A new coumarin from Murraya alata activates TRPV1 channel

Abstract One new coumarin, muralatin R, was isolated from the leaves of Murraya alata Drake (Rutaceae). Its structure was elucidated by extensive analysis of the NMR and MS data, along with the specific rotation comparison. Muralatin R was found to be capable of activating the transient receptor potential vanilloid 1 (TRPV1) channel through desensitization mechanism. The results supply reference for clarification of the therapeutic basis and mechanism of action of Murraya plants for treating psychogenic pain or somatoform pain disorders. Graphical Abstract


Introduction
Based on the empirical observations and folklore, the traditional Chinese medicine (TCM) increasingly gains attentions for the treatment of both acute and chronic pain due to its low toxicity and non-addictive property (Nahin et al. 2009). While, owing to the complex nature of TCM, its active ingredients and mechanism of actions remain largely unknown.
Many plants of the Murraya genus (family Rutaceae) have been used widely as flavorings and herbal medicines for the treatment of psychogenic pain or somatoform pain disorders, including toothache, gastralgia, lumbago, rheumatic pain, etc. (Song 1999). Previous phytochemical and pharmacological investigations on Murraya plants proved that coumarins functioned as the main bioactive agents responsible for their analgesic properties (Wu et al. 2010;Zou et al. 2000). In a previous pharmacological study, the extracts of eight Murraya species including M. alata Drake, M. exotica L., M. tetramera Huang, M. koenigii (L.) Spreng, M. microphylla (Merrill & Chun) Swingle, M. paniculata (L.) Jack, M. kwangsiensis (Huang) Huang, and M. kwngsiensis Huang var. macrophylla Huang were determined for their anti-nociceptive effects on abdominal writhing induced by acetic acid in mice, and M. alata was found to show the highest inhibitory activity on pain (Zou et al. 2000). These findings promoted us to further investigate the active ingredients in this plant and to elucidate their mechanisms. Recently, we have defined a novel coumarin derivative as a selective transient receptor potential vanilloid 1 (TRPV1) activator (Wei et al. 2016). This compound can specifically activate the TRPV1 channel and reverse the inflammatory pain in mice through channel desensitization. As a continuation of our search for new TRPV1 modulators from this plant, the 95% aqueous ethanol extract of the leaves of M. alata was investigated and a new coumarin, named muralatin R was identified using a combination of fluorescent calcium assay and electrophysiology as screening means. Muralatin R activates and desensitizes TRPV1 current. The structure of muralatin R was elucidated following a comprehensive analysis of the NMR and HRESIMS spectroscopic data.

