, T13) spring wounded, ripe seeds (8.895%, T16) spring wounded, immature outer shell (7.15%, T1) in spring, mature leaves (6.62%, T18) summer wounded, mature outer shell (6.195%, T15) spring wounded and mature cluster without fruit (5.195%, T14) spring wounded of the P. khinjuk respectively. 44
Results and Discussion
Chapter four
No significant differences were observed between P. khinjuk parts (immature clusters without fruit (11.84%, T2) in spring, ripe outer shell (11.80%, T19) summer wounded, gum (11.535%, T4) in spring and ripe seeds (11.385%, T17) summer wounded, gum (11.325%, T21) in summer. Immature cluster without fruit (13.875%, T6) in spring, unripe outer shell (12.830%, T7) in spring and immature leaves (11.445%, T5) in spring of P. eurycarpa respectively contain high amount of cinnamic acid than other parts at the different growth stage, while other value arranged from large to small; unripe seeds (10.54%, T27) in spring, mature outer shell (9.97%, T11) spring wounded, mature leaves (8.425%, T22) summer wounded, gum (8.075%, T26) summer collection, mature cluster without fruit (7.79%, T10) spring wounded, ripe seeds (6.285%, T12) spring wounded, mature leaves (6.005%, T9) spring wounded, mature outer shell (5.205%, T25) summer wounded, gum (4.795%, T8) in spring collection, mature cluster without fruit (4.550%, T23) and ripe seeds (3.490%, T24) summer wounded that contain little amount of cinnamic acid concentration. No significant differences were observed between P. eurycarpa parts in mature leaves (8.425%, T22) summer wounded, gum (8.075%, T26) summer collection, mature cluster without fruit (7.79%, T10) spring wounded) and mature outer shell (5.205%, T25) summer wounded, gum (4.795%, T8) in spring collection. Excess light and UV-radiation are hazardous natural stress factors, and plants have evolved a range of avoidance and tolerance strategies employing versatile tools against these constraints. Some plant outlines the contribution of non-photosynthetic pigments to the protection of plants from excess light and UV-radiation, as well as the mechanisms involved. A large pool of secondary metabolites, belonging mainly to the highly diversified array of Flavonoids (C6–C3–C6 types), and the closely related anthocyanins (flavylium salts, C6–C3–C6+ types), as well as betacyanins are often referred to as non-photosynthetic pigments and another group of secondary metabolites, cinnamic acid derivatives C6–C3 types (Aglika, 2005). Phenolic compounds exhibit a wide range of such physiological properties as antiallergenic, antiartherogenic, anti-inflammatory, antimicrobial, antioxidant, anti-thrombotic, cardio protective and vasodiolatory effects (Balasundram et al., 2006; Andersen and Markham, 2006 and Rajaei et al., 2010). The beneficial effects derived from phenolic compounds have been attributed to their antioxidant activity. These compounds could be a major determinant of antioxidant potentials of foods, and a natural source of antioxidants (Balasundram et al., 2006). The 44
Results and Discussion
Chapter four
research on phenolic compounds has been growing lately because of the increasing worldwide demand for phenolic compounds and their increasing application in food industry (Rodrigues and Pinto, 2006). The analysis of phenolic compounds in plant samples is difficult because of the great variety of their structure and the lack of appropriate standards (Huang et al., 2007; Magalhães et al., 2009). Moreover, the identification of anthocyanins may help in a better understanding of environment-induced changes in plant secondary metabolism (Harborne, 1993). and of relation between the chemical composition of vegetable tissues and specific biological/pharmacological activities (Larson, 1995). Optimization of extraction conditions (extraction temperature, number of extraction cycles, matrix particle size and solid-to-solvent ratio, flush volume, pressure, and static time) which are directly dependent on the availability of the extraction technology. Optimization of extraction parameters not only increases extraction efficiency of the analyte of interest but also reduces the solvent consumed and the waste generated during an extraction process (Devanand, 2006). The present study shows that all types of phenolic compounds are affected by seasonal changes from spring to summer according to wounded and unwounded stems of the two Pistacia spp. occurring during plant growth ripe and unripe, highest phenolic compounds is tannin otherwise the lowest level is cinnamic acid. These results were in agreement with the results of Taran et al., (2010) and Mahbubeh et al., (2013). But in this study present there are some differences disagreement with Ali Asghar et al., (2014) because different in climate, soil properties, extraction methods (Liazid et al, 2007) plant parts or species (Antolovich et al., 2000) Temperature (Vallejo et al, 2003) Solvent (Nuutila et al., 2003) and time of harvesting (Naczk and Shahidi, 2006). The phenolic content in plants includes the different factors that affect the content in plant bioactive compounds, such as light, temperature, mineral nutrition, pathogens, mechanical damage, plant-growth regulators, and other factors (Dinelli et al, 2006).
44
Chapter four
Results and Discussion
4.4. Volatile oil compounds Essential oil or volatile oil is one type secondary metabolites that is produced and released by some type of aromatic plant, in this study the extraction of volatile oil is from two types of pistacia spp. From different plan parts (leaves, outer shell, seeds, cluster without fruits and gum) in different growth stage (ripe and unripe) and (wounded and unwounded) parameters by Clevenger apparatus, after purification and dehydration by Na2SO4, and the detection of volatile oil compounds is by HPLC according to eleven standards. Appendix (12 – 19) represents the chromatograms of all parts of the two Pistacia spp. of the volatile oil compounds extracted according to eleven standards reference compounds available. Eleven peaks were detected including the following volatile oil compounds (α–Pinene, Phellandrine, β – Pinene, limonene, Sabienene, Aldehyde – citral, 3– Carene, Myrecene, Terpinene, Carveol and Myretenol). In addition some minor peaks which could not be identified were also shown in some chromatograms.
4.4.1 α– Pinene Figure (32) shows the percentage concentration of α–Pinene in some parts of the two pistacia spp., the significant differences between high concentrations from the gum of P. eurycarpa spring collection (55.762%, T1) with the lowest concentration present in the immature leaves of P. eurycarpa spring collected (30.00%, T5) while significant differences appear between Gum of P. khinjuk in summer collection (53.515%, T9) with cluster without fruit of P. khinjuk in spring (32.21%, T6) Gum of P. eurycarpa spring is collected (55.762%, T1), and significant differences were observed with ripe cluster without fruit summer wounded (54.585%, T11) and unripe cluster without fruit of in spring (52.682%, T4) rich in α–Pinene than other parts of the same species , while gum (47.725%, T8) in summer, unripe outer shell (45.735%, T3) ripe cluster without fruit of spring wounded (39.785%, T10) and immature leaves of P. eurycarpa in spring (30.00%, T5), poor in α–Pinene. No significant differences are found between Gum P. eurycarpa of spring collection (55.762%, T1), ripe cluster without fruit summer wounded (54.585%, T11) and unripe cluster without fruit of in spring (52.682%, T4) Gum of P. khinjuk in summer collection (53.515%, T9) contain high amount of α– Pinene than all parts, otherwise immature leaves (36.59%, T7) and cluster without fruit of P. khinjuk in spring (32.21%, T6) poor in α - Pinene than other parts, on the other hands 56
Chapter four
Results and Discussion
no significant differences are found between spring wounded and summer wounded of cluster without fruits of P. khinjuk. Both parts contain (45.325%, T12 and T13) of α – Pinene. Meaning wounded not affected on the production of α – Pinene in these parts. The chemical substance α - Pinene is a component of many essential oils and has antiinflammatory, antimicrobial properties (Rivas et al., 2012 and Wilder man et al., 2013). and skin burn wound healing activity (Haghdoost et al., 2013).
