Material Basis for Inhibition of Dragon's Blood on Capsaicin-Induced

European Journal of Pharmacology 702 (2013) 275–284

Contents lists available at SciVerse ScienceDirect

European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia Material basis for inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in rat dorsal root ganglion neurons

Li-Si Wei a,1, Su Chen a,1, Xian-Ju Huang b, Jing Yao c, Xiang-Ming Liu a,n a College of Biological & Medical Engineering, South-Central University for Nationalities, Minyuan Road 708, Wuhan, Hubei 430074, China b College of Pharmacy, South-Central University for Nationalities, Wuhan 430074, China c School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430030, China article info abstract

Article history: The effects of dragon’s blood and its components cochinchinenin A, cochinchinenin B, loureirin B as Received 26 May 2012 well as various combinations of the three components on capsaicin-induced TRPV1 receptor currents Received in revised form were studied in acutely dissociated DRG neurons using both voltage and current whole-cell patch 20 January 2013 clamp technique. The results indicated that dragon’s blood and its three components concentration- Accepted 31 January 2013 dependently reduce the peak amplitudes of capsaicin-induced TRPV1 receptor currents. There was no Available online 8 February 2013 signiﬁcant difference between the effects of dragon’s blood and the combination wherein the three Keywords: components were present in respective mass fractions in dragon’s blood. The respective concentrations Dragon’s blood of the three components used alone were all higher than the total concentration of three components Dorsal root ganglion (DRG) neurons used in combination when the percentage inhibition of the peak amplitude was 50%. The proportion of Transient receptor potential cation channel, three components was adjusted and the total concentration reduced, the resulting combination still subfamily V, member 1 (TRPV1) Interaction inhibit the currents with a lower IC50 value, and inhibit capsaicin-induced membrane depolarization on Analgesic drugs current clamp. The combination of three components not only increase the capsaicin IC50 value, but also reduce the capsaicin maximal response. These result suggested that analgesic effect of dragon’s blood may be partly explained on the basis of silencing pain signaling pathways caused by the inhibition of dragon’s blood on capsaicin-induced TRPV1 receptor currents in DRG neurons and could be due to the synergistic effect of the three components. Antagonism of the capsaicin response by the combination of three components is not competitive. The analgesic effect of dragon’s blood was also conﬁrmed using animal models. & 2013 Published by Elsevier B.V.

1. Introduction components had the same effect as dragon’s blood (Liu et al., 2006). Moreover, extracellular microelectrode recordings were Dragon’s blood from Dracaena cochinchinensis is one of the used to indicate that the above combination of the three compo- renowned traditional medicines. It is a multi-component mixture nents was material basis for inhibition of dragon’s blood on with analgesic activity and has got several therapeutic uses evoked discharges of wide dynamic range neurons in spinal dorsal (Gupta et al., 2008; Zhong, 2010). Its molecular targets and active horn of rats (Guo et al., 2008). components should be determined with strict scientiﬁc method During the past decade, one relatively new molecular target, to explore lead compounds for novel analgesic drugs from TRPV1 receptor has attracted signiﬁcant attention in the pharma- dragon’s blood. In previous studies, we have observed that ceutical industry. TRPV1 receptor is widely distributed in the central modulation of dragon’s blood on the tetrodotoxin-sensitive and peripheral nervous systems. It is activated not only by chemical (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents in factors such as capsaicin and acids, but also by a physical factor such dorsal root ganglion (DRG) neurons using patch clamp technique. as hot temperature (Z42 1C) (Helliwell et al., 1998; Patapoutian Three compounds, i.e., cochinchinenin A, cochinchinenin B, and et al., 2003). TRPV1 receptor is considered an integrator of noxious loureirin B (Fig. 1) were the active components in dragon’s blood stimuli in peripheral nociceptor terminals and therefore may be at a interfering with pain messages. The combination of the three crossroads for pain transmission pathways. The logical strategy for development of novel analgesics is to target the beginning of pain transmission pathway and aim potential treatments directly at

n nociceptors. The TRPV1 receptor antagonists may deliver broad Corresponding author. Tel.: þ86 27 67843892; fax: þ86 27 67841231. E-mail address: [email protected] (X.-M. Liu). spectrum efﬁcacy in nociceptive pain via silencing pain signaling 1 Contributed equally to this work. pathways (Kym et al., 2009). Therefore, the discovery of new TRPV1

0014-2999/$ - see front matter & 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ejphar.2013.01.052 276 L.-S. Wei et al. / European Journal of Pharmacology 702 (2013) 275–284

Table 1 Preparation of solutions and drugs.

Components Solutions

Individual Combination

123ABCIII

Cochinchinenin A (mmol/l) 38 / / 38 38 / 38 3.8 Cochinchinenin B (mmol/l) / 19 / 19 / 19 19 1.9 Loureirin B (mmol/l) / / 8 / 8 8 8 8

inﬂuence of the efﬁcacy, another solution of combination II was shown in Table 1. To obtain the concentration–response curves of the individual components and combinations of the three Fig. 1. Three compounds of dragon’s blood, cochinchinenin A, cochinchinenin B, components, the above relevant solutions were diluted and added and loureirin B cochinchinenin A: 40-Hydroxy-2,6-dimethoxy dihydrochalcone with 1 mmol/l capsaicin to observe the actions. (C17H18O4). cochinchinenin B: 6-hydroxy-7-methoxy-3-(40-hydroxybenzyl) chromone (C18H20O5). loureirin B: 1-(4-Hydroxyphenyl)-3-(2,4,6-trimethoxy- 2.2. Working solutions for patch clamp experiments phenyl)propan-1-one (C17H18O4). receptor antagonists becomes an attractive destination in the External solution used to record capsaicin receptor currents treatment of pain. in DRG neurons contained (mmol/l): NaCl 145.0; HEPES 10.0;