Results and discussion
The 95% aqueous EtOH extract of the leaves of M. alata was suspended in H 2 O and partitioned with petroleum ether and CHCl 3 , successively. The CHCl 3 -soluble portion was subjected repeatedly to silica gel CC followed by preparative TLC to afford the new coumarin, named muralatin R.
Muralatin R was obtained as a light yellow oil with ½a 24 D þ11.2 (c 0.11, MeOH). Its molecular formula was established as C 22 H 30 O 8 based on the 13 C NMR spectroscopic data and the HRESIMS [M þ Na] þ peak at m/z 445.1836 (calcd for C 22 H 30 O 8 Na, 445.1838). The characteristic absorptions at 213 and 308 nm in the UV spectrum and the presence of a pair of representative doublets at d H 6.22 and 7.89 (each 1 H, d, J ¼ 9.7 Hz) in 1 H NMR spectrum revealed a coumarin skeleton in muralatin R (Lv et al. 2015). Fragment ions at m/z 405.1912 and 321.1340 indicated the presence of hydroxy and isovaleryloxy groups, respectively. The NMR spectroscopic data (Table S1) of muralatin R were similar to those of murrayatin (Barik et al. 1983), except for the presence of two additional methoxy groups [d H 3.94 (3 H, s), 3.81 (3 H, s)], which were deduced to be located at C-5 and C-6, respectively, base on the HMBC correlations from H-4 to C-5/C-9/C-10 and from the protons of OCH 3 -5, OCH 3 -6, and OCH 3 -7 to C-5, C-6, and C-7, respectively. The HMBC correlations from H-1 0 to C-7/C-8/C-9 further supported the location of the side chain at C-8. The absolute configuration of C-2 0 was determined as S by comparison of the sign of the specific rotation with those of (S)-5-methoxymurrayatin (Kinoshita et al. 1996). Hence, the structure of muralatin R was assigned as 8-[(2S)-3-hydroxy-2-(3-methylbutanoyloxy)-3-methylbutyl]-5,6,7-trimethoxycoumarin, as shown in Fig. 1A.
TRPV1 channel, also known as capsaicin receptor, is a nonselective cation and heat-activated channel with temperature threshold above 43 C (Caterina et al. 1997). The expression of TRPV1 has been primarily demonstrated in pain pathways (Szallasi et al. 2007) and the mice lacking TRPV1 show dramatic reduction of pain hypersensitivity (Armero et al. 2012;Niiyama et al. 2007), indicating that targeting TRPV1 by desensitizing the channel function can serve as an attractive strategy for pain therapy. As shown in Figure 1B, muralatin R (500 lM) was found to be able to significantly increase the intracellular calcium level in TRPV1 expressing cells, as compared with the positive control capsaicin (1 lM) that specifically activates TRPV1. The increased calcium signal can be inhibited by TRPV1 channel blocker ruthenium red ( Figure 1C). These results indicate that coumarin muralatin R is a novel TRPV1 channel agonist. Bath application of muralatin R (500 lM) elicited the activation of TRPV1 current and the effect could be washed out before further activation of TRPV1 by capsaicin (1.0 lM) ( Figure 1D). The current induced by muralatin R (500 lM) was also blocked by 1.0 lM JNJ-17203212, a competitive TRPV1 antagonist ( Figure 1E). Fitting the data from dose-dependent activation of TRPV1 by muralatin R with Hill equation yielded an EC 50 value of 237.6 ± 23.2 lM (n ¼ 4 to 6) and a Hill coefficient of 1.66 ( Figure 1F).
As we have mentioned in our previous study (Wei et al. 2016), the 8-alkylated coumarin and capsaicin have structural similarities. They both possess the aromatic A-region and the double bond-participating junction B-region. Lengthening the C-8 substituent group of muralatin L or introducing an appropriate hydrophobic chain might promote TRPV1 activation. Compared with muralatin L, muralatin R contains an FlexStation3 fluorescent calcium assay. The screening scheme consists of 180 or 120 seconds for test compounds followed by bath application of capsaicin (left panel) that activates TRPV1 or 10 lM ruthenium red (right panel) that blocks TRPV1. D. Whole-cell current of TRPV1 in response to 500 lM muralatin R and 1 lM capsaicin. E. The TRPV1 current evoked by 500 lM muralatin R was inhibited by 1 lM TRPV1 potent antagonist JNJ-17203212. F. Curve fitting represents dose-dependent activation of TRPV1 by muralatin R with an EC 50 value at 237.6 lM, and each data point is expressed as mean ± S.E. of 4 to 6 independent tests. additional isovaleryloxyl group. Though they have the similar EC 50 values, it is obvious that muralatin R exhibited a stronger capability for channel desensitization than muralatin L (Figure 1E), indicating a better potential for the development of capsaicinlike analgesics.