4.4.2 Phellandrine Figure (33) reveals the percentage concentration of Phellandrine and significant differences were observed among two pistacia spp. between largest concentration mature cluster without fruits of P. khinjuk spring wounded (23.03%, T12) and the lowest concentration value present in gum of P. eurycarpa spring collection (7.185%, T1). Significant differences were observed between the largest amount of spring wounded clusters without fruits of P. khinjuk (23.03%, T12) with lowest level immature leaves (7.74%, T7) in spring, while in P. eurycarpa significant differences appear between ripe cluster without fruits (16.62%, T11) summer wounded with gum (7.185%, T1) in spring. Spring wounded cluster without fruits of P. khinjuk (23.03%, T12) contain large concentration of Phellandrine compounds than other parts and significant differences were observed with other parts, ripe cluster without fruits (9.31%, T13) summer wounded, while no significant differences were observed between gum of P. khinjuk from spring (10.83%, T9) to summer (10.60%, T2) and unripe cluster without fruits in spring (7.83%, T6) and immature leaves (7.74%, T7) in spring that contain smallest value of Phellandrine. Arrangement concentration of Phellandrine compounds from largest to smallest in P. eurycarpa; ripe cluster without fruits (16.62%, T11) summer wounded and significant differences were observed with other parts, unripe outer shell (12.033%, T3) in spring, unripe cluster without fruits (11.265%, T4) in spring, gum (9.623%, T8) in summer, immature leaves (9.055%, T5) in spring, ripe cluster without fruits (7.832%, T10) spring wounded and gum (7.185%, T1) in spring. Phellandrene is the name for a pair of organic compounds that have a similar molecular structure and similar chemical properties. Alpha Phellandrene and beta phellandrene are cyclic monoterpenes and are double-bond isomers. The phellandrenes are used in fragrances because of their pleasing aromas. Phellandrenes is mainly found in the essential oils of many species of Anacardiaceae family (Ghasemi and Alghaee, 2011).
55
Chapter four
Results and Discussion
4.4.3 β– Pinene Figure (34) represent the percentage concentration of β – Pinene from the two pistacia spp., the significant differences were observed between largest concentration of cluster without fruits of P. khinjuk summer wounded (19.72%, T13) and the lowest concentration value of outer shell of P. eurycarpa spring collection (4.29%, T3). Significant differences were observed between largest values of ripe cluster without fruits of P. khinjuk in summer (19.72%, T13) summer wounded with gum of same species in spring (5.425%, T2) while in P. eurycarpa significant differences were observed between highest concentration in ripe cluster without fruits (12.345%, T11) summer wounded with lowest concentration unripe outer shell in spring (4.29%, T3). Ripe cluster without fruits of P. khinjuk (19.72%, T13) summer wounded and significant differences were observed with other parts, ripe cluster without fruits (16.680%, T12) summer wounded, unripe cluster without fruits (10.775%, T6) and gum (9.295%, T9) in summer respectively contain high amount of β – Pinene, but the little amount of β – Pinene present in immature leaves (7.26%, T7) in spring collection and gum of same species at the same time (5.425%, T2). The highest percentage concentration value of β – Pinene in the P. eurycarpa present in ripe cluster without fruits (12.345%, T11) summer wounded and significant differences were observed with other parts, immature leaves of P.eurycarpa (12.175%, T5) in spring, gum (11.975%, T1) in spring and gum (11.465%, T8) in summer, while the smallest value appear in unripe outer shell of the P. eurycarpa in spring (4.29%, T3). significant differences are not found between in ripe cluster without fruits (12.345%, T11) summer wounded, immature leaves of P. eurycarpa (12.175%, T5) in spring, gum (11.975%, T1) in spring and gum (11.465%, T8) in summer. β – Pinene has antimicrobial and anti-inflammatory activity against some gram negative and gram positive bacteria (Rivas et al., 2012)
4.4.4 Limonene Figure (35) shows the percentage concentration of limonene in all parts of the two Pistacia spp., the significant differences are found between concentration values of cluster without fruits of P. khinjuk spring wounded (13.085%, T6) and the lowest concentration value of gum P. khinjuk summer collection (1.755%, T9) while significant differences are found between highest concentration values in gum of the P. eurycarpa in
56
Chapter four
Results and Discussion
summer (10.253%, T8) with smallest value in immature cluster without fruits in spring (1.980% T4). Concentration of limonene is present in P. khinjuk from higher to lower in unripe cluster without fruits in spring (13.085%, T6) spring wounded and significant differences are found with other parts; immature leaves (11.71%, T7) in spring and ripe cluster without fruits (11.103%, T13) summer wounded, gum (8.245%, T2) in spring, ripe cluster without fruits (3.78%, T12) spring wounded and gum of this species in summer collection (1.755%, T9) very poor in limonene than all parts. No significant differences are present between P. khinjuk parts such as: immature leaves (11.71%, T7) in spring and ripe cluster without fruits (11.103%, T13) summer wounded. Gum of P. eurycarpa in summer (10.253%, T8) contain large amount of limonene than other parts and respectively arrangement with outer shell (8.42%, T3) in spring, immature leaves (7.65%, T5) in spring, ripe cluster without fruits (6.655%, T10) spring wounded, ripe cluster without fruits (2.715%, T11) summer wounded, while cluster without fruits in spring (1.980% T4) is the smallest value than other parts. Significant differences are found between all above parts. Limonene is a very active compound against some types of bacteria (Aggarwal et al., 2002) and has anti-tumour activity (Bonithon-Kopp et al., 2000).
4.4.5 Sabinene Figure (36) shows the percentage concentration of Sabinene from parts of the two Pistacia spp., the significant differences present between concentration value of highest level of P. khinjuk immature leaves in spring (12.69%, T7) and lowest concentration value present from gum of P. khinjuk summer collection (1.865%, T9) while in P. eurycarpa significant differences are present between concentration value of highest amount immature outer shell (9.650%, T3) in spring with lowest concentration in ripe cluster without fruits in summer (7.335%, T11) summer wounded. Highest level of Sabinene is present in the P. khinjuk mature leaves (12.69%, T7) spring wounded and significant differences are found with other parts, gum (11.335%, T2) in spring and ripe cluster without fruits (8.875%, T13) summer wounded of the same species respectively, otherwise immature cluster without fruits (3.54%, T6) in spring, ripe cluster without fruits (3.025%, T12) spring wounded and gum (1.865%, T9) in summer contain lowest level of Sabinene.
