CB2 Ligands from

Article pubs.acs.org/jnp

Identification of CB1/CB2 Ligands from Zanthoxylum bungeanum Katina S. S. Dossou,†,# Krishna P. Devkota,‡,# Cynthia Morton,§ Josephine M. Egan,† Guanghua Lu,⊥ John A. Beutler,‡ and Ruin Moaddel*,† †

Biomedical Research Center, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Suite 100, Baltimore, Maryland 21224, United States ‡ Molecular Targets Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, United States § Section of Botany, Carnegie Museum of Natural History, 4400 Forbes Avenue Pittsburgh, Pennsylvania 15213, United States ⊥ Key Laboratory of the Ministry of Education in China on the Standardization of Chinese Materia Medica, School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, People’s Republic of China S Supporting Information *

ABSTRACT: In order to study cannabinoid receptor ligands, a novel plate-based assay was developed previously to measure internalization of CB1/CB2 receptors by determining the change in the intracellular levels of the radiolabeled agonists. This plate-based assay was also used for screening against complex matrices, specifically, in the present study screening for CB1/CB2 receptor activity of extracts for several species of the plant genus Zanthoxylum. The objective of this screen was to identify novel antagonists of the CB1 receptor, which simultaneously displayed agonist activity against the CB2 receptor, since compounds matching this criterion could be potential candidates for the treatment of type-1 diabetes. As a result, two Z. bungeanum extracts were deemed active, leading to the identification of eight compounds, of which compound 7 [(2E,4E,8E,10E,12E)-N-isobutyl-2,4,8,10,12-tetradecapentaenamide, γ-sanshool] was obtained as a promising lead compound.

C

mediated insulin secretion.9 Intriguingly, ECs also induce cell cycle arrest and apoptosis by inhibiting the PI3K-AKT cascade in various cancer cells.10−12 Of importance to metabolism, ECs influence insulin action through regulation of insulin receptor signaling in insulinsensitive tissues such as muscle, liver, and the islets of Langerhans, and blockade of CB1 receptors inhibits apoptosis in β-cells under stress from obesity and streptozotocin toxicity.13 Therefore, it is possible that CB1 receptor blockade might be useful in preventing β-cell apoptosis in type-1 diabetes. However, type-1 diabetes is a T-cell-mediated autoimmune disease, and insulin itself is an antigen responsible for triggering the autoimmune response. It follows that if CB1 receptor blockade results in improved insulin secretion in type1 diabetes, autoimmune destruction might increase in intensity. CB2 receptors are present on T-cells,14 and their activation by exogenous ligands has been shown to reduce T-cell activation.15 Therefore, a compound with dual functionCB1 receptor inhibition combined with CB2 activationmight be useful in type-1 diabetes. Previously, it was shown that crude extracts of Zanthoxylum clava-herculis L. (Rutaceae) have activity at the CB2 receptor using a CB1/CB2 open tubular column.16 Subsequently, a

annabinoid (CB) receptors are 40 kDa integral membrane G-protein-coupled receptors (GPCR) containing seven transmembrane domains. To date, three subtypes of cannabinoid receptors have been identified: CB1, CB2, and GPR55.1−3 These three G-protein-coupled receptors play important roles in many physiological processes, including metabolic homeostasis, craving, pain, anxiety, bone growth, and immune function.4,5 CB2 receptors are expressed mainly in immune cells, such as macrophages, lymph nodes, and microglia, although they have also been detected in nonimmune cells,2,6 while CB1 receptors are abundant in the central nervous system, particularly in regions involved in cognition, short-term memory, and motor function and movement.7,8 They are also expressed in peripheral areas, such as the pituitary gland, immune cells, heart, lung, bone marrow, islets of Langerhans, hepatocytes, skeletal muscle, and gastrointestinal tissues.2 CB1 receptors and the necessary enzymes for catalyzing the biosynthesis and degradation of endocannabinoids (ECs), 2arachidonoylglyceraol (2-AG) and anandamide (AEA), are present in β-cells of the islets of Langerhans.9 ECs are lipid transmitters synthesized “on demand” by Ca2+-dependent enzymes in the brain and the periphery. Additionally, β-cells synthesize ECs in a glucose-dependent manner, in synchrony with depolarization of the cell. Thus blocking CB1 receptors with inverse agonists in mice leads to increased glucose© 2013 American Chemical Society and American Society of Pharmacognosy