Nowadays, a number of small molecule TRPV1 receptor D-glucose 10.0; KCl 5.0; CaCl2 2.0; MgCl2 1.0. The external solution antagonists are undergoing clinical trials in patients with inflam- was adjusted to pH 7.4 with 1 mol/l NaOH. Internal solution matory or neuropathic pain. Clinical development of TRPV1 contained (mmol/l): KCl 140.0; EGTA 10.0; HEPES 10.0; Na2TAP receptor antagonists is, however, facing new challenges, some 10.0; CaCl2 1.0; MgCl2 2.0. The internal solution was adjusted to of these antagonists showed worrisome adverse reactions (e.g., pH 7.3 with 1 mol/l KOH. hyperthermia) in men, leading to their withdrawal from the Capsaicin was dissolved into stock solution of 10 mmol/l by clinical trials (Gavva et al., 2008; Gunthorpe and Chizh, 2009; dehydrated alcohol, and then diluted into 1 mmol/l by external Joshi and Szallasi, 2009). So, it has significance to search for new solution described above. Capsazepine, the competitive antago- TRPV1 receptor antagonists among the active components in nist of TRPV1 receptor, was prepared at concentration of 10 mmol/ dragon’s blood, which has displayed very low toxicity and no l using the same method. HEPES, EGTA, Na2TAP, capsaicin and apparent side-effects (Zeng and He, 1999). capsazepine were obtained from Sigma Company (USA). All other Our previous studies have indicated that two components, chemicals were of analytical grade unless otherwise stated. cochinchinenin B and loureirin B weakly inhibit capsaicin- induced TRPV1 receptor current, respectively (Wang et al., 2007, 2.3. Whole-cell patch-clamp experiments 2008). The result suggested that the pharmacological actions of dragon’s blood and the combinations of its active components on One-month-old male or female SPF Wistar rats (100–150 g) TRPV1 receptor currents should be further investigated. Thus we were provided by Laboratory Animal Center, Tongji Medical may gain a more comprehensive understanding of the mechanism College of Huazhong University of Science and Technology. The behind analgesic activity of dragon’s blood and further identify its animals were cared in accordance with the Regulations of active components, and provide lead compounds for the devel- Experimental Animal Administration issued by the State Com- opment of novel analgesic drugs. mittee of Science and Technology of the People’s Republic of China on November 14, 1988. After the rats were decapitated, DRG neurons were dissected and taken out. A volume of 5 ml cell 2. Materials and methods suspension was obtained using enzymatic dissociation (Huang et al., 2007). The suspension was filtrated through 200 mesh 2.1. Preparation of solutions and drugs gauze and transferred into a 3.5 mm culture dish to keep still. The solution was replaced by the external solution twice after the Dragon’s blood, cochinchinenin A, cochinchinenin B, and lour- cells were adhered to the disk. Whole-cell patch clamp recordings eirin B were provided by Guangxi Institute of Traditional Medical were carried out using an EPC-9 amplifier (HEKA, Germany). After and Pharmaceutical Sciences. Dragon’s blood and its three compo- 3–5 GO seal formation between a pipette and DRG membrane, nents were authenticated and extracted, respectively by Prof. Lu the membrane was ruptured and membrane capacitance was Wenjie. The purity of each component is 98%. The mass fractions of compensated (Liu et al., 2006). Only smaller DRG neurons were cochinchinenin A, cochinchinenin B and loureirin B in the final used for experiments as this kind of cells usually expressed a products of dragon’s blood are 22%, 11%, and 5%, respectively. higher percentage of TRPV1 receptor currents (Bevan and An appropriate amount of dragon’s blood was dissolved in Szolcsańyi, 1990; Caterina et al., 1997). TRPV1 receptor currents external solution to obtain a 0.005% (m/v) concentration. Accord- were evoked by given stimuli at a holding potential. After control ing to the mass fractions of the three components in the final currents were recorded, capsaicin (1 mmol/l) was firstly delivered products of dragon’s blood, the solutions of the individual to the cell over 7–10 s using a rapid solution exchange system components, the concentrations of which corresponded to the (DAD-12, ALA, USA). The residual drug was then washed out with above concentration of dragon’s blood solution, were prepared. the external solution after the peak value emerged. The normal The solutions of various combinations of the components were TRPV1 receptor current in DRG was recorded. Then the currents also prepared (Table 1). in the presence of drugs were recorded using the same method. To explore the possibility of decrease in the total concentra- Lastly capsaicin (1 mmol/l) was delivered again and the TRPV1 tion of the three components used in the combination I without receptor currents were recorded to observe the recovery extent. L.-S. Wei et al. / European Journal of Pharmacology 702 (2013) 275–284 277

The intermission of three deliveries should be longer than 30 s The percentage inhibition of capsaicin-induced TRPV1 receptor (Chen et al., 2004). All the experiments were performed at 22–25 1C. current produced by drug was calculated using Eq. (1):