56
Chapter four
Results and Discussion
No significant differences are found between P. khinjuk parts, immature cluster without fruits (3.54%, T6) in spring, ripe cluster without fruits (3.025%, T12) in summer spring wounded and gum (1.865%, T9) in summer. No significant differences are found between immature outer shell (9.650%, T3) in spring and gum (9.365%, T1) in spring of P. eurycarpa that contain large concentration of Sabienene than other parts, while the amount of Sabienene in some parts of the P. eurycarpa and no significant differences are found between the below parts; unripe cluster without fruits (8.845%, T4) in spring, mature cluster without fruits (8.085%, T10) spring wounded, immature leaves (8.080%, T5) in spring, gum (7.648%, T8) in summer and ripe cluster without fruits (7.335%, T11) summer wounded. The essential oil remarkably inhibited the growth of tested Gram-positive and Gramnegative bacteria except for Pseudomonas aeruginosa that contain Sabinene compounds (Zeki et al., 2001).
4.4.6 Aldehyde – citral Figure (37) shows the total Aldehyde – citral of the two pistacia spp. parts (leaves, outer shell, seeds cluster without fruit, and gum), significant differences are found between the highest concentration of Aldehyde-citral in the gum of P. khinjuk summer collection (9.295%, T9) and the lowest level appear in the cluster without fruits of P. eurycarpa spring wounded (2.5%, T4). Significant differences are found between the highest concentrations of Aldehydecitral in the gum of P. khinjuk (9.295%, T9) summer collection with lowest level of unripe cluster without fruits (4.020%, T6) in spring, while in P. eurycarpa mature cluster without fruits (8.87%, T10) spring wounded contain high amount of Aldehyde – citral and significant differences were observed with the lowest level unripe cluster without fruits (4.020%, T6) in spring. The largest concentration of Aldehyde – citral is present in the gum of P. khinjuk in summer collection (9.295%, T9) and significant differences are found between all parts that respectively arrangements from high to low concentration; gum (7.003%, T2) in spring, immature leaves (6.045%, T7) in spring, ripe clusters without fruits (5.45%, T12) spring wounded, ripe clusters without fruits (4.695%, T13) summer wounded and the lowest level is present in unripe cluster without fruits (4.020%, T6) in spring. Mature cluster without fruits (8.87%, T10) in summer, spring wounded contain high amount of Aldehyde – citral, and no significant differences were observed exist between
56
Chapter four
Results and Discussion
gum of P. eurycarpa in summer collection (7.225%, T8) with immature leaves (7.115%, T5) in spring of the same species. While no significant differences were observed between clusters without fruits (6.148%, T11) summer wounded and gums of P. eurycarpa in summer (5.870%, T1), Otherwise the amount of Aldehyde – citral is in immature outer shell (5.2%, T3) in spring, but the smallest value is present in unripe cluster without fruits (4.020%, T6) in spring. Aldehyde-citral constitutes an important flavor component and has enoate reductase activity of whole cells of yeasts, bacteria and fungi (Mélanie et al., 2006).
4.4.7 (3 – Carene) Figure (38) represents the percentage concentration of 3– Carene of the two pistacia spp. The significant differences were observed between highest values from unripe cluster without fruit of P. khinjuk spring collection (9.13%, T6) and the lowest concentration value of gum of P. khinjuk summer collection (2 %, T9), while in P. eurycarpa the significant differences are found between highest value in immature leaves (8.21%, T5) in spring with lowest value in immature cluster without fruit (3.585%, T4) in spring. Arrangement of 3– Carene concentration of the P. khinjuk from largest to smallest are; immature cluster without fruit (9.130%, T6) in spring and have significant differences were observed with other parts, mature cluster without fruit of (7.890%, T12) spring wounded, immature leaves (7.10%, T7) in spring, gum (4.158%, T2) in spring, ripe cluster without fruit (2.950%T13) summer wounded and gum (2%, T9) in summer collection. Immature leaves of P. eurycarpa contain high amount of 3-carene (8.21%, T5) in spring and significant differences were observed with other parts, gum (7.815%, T1) in spring, immature outer shell (6.560%, T3) in spring, gum (6.120%, T8) in summer, ripe cluster without fruit (6.050%, T11) summer wounded, ripe cluster without fruit (5.375%, T10) spring wounded, and immature cluster without fruit (3.585%, T4) in spring of the same species respectively. Significant differences disappear between immature leaves of P. eurycarpa contain (8.21%, T5) in spring with gum (7.815%, T1) in spring and gum (6.120%, T8) in summer with ripe cluster without fruit in summer (6.050%, T11) summer wounded. 3-carene is one type of Phytochemical compounds that is very active as antiviral against some types of macrophage (Anne and Margot, 1997).
67
Chapter four
Results and Discussion
4.4.8 Myrecene Figure (39) reveals the percentage concentration of Myrecene in all parts of the two pistacia spp. The significant differences present between concentration value of highest level in the mature cluster without fruits of P. eurycarpa summer wounded (7.51%, T11) and the lowest concentration value present in gum of P. khinjuk summer collection (1.1%, T9). Significant differences were observed between concentration values of highest level in the ripe cluster without fruits of P. eurycarpa summer wounded (7.51%, T11) with smallest level in ripe cluster without fruits (2.835%, T10) spring wounded, while in P. khinjuk significant differences are found between unripe cluster without fruits (5.905%, T6) in spring with gum (1.1%, T9) in summer collection. The high concentration of Myrecene presents in ripe cluster without fruits of P. eurycarpa (7.51%, T11) summer wounded and have significant differences with immature leaves (6.870%, T5) in spring, gum (6.4%, T8) in summer, gum (6.385%, T1) in spring of the same species, the lowest concentration appear in unripe cluster without fruits (5.77%, T4), immature outer shell (3.935%,T3) and ripe cluster without fruits (2.835%, T10) spring wounded, and not significant differences are there between immature leaves (6.870%, T5) in spring, gum (6.4%, T8) in summer collection and gum (6.385%, T1) in spring collection. Large amount of Myrecene are present in P. khinjuk from largest to smallest; unripe cluster without fruits (5.905%, T6) in spring and have significant differences
with
immature leaves (5.710%, T7) in spring, mature cluster without fruits (4.175%, T12) spring wounded, ripe cluster without fruits (3.965%, T13) summer wounded with gum (3.695%, T2) in spring, but the smallest value present in gum (1.1%, T9) in summer, and no significant differences appear between (unripe cluster without fruits (5.905%, T6) in spring with unripe leaves (5.710%, T7) in spring) and (mature cluster without fruits (4.175%, T12) spring wounded with ripe cluster without fruits (3.965%, T13) summer wounded). Myrecene is the one type of monoterpene that is present in some volatile oils, and have Antioxidants and Antigenotoxicity activity (Mitić-Ćulafić et al., 2009).
