Berberis vulgaris Q. Dioscorea Q. and Calcarea carb 200 for ...

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ARTICLE IN PRESS

Journal of Crystal Growth 283 (2005) 508–513 www.elsevier.com/locate/jcrysgro

In vitro dissolution of gallbladder stone by edible leaves, fruits and homoeopathic medicines Ishwar Dasa,, Yogendra Singhb, Shoeb A. Ansaria, Namita R. Agrawalc a

Department of Chemistry, DDU Gorakhpur University, Gorakhpur 273 009, India b Department of Chemistry, U.P. College, Varanasi, India c Department of Chemistry, St. Andrews College, Gorakhpur-273009, India Received 7 April 2005; accepted 9 June 2005 Available online 1 August 2005 Communicated by M. Schieber

Abstract Gallbladder stone sample of a female patient was analysed by diagnostic, spectroscopic methods and by differential scanning calorimetry (DSC). Besides cholesterol as the major constituent, bilirubin, creatinine and blood urea were also found to be present in the sample. Bile acid (ursodeoxycholic acid) was used to study its effect on the dissolution of cholestrol present in the stone. Extracts of edible leaves and fruits (amla, lemon and mausammi) and the homoeopathic medicines Berberis vulgaris Q. Dioscorea Q. and Calcarea carb 200 in the concentration range 0–3% (v/v) were found to be effective in the dissolution process in the following sequences: B. vulgaris Q. and Dioscorea Q.4C. carb 200, lemon4mausammi, amla was found to be more effective than jamun and tulsi leaf extracts in the given concentration range. r 2005 Elsevier B.V. All rights reserved. PACS: 81.40.z Keywords: A1. Dissolution; B1. Bile acid; B1. Cholesterol; B1. Edible leaves; B1. Fruits; B1. Gallbladder stone; B1. Homoeopathic medicines

1. Introduction Carcinoma of the gallbladder is the ﬁfth most common cancer of the digestive tract [1]. It is more Corresponding author. Tel.: +91 551 2338731;

fax: +91 551 2342880. E-mail address: [email protected] (I. Das).

common in women and most often found in patients of age group of 60 years. Carcinoma and gallstones coexist in 60–90% of patients in western nations, but it is not clear whether there is any causal relationship. Eighty per cent of gallstones are predominantly composed of cholesterol. Gallstone patients secrete bile that is saturated with cholesterol. Many calculi occur due to loss of

0022-0248/$ - see front matter r 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jcrysgro.2005.06.006

ARTICLE IN PRESS I. Das et al. / Journal of Crystal Growth 283 (2005) 508–513

2. Materials and methods

Table 1 Diagnostic analysis of alcoholic solution of gallbladder stone sample of a 55-year-old female patient. A 100 mg stone sample was mixed with 10 ml ethanol, ﬁltered and the ﬁltrate was used for analysis Constituent

Value in mg%

Cholesterol Bilirubin Blood urea Blood urea nitrogen Creatinine Uric acid Globulin Calcium

333.46 0.86 38.21 17.84 1.26 12.8 0.9 4.70

1.500

ABS

inhibitors of crystal formation. Chenodeoxycholic and ursodeoxycholic acids are two naturally occurring bile acids that may be given orally for gallstone dissolution and reduction in cholesterol saturation in the bile. Ursodeoxycholic acid causes no adverse effect but it is a little expensive. Although the kinetics of dissolution of calcium oxalate hydrates have been studied by several workers [2–7], the dissolution of gallbladder stones by natural inhibitors has not been reported so far. In the present paper, a gallbladder stone sample was analysed by diagnostic, spectroscopic and other methods, and the role of extracts of edible leaves of tulsi (well known for antibacterial and antitubercular properties, also known as Ocimum sanctum), jamun (Syzygium cumini L., a wellknown liver stimulant, also used in the treatment of diabetese and mouth ulcers), dry amla (Amblica officinalis), juices of fruits of citrus group. viz lemon (Citrus aurantifolia) and mausammi (Citrus sinensis) and homoeopathic medicines Berberis vulgaris Q., Dioscorea Q. and Calcarea carb 200 in the dissolution of gallbladder stones has been studied in vitro.

509

0.000 200

400

600

800

nm

Surgically removed gallbladder stones, leaves of tulsi and jamun, lemon and mausammi fruit juices, homoeopathic medicine, B. vulgaris Q., Dioscorea Q. and C. carb 200 (Hahnemann’s laboratories) were used in the investigation. 2.1. Procedure 2.1.1. Collection treatment and analysis of gallbladder stones Surgically removed stone samples from a 55year-old female patient were collected immediately after surgery, washed with distilled water several times and used for further investigation. Gallbladder stone (F/55) was analysed quantitatively for its constituents by diagnostic methods particularly for the presence of cholesterol, bilirubin, creatinine, uric acid, globulin, calcium, etc. by employing standard pathological techniques. Results are recorded in Table 1.

Fig. 1. UV–vis spectrum of ethanolic ﬁltrate of gallbladder stone sample obtained from a 55-years-old female patient.

