Food Chemistry 157 (2014) 213–220

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Sulphur-containing compounds of durian activate the thermogenesis-inducing receptors TRPA1 and TRPV1 Yuko Terada a, Takashi Hosono b, Taiichiro Seki b, Toyohiko Ariga b, Sohei Ito a, Masataka Narukawa a, Tatsuo Watanabe a,⇑ a b

Graduate School of Pharmaceutical and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan Department of Applied Life Sciences, Nihon University Graduate School of Bioresource Sciences, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan

a r t i c l e

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Article history: Received 16 August 2013 Received in revised form 16 January 2014 Accepted 5 February 2014 Available online 15 February 2014 Keywords: Durian Durio zibethinus Murr. TRPA1 TRPV1 Sulphur-containing compound Thermogenesis

a b s t r a c t Durian (Durio zibethinus Murr.) is classified as a body-warming food in Indian herbalism, and its hyperthermic effect is empirically known in Southeast Asia. To investigate the mechanism underlying this effect, we focused on the thermogenesis-inducing receptors, TRPA1 and TRPV1. Durian contains sulphides similar to the TRPA1 and TRPV1 agonists of garlic. Accordingly, we hypothesized that the thermogenic effect of durian is driven by sulphide-induced TRP channel activation. To investigate our hypothesis, we measured the TRPA1 and TRPV1 activity of the sulphur-containing components of durian and quantified their content in durian pulp. These sulphur-containing components had a stronger effect on TRPA1 than TRPV1. Furthermore, sulphide content in the durian pulp was sufficient to evoke TRP channel activation and the main agonist was diethyl disulphide. From these results, we consider that the bodywarming effect of durian is elicited by TRPA1 activation with its sulphides, as can be seen in spices. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) belong to the TRP family and are Ca2+-permeable nonselective cation channels (Caterina et al., 1997; Dhaka, Viswanath, & Patapoutian, 2006). Both of these TRP channels are activated by various pungent compounds contained in spices and herbal products. The most representative agonist for TRPV1 is capsaicin (the pungent component of hot pepper) (Caterina et al., 1997). For TRPA1, allyl isothiocyanate (AITC) (the spicy compound generated in crushed mustard) and cinnamaldehyde (the irritant component of cinnamon) are well known agonists (Bandell et al., 2004; Jordt et al., 2004). Moreover, diallyl sulphides (contained in the crushed garlic) (Bautista et al., 2005; Koizumi, Iwasaki, et al., 2009), piperine (a component of pepper) (McNamara, Randall, & Gunthorpe, 2005; Okumura et al., 2010), gingerols, and shogaols (constituents of ginger) (Bandell et al., 2004) act on both TRPA1 and TRPV1. These TRP channels are expressed in sensory neurons in the whole body, including trigeminal ganglion neurons and dorsal root ganglion neurons (Story et al., 2003; Tominaga et al., 1998). In the digestive tract, activation of these TRP channels induces adrenaline ⇑ Corresponding author. Tel.: +81 54 264 5543; fax: +81 54 264 5550. E-mail addresses: [email protected] (Y. Terada), hosono.takashi@ nihon-u.ac.jp (T. Hosono), [email protected] (T. Seki), [email protected] (T. Ariga), [email protected] (S. Ito), [email protected] (M. Narukawa), [email protected] (T. Watanabe). http://dx.doi.org/10.1016/j.foodchem.2014.02.031 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

secretion from the adrenal medulla and enhances thermogenesis. The best-known TRPV1 agonist, capsaicin, evokes adrenaline secretion and heat generation in rats (Iwai, Yazawa, & Watanabe, 2003) and humans (Henry & Emery, 1986; Yoshioka, St-Pierre, Suzuki, & Tremblay, 1998). The TRPA1 agonists AITC, cinnamaldehyde, and diallyl disulphides cause adrenaline secretion and elevate body temperature in rodents (Iwasaki, Tanabe, et al., 2008; Masamoto, Kawabata, & Fushiki, 2009; Oi et al., 1999). In traditional Chinese medicine, spices containing TRPA1 and TRPV1 agonists (e.g., hot pepper, mustard, cinnamon, garlic, and pepper) are classified as foods of body-warming nature (Tokui, Minari, Cho, & Kaku, 2003). Durian (Durio zibethinus Murr.) is one of the popular tropical fruits in Southeast Asia, and it is recognized as a ‘‘warm fruit’’ that has a warming effect on the human body. In a report regarding tropical fruit published by the USDA (United States Department of Agriculture), the physiological effect of durian is described as follows. ‘‘Eating a durian gives a feeling of internal warmth, followed by a glowing sensation difficult to describe. Apparently, it is for this reason that people have ascribed aphrodisiacal qualities to the durian’’ (Malo & Martin, 1979). In addition, durian is the first in the list of heat-generating foods in Indian herbalism. The hyperthermic effect of durian is so strong that it takes 12 mangosteens, which are representative body-cooling foods in Indian herbalism, to cancel the body-warming effect elicited by one durian fruit (Iwasa, 1974). The thermogenic effect of durian is empirically well known; however, mechanism underlying this effect is unknown.