Received: June 14, 2013 Published: October 31, 2013 2060

dx.doi.org/10.1021/np400478c | J. Nat. Prod. 2013, 76, 2060−2064

Journal of Natural Products

Article

On the basis of these results, further separation of Z. bungeanum (Sichuan) fraction A was conducted. From this fraction, eight major UV-absorbing peaks were collected and characterized as 2′,3′-dihydroxy-α-sanshool (1),18 hydroxy-αsanshool (2),19 hydroxy-β-sanshool (3),19 hydroxy-γ-sanshool (4),19 hydroxy-γ-isosanshool (5),20 isobungeanool (6),20 γsanshool (7),21 and tetrahydrobungeanool (8) (Figure S9, Supporting Information).20 While these sanshools have all been previously identified from other Zanthoxylum species, this is the first time 1 has been isolated from Z. bungeanum. All compounds were characterized by comparing their spectroscopic data with literature values. The identification of sanshools with cannabinoid activity is not unprecedented, as alkylamides from Echinacea spp. have been identified previously as cannabinoid ligands, with particularly high affinity for the CB2 receptor.22 As can be seen in Table 1, 3 possessed the greatest antagonism against the CB1 receptor, while 8 had the greatest antagonistic activity against CB2 receptors. Thus, the functional activity of these sanshool derivatives may depend on several factors including the stereoisomerism of the C-6, C-7 (1−3) or C-8, C-9 (4−8) double bonds, the length of the carbon chain [C12 (1−3) or C14 (4−8)], the unsaturation level of the linear carbon chain, and the presence of a hydroxy group on the alkylamine portion of the molecule. The stereoisomerism at C-6 had a pronounced effect on the antagonistic activity of 3 vs 2, where the cis configuration of 2 resulted in a 700-fold decrease in the IC50 for the CB1 receptor, while inducing a 2-fold increase in activity against the CB2 receptor (Table 1). While the cis-isomer of C-6 proved less potent than in 2, an opposite effect was observed when 4 and 5 were compared. In this case, the C-8 cis-isomer 4 was 10-fold more potent for CB1 but displayed no difference for CB2 (197−224 nM) (Table 1), indicating the position of the cis double bond may play a major role. However, there were several additional differences including a longer aliphatic carbon chain (e.g., C14 vs C12), the absence of a double bond at the C-6 position, or a double bond at the C-12 position. The nature of the C-8, C-10, and C-12 positions does not seem to affect the CB1 activity greatly, cf., 8 and 5 (4600 vs 1392 nM), while saturation resulted in a significant increase in activity against the CB2 receptor (224 vs 1.8 nM). For 4 (C-8 cis), the replacement of the C-10 and C-12 double bonds with a C-11 double bond (isobungeanool, 6) resulted in a significant decrease in antagonistic activity against both the CB1 and CB2 receptors, namely, 2400 vs 184 nM and 9100 vs 197 nM, respectively, indicating that the position of unsaturation may play an important role for the sanshools with a cis configuration at C-8

novel plate-based assay was developed to measure the ligand effect on internalization of the CB1/CB2 receptors by determining the change in intracellular levels of a radiolabeled agonist in order to determine the functional activity of the ligand.17 It was demonstrated that the plate-based assay was able to screen complex matrices, namely, several extracts of the plant genus Zanthoxylum. Five extracts of Asian Zanthoxylum species [Zanthoxylum armatum DC, Zanthoxylum bungeanum Maxim. (three distinct samples), and Zanthoxylum piperitum DC] were screened against both the CB1 and CB2 receptors for antagonistic and agonistic activity.17 The objective of this screen was to identify antagonists of the CB1 receptor that displayed simultaneously agonist activity against the CB2 receptor. Such selective compounds could be potential candidates for the treatment of type-1 diabetes. Herein, are reported the isolation and identification of eight compounds from Z. bungeanum and the identification of one compound (7, γ-sanshool) that satisfies selectivity criteria for potential therapeutic application as a type1 diabetes drug.