4.4.9 Terpinene Figure (40) display the percentage concentration of Terpinene and significant differences appear between largest value among two pistacia spp between immature 67
Chapter four
Results and Discussion
leaves of P. eurycarpa in spring (6.977%, T5) and the smallest percentage concentration of mature cluster without fruits of P. khinjuk summer wounded (1.46%, T13). Significant differences are found between largest values immature leaves in P. eurycarpa in spring (6.977%, T5) with lowest level in gum (2.425%, T2) in spring, while in P. khinjuk significant differences appear between largest amounts in immature cluster without fruits (3.765%, T6) in spring with smallest value of Terpinene present in the ripe cluster without fruits (1.460%, T13) summer wounded. Large amounts of Terpinene is present in leaves in P. eurycarpa in spring (6.977%, T5) and significant differences were observed with mature cluster without fruits (4.435%, T11) summer wounded and mature cluster without fruits (3.880%, T10) spring wounded, immature outer shell (3.15%, T3) in spring, gum (2.77%, T8) in summer collection, unripe cluster without fruits (2.620%, T4) in spring and gum (2.425%, T2) in spring of the same species respectively, while no significant differences appear among unripe cluster without fruits (2.620%, T4) in spring and gum (2.425%, T2) in spring. No significant differences appear between largest value of cluster without fruits of P. khinjuk (3.765%, T6) in spring and ripe cluster without fruits (3.685%, T12) in summer (spring wounded) that contain high amount of Terpinene, and no significant differences appear between moderate value gum (3.32%, T9) in summer with unripe cluster without fruits (3.24%, T7) in spring, otherwise smallest value of Terpinene present in gum (2.275%, T2) and ripe cluster without fruits (1.460%, T13) summer wounded. Anticholinesterase and antioxidant activities of terpene present in some type of aroma plants (Mehmet, 2012).
4.4.10 Carveol Figure (41) represents the percentage concentration of Carveol of the two pistacia spp. The high significant differences appear between highest values from immature outer shell of P. eurycarpa spring collection (6.4%, T3) and the lowest concentration value of mature cluster without fruits of P. eurycarpa summer wounded (2.7%, T11) while Gum of P. khinjuk (4.020%, T2) in spring contain high amount of Carveol and significant differences are found with smallest value present in the cluster without fruits in summer (2.865%, T13) summer wounded. Highest values of Carveol are present in the immature outer shell (6.40%, T3) of the P. eurycarpa in spring and significant differences are found with other parts, gum (5.780%, T1) and unripe cluster without fruits (4.173%, T4) respectively of the same species and
67
Chapter four
Results and Discussion
the moderate value present in the gum (3.292%, T8) in summer collection, immature leaves (3.280%, T5) in spring and ripe cluster without fruits (3.055%, T10) spring wounded of the P. eurycarpa, otherwise the smallest concentration present in the cluster without fruits (2.7%, T11) summer wounded, No significant difference present between immature outer shell (6.40%, T3) of the P. eurycarpa in spring with gum (5.780%, T1) in spring collection and gum (3.292%, T8) in summer, immature leaves (3.280%, T5) in spring and ripe cluster without fruits (3.055%, T10) spring wounded. Gum of P. khinjuk (4.020%, T2) in spring contain high amount of Carveol, and no significant differences are found between immature cluster without fruits (3.590%, T6) in spring, mature cluster without fruits (3.575%, T12) spring wounded, gum (3.10%, T9) in summer collected and immature leaves (3.055%, T7) in spring. Otherwise the smallest value is present in the cluster without fruits (2.865%, T13) in summer, summer wounded. Carveol has toxicological and dermatologic activity (Bhatia et al., 2008)
4.4.11 Myretenol Figure (42) shows the percentage concentration of Myretenol in all parts of the two Pistacia spp., significant differences present between highest values from gum of P. eurycarpa in spring collection (6.055%, T1) and the lowest concentration present in the gum of P. khinjuk summer collection (2 %, T9). High significant differences are present between highest values from gum of P. eurycarpa at spring (6.055%, T1) with lowest level in ripe cluster without fruits in summer (2.175%, T11) summer wounded, while in P. khinjuk high significant differences appear between largest value unripe cluster without fruits (5.925%, T6) in spring with smallest value in gum (2.00%, T9) in summer collection. High amount of Myretenol are present in the gum of P. eurycarpa in spring (6.055%, T1), immature outer shell (5.915%, T3) in spring, immature leaves (4.95%, T5), gum (4.215%, T8) in summer, ripe cluster without fruits (3.610%, T10) spring wounded, unripe cluster without fruits (3.010%, T4) in spring and ripe cluster without fruits (2.175%, T11) summer wounded, respectively and significant differences are present between all above P. eurycarpa parts from unripe to ripe samples. The arrangement of concentration value of Myretenol of P. khinjuk from large to small is present in the unripe cluster without fruits (5.925%, T6) in spring, gum (4.845%, T2) in spring, immature leaves (3.805%, T7) in spring, mature cluster without fruits (3.4%, T12) spring wounded, mature cluster without fruits (3.210%, T13) in summer
67
Chapter four
Results and Discussion
(summer wounded) and gum (2.00%, T9) in summer. And have significant differences are present between all above P. khinjuk parts from unripe to ripe samples. Otherwise no significant differences are present between (immature clusters without fruits of P. khinjuk (5.925%, T6) in spring with immature outer shell of P. eurycarpa (5.915%, T3) in spring), (immature leaves of P. eurycarpa (4.95%, T5) in spring with gum of P. khinjuk (4.845%, T2) in spring), (gum of P. eurycarpa (4.215%, T8) in summer with immature leaves of P. khinjuk (3.805%, T7) in spring), (mature cluster without fruits of P. eurycarpa (3.610%, T10) spring wounded with mature cluster without fruits of P. khinjuk (3.4%, T12) spring wounded), (immature cluster without fruits of P. khinjuk (3.210%, T13) summer wounded with immature cluster without fruits of P. eurycarpa (3.010%, T4) in spring), and (mature cluster without fruits of P. eurycarpa (2.175%, T11) summer wounded with gum of P. khinjuk (2.00%, T9) in summer). Myretenol is the smallest concentration compounds in volatile oils of the two pistacia ssp. Myrtenol have antibacterial and antifungal activities (Bénédicte et al., 2011). The present study show that all types of volatile oil compounds are affected by seasonally changes from spring to summer according to (wounded and unwounded) stems of the two Pistacia spp. occurring during plant growth (ripe and unripe). There are many qualitative and quantitative variations between the content of essential oils. These variations are related to several parameters like plant species and plant parts, harvesting time, geographical origin, and climatic conditions (Alma et al., 2004). The chemical composition of the Essential oils depends on climatic, seasonal, soil condition, geographic, harvest period and distillation technique (Kan et al., 2000). Soil pH affects significantly oil yield and oil quality. Selection of the optimum site coupled with a suitable climate plays an important role as they will provide a guarantee for optimum crop and essential oil quality (Figueiredo et al., 2005) Most plants require well-drained soils to prevent their roots from rotting and the plant from being damaged, thus adversely affecting essential oil production. Lack of water, for example (Yanive, and Palevitch, 1982) The timing of the harvest of the plant or herbal crop is one of the most important factors affecting the quality of the essential oil (Nguyen et al., 2004). The ability to accumulate essential oils is not omnipresent in plants but scattered throughout the plant kingdom; in many cases, however, very frequent within—or a typical character of—certain plant families (Vetter et al., 1997).
67
Chapter four
Results and Discussion
These results were in agreement with the results of Demirci et al., (2001); Taran et al., (2010); Faraidoon et al., (2013); Mahbubeh et al., (2013) and Ali et al., (2014). But this study disagrees and has some differences with Tohidi et al., (2011) and Ghasemi and Aghaee (2011) because different in climate, soil properties, solvent and time of harvesting.