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Durian is characterized by its intensely pungent smell. The odour is reminiscent of garlic, onion, or strong cheese (Malo & Martin, 1979). The unique sulphurous odour of durian is provided by sulphur-containing compounds, such as dialkyl polysulphides and thiols. Among these compounds, diethyl disulphide (DEDS) and diethyl trisulphide (DETS) are the predominant sulphur-containing volatile compounds in various types of durians (Voon, Sheikh Abdul Hamid, Rusul, Osman, & Quek, 2007; Chin et al., 2007; Laohakunjit, Kerdchoechuen, Matta, Silva, & Holmes, 2007) (Fig. 1). These sulphides are similar to diallyl sulphides, the TRPA1 and TRPV1 agonists found in garlic (Fig. 1) (Koizumi, Iwasaki, et al., 2009). Garlic is classified as a body-warming food in traditional Chinese medicine. Furthermore, diallyl disulphide induces a hyperthermic effect (Oi et al., 1999). Accordingly, we hypothesized that body-warming effect of durian is brought about by sulphide-induced TRPA1 and TRPV1 activation. Until now, some non-pungent or low pungent agonists for these channels have been identified, i.e., TRPV1 and TRPA1 agonists capsiate (contained in CH-19 sweet) (Kobata, Todo, Yazawa, Iwai, & Watanabe, 1998) and miogatrial (a low-pungent component of Japanese ginger) (Iwasaki et al., 2009), and a TRPV1 agonist monoacylglycerols (found in wheat) (Iwasaki, Saito, et al., 2008). Although they have little or no stimulant effect, they activate TRP channels to an equal extent as irritant agonists. In fact, the heat generation effects of capsiate and monoaclyglycerols in humans or mice have previously been demonstrated (Iwasaki et al., 2011; Ohnuki et al., 2001). These findings suggest that, although durian is not pungent, durian components may induce hyperthermia using the same pathway as spices. To test our hypothesis, we first measured the TRPA1 and TRPV1 activity of durian sulphur components. Second, we quantified the sulphur agonists from durian pulp using head-space SPME-GC– MS. Lastly, we prepared an essential oil from durian pulp and examined its TRPA1 agonism and its agonist contents. We found that sulphur-containing compounds of durian activate both TRPA1 and TRPV1 but exert a stronger effect on TRPA1 than on TRPV1. GC/MS analysis indicated that the agonist content in durian pulp is sufficient to evoke TRP channel activation, and the main agonist is DEDS. Furthermore, the essential oil of durian activated TRPA1. These findings indicate that, similar to spices, durian exhibits hyperthermic effect through the activation of TRPA1 with its sulphides. 2. Material and methods 2.1. Chemicals Capsaicin, capsazepine (CPZ), diethyl disulphide (DEDS) (99% purity), 1-propanethiol (PT), thiophene, L-a-lysophosphatidylcholine (LPC) from Glycine max (soybean), and ruthenium red were

A

S

S

S

Diethyl disulphide (DEDS)

S

S

Dipropyl disulphide (DPDS)

S

S

S Diallyl sulphide (DAS)

1-Propanthiol (PT)

S

S

Dipropyl trisulphide (DPTS)

Diethyl trisulphide (DETS)

B

SH

S

S

S

Diallyl disulphide (DADS)

S

S

S

Diallyl trisulphide (DATS)

Fig. 1. Chemical structure of sulphur-containing compounds of durian and garlic. Sulphides and thiols of durian (A), sulphide agonists of TRPA1 and TRPV1 contained in garlic (B).

purchased from Sigma–Aldrich (St. Louis, MO, USA). Allyl isothiocyanate (AITC) and l-menthol were obtained from Wako Pure Chem. Ind. (Osaka, Japan). HC-030031 was obtained from ChemBridge (San Diego, CA, USA). Dipropyl disulphide (DPDS) was purchased from LKT Laboratories (Saint Paul, MN, USA). Diethyl trisulphide (DETS) and dipropyl trisulphide (DPTS) were synthesized according to the method Milligan and Swan (1961) and purified by HPLC. The purity of DETS and DPTS was higher than 99%. The chemical structures of the five sulphur volatiles for which we measured TRPA1 and TRPV1 agonism are shown in Fig. 1. 2.2. Plant materials We obtained three durian fruits (D. zibethinus Murr., cultivar: D24) from a local market in Singapore in July 2011. The fruits were cultivated in Malaysia. There are various cultivars of durian and we selected D24 because D24 is one of the major cultivars and it is often used for analysis of aromatic compounds (Chin, Nazimah, Quek, Che Men, & Mat Hashim, 2005; Chin et al., 2007; Voon et al., 2007). When the husk of the durian began to crack and release a strong fruity smell, we judged that the durian had ripened well and cut it open. The pulp was removed from the fruits and stored at 80 °C until analysis. 2.3. Cloning and expression of human or mouse TRP channels Human TRPV1 (hTRPV1), hTRPA1, and mouse TRPM8 (mTRPM8) were stably expressed in HEK293T cells (TRPV1) or HEK T-REx™ cells (TRPA1 and TRPM8). hTRPV2 and hTRPV3 were transiently expressed in HEK293T cells. hTRPV1, hTRPA1, and mTRPM8 cDNA was amplified by RT-PCR from first-strand cDNA from human brain (hTRPV1) (Agilent Technologies, Santa Clara, CA, USA), human WI38 cells (hTRPA1), and mouse dorsal root ganglion cells (mTRPM8). The following primers were used for cloning: TRPV1 forward primer 50 -GCAAGGATGAAGAAGAAATGGA-30 and reverse primer 50 -TCACTTCTCCCCGGAAGCGC-30 ; TRPA1 forward primer 50 -TGGGTCAATGAAGTGCAG-30 and reverse primer 50 -GAAGGTCTGAGGAGCTAAGGC-30 ; mTRPM8 forward primer: 50 ATGTCCTTCGAGGGAGCCAG-30 ; reverse primer: 50 -CGCCAGCCTTACTTGATGTT-30 . hTRPV2 (SKU: SC320356) and hTRPV3 (SKU: SC316997) were purchased from Origene (Rockville, MD, USA). Except for TRPV1 cDNA, the cDNA for the channels was subcloned into pcDNA4/TO (Invitrogen, Carlsbad, CA, USA). TRPV1 cDNA was subcloned into pcDNA3 (Invitrogen) and then transfected into HEK293T cells using SuperFect transfection reagent (Qiagen, Hilden, Germany). After culturing cells in the presence of 750 lg/mL G418, we obtained a HEK293T cell line that stably expressed TRPV1. The stable expression of full-length TRPA1 or TRPM8 in HEK T-REx™ cells was induced using the tetracycline-inducible T-REx™ expression system (Invitrogen). The plasmids were transfected into HEK T-REx™ cells by using Lipofectamine 2000 reagent (Invitrogen). HEK T-REx™ cells stably maintaining the TRPA1 or TRPM8 gene were selected using 500 lg/mL zeocin and 10 lg/mL blasticidin and grown according to the manufacturer’s instructions.hTRPV2 or hTRPV3 plasmids were transiently transfected into HEK293T cells using Lipofectamine LTX reagent (Invitrogen). The cells were sub-cultured every week, and the highest passage number was 40. 2.4. Measurement of intracellular Ca2+ concentration The intracellular Ca2+ concentration ([Ca2+]i) was measured using a Flex Station™ II system (Molecular Devices, Sunnyvale, CA, USA) at 37 °C. The TRPV1-, TRPA1-, or TRPM8-expressing cells were seeded in 96-well plates 24 h before the assay. To obtain TRPA1- or TRPM8-expressing HEK cells, 1 lg/mL tetracycline was