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RESULTS AND DISCUSSION In previous work,17 a novel plate-based assay was developed that measured internalization of CB1/CB2 receptors by determining the change in the intracellular levels of the radiolabeled agonists [3H]-Win 55,212-2 (for CB1) and [3H]CP 55,940 (for CB2). The method was validated by determining IC50 values of numerous known CB1 and CB2 antagonists and demonstrating that they correlate with values determined by other methods. It was then demonstrated that this plate-based assay was able to identify CB1/CB2 modulators present in extracts of three species of Zanthoxylum. Of the five samples tested for CB1/CB2 activity, only two Z. bungeanum extracts (from Sichuan and Gansu Provinces, People’s Republic of China, respectively) were studied further. Fractionation of the Z. bungeanum (Sichuan) extract was guided using both CB1 and CB2 plate-based assays. From the Z. bungeanum (Sichuan) extract, five fractions (A− E) were tested for activity at both the CB1 and CB2 receptor. Of the five subfractions evaluated only A and B had strong activity at the CB1 receptors, while B and E had limited activity at the CB2 receptors.

Table 1. IC50 Values of Compounds 1−8 for Inhibition of the Internalization of Uptake of 10 nM [3H]-Win 55,212-2 and 2 nM [3H]-CP 55,940 for the CB1 and CB2 Receptors, Respectively

a

compound

CB1 IC50a (nM)

inhibition of internalization (%)

CB2 IC50a (nM)

inhibition of internalization (%)

CB1/CB2

1 2 3 4 5 6 7 8

41 [28−60] 7400 [6060−9117] 9 [3.1−29] 184 [86−392] 1392 [127−15200] 2400 [1900−3043] 100 [82−120] 4600 [3100−5300]

76 29 40 31 39 45 37 30

168 [124−227] 59 [47−74] 131 [120−143] 197 [102−380] 224 [180−278] 9100 [1600−50000] 664 [418−1054] 2 [0.16−20]

65 29 43 65 34 62 57 73

0.24 1254 0.07 0.9 6.2 0.26 0.15 2556

95% confidence interval. 2061



Article

orphan GPCR panel, the compound activity was calculated as the percentage change in assay signal over basal activity. While 7 did not result in any activity representative of a strong agonist (>100% activity, representing a 2-fold increase over basal activity) for any of the 75 orphan GPCRs tested, it did result in a significant increase (>50% and 3 SD from the mean basal activity) for GPR55 (a novel cannabinoid receptor), GPR132 (a high-affinity receptor for lysophosphatidylcholine), and GPR151 (galanin receptor 4) with agonistic activities of greater than 50% (Table 2), indicating that 7 may have affinity for these receptors.

for both the CB1 and CB2 receptors. The modification of the 2′-methylpropan-2′-ol group of the sanshools also resulted in significant changes in the activity against both the CB1 and CB2 receptors. The removal of the 2′-hydroxy group from 5 as in 7 resulted in a significant increase in antagonistic activity against the CB1 receptor (1392 vs 99.6 nM) (Figure 1A).

Table 2. Screening of Compound 7 against 165 Known GPCRs for Agonist/Antagonist Activitya GPCR

% activity

% inhibition

CNR1 CNR2 PRLHR FSHR GPR55 GPR132 GPR151 HRH4 PTGER2 RXFP3

5 64 5 5 67 61 76 18 7 18

72 15 52 68 N/A N/A N/A −54 −52 −81

a

A DiscoverRX gpcrMAX panel was used for this investigation (active: CNR1, CNR2, PRLHR, FSHR, HRH4, PTGER2, RXFP3) and 75 orphan GPCRs for agonist activity using the DiscoverRX orphanMAX panel (active: GPR55, GPR132, GPR151). For a complete list of GPCRs, see the Supporting Information.