66
Chapter four
Results and Discussion
α - Pinene 70
bc
bc
a
a
b
T12
T13
de f
e
40 f
Percentage
cd
bc
50
a
a
60
30
20
10
0 T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
Parts of plant Figure 32: the percentage concentration of α – Pinene in different parts of the two Pistacia spp. at different growth stage * (T1 = gum of P. eurycarpa , T2 = gum of P. khinjuk, T3 = outer shell of P. eurycarpa, T4 = cluster without fruit of P. eurycarpa , T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. khinjuk , T7 = leaf of P. khinjuk , T8 = gum of P. eurycarpa , T9 = gum of P. khinjuk , T10 = cluster without fruit of P. eurycarpa spring wounded , T11 = cluster without fruit of P. eurycarpa summer wounded ,T12 = cluster without fruit of P. khinjuk spring wounded, T13 = cluster without fruit of P. khinjuk summer wounded)
* T1 – T7 Unripe or spring samples. * T8 – T13 Ripe or summer samples. * Different colour or letters has significant difference between them. According to Duncan test at p< 0.05
* T1, T3-T5, T8, T10 and T11 samples of P. eurycarpa, T2, T6, T7, T9, T12 and T13 samples of P. khinjuk
65
Chapter four
Results and Discussion
Phellandrene 30
a
25
b fg
fg T7
def
g
T6
fg
T4
10
cde
T3
def
cd
T2
efg
c
15 cde
Percentage
20
5
0 T1
T5
T8
T9
T10
T11
T12
T13
Parts of plant Figure 33: the percentage concentration of Phellandrene in different parts of the two Pistacia spp. at different growth stage * (T1 = gum of P. eurycarpa , T2 = gum of P. khinjuk, T3 = outer shell of P. eurycarpa, T4 = cluster without fruit of P. eurycarpa , T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. khinjuk , T7 = leaf of P. khinjuk , T8 = gum of P. eurycarpa , T9 = gum of P. khinjuk , T10 = cluster without fruit of P. eurycarpa spring wounded , T11 = cluster without fruit of P. eurycarpa summer wounded , T12 = cluster without fruit of P. khinjuk spring wounded, T13 = cluster without fruit of P. khinjuk summer wounded)
* T1 – T7 Unripe or spring samples. * T8 – T13 Ripe or summer samples. * Different colour or letters has significant difference between them. According to Duncan test at p< 0.05 * T1, T3-T5, T8, T10 and T11 samples of P. eurycarpa, T2, T6, T7, T9, T12 and T13 samples of P. khinjuk .
66
Chapter four
Results and Discussion
β - Pinene
a
25
de
cd
cd
c
c
c
15
c 10
g
f
ef
Percentage
b
20
g
5
0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
Parts of plant Figure 34: the percentage concentration of β - Pinene in different parts of the two Pistacia spp. at different growth stage * (T1 = gum of P. eurycarpa , T2 = gum of P. khinjuk, T3 = outer shell of P. eurycarpa, T4 = cluster without fruit of P. eurycarpa , T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. khinjuk , T7 = leaf of P. khinjuk , T8 = gum of P. eurycarpa , T9 = gum of P. khinjuk , T10 = cluster without fruit of P. eurycarpa spring wounded , T11 = cluster without fruit of P. eurycarpa summer wounded , T12 = cluster without fruit of P. khinjuk spring wounded, T13 = cluster without fruit of P. khinjuk summer wounded)
* T1 – T7 Unripe or spring samples. * T8 – T13 Ripe or summer samples.
* Different colour or letters has significant difference between them. According to Duncan test at p< 0.05 * T1, T3-T5, T8, T10 and T11 samples of P. eurycarpa, T2, T6, T7, T9, T12 and T13 samples of P. khinjuk.
66
Chapter four
Results and Discussion
Sabinene
a
14
c
c 8
c
c
Percentage
c
c
bc
bc
10
ab
12
6
d
d
d
4
2
0 T1
T2
T3
T4
T5
T6
T7
T8
T9
10
T11
T12
T13
Parts of plant Figure 36: the percentage concentration of Sabinene in different parts of the two Pistacia spp. at different growth stage * (T1 = gum of P. eurycarpa , T2 = gum of P. khinjuk, T3 = outer shell of P. eurycarpa, T4 = cluster without fruit of P. eurycarpa , T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. khinjuk , T7 = leaf of P. khinjuk , T8 = gum of P. eurycarpa , T9 = gum of P. khinjuk , T10 = cluster without fruit of P. eurycarpa spring wounded, T11 = cluster without fruit of P. eurycarpa summer wounded , T12 = cluster without fruit of P. khinjuk spring wounded, T13 = cluster without fruit of P. khinjuk summer wounded)
* T1 – T7 Unripe or spring samples. * T8 – T13 Ripe or summer samples. * Different colour or letters has significant difference between them. According to Duncan test at p< 0.05
* T1, T3-T5, T8, T10 and T11 samples of P. eurycarpa, T2, T6, T7, T9, T12 and T13 samples of P. khinjuk .
66
Chapter four
Results and Discussion
Limonene 16
b
a
14
b
b
12
cd
T3
cd
cd
T2
de
8 e
Percentage
10
fg
6
g
g
fg
4
2
0
T1
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
Parts of plant Figure 35: the percentage concentration of Limonene in different parts of the two Pistacia spp. at different growth stage * (T1 = gum of P. eurycarpa , T2 = gum of P. khinjuk, T3 = outer shell of P. eurycarpa,
T4 = cluster without fruit
of P. eurycarpa , T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. khinjuk , T7 = leaf of P. khinjuk , T8 = gum of P. eurycarpa , T9 = gum of P. khinjuk , T10 = cluster without fruit of P. eurycarpa spring wounded , T11 = cluster without fruit of P. eurycarpa summer wounded , T12 = cluster without fruit of P. khinjuk spring wounded, T13 = cluster without fruit of P. khinjuk summer wounded)
* T1 – T7 Unripe or spring samples. * T8 – T13 Ripe or summer samples.
* Different colour or letters has significant difference between them. According to Duncan test at p< 0.05 * T1, T3-T5, T8, T10 and T11 samples of P. eurycarpa, T2, T6, T7, T9, T12 and T13 samples of P. khinjuk .
67
Chapter four
Results and Discussion
Aldehyde -citral 12
a
a
10
bc
bc
b
b
d
cd
cd
c
bc
percentage
6
b
8
e
4
2
0 T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
Parts of plant Figure 37: the percentage concentration of Aldehyde-citral in different parts of the two Pistacia spp. at different growth stage * (T1 = gum of P. eurycarpa, T2 = gum of P. khinjuk, T3 = outer shell of P. eurycarpa, T4 = cluster without fruit of P. eurycarpa , T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. khinjuk , T7 = leaf of P. khinjuk , T8 = gum of P. eurycarpa , T9 = gum of P. khinjuk , T10 = cluster without fruit of P. eurycarpa spring wounded , T11 = cluster without fruit of P. eurycarpa summer wounded , T12 = cluster without fruit of P. khinjuk spring wounded, T13 = cluster without fruit of P. khinjuk summer wounded)
* T1 – T7 Unripe or spring samples. * T8 – T13 Ripe or summer samples.