Y. Terada et al. / Food Chemistry 157 (2014) 213–220

added to induce the expression of these TRP channels. TRPV2- or TRPV3-expressing cells were seeded in 96-well plates 6 h after transfection and incubated for 18 h at 37 °C before calcium imaging. The cells were loaded with 3 lM Fluo-4 AM (Molecular Probes, Eugene, OR, USA) for 1 h at 37 °C in a loading buffer (5.37 mM KCl, 0.44 mM KH2PO4, 137 mM NaCl, 0.34 mM Na2HPO47H2O, 5.56 mM D-glucose, 20 mM HEPES, 1 mM CaCl2, 0.1% BSA, and 2.5 mM probenecid at pH 7.4). Probenecid is an agonist for TRPV2, so we used probenecid-free buffer for TRPV2. When measuring TRPV3 activity, the temperature for the assay and the loading was set at 25 °C. We used the following agonists as positive controls for each TRP channel: 100 lM AITC for TRPA1; 10 lM CAP for TRPV1; 100 lM menthol for TRPM8; 30 lM LPC for TRPV2; 300 lM menthol for TRPV3. We used the following five durian components that were commonly identified by several reports: diethyl disulphide (DEDS), dipropyl disulphide (DPDS), diethyl trisulphide (DETS), dipropyl trisulphide (DPT), and 1-propanthiol (PT) (Chin et al., 2005,2007; Jiang, Choo, Omar, & Ahamad, 1998; Laohakunjit et al., 2007; Voon et al., 2007). For TRPA1 antagonism experiments, HC-030031 (30 lM) was co-administered with the sulphide compounds (30 lM), PT (3 mM), or the essential oil of durian (containing 20 lM DEDS). For TRPV1, CPZ (30 lM) was co-treated with each of the sulphides (100 lM) or PT (3 mM). 100 lM DEDS was administered with 5 lM ruthenium red for TRPV2 and TRPV3, or with 10 lM BCTC for TRPM8. To obtain the concentration–response curves for TRPA1 activation, we used 0.01 lM–1 mM DEDS, DPDS, and DPTS; 0.01–300 lM DETS; 500 lM–5 mM of PT; 0.2–45 lM DEDS containing the essential oil; and 0.01–100 lM AITC. For TRPV1, 1 lM–1 mM DEDS, DPDS, DETS, and DPTS; 100 lM– 5 mM PT; and 0.1 nM–10 lM CAP were administered. All of the compounds did not show a non-selective effect on HEK cells not expressing TRP channels in these concentrations. The test compounds were dissolved in DMSO and added to the loading solution. Sulphur-containing compounds and AITC were added to the loading buffer just before the experiment, as they are volatile and unstable under water. The final concentration of DMSO was 0.1–0.4% for TRPA1, TRPM8, TRPV2, and TRPV3, and 0.1–1% for TRPV1. LPC was dissolved in methanol, and its final concentration was 0.1% for TRPV3. These solvents did not affect HEK cells in the above concentrations. To each well, 5 lM ionomycin was added to elicit the maximum fluorescence intensity. The data values for the test compounds except for TRPV2 were expressed as a percentage of the response to 5 lM ionomycin. The calcium responses of TRPV2 were smaller than those of the other TRP channels. Therefore, the obtained responses (F) were normalized to the fluorescence at baseline (F0) and expressed as the DF/F value (%): DF/F (%) = (F  F0)/F0  100. Curve fitting and parameter estimation were performed using Prism 5a software (Graph Pad Software, San Diego, CA, USA). 2.5. Extraction of aromatic compounds from durian pulp using headspace-solid-phase micro extraction (HS-SPME) In the present study, we focused on sulphides contained in durian. They are highly volatile and there is the risk for the loss of minor sulphur constituents by evaporation. There has been a report that analyzes odours of durian using a GC–MS and an organic solvent-extracts sample that were extracted with dichloromethane/ pentane and distillated. On this study, only disulphides (methyl ethyl disulphide and diethyl disulphide) have been mainly detected as sulphides (Hugo, Wim, & Anton, 1996). Other reports using SPME have identified more (nine to eleven) kinds of sulphides from durian (Chin et al., 2007; Voon et al., 2007).