2.1.2. UV– vis spectral studies UV–vis spectrum of alcoholic portion of gallbladder stones was taken with the help of a HITACHI UV–vis spectrophotometer. Result is shown in Fig. 1. 2.1.3. Infra red spectral studies Infrared spectrum of gallbladder stones was recorded using ‘JASCO-FTIR-5300’ spectrophotometer. Pellet was prepared in KBr. 2.1.4. Differential scanning calorimetry (DSC) Thermal characteristics of the stone sample was investigated by DSC upto 800 1C using NETZSCH

ARTICLE IN PRESS I. Das et al. / Journal of Crystal Growth 283 (2005) 508–513

510

Peak: 495.6 °C

Mass Change: -8.38%

100

Peak: 441.2 °C

DSC/ (mW/mg) ↑ exo 10

80 5 60 Mass Change: -22.77%

TG/%

Peak: 67.2 °C

0 Peak: 360.9 °C

40

Residual Mass: 2.88 % (800.8 °C)

-5 Peak: 148.9 °C Mass Change: -63.67%

20

-10 Mass Change: 1.16%

0 100

200

300

400 500 Temperature/°C

600

700

800

Fig. 2. DSC thermogram of the gallbladder stone at the heating rate of 32 1C/10 min.

STA 409 PG/PC. A total of 10.16 mg of the sample was taken. Results are shown in Fig. 2. 2.1.5. Preparation of leaf extracts Extracts of fresh tulsi (O. sanctum) and jamun (S. cumini L.) leaves were prepared. Leaves were washed and crushed with distilled water (1:1 w/v) in a mixer and grinder at room temperature. The crushed materials were squeezed, ﬁltered and used as such for further investigation. 2.1.6. Preparation of amla (A. officinalis) extract To prepare extract of amla, dry powder of amla was taken and admixed with distilled water (20 g/ 100 ml), boiled and ﬁltered. The extract thus obtained was used as such. 2.1.7. Preparation of fruit juices Juices of lemon and mausammi were used as such.

2.2. Dissolution of gallbladder stone 2.2.1. Flame photometric studies Dissolution of gallbladder stone in aqueous extracts of leaves, fruits and aqueous solutions of B. vulgaris Q., Dioscorea Q. and C. carb 200 were studied by measuring the calcium content in the ﬁltrate at different experimental conditions with the help of a ﬂame photometer, attached with a calcium ﬁlter (Toshniwal, India). A total of 25 mg of gallbladder stone powder was mixed with 10 ml distilled water and fruit juices (0–3% v/v) were added in separate test tubes, wrapped with paraﬁlm, stirred thoroughly and kept for 7 days. Calcium content in the ﬁltrate was determined. Results are shown in Fig. 3. Similar experiments were also performed with the extracts of tulsi (O. sanctum) and jamun (S. cumini L.) leaves, amla (A. officinalis) and homoepathic medicines (B. vulgaris Q., Dioscorea Q. and C. carb 200). Results are shown in Figs. 4 and 5.

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Fig. 3. Dissolution of gallbladder stone in juices of (J) lemon and (D) mausammi.

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Fig. 5. Dissolution of gallbladder stone in homoeopathic medicines () Berberis vulgaris Q., (J) Dioscorea Q. and (D) Calcarea carb 200 of different concentrations in the range 0–3% (v/v). A total of 25 mg of gallbladder stone was mixed with the aqueous solution of the medicine and kept for 7 days. Calcium content in the ﬁltrate was measured employing a Flame photometer.

studied for comparison. Results are shown in Figs. 6 and 7.

Fig. 4. Dissolution of gallbladder stone in extracts of (J) jamun leaf, (D) tulsi leaf and () amla of different concentrations. A total of 25 mg of gallbladder stone was mixed with the extract and calcium content in the ﬁltrate was measured after 7 days ﬂame photometrically.

Dependence of dissolution of gallbladder stone (F/55) on time in presence of fruit juices and the aqueous solution of B. vulgaris Q. was also

2.2.2. Dissolution of cholesterol in gallbladder stone by bile acid Dissolution of cholesterol present in the gallbladder stones in an aqueous solution of bile acid (ursodeoxycholic acid) using the ‘Precichem Cholesterol CHOD. POD method.’ For this purpose, the stone sample was put in an aqueous solution of the bile acid (340 mg/100 ml). Cholesterol content in the aqueous part was measured at different time intervals. Results are shown in Fig. 8.