* Different colour or letters has significant difference between them. According to Duncan test at p< 0.05
* T1, T3-T5, T8, T10 and T11 samples of P. eurycarpa, T2, T6, T7, T9, T12 and T13 samples of P. khinjuk
67
Chapter four
Results and Discussion
3 - Carene 12
ab cd
cd
bcd
bc
ab
de
6
ef
Percentage
8
ab
a
10
g
fg
fg
4
2
0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
Parts of plant Figure 38: the percentage concentration of 3 – Carene in different parts of the two Pistacia spp. at different growth stage * (T1 = gum of P. eurycarpa , T2 = gum of P. khinjuk, T3 = outer shell of P. eurycarpa, T4 = cluster without fruit of P. eurycarpa , T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. khinjuk , T7 = leaf of P. khinjuk , T8 = gum of P. eurycarpa , T9 = gum of P. khinjuk , T10 = cluster without fruit of P. eurycarpa spring wounded , T11 = cluster without fruit of P. eurycarpa summer wounded , T12 = cluster without fruit of P. khinjuk spring wounded, T13 = cluster without fruit of P. khinjuk summer wounded)
* T1 – T7 Unripe or spring samples. * T8 – T13 Ripe or summer samples.
* Different colour or letters has significant difference between them. According to Duncan test at p< 0.05 * T1, T3-T5, T8, T10 and T11 samples of P. eurycarpa, T2, T6, T7, T9, T12 and T13 samples of P. khinjuk.
67
Chapter four
Results and Discussion
Myrecene 10 9
ab
ab
b
b
cd
d
cd
4
cd
5 cd
Percentage
6
b
ab
7
a
8
3
e
2 1 0
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
Parts of plant Figure 39: the percentage concentration of Myrecene in different parts of the two Pistacia spp. at different growth stage * (T1 = gum of P. eurycarpa, T2 = gum of P. khinjuk, T3 = outer shell of P. eurycarpa, T4 = cluster without fruit of P. eurycarpa , T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. khinjuk , T7 = leaf of P. khinjuk , T8 = gum of P. eurycarpa , T9 = gum of P. khinjuk , T10 = cluster without fruit of P. eurycarpa spring wounded , T11 = cluster without fruit of P. eurycarpa summer wounded , T12 = cluster without fruit of P. khinjuk spring wounded, T13 = cluster without fruit of P. khinjuk summer wounded)
* T1 – T7 Unripe or spring samples. * T8 – T13 Ripe or summer samples. * Different colour or letters has significant difference between them. According to Duncan test at p< 0.05 * T1, T3-T5, T8, T10 and T11 samples of P. eurycarpa, T2, T6, T7, T9, T12 and T13 samples of P. khinjuk .
67
Chapter four
Results and Discussion
Terpinene
a
8
7
6
b bcd
cdef
cdef def
ef fg
ef
3
bc
bcd
4 cdef
Percentage
5
g
2
1
0 T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
Parts of plant
Figure 40: the percentage concentration of Terpinene in different parts of the two Pistacia spp. at different growth stage * (T1 = gum of P. eurycarpa , T2 = gum of P. khinjuk, T3 = outer shell of P. eurycarpa, T4 = cluster without fruit of P. eurycarpa , T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. khinjuk , T7 = leaf of P. khinjuk , T8 = gum of P. eurycarpa , T9 = gum of P. khinjuk , T10 = cluster without fruit of P. eurycarpa spring wounded , T11 = cluster without fruit of P. eurycarpa summer wounded , T12 = cluster without fruit of P. khinjuk spring wounded, T13 = cluster without fruit of P. khinjuk summer wounded)
* T1 – T7 Unripe or spring samples. * T8 – T13 Ripe or summer samples. * Different colour or letters has significant difference between them. According to Duncan test at p< 0.05 * T1, T3-T5, T8, T10 and T11 samples of P. eurycarpa, T2, T6, T7, T9, T12 and T13 samples of P. khinjuk .
67
Chapter four
Results and Discussion
Carveol 8
6
a
a
7
T9
T10
d
3
cd
bcd
bcd bcd
bcd
bcd
bcd
4
bcd
b
bc
Percentage
5
2
1
0 T1
T2
T3
T4
T5
T6
T7
T8
T11
T12
T13
Parts of plant Figure 41: the percentage concentration of Carveol in different parts of the two Pistacia spp. at different growth stage * (T1 = gum of P. eurycarpa , T2 = gum of P. khinjuk, T3 = outer shell of P. eurycarpa, T4 = cluster without fruit of P. eurycarpa , T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. khinjuk , T7 = leaf of P. khinjuk , T8 = gum of P. eurycarpa , T9 = gum of P. khinjuk , T10 = cluster without fruit of P. eurycarpa spring wounded , T11 = cluster without fruit of P. eurycarpa summer wounded , T12 = cluster without fruit of P. khinjuk spring wounded, T13 = cluster without fruit of P. khinjuk summer wounded)
* T1 – T7 Unripe or spring samples. * T8 – T13 Ripe or summer samples.
* Different colour or letters has significant difference between them. According to Duncan test at p< 0.05
* T1, T3-T5, T8, T10 and T11 samples of P. eurycarpa, T2, T6, T7, T9, T12 and T13 samples of P. khinjuk .
66
Chapter four
Results and Discussion
Myretenol 8
abc
abc
ab
a 6
cd
cd
5
d
de
d
4
T12
T13
de
Percentage
ab
7
ef
ef
3
2
1
0 T1
T2
T3
T4
T5
T6 T7 T8 Parts of plant
T9
T10
T11
Figure 42: the percentage concentration of Myretenol in different parts of the two Pistacia spp. at different growth stage * (T1 = gum of P. eurycarpa , T2 = gum of P. khinjuk, T3 = outer shell of P. eurycarpa, T4 = cluster without fruit of P. eurycarpa , T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. khinjuk , T7 = leaf of P. khinjuk , T8 = gum of P. eurycarpa , T9 = gum of P. khinjuk , T10 = cluster without fruit of P. eurycarpa spring wounded , T11 = cluster without fruit of P. eurycarpa summer wounded , T12 = cluster without fruit of P. khinjuk spring wounded, T13 = cluster without fruit of P. khinjuk summer wounded)
* T1 – T7 Unripe or spring samples. * T8 – T13 Ripe or summer samples. * Different colour or letters has significant difference between them. According to Duncan test at p< 0.05 * T1, T3-T5, T8, T10 and T11 samples of P. eurycarpa, T2, T6, T7, T9, T12 and T13 samples of P. khinjuk.