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Therefore, we applied SPME to avoid this risk of losing sulphide compounds. An 85-lm carboxen on polymethylsiloxane (CAR/PDMS) (Supelco, Bellefonte, PA, USA) was used as the SPME. The CAR/ PDMS fibre is suitable for extracting sulphur compounds and is often used for analyzing sulphur volatiles in foods such as cheese and hum (Burbank & Qian, 2005; Garcia-Esteban, Ansorena, Astiasaran, & Ruiz, 2004). To quantify the sulphur compounds of the durian pulp, we used thiophene as an internal standard (IS). Thiophene was dissolved in dimethyl sulfoxide (DMSO) to make a 10 mM solution. It has been shown that the flavour profile from cut pieces of durian does not significantly differ from that of whole durian pulp (Chawengkijwanich, Sa-nguanpuag, & Tanprasert, 2008). This report has suggested that headspace volatiles emitted by cut pieces closely represent that by whole durian, and only cut pieces will be used for investigation of volatiles. Thus, we used a part of durian pulp in SPME. Cut pieces of the frozen durian pulp (approximately 10 g) were randomly sampled and ground them well under liquid nitrogen. Then, the ground durian powder (0.5 g) was quickly transferred into a 10-mL vial. After the addition of 50 nmol of thiophene (10 mM, 5 lL) to the powder, the vial was immediately crimp-sealed with a Teflon septum and vortexed well to disperse IS uniformly in the paste. After equilibration for 30 min at 37 °C in a water bath, head-space sampling was performed at the same temperature for 3 h. Each analytical sample was extracted twice.

2.6. Gas mass spectrometry (GC–MS) conditions The GC–MS conditions were optimized by referring to other research papers (Chawengkijwanich et al., 2008; Jiang et al., 1998; Voon et al., 2007). An Agilent 6890N gas chromatography system (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with 5975 inert mass selective detector (electron ionization mass spectrometer) (Agilent) was used. Volatile compounds were separated using a HP-5MS capillary column (30 m  0.25 mm, 0.25 lm film thickness) (J&W Scientific, Folsom, CA, USA) with the injector and detector maintained at 280 °C. Desorption of the analytes from the SPME was performed at the injection port at 280 °C for 2 min. The injection port was operated in splitless mode with purified helium as the carrier gas flowing at 1 mL/min. The oven temperature was increased from 40 to 200 °C at a rate of 5 °C/min and 200 to 280 °C at 40 °C/min, and maintained at 280 °C for 5 min. The mass spectrometer was set as follows: electron ionization mode with an ionization voltage of 70 eV, ion source temperature of 230 °C, and full scan mode (m/z 40–250). The quadrupole was set at 150 °C. Mass spectra data were analysed using Agilent G1701DA MSD Productivity ChemStation software (version E 01.00.237). Identification of the four sulphur-containing compounds (DEDS, DPDS, DPTS, and PT) was performed by injecting 5 lL of each 10 mM standard DMSO solution using headspace SPME under the same conditions used for the samples. Identification of other compounds was accomplished by matching mass spectra with the NIST library (version 11.0) (Palisade Corp., Newfield, NY, USA). Quantification was performed by comparing the peak areas of the analytes to those of thiophene added as an internal standard to the samples. To compare sulphur compound content and the EC50 values (lM) for TRPA1 and TRPV1, the contents were expressed as lmol/kg of the durian pulp. Assuming that the density of the durian pulp is 1, 1 lmol/kg of the durian pulp is equal to 1 lM. The results were expressed as follows: peak area/internal standard (IS) area  100 (lmol/kg durian pulp).

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2.7. Isolation of the essential oil from the durian pulp and quantification of DEDS

3. Results 3.1. Effects of sulphur-containing compounds of durian on TRPA1 and TRPV1

The frozen durian pulp (100 g) was broken into 2–3 cm pieces with a hammer and quickly transferred into a 1 L Kjeldahl flask with a magnetic stirring bar. One litre of distilled H2O was added, the pulp was distilled for 6 h in a Clevenger apparatus under stirring, and the essential oil (we called it by a general name, even though it might contain non-essential oil substances) was obtained on the surface of the water. DEDS in the essential oil was quantified using HPLC in an ODS column (J’ sphere ODS H-80 4.6  150 mm, YMC, Kyoto, Japan) and UV detection at 254 nm using acetonitrile/H2O = 80:20 (v/v) as the eluting solvent at a flow rate of 1.0 mL/min. The DEDS content in the essential oil was estimated using a calibration curve for authentic DEDS.

We evaluated the TRPA1 and TRPV1 activity of five sulphurcontaining volatiles of durian. Fig. 2 (A and B: TRPA1, C and D: TRPV1) shows the Ca2+ responses induced by each of the five sulphur-containing compounds in TRPA1- or TRPV1-expressing cells: DEDS, DPDS, DETS, DPTS, and PT increased [Ca2+]i in both TRPA1and TRPV1-expressing cells. The addition of the TRPA1 antagonist HC-030031 (30 lM) or TRPV1 antagonist CPZ (30 lM) significantly attenuated the Ca2+ influx induced by these sulphur compounds. In addition, these compounds scarcely affected [Ca2+]i in cells not expressing TRPA1 or TRPV1 (Fig. 2A and C). These results indicate that sulphur compounds of durian (DEDS, DPDS, DETS, DPTS, and PT) activate both TRPA1 and TRPV1. The concentration–response curves for the five sulphur-containing compounds against TRPA1 and TRPV1 are also shown in Fig. 2 (B: TRPA1, D: TRPV1). The EC50 values (lM) and maximal responses (%) of these compounds are shown in Table 1. The maximal responses are shown as a percentage of the response to 5 lM ionomycin. For TRPA1 activity, among all compounds tested, DETS showed the strongest activation efficacy, and its EC50 value and maximal

2.8. Extraction of durian with organic solvents Lyophilized durian pulp (20 g) was extracted successively with 400 mL of hexane, ethyl acetate, and methanol each. The organic solvents were completely evaporated, then the obtained extracts were dissolved into DMSO and their TRPA1 activity at 100 and 300 lM was measured.