General experimental procedures
Optical rotation was measured on Rudolph Autopol III automatic polarimeter (Rudolf Research, Fairfield, New Jersey, USA). UV spectrum was recorded on a Shimadzu UV-2450 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). IR spectrum was recorded on a Thermo Nicolet Nexus 470 FT-IR spectrometer (Thermo Nicolet, Vernon Hills, Illinois, USA). NMR spectra were recorded on a Varian INOVA-500 NMR spectrometer (Agilent Technologies, Santa Clara, California, USA), using CDCl 3 as solvent, and the chemical shifts were referenced to the solvent residual peak. HRMS data was acquired on a Waters Xevo G2 Q-TOF spectrometer fitted with an ESI source. Column chromatography (CC) was performed on silica gel (100 À 200 mesh, Qingdao Marine Chemical Inc., China). TLC analysis and preparative TLC were carried out on precoated silica gel GF254 plates (Qingdao Marine Chemical Inc., China). Spots were visualized under UV light (254 and 365 nm) or by heating after spraying with 2% vanillin-H 2 SO 4 solution. All the solvents were of analytical grade.

Plant material, extraction, and isolation
The leaves of Murraya alata Drake were collected in Sanya, Hainan Province, China, in August 2011. The plant material was identified by Professor P.-F. Tu. A voucher specimen (No. YY201108) has been deposited at the Herbarium of the Peking University Modern Research Center for Traditional Chinese Medicine.

Cell culture and transient transfection of cells
HEK-293T cells were maintained in media containing 90% Dulbecco's modified Eagle's medium and 10% fetal bovine serum in 5% CO 2 at 37 C. HEK-293T cells were plated onto glass coverslips for subsequent patch recordings. Cells were transiently transfected with human TRPV1 cDNA using Lipofectamine 2000 (Invitrogen). The accession number of the cDNA was NM_080704.3. hTRPV1 was constructed by primers ATCGATGAAGAAATGGAGCAGCA (forward) and ACTGTCACTTCTCCCCGGAAGC (reverse) using LA Taq (TAKARA) and subcloned into BglII and SalI restriction sites of the pIRES2-EGFP vector. Attachment of EGFP, used as a marker for identification of the transfected cells, did not affect channel function. 4 lg of individual cDNA was used. All restriction enzymes were purchased either from Invitrogen or Takara, and insert of cDNA clone was verified by sequencing. Electrophysiological experiments were performed between 18 and 36 hours after transfection (Wei et al. 2016).

Intracellular calcium measurement by FlexStation 3 multi-mode microplate reader assay
Changes in intracellular calcium level ([Ca 2þ ] i ) in a population of cells were measured by fluorescent calcium-sensitive dyes using the Calcium 5 Assay Kit in FlexStation 3 Microplate Reader (Molecular Devices) format. HEK-293T cells were plated at a density of $30,000 cells/well in 96-well black-walled plates (Thermo) covered with poly-Dlysine. Cells were loaded with dyes from the FLIPR Calcium 5 Assay Kit for 1 h at 37 C in the presence of 2.5 mM probenecid. Loading and imaging were performed in Hanks' balanced salt solution (137 mM NaCl, 5.4 mM KCl, 0.4 mM KH 2 PO 4 , 0.1 mM Na 2 HPO 4 , 1.3 mM CaCl 2 , 0.8 mM MgSO 4 , 5.5 mM glucose, 4 mM NaHCO 3 , and 20 mM HEPES, pH 7.4). Fluorescence intensity was measured at an interval of 1.6 s at 525 nm, using an excitation wavelength at 485 nm and an emission cut-off wavelength at 515 nm (Lei et al. 2013).

Electrophysiology
Whole-cell patch clamp recordings were performed using a HEKA EPC10 amplifier with PatchMaster software (HEKA). Patch pipettes were pulled with borosilicate glass using a puller (DMZ-Universal) and fire polished to a resistance of 3 -5 MX. Both pipette solution and bath solution contained 130 mM NaCl, 0.2 mM EDTA, and 3 mM HEPES for the other electrophysiology recording of HEK-293T cells. Membrane potential was held at 0 mV. Current was elicited by a 300-ms step to þ80 mV and followed by a 300-ms step to -80 mV at 1-s intervals (Wei et al. 2016).

Statistical analysis
All data were expressed as mean ± S.E. Statistical significance was assessed by Student's t test using Prism 5.0 software. A value of p < 0.05 was considered to represent statistical significance. EC 50 is the concentration for half-maximal effect.