Compound 7 was also screened against 165 known GPCRs for activity. The activity was normalized to the min and max controls (Table 2 and Supporting Information), and potential interactions were identified when % activity/inhibition was greater than 50%. Significance was determined as >3 SD from the mean basal or EC80 activity. As seen in Table 2, 7 displayed activity at several receptors, including the CB1 (CNR1) and CB2 (CNR2) receptors. Based on the DiscoveRx gpcrMAX panel, 7 is an agonist for the CB2 receptor (CNR2) with 64% activity and an antagonist for the CB1 receptor (CNR1). While it has no agonistic activity, it showed a significant inhibition of the CB1 receptor (72%) (Table 2). These results are consistent with results observed in initial testing, where it was determined that 7 is a strong agonist for the CB2 receptor (EC50 of 41.7 nM) and a strong antagonist for the CB1 receptor (IC50 100 nM). In addition to confirming its interactions with CB1 and CB2 receptors, 7 displayed antagonistic activity at the folliclestimulating hormone receptor (FSHR) with 68% inhibition and at the prolactin-releasing hormone (PRLHR) with 52% inhibition (Table 2). The effect of 7 on FSHR is not unexpected; it has been previously reported that Δ 9tetrahydrocannabinol, a high-affinity agonist for both the CB1 and CB2 receptors, displayed specific inhibition of the FSH receptor.23 Also, it is already well established that altering the CB system leads to changes in the hypothalamic/pituitary axis by altering GnRH and LH/FSH secretion and synthesis, thereby causing a decrease in fertility. In the case where negative inhibition was observed (cf., HRH4, PTGER2, RXFP3), this may be indicative of agonist or positive allosteric modifiers (PAM) activity; however, the activity is modest but may warrant further investigation. Taken together, these results

Figure 1. Dose−response curve of compound 7 on the inhibition of the uptake of [3H]-Win 55,212-2 for the CB1 receptor (a) and uptake of [3H]-CP-55, 940 for the CB2 receptor (b). Each experiment was run using 0.5-fold serial dilutions, starting at 5.0 μM for CB1 and 10 μM for CB2.

However, two compounds showed less pronounced and opposite effects on the CB2 receptor (224 vs 664 nM) (Figure 1B), indicating the importance of the 2′-hydroxy group. The addition of a second hydroxy group at the 3′-carbon (1) resulted in a significant increase in antagonistic activity against the CB1 receptor (7400 vs 41 nM), with a smaller and opposite effect on the CB2 receptor (59 vs 168 nM), indicating that the presence of hydroxy groups in the 2′-methylpropan-2′-ol moiety can have pronounced effects on the antagonistic activity of the particular sanshool derivative under investigation. For the eight compounds isolated, an objective was to identify a potent CB1 antagonist that also displayed CB2 agonistic activity. Only isobungeanool 6 and γ-sanshool 7 were weak antagonists for the CB2 receptor. Of these two compounds, only 7 also displayed strong antagonistic properties against the CB1 receptor. Therefore, it was determined that 7 has agonistic effects on the CB2 receptor with an EC50 of 41.7 nM. We also sought to define the broader specificity for 7 among other G-protein-coupled receptors by screening it against 75 orphan GPCR targets for agonist activity using the Discover Rx orphanMAX panel (Table S1, Supporting Information) and 165 GPCR for agonist/antagonist activity using the DiscoveRx gpcrMAX panel (Table S2, Supporting Information). For the 2062