100

% Activity to 5 μM Ionomycin

B

TRPA1 Sample only Sample + HC-030031 TREx-HEK

75 3000 μM

50

25 *** *** *** ***

0

C

AITC

DEDS

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DPDS

TRPA1 100

DETS

*** ***

DPTS

% Activity to 5 μM Ionomycin

A

AITC DEDS 75

DETS 50

PT

25

0 -8

PT

D

TRPV1

-7

-6 -5 -4 Concentration (Log M)

-3

-2

TRPV1 100

Sample + Capsazepine 75

HEK293T

50 3000 μM

25 *** ***

***

***

***

***

500 μM ***

***

***

% Activity to 5 μM Ionomycin

Sample only % Activity to 5 μM Ionomycin

DPTS

*** ***

100

0

DPDS

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CAP DEDS DPDS DETS

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DPTS PT

25

***

0 CAP

DEDS

DPDS

DETS

DPTS

PT

-10

-9

-8

-7

-6

-5

-4

-3

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Concentration (Log M)

Fig. 2. TRPA1 and TRPV1 activation by sulphur-containing compounds of durian. Fluo-4 Ca2+ responses of HEK cells expressing human TRPA1 (A and B) and TRPV1 (C and D) are described. (A and C) Effect of co-administration of TRPA1 or TRPV1 antagonist with the compounds and responses of HEK cells not expressing TRP channels. (B and D) Concentration–response curves for TRPA1 and TRPV1 of the compounds. Data values for these compounds are each expressed as a percentage of the response to 5 lM ionomycin. Each data point represents the mean ± SEM (n = 6–8). Filled columns show Ca2+ responses by the durian components. The sulphides were added at 30 lM for TRPA1 (A) and 100 lM for TRPV1 (C). PT was used at 3000 lM for both TRP channels, and DPTS was administered at 500 lM for TRPV1. Hatched columns indicate TRPA1 or TRPV1 activation by these compounds with the co-addition of 30 lM HC-030031 (A) or 30 lM capsazepine (C). Unfilled columns show the responses to these compounds of HEK cells not expressing the TRP channels (TRPA1: HEK T-REx™ cells, TRPV1: HEK293T cells) (A and C). ⁄⁄⁄ indicates p < 0.0005 (unpaired t-test). Concentration–response curves for TRPA1 (B) and TRPV1 (D) of five durian components.

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Y. Terada et al. / Food Chemistry 157 (2014) 213–220 Table 1 TRPA1 and TRPV1 agonists contained in durian and spices. TRPA1

AITC CAP DEDS DPDS DETS DPTS PT

TRPV1 *

EC50 (lM)

TOP (%)

EC50 (lM)

TOP (%)

0.5 – 45 4.6 1.3 18 2543

87 – 88 68 86 74 61

– 0.0035 20 31 26 322 2337

– 60 25 16 22 13 30

EC50 (lM)

TOP (%)*

EC50 (lM)

TOP (%)*

254 7.5 0.5 29 6 35 7 52

99 97 92 77 90 102 106 93

151 37 43 0.6 – 0.07 0.1 29

55 41 50 58 – 99 41 20

TRPA1

DAS DADS DATS Piperine Cinnamaldehyde 6-Gingerol 6-Shogaol a-Sanshool

*

TRPV1

Mean contents

Range of contents

(lmol/kg pulp)

(lmol/kg pulp)

– – 335.4 ± 53.8 17.7 ± 3.7 Not detected 2.0 ± 0.8 6.0 ± 5.9

– – 175.5–500.0 7.3–29.1

Spice

References

Garlic

Koizumi, Iwasaki, et al. (2009)

Pepper Cinnamon Ginger Steamed ginger Japanese pepper

Okumura et al. (2010) Koizumi (2009) Iwasaki et al. (2006), Koizumi (2009) Iwasaki et al. (2006), Koizumi (2009) Sugai et al. (2005), Koizumi (2009)

0.4–5 0–35.7

Sulphur compounds in durian are highlighted in bold in the table. Percentage of the response to 5 lM ionomycin. AITC, allyl isothiocyanate; CAP, capsaicin; DEDS, diethyl disulphide; DPDS, dipropyl disulphide; DETS, diethyl trisulphide; DPTS, dipropyl trisulphide; PT, 1-propanthiol.

*

response were nearly equal to those of AITC. The sulphides activated TRPA1 at a concentration that was from 100 to 2000 fold lower than the concentration of PT. The EC50 values of the sulphides (DPDS, DPTS, and DEDS) were 10–90 times larger than those of AITC, and the EC50 value of PT was 5,000 times higher than that of AITC. The maximal responses to AITC relative to PT were 70– 101% (Table 1). TRPV1 activity: The EC50 values of these sulphur compounds were 60–10,000,000 times higher than those of CAP, and the relative maximum activity of these compounds was less than half of that of CAP. The maximal responses relative to the response to CAP were 22–50% (Table 1). For both TRPA1 and TRPV1, the activation potency of the sulphides was 100–1000 times stronger than that of PT. All sulphides acted on not only TRPA1 but also TRPV1, and they activated TRPA1 to a greater extent than TRPV1. Their EC50 values for TRPV1 were 10 times higher than those for TRPA1, and the maximal responses for TRPV1 were less than half of those for TRPA1. 3.2. Quantification of sulphide agonists for TRPA1 and TRPV1 of durian pulp Next, we quantified the sulphide agonists of durian pulp (cultivar: D24) to determine whether TRPA1 and TRPV1 can be activated by eating durian. Because the sulphides agonists are highly volatile, we extracted them by SPME from crushed durian pulp and analysed them by GC/MS. To quantify each aromatic compound, thiophene was added to the pulp as an IS. We identified the following seven sulphides: DEDS, ethyl propyl disulphide, DPDS, cis-dimethyl-1, 2, 4-trithiolane, trans-dimethyl1, 2, 4-trithiolane, 1, 1-bis (ethylthio)-ethane, and DPTS. Among them, DEDS was the main component, and it occupied one quarter of all volatiles (Fig. 3 and Table 2). A list of all quantified volatiles and their contents are displayed in Table 2, and one of the representative chromatograms and detected sulphur compounds are shown in Fig. 3. The sulphur compound contents and EC50 values (lM) for the TRP channels are displayed in Table 1. The volatile contents are shown as lmol/kg durian pulp to compare the sulphide contents and their EC50 values (lM) for TRPA1 and TRPV1 (Table 1). We used three durian fruits, and each of the fruits was