Inhibition% ¼½ðIcapIdugÞ=Idug100% ð1Þ 2.4. Evaluation of analgesic activity of dragon’s blood by using where Icap is the peak amplitude of TRPV1 receptor currents animal models induced by 1 mmol/l of capsaicin, Idug is the peak amplitude of TRPV1 receptor currents induced by mixed solution of 1 mmol/l of The analgesic activity of dragon’s blood was tested by using hot capsaicin and the drug. plate method, tail flick method and acetic acid-induced writhing Drug concentration–response curve was determined by plot- response in rats. In each test, twenty rats weighing 200–250 g were ting the percentage inhibition of the capsaicin-induced TRPV1 randomly selected and divided into control group and dragon’s receptor current as a function of the drug concentration. Data blood group with 10 rats in each group. The rats of control group were fitted with Hill Eq. (2): received an intraperitoneal injection of 2 ml/kg normal saline and h the rats of dragon’s blood group received an intraperitoneal injection Inhibition%=Inhibitionmax% ¼ 1=½1þðIC50=cÞ ð2Þ of 5 mg/kg dragon’s blood (2 ml/kg of injection volume). where Inhibition% is the percentage inhibition on peak amplitude of capsaicin-induced TRPV1 receptor current produced by drug, 2.4.1. Hot plate test Inhibitionmax% is the maximum of Inhibition% also called maximum efficacy. Here the value of Inhibitionmax% was set at 1. c is Hot plate analgesia meter (Huaibei zhenghua biological equip- the drug concentration, h is Hill coefficient and IC50 is the half ment Co., Ltd, China) was used. The temperature of the metal surface maximal inhibitory concentration of the drug. waskeptat(5270.5) 1C. Rats were placed on the hot plate, and the time until either licking of the hind paw or jumping occurred was 2.7. Assessment of drug interaction recorded. Animals were tested before and 30 min after drug administration. A cutoff time of 60 s was set to avoid tissue damage. Three major interactional effects can occur when two or more The maximal possible effect (MPE) was defined as the lack of a drugs interact with each other: additive, antagonistic, and synergis- nociceptive response during the exposure to the heat stimulus. The tic effects. When the concentration–response curves of drug A and percentage of MPE was calculated according to the formula: [(test- drug B have the same maximal effect Emax and the different Hill baseline)/(cutoff-baseline)] 100, where test and baseline were the coefficients, the concept of dose equivalence lead to the equations of latencies obtained before and after drug injection. the two different additive isoboles for assessing drug interaction derived by Tallarida (2006). Similarly, based on the concept of dose equivalence, the following equations of the three additive surfaces 2.4.2. Tail-flick test corresponding to the combined effect Ei produced by three drugs The tail-flick test was carried out in rats using a tail flick (e.g., cochinchinenin A, cochinchinenin B and loureirin B ) were analgesia meter (Huaibei zhenghua biological equipment Co., Ltd, proposed (Guo et al., 2008) when the concentration–response curves China). The tail-flick latency is defined by the time from onset of of three drugs have the same Emax and the different Hill coefficients, stimulation to a rapid flick/withdrawal of the tail from a radiant hb=hc heat source. The heat source was adjusted to produce a baseline c ¼ CiC50=ðA50=aÞC50=ðB50=bÞ ð3Þ tail-flick latency of 3 to 5 s. A cutoff time of 12 s was set to avoid hb=ha ha=hc tissue damage. Animals were tested before and 30 min after drug c ¼ C50 ½ðAiaÞ=A501=ðB50=bÞ ð4Þ administration. The increase in tail-flick latency was defined as ha=hb hb=hc antinociception and calculated as MPE% according to the formula: c ¼ C50 ½ðBibÞ=B501=ðA50=aÞ ð5Þ [(test-baseline)/(cutoff-baseline)] 100, where test and baseline where a, b,andc were the concentrations of cochinchinenin A, were the latencies obtained before and after drug injection. cochinchinenin B and loureirin B in the combination that produces

the speciﬁed effect Ei, Ai, Bi and Ci were the concentrations of the three components which produce the same effect E when used alone. A , 2.4.3. Writhing test i 50 B and C were the IC value of the three components, respectively. Thirty minute after an intraperitoneal injection of normal 50 50 50 h , h and h were the Hill coefﬁcients of the three components, saline or dragon’s blood, the rats were given an intraperitoneal a b c respectively. In Eq. (3), combination of cochinchinenin A (concentra- injection of 2% acetic acid solution (2 ml/kg of injection volume). tion of a) and cochinchinenin B (concentration of b) yielded the same The rats were placed individually into glass beakers and the effect as that of loureirin B whose concentration was (C c). In Eq. (4), number of writhing was counted for 15 min. For scoring purposes, i combinationofcochinchineninB(concentrationofb) and loureirin B a writhe is indicated by stretching of the abdomen with simulta- (concentration of c) yielded the same effect as that of cochinchinenin neous stretching of at least one hind limb. A whose concentration was (Aia). In Eq. (5), combination of cochinchinenin A (concentration of a) and loureirin B (concentration 2.5. Data analysis of c) yielded the same effect as that of cochinchinenin B whose

concentration was (Bib). The above parameters and concentration– Data were analyzed using Pulse Fit software (Version 8.5, response curves of the three components were obtained, respectively HEKA, Germany) and Igor Pro software (Version 4.09, Wave- by ﬁtting the Hill Eq. (2) to the corresponding experimental data.

Metrics, USA). Data were expressed as the mean7S.E.M. The Then the values of Ai, Bi and Ci were evaluated according to number of cells (n) for each condition was indicated. Unpaired concentration–response curves. The above parameters, along with t-test was used to determine the statistical significance of differ- Ai, Bi and Ci, were substituted into Eqs. (3)–(5), respectively. In the ences between two sets of data. Analysis of variance (ANOVA) with three-dimensional space whose each axis represents the concentra- least significant difference (LSD) multiple comparison was used to tion of one of the three components, the figure describing the determine whether there were any significant differences among positional relationship between the experimental point with coordi- more than two sets of data. Po0.05 was considered statistically nates (a, b, c) and three additive surfaces corresponding to the effect Ei significant. was obtained with Matlab software at last. 278 L.-S. Wei et al. / European Journal of Pharmacology 702 (2013) 275–284

If the experimental point was below all the three additive surfaces, voltage stimulation under the mode of voltage clamp. Wash the interactional effect was judged to be synergistic. If the point was immediately with extracellular fluid after the currents reached a among the three additive surfaces, the interactional effect was judged peak. After 30 s washout, the combined solution of 1 mmol/l of to be additive. If the point was above all the three additive surfaces, capsaicin and 10 mmol/l of capsazepine was ejected on the cell, the interactional effect was judged to be antagonistic. and record the TRPV1 receptor currents for the second time. The result showed that the currents induced by 1 mmol/l of capsaicin were almost entirely blocked by 10 mmol/l of capsazepine (Fig. 2), 3. Results consistent with activation of TRPV1 receptor. The effect of dragon’s blood on the TRPV1 receptor current induced by capsai- 3.1. Effects of dragon’s blood on capsaicin-induced TRPV1 receptor cin (1 mmol/l) was illustrated in Fig. 3A. t-test indicated that the currents in DRG neurons average peak amplitudes of TRPV1 receptor currents were significantly reduced after application of dragon’s blood. The effects At a holding potential of 60 mV, the TRPV1 receptor currents of different concentrations of dragon’s blood solutions on in DRG neurons were evoked by 1 mmol/l of capsaicin without any capsaicin-induced TRPV1 receptor currents were shown in Table 2. The concentration–response data were fitted to Eq. (2)

(Fig. 3B), giving a IC50 value of (0.0000270.000005)% (m/v) and a Hill coefﬁcient of 0.5770.07 (n¼7).