Article

completion, all the shortest trees were saved, and a strict consensus or majority rule tree was computed. Relative support for individual clades was estimated with the bootstrap method.26 One thousand pseudoreplicates were performed with uninformative characters excluded. Ten random-taxon-addition heuristic searches for each pseudoreplicate were performed, and all minimum-length trees were saved for each search. To reduce bootstrap search times, branches were collapsed if their minimum length was zero (“amb-”). A final cladogram is presented in Figure S10 (Supporting Information). Extraction and Isolation. Dried pericarp (25 g) of Z. bungeanum (Sichuan collection) was ground and soaked in 2 L of 70% EtOH/ H2O overnight. It was then heated at 70 °C for 5 min and filtered. The filtrate was concentrated in vacuo to yield 2.1 g of a crude extract, of which 500 mg was chromatographed on a 2.5 × 40 cm Sephadex LH20 column eluted with CH2Cl2/MeOH (1:1, v/v), with 200 drop fractions collected in each tube. On the basis of the UV-absorption profile (254 nm), five major fractions were pooled. Among these, the fraction that showed the greatest CB1/CB2 activity was purified by HPLC. HPLC separation was performed on 125 mg of fraction B2 dissolved in 10 mL of MeOH and injected in 200 μL aliquots (ca. 2.5 mg/injection) onto a 250 × 10 mm Varian Dynamax Microsorb 60-8 C18 HPLC column. The detector wavelength was 225 nm, and the solvent flow rate was 4.0 mL/min. Elution began with 70% MeOH/ H2O, isocratic for 3 min, then a linear gradient of 70% to 100% MeOH for 25 min. The column was flushed with 100% MeOH for 5 min. Eight major UV absorbing peaks were collected and evaporated in vacuo to yield compounds 1 (1.7 mg), 2 (48.5 mg), 3 (10.8 mg), 4 (10.7 mg), 5 (3.1 mg), 6 (2.4 mg), 7 (0.6 mg), and 8 (0.6 mg), eluting at ca. 11.3, 11.6, 11.9, 14.6, 14.9, 15.2, 17.6, and 19.7 min, respectively. The compounds were identified by comparison to literature values. A similar extraction procedure on 25 g of dried pericarp of Z. bungeanum (Gansu collection) yielded 2.1 g of a crude extract. A 500 mg portion was chromatographed on a 2.5 × 40 cm Sephadex LH-20 column and eluted with CH2Cl2/MeOH (1:1, v/v), with 200 drop fractions collected in each tube. Based upon the UV absorption (254 nm), five fractions were collected and tested for CB1/CB2 activity. Plate-Based Assay. A plate-based assay was utilized, as described in a previous report.14 Chinese hamster ovary (CHO) cells stably transfected with cDNA encoding human cannabinoid CB1 or CB2 receptors were kindly donated by Paul Hollenburg (University of Michigan Medical School). Briefly, CHO cells, transfected with CB1 or CB2, were cultured in 96-well plates at a density of 2.0 × 105 cells/ mL for 24 h in a medium consisting of DMEM/F12 (1:1) with Lglutamine and 2.438 g/L sodium bicarbonate supplemented with 1% penicillin/streptomycin solution and 10% fetal bovine serum (v/v). To assess antagonist effects mediated by CB1 and CB2 receptor internalization, the cells were preincubated with 2-fold serial dilutions of each compound tested for 1 h prior to treatment with 10 nM of the radioligand tracer, [3H]-Win 55,212-2, and 2 nM of [3H]-CP 55,940, respectively. CHO-CB1/CB2 cells were incubated for 1 h with the radiolabeled agonist following a preincubation of 1 h with the tested antagonist. Then, the medium was discarded, the cells were washed twice with PBS, and 50 μL of 1.0 N NaOH was used to lyse the cells and determine agonist uptake (radioligand tracer) after 1 h of plate shaking. Radioactivity was measured by liquid scintillation counting. The data from each plate were normalized to the amount of radioligand tracer internalized in the absence of the test compound. Positive controls were also run for both the CB1 receptor using JD5037 and AM-251 and CB2 receptors using JD5037. The IC50 values obtained were within the expected range. Data were analyzed with nonlinear regression and the sigmoid dose−response curve using GraphPad Prism software. The results are presented as the means ± SE (n = 6). GPCR Profiling. Compound 7 was screened against 165 known Gprotein-coupled receptors for agonist/antagonist activity using the DiscoverRx gpcrMAX panel and 75 orphan GPCRs for agonist activity using the DiscoverRx orphanMAX panel. Full details are found in the Supporting Information.

suggest that 7 is a potential candidate for the treatment of type1 diabetes.

