- Email: [email protected]
Studies on antidepressant and antinociceptive effects of ethyl acetate extract from Piper laetispicum and structure–activity relationship of its amide alkaloids
Fitoterapia 82 (2011) 1086–1092
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Studies on antidepressant and antinociceptive effects of ethyl acetate extract from Piper laetispicum and structure–activity relationship of its amide alkaloids Hui Xie a, 1, Ma-cheng Yan b, 1, Di Jin c, Jia-jia Liu d, Min Yu c, Dong Dong a, Cheng-cheng Cai a, Sheng-Li Pan a,⁎ a b c d
School of Pharmacy, Fudan University, Shanghai, 201203, China Pharmaceutical Preparation Section, Punan Hospital, Pudong New District, Shanghai, 200127, China Department of Pharmacognosy, College of Pharmacy, Jiamusi University, Jiamusi, 154007, China Academy of Chinese Medical, Yunnan University of Traditional Chinese Medicine, Kunming, 650500, China
a r t i c l e
i n f o
Article history: Received 12 April 2011 Received in revised form 8 July 2011 Accepted 8 July 2011 Available online 23 July 2011 Keywords: Piper laetispicum Ethyl acetate extract Antidepressant Antinociceptive Amide alkaloid Structure–activity relationship
a b s t r a c t Piper laetispicum C.DC. (Piperaceae), is an endemic climbing, glabrous plant distributed in the southern part of China. A novel alkaloid amide, Laetispicine, from it has been proven to possess antidepressant activity. In this present study, antidepressant and antinociceptive effects of the ethyl acetate extract (EAE) of P. laetispicum have been studied in forced swimming, open field, acetic acid writhing and formalin tests in KM mice. A significantly antidepressant-like effect was showing at doses of higher than 60 mg/kg, which was not due to an increase in locomotive activity. The EAE also presented an analgesic effect, in our studies. At lower doses (30 mg/kg) the antinociceptive effect was likely mediated via peripheral inflammation and changes in central processing, and at higher doses (120 mg/kg) that was due to both central and peripheral pathways. We also quantitatively analyzed the major components of EAE by HPLC and approached the structure–activity relationship between structure of amide alkaloids and its antidepressant activities. The antidepressant effective components of EAE might be Leatispiamide A and Laetispicine. In their molecular structures, the isolated double bond from benzene ring and conjugated double bond located at 2–3 and 4–5 were necessary for its antidepressant activity. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Depression, with a lifetime incident of 15–25% [1–3], has been ranked by WHO as one of the most burdensome diseases in the world. Its symptoms are intense feelings of sadness, hopelessness, and despair; the inability to experience pleasure in usual activities; changes in sleep patterns and appetite; loss of energy; and suicidal thoughts [4]. Although, numerous antidepressants are clinically prescribed, including tricyclic antidepressants (TCAs), selective serotonin reuptake inhibi⁎ Corresponding author. Tel.: + 86 21 51980137. E-mail address: [email protected] (S.-L. Pan). 1 The authors contributed equally to the paper. 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.07.006
tors (SSRIs), serotonin-noradrenergic reuptake inhibitors (SNRIs), and other atypical antidepressant drugs-monoamine oxidase inhibitors (MAOIs) and so on [2], the heterogeneity of clinical responses and side-effects were noticed and have become the major problems, such as sedation, apathy and fatigue, sleep disturbance, cognitive impairment, sexual dysfunction, etc. [5]. Concurrently, a lot of herbal medicines have been researched as alternative therapies for depression, such as Hypericum perforatum L. (St. John's wort), Nelumbo nucifera Gaertn, Ginkgo biloba L., Panax ginseng C.A. Meyer, Piper methysticum Forst and Chaihu-Shugan-San etc.[6–9]. Piper laetispicum C.DC. (Piperaceae) is an endemic climbing, glabrous plant distributed in the southern part of
H. Xie et al. / Fitoterapia 82 (2011) 1086–1092
China, which was usually used for invigorating circulation and reducing stasis, detumescence and analgesia, and is popularly known as Xiao Chang-feng, Shan Hu-jiao, and Ye Hu-jiao in folk terms [10]. Previous studies of our group demonstrated that Laetispicine (N-isobutyl-(3,4-methylendioxyphenyl)-2E, 4E, 9E-undecatrienoamide), a novel amide alkaloid from P. laetispicum, presents antidepressant activity [11,12]. In this paper, pharmacological effects of the ethyl acetate extract of P. laetispicum have been researched, major components from the ethyl acetate extract have been examined by HPLC and the relationship between structure of amide alkaloids and its activities has been discussed. Those were designed to clue a novel alternative antidepressant. 