3. Results and discussion Surgically removed gallbladder stone sample of a 55-years-old female patient was collected immediately after the surgery, washed with distilled

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Fig. 6. Dissolution of gallbladder stone in (J) amla extract, () lemon juice and (D) mausammi juice. A total of 25 mg gallbladder stone was mixed with extract in different test tubes and calcium content in respective ﬁltrates were measured at different time intervals.

water several times and analysed quantitatively for its constituents. As recorded in Table 1, cholesterol, bilirubin, blood urea, blood urea nitrogen, creatinine, uric acid contents in the sample were found to be 333.46, 0.86, 38.21, 17.84, 1.26, 12.8 mg%, respectively. The presence of cholesterol was also indicated by characteristic peaks in the IR and GCMS and its melting point at 148.1 1C in DSC studies (Fig. 2). The stone sample was found to be thermally stable upto 300 1C beyond which it decomposes in two steps as indicated by exotherms at 441.2 and 495.6 1C. The corresponding weight losses were 63.67% and 22.77%, respectively. The gallbladder stones contain various components, it is therefore difﬁcult to interpret the results completely. However, a peak corresponding to cholesterol which is a major constituent of gallbladder stone appears at m/e value ¼ 386. The presence of bilirubin, urea and uric acid is indicated in the IR spectrum. Fig. 1 showed a peak at 445 nm in the UV–vis spectrum, characteristic of bilirubin (lmax for bilirubin is 453 nm [8]). Since cholesterol is one of the major

Fig. 7. Dissolution of gallbladder stone in Berberis vulgaris Q. as a function of time. A total of 25 mg stone sample was mixed with 10 ml distilled water and 100 ml of the medicine in different tubes. Calcium content in the ﬁltrate of each test tube was measured using a ﬂame photometer at different time intervals.

constituents of gallbladder stone, dissolution of gallbladder stone in an aqueous solution of the bile acid (ursodeoxycholic acid) (Structure I) was monitored by measuring cholesterol content in the ﬁltrate diagnostically at different time (upto 12 days).

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ﬁltrate. Results are plotted in Figs. 6 and 7. Results showed that all these extracts and juices were capable of dissolving the stone. Ca2+ ions are obtained due to ionization of CaC2O4 which combines with the carbohydrate or protein present in the extracts to form Ca2+ –carbohydrate or Ca2+– protein complex. Thus, a decrease in [Ca] is observed. However, at higher extract concentration, the complex gets partly dissolved showing an increase in [Ca]. The process continues and a chaotic trend is observed as shown in Figs. 3–6. [Calcium] in the ﬁltrate varies with time periodically in each case. The dissolution in B. vulgaris Q. was found to be maximum. The dissolution in lemon juice was found to increase linearly with time (Fig. 6). It has been found that the relative order of dissolution of gallbladder stone is as follows: B: vulgaris Q: and Dioscorea Q:4C: carb 2004 lemon4mausammi amla4jamun and tulsi: Fig. 8. Dissolution of cholesterol, present in gallbladder stone by ursodeoxycholic acid (bile acid) as a function of time. Conditions: One piece of stone sample (360 mg) was suspended in an aqueous solution of ursodeoxycholic acid (340 mg / 100 ml) and [cholesterol] was determined in the ﬁltrate as a function of time.

As shown in Fig. 8. the cholesterol content in the ﬁltrate was found to increase with time upto 8 days, after that the trend was reversed. It may be due to complete consumption of the bile acid in 8days. Dissolution of stone in aqueous leaf extracts of tulsi, jamun and fruits amla, lemon, mausammi and homoeopathic medicines B. vulgaris Q., Dioscorea Q. and C. carb 200 has been studied with the help of a ﬂame photometer. Two types of experiments were performed. A known amount of crushed gallbladder stone (25 mg) was mixed with 10 ml distilled water and extract of different concentrations was put for 7 days and ﬁltered. Calcium content in each ﬁltrate was measured with the help of a ﬂame photometer. Results are shown in Figs. 3–5. In the second experiment gallbladder stone sample in equal amount was taken in six test tubes. In each test tube lemon, amla, mausammi and B. vulgaris Q. were added, stirred and allowed to stand. Material of each test tube was ﬁltered on alternate days and calcium content was measured in the

Acknowledgments Authors wish to thank Prof. M.L. Srivastava, Dean, Faculty of Science and Head, Chemistry Department, DDU Gorakhpur University, India, for providing necessary laboratory facilities and Dr. Sujeet Kumar Gupta for helpful discussion. Thanks are also due to Prof. A.K. Singh, Chemistry Department, IIT, Powai, Mumbai, for providing GCMS results. References [1] S.L. Robbins, R.S. Cortran, V. Kumar, T. Collins (Eds.), Pathogenic Basis of Disease, sixth ed, Harcourt Asia PTE Ltd., Singapore, 1999. [2] B. Tomazic, G.H. Nancollas, J. Crystal Growth 46 (1979) 355. [3] S. Atanassova, K. Neykov, I. Gutzow, J. Crystal Growth 160 (1996) 148. [4] I. Das, S.K. Gupta, V.N. Pandey, S.A. Ansari, J. Crystal Growth 267 (2004) 654. [5] I. Das, S.K. Gupta, S.A. Ansari, V.N. Pandey, R.P. Rastogi, J. Crystal Growth 273 (2005) 646. [6] F. Atmani, S. Khan, BJU Int. 85 (2000) 621. [7] A.M. Freitas, N. Schor, M.A. Boim, BJU Int. 89 (2002) 829. [8] Fluka Chemica – Biochemica, Industriestrasse 25, CH-9470 Buchs, Switzerland, 1993, p.180.