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR experiments were performed on a Bruker 600 MHz NMR spectrometer. 1H and 13C spectra were referenced to deuterated solvent peaks. LC-MS data were obtained using a Hewlett-Packard 1100 MSD instrument. The Sephadex LH-20 columns attached to a model UA-6 UV detector and Foxy 200 fraction collector (Teledyne Isco) were used for fractionation, whereas compound purification was performed using a Varian ProStar 210/215 HPLC equipped with a Varian ProStar 325 UV−vis detector, operating under Star 6.41 chromatography workstation software. All solvents and chemicals were of analytical grade. All compounds tested were dissolved in DMSO. The treatment solutions were obtained from stock solutions by using 0.5-fold serial dilutions with final higher concentration of DMSO less than 0.1% in the medium. Plant Material. The pericarp of Z. bungeanum was collected by one of the authors (G. Lu) in Hanyuan County, Sichuan Province, People’s Republic of China, in October 2011. A voucher specimen is deposited in the herbarium of the Chengdu University of TCM, number 00055244. DNA Extraction. Plant material of three additional Zanthoxylum species was extracted, for which the voucher information is listed in the Supporting Information. The total genomic DNA was extracted from (0.5−1.0 g) fresh or dried leaf material. Leaves were ground with a mortar and pestle and subsequently treated with the DNEasy plant DNA extraction kit from Qiagen (Qiagen, Valencia, CA, USA), following the manufacturer’s protocol. Internal Transcribed Spacer (ITS). The amplification of the ITS gene was performed successfully using oligonucleotide primers ITS1/ ITS4 to acquire the entire region.24 The DNA fragment amplified using these two primers is approximately 800bp long and includes ITS1, ITS2, and the 5.8S ribosomal gene. The basic mix contained the following: 38 μL of H2O, 5 μL of 10% Mg-free buffer solution, 3−6 μL of 25 mM MgCl2, 1 μL of 10 mM dNTPs, 0.5 μL of each primer (10 nM), and 0.25 μL of Taq DNA along with 1.5 μL of DNA template for each reaction. The thermal cycler was programmed to perform an initial 1 cycle of denaturation at 95 °C for 2 min followed by 24 cycles of 30 s at 55 °C, 72 °C for 1 min 30 s, and 95 °C for 30 s. This was followed by a 10 min extension at 72 °C to allow completion of unfinished DNA strands, which in turn linked to a soak file at 4 °C. Cycle Sequencing. The PCR products were cleaned using the Qiagen QIAquick PCR purification kit (Qiagen, Inc., Chatsworth, CA, USA) following the protocol provided by the manufacturer. Cleaned products were then directly sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Ready Kit with AmpliTaq DNA Polymerase (Applied Biosystems Inc., Foster City, CA, USA). Unincorporated dye terminators were removed using the QIAGEN DyeEx dye-terminator removal system (Qiagen, Inc.) following the manufacturer’s recommendations. Samples were then loaded into an ABI 3100 DNA sequencer. The sequencing data was analyzed and edited using the Sequencer software program (Gene Codes Corporation, Ann Arbor, MI, USA). Phylogenetic Analysis. Boundaries of the ITS nuclear gene were determined by comparison with sequences in GenBank. The following two alignment criteria and methodology were used: (1) when two or more gaps were not identical but overlapping, they were scored as two separate events and (2) phylogenetically informative indels (variable in two or more taxa) were scored as one event at the end of the data set. All DNA sequences reported in the analyses have been deposited in GenBank (Supporting Information). All phylogenetic analyses employed maximum-parsimony with the heuristic search option in PAUP* 4.0b825 with uninformative characters excluded. Searches were conducted with 100 randomtaxon-addition replicates with TBR branch swapping, steepest descent, and MulTrees selected with all characters and states weighted equally and unordered. All trees from the replicates were then swapped onto 2063