65
Chapter four
Results and Discussion Anthocyanin
ef
T24
bc
T23
ef
bc
T19
bc
cd
de ef
T18
f
10
ef
de
cd
T12
ef
bc
T11
de
bc
cd
bc
T6
cd T5
ab
bc
bc
bc
15
cd
bc
Percentage
20
ab
a
25
5 0 T1
T2
T3
T4
T7
T8
T9
T10
T13
T14
T15
T16
T17
T20
T21
T22
T25
T26
T27
Plant parts
Figure 29: the percentage concentration of Anthocyanin in different parts of the two Pistacia spp. (T1 = outer shell of P. khinjuk, T2 =cluster without fruit of P. khinjuk, T3 = leaf of P. khinjuk, T4 = gum of P. khinjuk, T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. eurycarpa, T7 = outer shell of P. eurycarpa, T8 = Gum of P. eurycarpa, T9 = leaf of P. eurycarpa, T10 = cluster without fruit of P. eurycarpa, T11 = outer shell of P. eurycarpa, T12 = seed of P. eurycarpa T13 = leaf of P.khinjuk,T14 = cluster without fruit of P. khinjuk, T15 = outer shell of p. khinjuk, T16 = seed of P. khinjuk T17 = seed of P. khinjuk, T18 = leaf of P. khinjuk, T19 = outer shell of P. khinjuk, T20 = cluster without fruit of P. khinjuk, T21 = Gum of P. khinjuk, T22 = leaf of P. eurycarpa, = T23 = cluster without fruit of P. eurycarpa, T24 = seed of P. eurycarpa, T25 = outer shell of P. eurycarpa, T26 = Gum of P. eurycarpa, T27 = seed of P. eurycarpa). * T1 – T8 and T27 unripe samples (at the spring sampling). *T9 – T26 ripe samples (at the end of summer sampling). But T9 –T16 spring wounded, T17 – T26 summer wounded. * Different colour or letters has significant difference between them. According to Duncan test at p< 0.05 * T1-T4 and T13-T21 samples of P. khinjuk, T4 –T12 and T22-T27 samples of P. eurycarpa.
62
Chapter four
Results and Discussion Anthocyanin
ef
T24
bc
T23
ef
bc
T19
bc
cd
de ef
T18
f
10
ef
de
cd
T12
ef
bc
T11
de
bc
cd
bc
T6
cd T5
ab
bc
bc
bc
15
cd
bc
Percentage
20
ab
a
25
5 0 T1
T2
T3
T4
T7
T8
T9
T10
T13
T14
T15
T16
T17
T20
T21
T22
T25
T26
T27
Plant parts
Figure 29: the percentage concentration of Anthocyanin in different parts of the two Pistacia spp. (T1 = outer shell of P. khinjuk, T2 =cluster without fruit of P. khinjuk, T3 = leaf of P. khinjuk, T4 = gum of P. khinjuk, T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. eurycarpa, T7 = outer shell of P. eurycarpa, T8 = Gum of P. eurycarpa, T9 = leaf of P. eurycarpa, T10 = cluster without fruit of P. eurycarpa, T11 = outer shell of P. eurycarpa, T12 = seed of P. eurycarpa T13 = leaf of P.khinjuk,T14 = cluster without fruit of P. khinjuk, T15 = outer shell of p. khinjuk, T16 = seed of P. khinjuk T17 = seed of P. khinjuk, T18 = leaf of P. khinjuk, T19 = outer shell of P. khinjuk, T20 = cluster without fruit of P. khinjuk, T21 = Gum of P. khinjuk, T22 = leaf of P. eurycarpa, = T23 = cluster without fruit of P. eurycarpa, T24 = seed of P. eurycarpa, T25 = outer shell of P. eurycarpa, T26 = Gum of P. eurycarpa, T27 = seed of P. eurycarpa). * T1 – T8 and T27 unripe samples (at the spring sampling). *T9 – T26 ripe samples (at the end of summer sampling). But T9 –T16 spring wounded, T17 – T26 summer wounded. * Different colour or letters has significant difference between them. According to Duncan test at p< 0.05 * T1-T4 and T13-T21 samples of P. khinjuk, T4 –T12 and T22-T27 samples of P. eurycarpa.
62
Chapter four
Results and Discussion
abcdef
cdefghij ijk
abcd
abcdef
k
efghijk
abc
abcd
cdefghij jk
ijk
hijk
bcdefghi
T7
ghijk
T6
ijk
T5
abcdefgh
ab
T4
fghijk
abcd
T3
abcdefgh
abc
T2
cdefghij
abcdef
a
abc
16 14 12 10 8 6 4 2 0
defghijk
Percentage
Cinnamic acid
T1
T8
T9
T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T20 T21 T22 T23 T24 T25 T26 T27 Plant parts
Figure 31: the percentage concentration of Cinnamic acid in different parts of the two Pistacia spp. (T1 = outer shell of P. khinjuk, T2 =cluster without fruit of P. khinjuk, T3 = leaf of P. khinjuk, T4 = gum of P. khinjuk, T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. eurycarpa, T7 = outer shell of P. eurycarpa, T8 = Gum of P. eurycarpa, T9 = leaf of P. eurycarpa, T10 = cluster without fruit of P. eurycarpa, T11 = outer shell of P. eurycarpa, T12 = seed of P. eurycarpa T13 = leaf of P.khinjuk,T14 = cluster without fruit of P. khinjuk, T15 = outer shell of p. khinjuk, T16 = seed of P. khinjuk T17 = seed of P. khinjuk, T18 = leaf of P. khinjuk, T19 = outer shell of P. khinjuk, T20 = cluster without fruit of P. khinjuk, T21 = Gum of P. khinjuk, T22 = leaf of P. eurycarpa, = T23 = cluster without fruit of P. eurycarpa, T24 = seed of P. eurycarpa, T25 = outer shell of P. eurycarpa, T26 = Gum of P. eurycarpa, T27 = seed of P. eurycarpa). * T1 – T8 and T27 unripe samples (at the spring sampling). *T9 – T26 ripe samples (at the end of summer sampling). But T9 –T16 spring wounded, T17 – T26 summer wounded. * Different colour or letters has significant difference between them. According to Duncan test at p< 0.05 * T1-T4 and T13-T21 samples of P. khinjuk, T4 –T12 and T22-T27 samples of P. eurycarpa.
64
Chapter four
Results and Discussion
Ellagic acid a
25
k
k
k
k
k
gh
cdef
l
5
jk
hi
gh
fgh
cdef
efg
fgh
efgh
cdef
cdef
T5
ij T4
efg
T3
cdef
T2
efgh
bc
T1
10
gh
b
15
bc
Percentage
20
0 T6
T7
T8
T9
T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T20 T21 T22 T23 T24 T25 T26 T27
Plant parts Figure 28: the percentage concentration of ellagic acid in different parts of the two Pistacia spp. (T1 = outer shell of P. khinjuk, T2 =cluster without fruit of P. khinjuk, T3 = leaf of P. khinjuk, T4 = gum of P. khinjuk, T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. eurycarpa, T7 = outer shell of P. eurycarpa, T8 = Gum of P. eurycarpa, T9 = leaf of P. eurycarpa, T10 = cluster without fruit of P. eurycarpa, T11 = outer shell of P. eurycarpa, T12 = seed of P. eurycarpa T13 = leaf of P.khinjuk,T14 = cluster without fruit of P. khinjuk, T15 = outer shell of p. khinjuk, T16 = seed of P. khinjuk T17 = seed of P. khinjuk, T18 = leaf of P. khinjuk, T19 = outer shell of P. khinjuk, T20 = cluster without fruit of P. khinjuk, T21 = Gum of P. khinjuk, T22 = leaf of P. eurycarpa, = T23 = cluster without fruit of P. eurycarpa, T24 = seed of P. eurycarpa, T25 = outer shell of P. eurycarpa, T26 = Gum of P. eurycarpa, T27 = seed of P. eurycarpa). * T1 – T8 and T27 unripe samples (at the spring sampling). *T9 – T26 ripe samples (at the end of summer sampling). But T9 –T16 spring wounded, T17 – T26 summer wounded. * Different colour or letters has significant difference between them. According to Duncan test at p< 0.05 * T1-T4 and T13-T21 samples of P. khinjuk, T4 –T12 and T22-T27 samples of P. eurycarpa.