3.2. Effect of three components used alone on capsaicin-induced TRPV1 receptor currents in DRG neurons

The effects of different concentrations of cochinchinenin A, cochinchinenin B and loureirin B solutions on capsaicin-induced TRPV1 receptor currents were shown in Table 3. The concentration–

response data were ﬁtted to Eq. (2), giving IC50 values and Hill coefﬁcients of 46.6473.27 mmol/l and 1.3970.119 (n¼7) for Fig. 2. Effects of capsazepine on capsaicin-induced TRPV1 receptor currents 7 7 ¼ in DRG neurons. 10 mmol/l capsazepine inhibits rapidly the current induced by cochinchinenin A, 718.32 67.6 mmol/l and 1.41 0.166 (n 7) for 1 mmol/l capsaicin, consistent with activation of TRPV1 receptor. The horizontal cochinchinenin B, and 4.1370.326 mmol/l and 1.7970.242 (n¼7) bar above each trace indicates period of drug application. for loureirin B (Fig. 4).

Fig. 3. Effect of dragon’s blood on capsaicin-induced TRPV1 receptor currents in DRG neurons. (A) Dragon’s blood inhibits rapidly and reversibly the current induced by 1 mmol/l capsaicin. The horizontal bar above each trace indicates period of drug application. (B) Concentration–response curves for inhibition of the capsaicin (1 mmol/l) response by dragon’s blood. Data points, percent change in peak amplitude in the presence of dragon’s blood (mean of seven experiments). Error bars, S.E.s. Error bars are not indicated when smaller than the size of the circle. The data points are ﬁtted with Eq. (2).

Table 2 Effects of dragon’s blood solutions on capsaicin-induced TRPV1 currents.

Drug Concentration Inhibition rate (%) (m/v) (%) (P value of test) (n¼7)

Dragon’s blood 0.005 90.974.7 (0.00532) 0.0005 83.474.8 (0.00312) 0.00005 67.475.7 (0.00222) 0.000005 29.174.7 (0.00357) 0.0000005 9.473.6 (0.00080) L.-S. Wei et al. / European Journal of Pharmacology 702 (2013) 275–284 279

3.3. Effect of combinations of three components on capsaicin- among which the proportion of cochinchinenin A, cochinchinenin B induced TRPV1 receptor currents in DRG neurons and loureirin B was 2.2:1.1:5. The result of t-test showed that there was no significant difference between the percentage inhibitions of The total concentration of three components in combination I combination I and II on capsaicin-induced TRPV1 receptor currents was 65 mmol/l, among which the proportion of cochinchinenin A, (P40.05). The two combination solutions were diluted 10, 100 and cochinchinenin B and loureirin B was 22:11:5. The total concen- 1000 times, respectively and the effects of different concentrations tration of three components in combination II was 13.7 mmol/l, of the solutions on capsaicin-induced TRPV1 receptor currents were observed to obtain concentration–response curves. All the solutions showed some degree of inhibition against the currents (Table 4 and Table 3 Fig. 5). The concentration–response data were fitted to Eq. (2), Effects of three components used alone on capsaicin-induced TRPV1 currents. giving IC50 values and Hill coefficients of 2.5570.43 mmol/l and 0.5370.01 (n¼7) for combination I, 1.0170.29 mmol/l and 0.467 Components Concentration Inhibition rate (%) 0.06 (n¼7) for combination II (Fig. 5). (lmol/l) (P value of t test) (n¼7)

Cochinchinenin 3800 73.175.1 (0.00312) 3.4. Comparison for the pharmacological effect of dragon’s blood A 380 70.274.8 (0.00212) and combinations of its components 190 65.775.4 (0.00562) 38 30.172.8 (0.00807) Three kinds of solutions combined with any two of the 19 18.275.3 (0.00030) components inhibit capsaicin-induced TRPV1 receptor currents 7 Cochinchinenin B 19000 80.2 5.0 (0.00448) in DRG neurons to some extent. The percentage inhibitions of 7 3800 78.3 3.4 (0.00712) A B C 750 40.471.3 (0.00021) combination , , and solutions on peak amplitudes of TRPV1 380 25.473.3 (0.00905) receptor currents were (35.2271.5)%, (68.3775.0)%, and 190 11.777.3 (0.03807) (66.6674.9)% (n¼7), respectively. ANOVA test demonstrated Loureirin B 200 83.074.4 (0.00782) that there were signiﬁcant difference among the percentage 40 70.771.4 (0.0183) inhibitions of combination I, A, B, C, and 0.005% (m/v) dragon’s 8 65.377.0 (0.00176) blood solutions on capsaicin-induced TRPV1 receptor currents 4.88 44.172.7 (0.00137) (F¼40.71, Po0.01). However, LSD test indicated that there was 1.44 11.977.6 (0.02676) no signiﬁcant difference between effects of combination I and 0.005% (m/v) of dragon’s blood (P40.05) (Table 5). For purposes of comparison, the concentration response curves of combination I, II and dragon’s blood were displayed in the same

Fig. 6 by converting molarity to percent mass/volume. The IC50 value of combination I and II were (0.00008170.000029)% (m/v) and (0.00003770.000020)% (m/v), respectively.