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(14) Galiègue, S.; Mary, S.; Marchand, J.; Dussossoy, D.; Carrière, D.; Carayon, P.; Bouaboula, M.; Shire, D.; Le Fur, G.; Casellas, P. Eur. J. Biochem. 1995, 232, 54−61. (15) Pandey, R.; Hegde, V. L.; Nagarkatti, M.; Nagarkatti, P. S. J. Pharmacol. Exp. Ther. 2011, 238, 819−828. (16) Moaddel, R.; Rosenberg, A.; Spelman, K.; Frazier, J.; Frazier, C.; Nocerino, S.; Brizzi, A.; Mugnaini, C.; Wainer, I. W. Anal. Biochem. 2011, 412, 85−91. (17) Dossou, K. S. S.; Devkota, K. P.; Kavanagh, P. V.; Beutler, J. A.; Egan, J. M.; Moaddel, R. Anal. Biochem. 2013, 437, 138−143. (18) Jang, K. H.; Chang, Y. H.; Kim, D. D.; Oh, K. B.; Shin, J. Arch. Pharm. Res. 2008, 31, 569−572. (19) Yasuda, I.; Takeya, K.; Itokawa, H. Phytochemistry 1982, 21, 1295−1298. (20) Xiong, Q.; Shi, D.; Yamamoto, H.; Mizuno, M. Phytochemistry 1997, 46, 1123−1126. (21) Kashiwada, Y.; Ito, C.; Katagiri, H.; Mase, I.; Komatsu, K.; Namba, T.; Ikeshiro, Y. Phytochemistry 1997, 44, 1125−1127. (22) Raduner, S.; Majewska, A.; Chen, J.-Z.; Xie, X.-Q.; Hamon, J.; Faller, B.; Altmann, K. H.; Gertsch, J. J. Biol. Chem. 2006, 281, 14192− 14206. (23) Heindel, J. J.; Keith, W. B. Toxicol. Appl. Pharmacol. 1989, 10, 124−134. (24) White, T. J.; Burns, T.; Lee, S.; Taylor, J. In PCR Protocols: A Guide to Methods and Amplifications; Innis, M. A.; Gelfand, D. H.; Sninsky, J. J.; White, T. J., Eds.; Academic Press: San Diego, 1990; pp 315−322. (25) Swofford, D. L. PAUP*: Phylogenetic Analysis using Parsimony (*and Other Methods), version 4.10b; Sinauer: Sunderland, MA, USA, 2002. (26) Felsenstein, J. Evolution 1985, 39, 783−791.

ASSOCIATED CONTENT

S Supporting Information *

Chemical structures, 1H and 13C NMR spectra of compounds 1−8, full GPCR panel results for 7, and a cladogram of ITS results for the plant samples. These materials are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (410) 558-8294. Fax: (410) 558-8695. Author Contributions #

K. S. S. Dossou and K. P. Devkota contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Intramural Research Program of the National Institute of Aging, NIH, and, in part, by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The work was supported, in part, by funds from the NIH Office of Dietary Supplements, grant OD-Y2-OD-1557-01. We thank Dr. C.-T. Che of the University of Illinois at Chicago for arranging for the collections of the Chinese Zanthoxylum species. We thank Drs. S. Tarasov and M. Dyba (Biophysics Resource Core, Structural Biophysics Laboratory, CCR) for assistance with mass spectrometry. We thank Drs. A. Tucker and R. J. Sahraoui (Delaware State University) for the image of Z. bungeanum in the graphical abstract.

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REFERENCES

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