61
Chapter four
Results and Discussion
T1
T2
fgh
fgh
gh
fgh
ab
abc fgh
defgh
defgh
bcdef
bcdefg
bcde
defgh
bcdef
bcdef
cdefg
h
fgh
bcd
T6
bcd
a cdefg
T5
abc
cdefg
bcdef
abc h
Percentage
Gallic acid 20 18 16 14 12 10 8 6 4 2 0
T3
T4
T7
T8
T9
T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T20 T21 T22 T23 T24 T25 T26 T27
Plant parts Figure 30: the percentage concentration of Gallic acid in different parts of the two Pistacia spp. (T1 = outer shell of P. khinjuk, T2 =cluster without fruit of P. khinjuk, T3 = leaf of P. khinjuk, T4 = gum of P. khinjuk, T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. eurycarpa, T7 = outer shell of P. eurycarpa, T8 = Gum of P. eurycarpa, T9 = leaf of P. eurycarpa, T10 = cluster without fruit of P. eurycarpa, T11 = outer shell of P. eurycarpa, T12 = seed of P. eurycarpa T13 = leaf of P.khinjuk,T14 = cluster without fruit of P. khinjuk, T15 = outer shell of p. khinjuk, T16 = seed of P. khinjuk T17 = seed of P. khinjuk, T18 = leaf of P. khinjuk, T19 = outer shell of P. khinjuk, T20 = cluster without fruit of P. khinjuk, T21 = Gum of P. khinjuk, T22 = leaf of P. eurycarpa, = T23 = cluster without fruit of P. eurycarpa, T24 = seed of P. eurycarpa, T25 = outer shell of P. eurycarpa, T26 = Gum of P. eurycarpa, T27 = seed of P. eurycarpa). * T1 – T8 and T27 unripe samples (at the spring sampling). *T9 – T26 ripe samples (at the end of summer sampling). But T9 –T16 spring wounded, T17 – T26 summer wounded. * Different colour or letters has significant difference between them. According to Duncan test at p< 0.05 * T1-T4 and T13-T21 samples of P. khinjuk, T4 –T12 and T22-T27 samples of P. eurycarpa.
63
Appendix
Volatile oil
Rt.
Sabienene
1.19
Limonene
2.11
β- Pinene
3.38
β- Myrecene
4.27
α- Pinene
5.35
Phellandrine total
6.17
3-Carene
7.41
Aldehyde-citral
8.19
Myretenol
9.16
Carveol
10.02
Terpineolene
11.04
Appendix 18: chromatogram of Rt. (min.) of different compounds of the gum in P. khinjuk
Volatile oil
Rt.
Sabienene
1.25
Limonene
2.24
β- Pinene
3.34
β- Myrecene
4.37
α- Pinene
5.29
Phellandrine total
6.22
3-Carene
7.45
Aldehyde-citral
8.30
Myretenol
9.42
Carveol
10.03
Terpineolene
11.10
Appendix 19: chromatogram of Rt. (min.) of different compounds from the outer shell in P. eurycarpa
Chapter four
Results and Discussion
Stilbene
defgh
fghij
cdef
bc
efghi
hijk
defgh
efghi
cde
cde
cdefg
hijkl
efghij
bc
efghi
ab
lm
kl T5
m
kl T4
jkl
T3
20 15
cde
T2
ijkl
efghi
25
fghij
30
n
Percentage
35
a
40
10 T1
T6
T7
T8
T9
T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T20 T21 T22 T23 T24 T25 T26 T27
Plant parts
Figure 26: the percentage concentration of Stilbene in different parts of the two Pistacia spp. (T1 = outer shell of P. khinjuk, T2 =cluster without fruit of P. khinjuk, T3 = leaf of P. khinjuk, T4 = gum of P. khinjuk, T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. eurycarpa, T7 = outer shell of P. eurycarpa, T8 = Gum of P. eurycarpa, T9 = leaf of P. eurycarpa, T10 = cluster without fruit of P. eurycarpa, T11 = outer shell of P. eurycarpa, T12 = seed of P. eurycarpa T13 = leaf of P.khinjuk,T14 = cluster without fruit of P. khinjuk, T15 = outer shell of p. khinjuk, T16 = seed of P. khinjuk T17 = seed of P. khinjuk, T18 = leaf of P. khinjuk, T19 = outer shell of P. khinjuk, T20 = cluster without fruit of P. khinjuk, T21 = Gum of P. khinjuk, T22 = leaf of P. eurycarpa, = T23 = cluster without fruit of P. eurycarpa, T24 = seed of P. eurycarpa, T25 = outer shell of P. eurycarpa, T26 = Gum of P. eurycarpa, T27 = seed of P. eurycarpa). * T1 – T8 and T27 unripe samples (at the spring sampling). *T9 – T26 ripe samples (at the end of summer sampling). But T9 –T16 spring wounded, T17 – T26 summer wounded. * Different colour or letters has significant difference between them. According to Duncan test at p< 0.05 * T1-T4 and T13-T21 samples of P. khinjuk, T4 –T12 and T22-T27 samples of P. eurycarpa.
59
Chapter four
Results and Discussion
Tannin a
35
mn
ijkl
ijk
o
ijkl
c
defgh
efghi
defgh
efghi
defgh
d
de
p
10
klm
mn
mn
T5
defg
T4
defgh
fghi
T3
ghij
d
T2
15
defg
defgh
20
def
25
b
Percentage
30
5 0
T1
T6
T7
T8
T9
T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T20 T21 T22 T23 T24 T25 T26 T27
Plant parts
Figure 25: the percentage concentration of Tannin in different parts of the two Pistacia spp. (T1 = outer shell of P. khinjuk, T2 =cluster without fruit of P. khinjuk, T3 = leaf of P. khinjuk, T4 = gum of P. khinjuk, T5 = leaf of P. eurycarpa, T6 = cluster without fruit of P. eurycarpa, T7 = outer shell of P. eurycarpa, T8 = Gum of P. eurycarpa, T9 = leaf of P. eurycarpa, T10 = cluster without fruit of P. eurycarpa, T11 = outer shell of P. eurycarpa, T12 = seed of P. eurycarpa T13 = leaf of P.khinjuk,T14 = cluster without fruit of P. khinjuk, T15 = outer shell of p. khinjuk, T16 = seed of P. khinjuk T17 = seed of P. khinjuk, T18 = leaf of P. khinjuk, T19 = outer shell of P. khinjuk, T20 = cluster without fruit of P. khinjuk, T21 = Gum of P. khinjuk, T22 = leaf of P. eurycarpa, = T23 = cluster without fruit of P. eurycarpa, T24 = seed of P. eurycarpa, T25 = outer shell of P. eurycarpa, T26 = Gum of P. eurycarpa, T27 = seed of P. eurycarpa). * T1 – T8 and T27 unripe samples (at the spring sampling). *T9 – T26 ripe samples (at the end of summer sampling). But T9 –T16 spring wounded, T17 – T26 summer wounded. * Different colour or letters has significant difference between them. According to Duncan test at p< 0.05 * T1-T4 and T13-T21 samples of P. khinjuk, T4 –T12 and T22-T27 samples of P. eurycarpa.
58