3.5. Effect of combination II on capsaicin-induced membrane depolarization of DRG neuron under the condition of current clamp

To verify whether the components of dragon’s blood inhibit capsaicin-induced TRPV1 receptor currents, combination II was selected to observe the capsaicin-induced changes in membrane potential of DRG neuron before and after applying the components. Capsaicin evoked a membrane depolarization and elicited action potential firing at 1 mmol/l (Fig. 7, top). Membrane potential recovered back to the normal level after capsaicin washout, the average of resting membrane potential was 57.676.3 mV (n¼7). However, 1 mmol/l capsaicin did not evoke action potential firing in the presence of combination II (Fig. 7,middle),themembrane potential of depolarization was 25.775.4 mV (n¼7). Capsaicin Fig. 4. Concentration–response curves of three components. Data points, percent (1 mmol/l) was able to evoke membrane depolarization and action change in peak amplitude in the presence of cochinchinenin A, cochinchinenin B, and loureirin B, respectively (mean of seven experiments). Error bars, S.E.s. Error potential firing after washout for 90 s (Fig. 7, bottom). In addition, bars are not indicated when smaller than the size of the point. Each set of data combination II alone failed to change membrane potential or cause points is fitted with Eq. (2). action potential firing in all observed DRG neurons. In summary,

Table 4 Effects of combinations of three components on capsaicin-induced TRPV1 currents in DRG neurons.

Combination Cochinchinenin A (lmol/l) Cochinchinenin B (lmol/l) Loureirin B (lmol/l) Inhibition rate (%)(P value of t test) (n¼7)

Combination I 38 19 8 84.874.9 (0.00004) Diluted 10-fold 3.8 1.9 0.8 59.674.0 (0.00001) Diluted 100-fold 0.38 0.19 0.08 35.576.1 (0.00039) Diluted 1000-fold 0.038 0.019 0.008 10.673.6 (0.00107) Combination II 3.8 1.9 8 79.376.6 (0.00212) Diluted 10-fold 0.38 0.19 0.8 49.176.6 (0.00562) Diluted 100-fold 0.038 0.019 0.08 32.575.0 (0.00807) Diluted 1000-fold 0.0038 0.0019 0.008 9.974.4 (0.00030) 280 L.-S. Wei et al. / European Journal of Pharmacology 702 (2013) 275–284

Fig. 5. Effects of combination I and II on capsaicin-induced TRPV1 receptor currents in DRG neurons. (A) Combination I and II inhibit rapidly and reversibly the current induced by 1 mmol/l capsaicin. The horizontal bar above each trace indicates the period of drug application. (B) Concentration–response curves for inhibition of the capsaicin (1 mmol/l) response by combination I and II. Data points, percent change in peak amplitude in the presence of combination I or II (mean of seven experiments). Error bars, S.E.s. Error bars are not indicated when smaller than the size of the point. Each set of data points is ﬁtted with Eq. (2).

Table 5 Comparisons for the percentage inhibition of dragon’s blood and that of the combinations.

Drug Concentration of individual component in the combination (lmol/l) Inhibition rates (%) 9yDyC9 in least square difference (LSD) method

Cochinchinenin A Cochinchinenin B Loureirin B

Combination I 38 19 8 84.8174.9 0.05187o0.09468 Combination A 38 19 / 35.2271.5 0.5419840.09468 Combination B 38 / 8 68.3775.0 0.2391040.09468 Combination C / 19 8 66.6674.9 0.2212040.09468

As Table 5 showed, the differences between the percentage inhibition produced by each combinations and 0.005% (m/v) of dragon’s blood are compared with LSD value

(0.09468) at the 5% level, where yD and yC are the percentage inhibition of dragon’s blood and combinations, respectively.

combination II was able to decrease capsaicin evoked depolarization in rat DRG neurons and its effects were partially reversible by washout, these results were consistent with those under voltage- clamp condition.

3.6. Effect of combination II on the concentration–response curve of capsaicin

Due to a lower IC50 value of combination II than that of combination I in inhibition of capsaicin-induced TRPV1 receptor currents, effect of combination II on concentration–response curves of capsaicin was investigated. Under the absence and presence of combination II, the peak amplitude of capsaicin- induced TRPV1 receptor currents would not increase when the concentration of capsaicin was above 10 mmol/l (P40.05), indicating a maximal effect of capsaicin. Furthermore, all peak amplitudes of capsaicin-induced TRPV1 receptor currents were standardized by the peak amplitude of TRPV1 receptor current induced 0.5 mmol/l capsaicin. Thus the cell-to-cell variability of capsaicin-induced TRPV1 receptor currents were eliminated. Pooled data were used to construct the concentration–response Fig. 6. Comparison of the concentration–response curves for dragon’s blood, curves for capsaicin in the absence and presence of combination II combinations I, and combination II. Here the concentration is expressed in (Fig. 8). In the absence of combination II, the maximum value the unit of the percent mass/volume. Data points, percent change in of capsaicin-induced standard currents was 2.2870.20, the IC50 peak amplitude in the presence of dragon’s blood, combinations I, and combina- value was 0.6970.06 mmol/l, and the Hill coefﬁcient was tion II (mean of seven experiments). Error bars, S.E.s. Error bars are not indicated 7 when smaller than the size of the point. Each set of data points is ﬁtted 1.54 0.23 (n¼7). In the presence of combination II, the max- with Eq. (2). imum value of capsaicin-induced standard currents was L.-S. Wei et al. / European Journal of Pharmacology 702 (2013) 275–284 281

Fig. 7. Effect of combination II on capsaicin-induced membrane potential of DRG neuron. Top, in control, 1 mmol/l capsaicin was applied and induced membrane depolarization enough to evoke action potential firings. The action potential firings were disappeared upon washout. Middle, in the presence of combination II, capsaicin was applied with the same protocol as in control. The capsaicin evoked depolarization was significantly decreased, there is no action potential firing. Bottom, after 90s washout of combination II and capsaicin, capsaicin was applied again in the same protocol as in control, the effect of capsaicin was partially recovered.