measured twice. The identified compounds were consistent among the three fruits but the contents varied; we also show the range of the contents in Tables 1 and 2. 3.3. Effect of essential oil and organic solvent-extracts of durian on TRPA1 We prepared approximately 25 lL of essential oil from 100 g of durian pulp. Because the quantity of the obtained essential oil was small and durian sulphides exerted a stronger effect on TRPA1 than on TRPV1, we measured only their TRPA1 agonism. DEDS was the predominant sulphide volatile of durian, so we quantified its content in the essential oil. The DEDS concentration of the essential oil was 11.2 mM, and the essential oil acted on TRPA1. This response was significantly suppressed by co-treatment with the TRPA1 antagonist HC030031 (Fig. 4A). The concentration-dependence curve of the essential oil (containing 0.3–50 lM DEDS) was similar to that of DEDS (Fig. 4B). We did not examine the effect of the essential oil on TRPA1 activity at higher concentration (>50 lM), because of the lower solubility of the essential oil in the measuring buffer. These results indicated that durian extract activates TRPA1 and DEDS is the major compound responsible for the TRPA1 activation. In regards to the organic solvent-extracts, neither of them exhibited TRPA1 activity (Fig. 4C). 3.4. Selectivity of DEDS for thermo-sensitive TRP channels To examine the selectivity of DEDS for thermo-sensitive TRP channels, we administered 100 lM DEDS to TRPM8-, TRPV2-, or TRPV3-expressing HEK cells. As a result, none of the channels responded to DEDS (Fig. 4D–F). From these results, the main target of DEDS among thermo-sensitive TRP channels is TRPA1. 4. Discussion We measured the TRPA1 and TRPV1 activity of four sulphides (DEDS, DPDS, DETS, and DPTS) and one thiol compound (PT) using human TRPA1- or TRPV1-expressing HEK cells (chemical structure

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Fig. 3. Representative GC–MS total ion chromatogram of durian volatiles and detected sulphur compounds. Peak numbers in a square are sulphur compounds.

Table 2 Identified durian volatiles obtained by headspace SPME. Peak No.

RT (min)

Compound

Mean contents (lmol/kg pulp)

Range of cotents (lmol/kg pulp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

3.8 5.0 6.2 6.9 8.0 10.0 10.6 12.8 15.5 16.4 16.5 19.2 21.6 21.8

PT Ethyl propanoate Methyl 2-methylbutanoate Propyl propanoate Ethyl 2-methylbutanoate DEDS Propyl 2-methylbutanoate Ethyl propyl disulphide DPDS cis-3,5-Dimethyl-1,2,4-trithiolane trans-3,5-Dimethyl-1,2,4-trithiolane Unknown 1,1-Bis (ethylthio)-ethane DPTS

6.0 ± 5.9 93.3 ± 7.1 93.2 ± 5.7 24.2 ± 7.6 311.1 ± 25.9 335.4 ± 53.8 61.2 ± 9.8 162.0 ± 29.1 17.7 ± 3.7 23.6 ± 6.4 64.0 ± 20.2 17.7 ± 5.2 12.1 ± 3.9 2.0 ± 0.8

0–35.7 74.2–109.0 74–108.9 11.1–53.7 261.2–434.4 175.5–500.0 30.4–91.6 74.8–250.2 7.3–29.1 7.6–46.9 21.7–146.6 6.0–34.2 3.5–25.1 0.4–5

Sulphur compounds are highlighted in bold in the table. DEDS, diethyl disulphide; DPDS, dipropyl disulphide; DPTS, dipropyl trisulphide; PT, 1-propanthiol.

shown in Fig. 1). It was shown that all sulphides affected TRPA1 more strongly than TRPV1. PT was a weak agonist for both TRP channels, and its EC50 values were 100–2000 times higher than those of the sulphides (Fig. 2B and D, Table 1). Diallyl suphide (DAS), diallyl disulphide (DADS), and diallyl trisuphide (DATS) have been identified as TRPA1 and TRPV1 agonists from garlic (Koizumi, Iwasaki, et al., 2009). Their EC50 values and maximal responses for human TRPA1 and TRPV1 are also shown in Table 1. Comparing these data in Table 1, each of the disulphides and trisulphides contained in durian and garlic have nearly equal potency against these two TRP channels. There is a common tendency that longer sulphide bonds are associated with stronger TRPA1 agonism. The effect of trisulphides is approximately 10 times stronger than that of disulphides. The mechanism of TRPA1 activation by DADS was previously reported as follows. The sulphide moiety of DADS directly bonds to cysteine residues within the cytoplasmic N terminus of TRPA1 and triggers channel activation (Cebi & Koert, 2007; Hinman, Chuang, Bautista, & Julius, 2006). From these reports, it is predicted that durian sulphides have the same TRPA1-activating mechanism as garlic sulphides.