1.6470.08, the IC50 value was 1.9570.28 mmol/l, and Hill coefﬁ- cient was 1.5570.28 (n¼7). The result indicated that combination II reduced the capsaicin-induced standard currents by 27.9%.

3.7. The effects of combination II inside and outside plasma membrane

To explore the binding site of components of dragon’s blood, the effects of combination II (0.38 mmol/l of cochinchinenin A, 0.19 mmol/l of cochinchinenin B, and 0.8 mmol/l of loureirin B) on capsaicin-induced TRPV1 receptor currents was tested when it was applied to the intracellular side and extracellular side, respectively. The inhibitory effect on capsaicin-induced TRPV1 receptor currents was observed after extracellular application of combination II, and the extracellular percentage inhibition was 49.176.6% (n¼7). In contrast, combination II produced change no on capsaicin-induced TRPV1 receptor currents after 20 min intracellular dialysis by pipette solution (P40.05, n¼7). In addition, the percentage inhibition was 51.3175.1% (n¼7) when combination II was simultaneously applied to the intracellular Fig. 8. Antagonism of the capsaicin response by combination II is not competitive. side and extracellular side, which similar to the extracellular Concentration–response curves for capsaicin in the absence and presence of combination II: 3.8 mmol/l of cochinchinenin A, 1.9 mmol/l of cochinchinenin B, percentage inhibition (P40.05). and 8 mmol/l of loureirin B. All responses are normalized to the peak current (n) induced by 0.5 mmol/l capsaicin. Data points, normalized peak currents (mean of 3.8. Assessment of interaction among the three components seven experiments). Error bars, S.E.s. Error bars are not indicated when smaller than the size of the circle. Each set of data points is ﬁtted with Eq. (2). The concentrations of the three components that produce the effect of combination I (84.81% inhibition of TRPV1 receptor current) when used alone were determined from the concentration–response Matlab software (Fig. 9). To statistically determine the posi- curves of the individual components (Fig. 4), and the resulting three tional relation between the experimental point Q1 (38, 19, 8) concentration values, the concentration values (38, 19, 8) of the and additive surfaces, the coordinate values of theoretical three components in combination I, and the parameter values additive points corresponding to the point Q1 were calculated reported above were substituted into Eqs. (3)–(5), respectively. together with the associated 95% conﬁdence intervals. The According to the three equations, the additive surface drawings theoretical additive points are the intersections of the additive corresponding to 84.81% inhibition were then generated with surfaces and the ray which starts at the origin and also passes 282 L.-S. Wei et al. / European Journal of Pharmacology 702 (2013) 275–284

Fig. 9. Additive surfaces corresponding to the combined effects produced by cochinchinenin A, cochinchinenin B, and loureirin B. (A) The positional relation between point

Q1 and the additive surfaces corresponding to the effect of combination I (EI ¼84.81%) and (B) The positional relation between point Q2 and the additive surfaces corresponding to the effect of combination II (EII¼79.26%).

Table 6

Coordinate values of theoretical additive points corresponding to the experimental points Q1 and Q2.

coordinate value of Q1 (mmol/l) Coordinate value of point Q1’s additive points [95% conﬁdence interval] (mmol/l)

Inhibition rate of combination I, EI¼84.81% 38 216.2426 233.288 197.46 [151.170, 281.314] [182.516, 284.06] [158.918, 236.011] 19 108.121 116.6443 98.732 [79.459, 118.005] [91.258, 142.030] [75.585, 140.657] 8 45.524 49.113 41.571 [31.825, 59.224] [38.424, 59.800] [33.456, 49.686]

Inhibition rate of combination II, EII¼79.26%

Coordinate value of Q2 (mmol/l) Coordinate value of point Q2’s additive points [95% conﬁdence interval] (mmol/l) 3.8 15.372 15.965 14.987 [12.647, 18.096] [13.751, 18.180] [12.962, 17.011] 1.9 7.686 7.982 7.493 [6.323, 9.048] [6.875, 9.090] [6.481, 8.505] 8 32.362 33.612 31.552 [26.626, 38.097] [28.949, 38.275] [27.289, 35.8143]

Table 7 3.9. Effects of dragon’s blood in three animal models The effects of 5 mg/kg dragon’s blood in three animal models.

Group MPE of hot plate MPE of tail-flick Number of writhing The results of hot plate test, tail flick test and writhing test test (%) test (%) in writhing test presented in Table 7 showed that 5 mg/kg Dragon’s Blood significantly raised the pain thresholds in hot plate test and tail- Control 2.673.1 1.772.6 34.274.3 flick test, and in writhing test it also lead to a 69.6% reduction in Dragon’s 70.878.6a 81.177.8a 10.474.3a the number of writhing in comparison with the control group. blood

a Compared with control, Po0.01. 4. Discussion

through the point Q1. A similar calculation was performed for The present study ﬁrst observed the inhibition of dragon’s the case of combination II (79.26% inhibition of TRPV1 receptor blood on capsaicin-induced TRPV1 receptor currents in DRG current) to obtain the coordinate values of theoretical additive neurons using whole-cell patch clamp technique. 0.005% (m/v) points corresponding to the experimental point Q2 (3.8, 1.9, 8) solution of dragon’s blood completely suppress capsaicin-induced (Table 6). TRPV1 receptor currents, indicating that it block up the transmis-