TRP activities of durian sulphides are compared to those of spice components, i.e., piperine, cinnamaldehyde, 6-gingerol, 6-shogaol, and a-sanshool (Table 1). The sulphides showed from nearly equal to fifty times stronger efficacy on TRPA1 than other agonists. In terms of TRPV1 activity, the sulphides have from 10 to 100 times lower potency and less than half maximal responses compared with those of the spice-derived compounds. Although we tried to examine the TRP activity of ethyl propyl disulphide, cis and trans isomers of dimethyl-1, 2, 4-trithiolane, and 1, 1-bis (ethylthio)-ethane, we could not purchase or synthesize these compounds. Their contents were lower than that of DEDS, so it was speculated that their contribution to total TRP agonism would be lower than that of DEDS. Next, to determine whether the sulphides are sufficiently contained in durian to induce TRPA1 and/or TRPV1 activation, we quantified their contents using head-space SPME-CG/MS. The detected aroma profiles (esters, ketones, sulphides, and aldehydes) in this study were consistent with those of previous studies (Chin et al., 2007; Voon et al., 2007). The very major sulphur compound was DEDS. The DEDS content (200–500 lmol/kg) was more than 10 times higher than its EC50 values (45 lM for TRPA1, 120 lM

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40

B Essential oil only Essential oil + HC-030031

30 20 10 ***

C 100

Essential oil 80

% Activity to 5 μM Ionomycin

50

% Activity to 5 μM Ionomycin

% Activity to 5 μM Ionomycin

A

DEDS

60 40 20

100

300 μM of extract

60 40 20 0

0

0

100 μM of extract

80

-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0

Hexane

Ethyl acetate

DEDS Concentration (Log M)

F/F

60

TRPV2 LPC only LPC + Ruthenium Red DEDS only

40

20 ***

0

E 100 80

F

TRPV3 Menthol only Menthol + Ruthenium Red DEDS only

60 40 ***

20 0

% Activity to 5 μM Ionomycin

80

% Activity to 5 μM Ionomycin

D

100

Methanol

TRPM8 Menthol only

80

Menthol + BCTC DEDS only

60 40 20 ***

0

Fig. 4. TRPA1 activity measuring of essential oil and extracts of durian and TRP channel selectivity of DEDS. The essential oil (A and B) and organic solvent-extracts (C) of durian induced Ca2+ influx in TRPA1-expressing HEK cells. Ca2+ responses elicited by DEDS in HEK cells expressing TRPV2 (D), TRPV3 (E), or TRPM8 (F). The data values for each of these compounds are expressed as a percentage of the response to 5 lM ionomycin or the DF/F value (for TRPV2). Each data point represents the mean ± SEM (n = 4– 6). ⁄⁄⁄ indicates p < 0.0005 (unpaired t-test). Filled column shows TRPA1 activation by the essential oil containing 20 lM DEDS. Hatched column indicates the response when 30 lM HC-030031 was co-treated with the essential oil (A). Concentration–response curves of the essential oil and DEDS for TRPA1. The X axis indicates the concentration (Log M) of DEDS (B). Dotted columns and filled columns indicate 100 and 300 lM of the extract-induced response in TRPA1-expessing cells, respectively (C). Filled columns show TRP channel agonism by positive controls (40 lM LPC for TRPV2, 300 and 100 lM menthol for TRPV3 and TRPM8). Slant-lined columns indicate the inhibitory effect of each antagonist for the positive controls (5 lM ruthenium red for TRPV2 and TRPV3, 10 lM BCTC for TRPM8). Unfilled columns show the responses evoked by 100 lM DEDS for each TRP channel-expressing HEK cells (D–F).

for TRPV1). DPDS and DPTS were contained at sufficient levels to evoke TRPA1 activation. Their contents and EC50 values were 10– 30 lmol/kg and 4.6 lM for TRPA1 and 31 lM for TRPV1 for DPDS, and 0.5–5 lmol/kg and 18 lM for TRPA1 and 322 lM for TRPV1 for DPTS. These results indicate that sulphide agonists are sufficiently contained in durian to trigger TRPA1 and/or TRPV1 activation. When we eat durian, TRPA1 and/or TRPV1 that express on the surfaces of the digestive system (i.e., the stomach and intestines) can be activated by these sulphides. Despite some reports showing DETS as one of the major sulphides in various cultivars of durian (Chin et al., 2007; Laohakunjit et al., 2007; Voon et al., 2007), DETS was not detected in our analysis. Such as disagreements of flavour profiles have also been reported in other studies (Laohakunjit et al., 2007; Voon et al., 2007). This may result from geographical differences, as it has also been reported that environment, cultural practices, agrichemicals, and nutrition affect the flavours of crops (Mattheis & Fellman, 1999). We obtained an essential oil from the durian fruit and measured its TRPA1 activity and DEDS content. We made clear that the essential oil acted on TRPA1, and its concentration–response curve plotted with the DEDS contents matched that of pure DEDS (Fig. 4A and B) These results showed that the main agonist of the essential oil is DEDS. Although the essential oil activated TRPA1, the organic solvent-extracts did not show any TRPA1 activation potency (Fig. 4C). The sulphides are highly volatile so it was speculated that they would be lost by evaporation.