As shown in Table 6, the coordinate values of Q1 and Q2 were sion of pain message for primary sensory neurons via direct all statistically smaller than those of their corresponding theore- action on TRPV1 receptor. tical additive points. Therefore, the points Q1 and Q2 were both The effects of three components (cochinchinenin A, cochinch- below the respective corresponding three additive surfaces inenin B, and loureirin B) and their combinations on the (Fig. 9), which indicated that interactional effect of the three capsaicin-induced TRPV1 receptor currents in DRG neurons of components in both combination I and II on capsaicin-induced rats were also observed. The result demonstrated that each TRPV1 receptor currents were both synergistic. individual component and the combinations of any two out of L.-S. Wei et al. / European Journal of Pharmacology 702 (2013) 275–284 283 the three components reduce capsaicin-induced TRPV1 receptor preliminary experiment showed that loureirin B inhibit the currents to some extent, but none of them had an equally inward current induced by extracellular lower pH value of DRG inhibition as that of 0.005% (m/v) dragon’s blood. According to neurons. Since TRPV1 receptor currents was also activated by the operational definition of the material basis for the efficacy extracellular lower pH value, and the area of vanillin activation of traditional medicine (Liu et al., 2006), the three individual sites on TRPV1 receptor was not the same as proton binding area components and the combinations of two components were (Wang and Cao, 2008), it was presumed that the function site regarded as the effective component/combinations but not the of loureirin B would be different from that of capsaicin and Hþ . corresponding material basis of dragon’s blood. However, the It should bind to TRPV1 receptor on this site and cause the combination of three components prepared in terms of the mass structural change of TRPV1 receptor, decreasing the affinity and fraction of three components in final products of dragon’s blood, activity of excitomotor, thus producing antagonistic effect. The which guaranteed that the quantity of each component in the mechanism may be similar to AMG9810, which made TRPV1 combination was quite in agreement with that in dragon’s blood, receptor in inactive or turn-off condition even vanillin activation generate similar effect as that of 0.005% (m/v) dragon’s blood. sites on TRPV1 receptor had been binded (Gavva et al., 2005). Therefore it was considered that the effective combination con- Many evidence indicated that besides capsaicin and resinifer- taining cochinchinenin A, cochinchinenin B, and loureirin B was atoxin, capsazepine, the competitive antagonist of TRPV1 recep- the material basis of dragon’s blood for the modulation of tor, could not antagonize the biological effect of other agonists capsaicin-induced TRPV1 receptor currents, and this modulation (Perkins and Campbell, 1992). But the specific agonists, capsaicin intervene in pain messages and thereby result in pain relief. This and resiniferatoxin were unable to be synthesized in the body of remarkable synergistic interaction among three components may vertebrates. The activation of TRPV1 receptor in the body may explain the mode of their modulation on the capsaicin-induced mainly depend on the nociceptive thermal stimulus and the TRPV1 receptor currents in DRG neurons. conduction of the acid stimulation in tissue. Therefore, TRPV1 The present study also discovered that the effect of loureirin B receptor antagonist should block up the activation of the receptor was stronger than that of cochinchinenin A or cochinchinenin B, induced by thermal stimulus as well as H þ , but not antagonize suggesting the chief role of loureirin B in the pharmacological effect the binding site of capsaicin (Yan and Zhang, 2002). Unlike of the combination. New combination could be made by altering the capsazepine, the three components of dragon’s blood antagonize proportion of the three components, i.e., reducing the relative TRPV1 receptor without competing with capsaicin for the same proportions of cochinchinenin A and cochinchinenin B but increas- site and would not lead to hyperthermia when they are used in ing the relative proportion of loureirin B. It still generate a strong the long run. Consequently, it may be a promising candidate in effect with lower total concentration of the three components. The developing new analgesic drugs targeting TRPV1 receptor. results showed that the combination II (namely the new combina- The present study shows that combination II is only work- tion above-mentioned) had a higher potency than combination I and ing outside plasma membrane, this may be because the intra- the components displayed higher synergistic reaction. Thus it should cellular application of combination II affects neither the capsaicin be presumed that the proportion of the combination should be response nor the inhibition effect of extracellularly applied further optimized to obtain more efficient combinations. combination II on the capsaicin response, which suggests that Our previous study has revealed that dragon’s blood modulate the binding sites of the three components in combination II are TTX-R sodium currents and the material basis of dragon’s blood located entirely outside of plasma membrane. This result may for the modulation is the combination of three components, provide the clue for further biophysical studies of channel/drug namely cochinchinenin A, cochinchinenin B and loureirin B, interaction. which synergistically modulate TTX-R sodium currents (Liu As for the possible molecular mechanisms of the synergistic et al., 2006). All these were in accordant with the present study effects of the three active compounds, at present we can only on capsaicin-induced TRPV1 receptor currents. It is noticeable propose the following hypotheses: by reason of the different that the IC50 value of dragon’s blood which inhibits TTX-R sodium chemical structures of the three components, each component current is three orders of magnitude higher than that of dragon’s has its own binding site on TRPV1 receptor. When the three blood which inhibits capsaicin-induced TRPV1 receptor currents, components bound to their respective binding sites on TRPV1 which suggests that in DRG neurons the first target of dragon’s receptor, the conformational changes of the binding sites impel blood is TRPV1 receptor rather than TTX-R sodium channel. The the combined inhibition effects of the three components on other difference is that although the three components inhibit TRPV1 receptor to be synergistically enhanced. There was no TTX-R sodium and capsaicin-induced TRPV1 receptor currents, antagonistic interaction could be observed probably because the the IC50 value of loureirin B which inhibits TTX-R sodium current concentration of each component was too low to bind all of its was of the same order of magnitude as that of the other two own sites on TRPV1 receptor. components, whereas the IC50 value of loureirin B inhibiting In conclusion, our study further proved that the combination capsaicin-induced TRPV1 receptor currents was two to three of three components from dragon’s blood according to the orders of magnitude lower than that of the other two compo- proportion by weight synergistically contributed to the corre- nents, which indicated that different mechanisms of dragon’s sponding analgesia material basis of dragon’s blood via the blood as influence on TTX-R sodium channel and TRPV1 receptor. inhibition of capsaicin-induced TRPV1 receptor currents. The addition of combination II do not only make the concentration–response curve of capsaicin-induced TRPV1 receptor currents move rightwards, but also reduce the maximal TRPV1 receptor currents induced by capsaicin, demonstrating that the Acknowledgments inhibition of dragon’s blood and its three components were not competitive. The receptor-ligand binding model was used to This work was supported by National Natural Science Founda- simulate the interaction of loureirin B and TRPV1 receptor. The tion of China [Grant 30973961]; Young Chenguang Foundation result indicated that the interaction of loureirin B and TRPV1 of Wuhan [Grant 201150431075]; Academic Team of South Central receptor was fitted with the simple model of one ligand and one University for Nationalities [Grant XTZ09010]; and Innovative receptor site. There may be only one binding site on TRPV1 Foundation Project for Students of South-Central University for receptor for loureirin B (Wang et al., 2007). In addition, our Nationalities [Grant KYCX110010E]. 284 L.-S. Wei et al. / European Journal of Pharmacology 702 (2013) 275–284