Finally, TRPV2-, TRPV3-, or TRPM8-expressing HEK cells were treated with DEDS to clarify the channel selectivity. The result showed that DEDS did not activate any of these channels (Fig. 4D–F). Therefore, the main target of durian sulphides for thermo-sensitive TRP channels is TRPA1. Protein content of raw durian pulp is 2% and this is relatively high among fruits (Malo & Martin, 1979). Specific dynamic action of protein is four times larger than that of carbohydrate and fat (McCue, 2006). Therefore, there is a possibility that protein of durian can be involved in hyperthermic effect of durian. It has been reported that ingestion of TRPA1 and TRPV1 agonists prevents fat deposit in the adipose tissue (Iwai et al., 2003; Tamura, Iwasaki, Narukawa, & Watanabe, 2012). However, fat content in durian is relatively high among fruits (1–4% in fresh durian pulp) (Malo & Martin, 1979), so it is not clear that if intake of durian suppresses body fat accumulation. TRPV1 and TRPA1 agonists, i.e., CAP (hot pepper), AITC (mustard), cinnamaldehyde (cinnamon), and DADS (garlic), facilitate heat generation by secretion of adrenaline and noradrenaline via activation of the TRP channels (Iwai et al., 2003; Iwasaki, Tanabe, et al., 2008; Masamoto et al., 2009; Oi et al., 1999). The effects of various sulphides on adrenaline and noradrenaline secretion have been evaluated in rats (Oi et al., 1999). We identified dialkyl sulphides as main TRP agonists from durian, and four kinds of dialkyl sulphides have been also tested by Oi et al. Although the effects of the dialkyl sulphides on catecholamine secretion are weaker than those of diallyl sulphides (garlic), plasma adrenaline and

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noradrenaline concentrations are significantly increased or tended to increase by administration of the sulphides. For example, administration of durian-derived TRPA1 agonists, DEDS and DPDS have the tendency to increase blood adrenaline only and both blood adrenaline and noradrenaline levels, respectively. Consequently, there is a possibility that dialkyl sulphides of durian enhance thermogenesis by inducing release of catecholamines. 5. Conclusion In this paper, we found that sulphur-containing compounds of durian activate the thermogenesis-inducing receptors TRPA1 and TRPV1. In addition, we showed that durian pulp contains a sufficient amount of sulphides to evoke TRPA1 and/or TRPV1 activation. Moreover, the essential oil of durian activated TRPA1, and the main agonist was DEDS. From these results, we consider that the hyperthermic effect of durian is produced by sulphide-induced TRPA1 activation. Acknowledgements This study was supported in part by the Global Center of Excellence (COE) Program of University of Shizuoka from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and Grants-in Aid for Scientific Research from JSPS (24 10919 to Y.T. and 24580194 to T.W.). References Bandell, M., Story, G. M., Hwang, S. W., Viswanath, V., Eid, S. R., Petrus, M. J., et al. (2004). Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron, 41, 849–857. Bautista, D. M., Movahed, P., Hinman, A., Axelsson, H. E., Sterner, O., Hogestatt, E. D., et al. (2005). Pungent products from garlic activate the sensory ion channel TRPA1. Proceedings of the National Academy of Sciences of the United States of America, 102, 12248–12252. Burbank, H. M., & Qian, M. C. (2005). Volatile sulfur compounds in Cheddar cheese determined by headspace solid-phase microextraction and gas chromatographpulsed flame photometric detection. Journal of Chromatography A, 1066, 149–157. Caterina, M. J., Schumacher, M. A., Tominaga, M., Rosen, T. A., Levine, J. D., & Julius, D. (1997). The capsaicin receptor: A heat-activated ion channel in the pain pathway. Nature, 389, 816–824. Cebi, M., & Koert, U. (2007). Reactivity recognition by TRPA1 channels. ChemBioChem, 8, 979–980. Chawengkijwanich, C., Sa-nguanpuag, K., & Tanprasert, K. (2008). Monitoring volatile compounds emitted by durian pulp (Durio zibethinus murr.) at mature and ripe stage using solid phase microextraction (SPME). Acta Horticulturae, 804, 321–326. Chin, S. T., Nazimah, S. A. H., Quek, S. Y., Che Men, Y. B., Abdul Rahman, R., & Mat Hashim, D. (2007). Analysis of volatile compounds from Malaysian durians (Durio zibethinus) using headspace SPME coupled to fast GC–MS. Journal of Food Composition and Analysis, 20, 31–44. Chin, S. T., Nazimah, S. A. H., Quek, S. Y., Che Men, Y. B., & Mat Hashim, D. (2005). Application of headspace solid-phase microextraction to the analysis of durian (Durio zibethinus) variety D24 volatile compounds. Journal of Physical Science, 16, 149–157. Dhaka, A., Viswanath, V., & Patapoutian, A. (2006). TRP ion channels and temperature sensation. Annual Review of Neuroscience, 29, 135–161. Garcia-Esteban, M., Ansorena, D., Astiasaran, I., & Ruiz, J. (2004). Study of the effect of different fiber coatings and extraction conditions on dry cured ham volatile compounds extracted by solid-phase microextraction (SPME). Talanta, 64, 458–466. Henry, C. J., & Emery, B. (1986). Effect of spiced food on metabolic rate. Human Nutrition. Clinical Nutrition, 40, 165–168. Hinman, A., Chuang, H. H., Bautista, D. M., & Julius, D. (2006). TRP channel activation by reversible covalent modification. Proceedings of the National Academy of Sciences of the United States of America, 103, 19564–19568. Hugo, W., Wim, E. K., & Anton, A. (1996). Sulfur-containing volatiles of durian fruits (Durio zibethinus Murr.). Journal of Agricultural and Food Chemistry, 44, 3291–3293. Iwai, K., Yazawa, A., & Watanabe, T. (2003). Roles as metabolic regulators of the non nutrients, capsaicin and capsiate, supplemented to diets. Proceedings of the Japan Academy Series B-Physical and Biological Sciences, 79, 207–212. Iwasa, S. (1974). Fruit-trees of Southeast Asia. Tokyo: Tropical Agriculture Research Center, Ministry of Agriculture and Forestry.

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