References Joshi, N.K., Szallasi, A., 2009. TRPV1 antagonists: the challenges for therapeutic targeting. Trends. Mol. Med. 15, 14–22. Kym, P.R., Kort, M.E., Hutchins, C.W., 2009. Analgesic potential of TRPV1 antago- Bevan, S., Szolcsańyi, J., 1990. Sensory neuron-specific actions of capsaicin: nists. Biochem. Pharmacol. 78, 211–216. mechanisms and applications. Trends. Pharmacol. Sci. 11, 331–333. Liu, X.M., Chen, S., Zhang, Y.X., Zhang, F., 2006. Modulation of dragon’s blood on Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D., Julius, D., tetrodotoxin-resistant sodium currents in dorsal root ganglion neurons and 1997. The capsaicin receptor: a heat-activated ion channel in the pain path- identification of its material basis for efficacy. Sci. China, Ser. C Life Sci. 49, way. Nature 389, 816–824. Chen, S.C., Chang, T.J., Wu, F.S., 2004. Competitive inhibition of the capsaicin 274–285. receptor-mediated current by dehydroepiandrosterone in rat dorsal root Patapoutian, A., Peier, A.M., Story, G.M., Viswanath, V., 2003. ThermoTRP channels ganglion neurons. J. Pharmacol. Exp. Ther. 311, 529–536. and beyond: mechanisms of temperature sensation. Nat. Rev. Neurosci. 4, Gavva, N.R., Treanor, J.J.S., Garami, A., Fang, L., Surapaneni, S., Akrami, A., Alvarez, F., 529–539. Bak, A., Darling, M., Gore, A., 2008. Pharmacological blockade of the Perkins, M.N., Campbell, E.A., 1992. Capsazepine reversal of the antinociceptive vanilloid receptor TRPV1 elicits marked hyperthermia in humans. PAIN 136, action of capsaicin in vivo. Brit. J. Pharmacol. 107, 329–333. 202–210. Tallarida, R.J., 2006. An overview of drug combination analysis with isobolograms. Gavva, N.R., Tamir, R., Qu, Y., Klionsky, L., Zhang, T., Immke, D., Wang, J., Zhu, D., J. Pharmacol. Exp. Ther. 319, 1–7. Vanderah, T.W., Porreca, F., 2005. AMG 9810 [(E)-3-(4-t-butylphenyl)-N-(2,3- Wang, C., Chen, S., Liu, X.M., 2007. Loureirin B inhibits capsaicin-induced currents dihydrobenzo [b] [1,4] dioxin-6-yl) acrylamide], a novel vanilloid receptor 1 in rat dorsal root ganglion neurons. Chin. Pharmacol. Bull. 2, 76–79. (TRPV1) antagonist with antihyperalgesic properties. J. Pharmacol. Exp. Ther. Wang, L., Cao, Y., 2008. Progress in vanilloid receptor 1 antagonist. Int. J. Pathol. 313, 474–484. Clin. Med. 28, 8–224. Gunthorpe, M.J., Chizh, B.A., 2009. Clinical development of TRPV1 antagonists: Wang, S.T., Chen, S., Guo, M., Liu, X.M., 2008. Inhibitory effect of cochinchinenin B targeting a pivotal point in the pain pathway. Drug. Discov. Today 14, 56–67. on capsaicin-activated responses in rat dorsal root ganglion neurons. Brain. Guo, M., Chen, S., Liu, X.M., 2008. Material basis for inhibition of dragon’s blood on Res. 1201, 34–40. evoked discharges of wide dynamic range neurons in spinal dorsal horn of Yan, M., Zhang, L.Y., 2002. Capsaicin receptor, a novel target for new peripheral rats. Sci. China, Ser. C Life Sci. 51, 1025–1038. acting analgesics. Chin. J. New Drug 11 756-756. Gupta, D., Bleakley, B., Gupta, R.K., 2008. Dragon’s blood: botany, chemistry and Zeng, X.Y., He, F., 1999. Studies on effects of anti-inflammation and acesodyne and therapeutic uses. J. Ethnopharmacol. 115, 361–380. toxicity of dragon’s blood Blood in Guangxi. China J. Chin. Mater. Med. 24, Helliwell, R.J.A., Mclatchie, L.M., Clarke, M., Winter, J., Bevan, S., McIntyre, P., 1998. 171–173. Capsaicin sensitivity is associated with the expression of the vanilloid Zhong, Y.P., 2010. Clinical application and research progress of traditional Chinese (capsaicin) receptor (VR1) mRNA in adult rat sensory ganglia. Neurosci. Lett. medicine dragon’s blood. Mod. J. Integr. Chin. Trad. Western Med. 19, 250, 177–180. 2469–2470. Huang, H., Ruan, H.Z., Cai, W.Q., Wu, X.G., Chen, P.H., 2007. A method to acutely isolate DRG neurons from adult rats for patch clamp technique. Acta Acade- miae Medicinae Militaris Tertiae 29, 460–461.