2. Materials and methods 2.1. Plant material and preparation of extract The stems of P. laetispicum were collected in 2006, from Hainan Province, China. The plant was identified by Prof. Sheng-li Pan, School of Pharmacy, Fudan University, where a voucher specimen (no. 060812) of the plant material has been deposited for further reference. Dried stems of P. laetispicum (10 kg) were powdered and percolated with 95% EtOH, and the solution was concentrated under reduced pressure to give crude extract (760 g). The crude extract was subsequently suspended in three volumes of water saturated ethyl acetate (M/V), stored for 30 min, and the upper phase was removed into a new tube. This treatment was repeated twice. All of the upper phase were pooled together and concentrated under reduced pressure. With a yield of 44.19%, the ethyl acetate extract (EAE) was obtained, which was used in activity tests and the isolation of its major amide alkaloids. The EAE was kept in a refrigerator at + 4 °C, and suspended in a vehicle (2% Tween-80 suspension in saline) using ultrasonic cleaner under 60 °C, while any pharmacological screening were taken. All dosages were expressed as milligrams of it per kilogram body weight. 2.2. Isolation of major amide alkaloids The EAE was chromatographed on a silica gel column by a gradient elution using mixtures of petroleum ether and acetone to afford six fractions. Fraction 2, 3, 4 was subsequently separated by repeated chromatography on silica gel column by chloroform: methanol (1:1). Finally, preparative liquid chromatography was used for isolation of Chingchingenamide (1), Leatispiamide A (2), Leatispiamide B (3) and Laetispicine (4). The solvent system was composed of A (methanol) and B (water), with an elution 85(A):15(B), V/V. Guineensine (5) was extracted and isolated from another plant of Genus Piper L., Piper boehmeriaefolium (Miq.)C. DC. var. tomkinense C.DC. The structure of these five compounds was fully characterized by UV, IR, NMR and MS [11,13–16]. The obtained amide alkaloids were kept in a refrigerator at + 4 °C, and suspended in a vehicle (2% Tween-80 suspension in saline) using ultrasonic cleaner under 60 °C, when used in any pharmacological screening. All dosages
1087
were expressed as milligrams of the respective drugs per kilogram body weight. 2.3. Quantitative analysis of major components of ethyl acetate extract by HPLC 2.3.1. Chromatographic instrumentation and conditions A liquid chr7omatograph 105 system (LC-10AT, Shimadzu), equipped with an UV–Vis detector (SPD-10A, Shimadzu) was used. The samples were separated on a Luna ODS column (250 × 4.6 mm, 5 μm, Phenomenex). Data analyses were carried out using ChemStation software (HW2000, Dianpu Co., Ltd, China).The solvent system was composed of A (methanol) and B (water), with an elution 77(A):23(B), V/V. The flow rate was 1 ml/min. Injection volume was 20 μl and the detection was conducted using the wavelength of 259 nm. 2.3.2. Standard solution A methanolic stock solution 5 ml containing Chingchingenamide (1) 9.36 mg, Leatispiamide A (2) 10.66 mg, Leatispiamide B (3) 4.75 mg and Laetispicine (4) 14.30 mg was prepared and stored out of light at 4 °C until required for analysis. 2.3.3. Calibration curve Calibration curve of each compound was constructed by plotting the peak area against the concentration. Each calibration curve contained seven different concentrations and was performed in triplicate. For the constituents detected by UV detection, their regression equations were calculated in the form of y = bx + a, where y and x were the peak area and sample concentration, respectively. 2.3.4. Precision Instrument precision was evaluated by carrying out intraday assay. Briefly, the mixed standard solution was continuously injected six times in 1 day. 2.3.5. Recovery To evaluate accuracy, recovery tests were carried out. The powders of P. laetispicum were spiked with the mixed standard solutions at high, middle and low concentration levels. Triplicate experiments at each level were performed. The total amount of each compound was calculated from the corresponding calibration curve, the detected amount of each compound was calculated by subtracting the amount of each compound contained in the herb from the total amount of each compound after spiking. The recovery of each compound was obtained using the following equation: Recovery (%) = (Amount detected) / (Amount spiked) × 100%. 2.4. Animals and grouping Adult KM mice were purchased from the Department of Experimental Animal Center of Fudan University, and were housed under a normal 12 h light/dark cycle with lights on at 07:00 a.m. The mice had free access to tap water and food. Ambient temperature and relative humidity were maintained at 22–25 °C and 55 ± 10%, respectively. Prior to the test procedure, mice were acclimatized to the laboratory for at
1088
H. Xie et al. / Fitoterapia 82 (2011) 1086–1092
least 5 days. All behavioral tests were conducted during the light cycle. The experiment procedures were conducted in compliance with the National Institutes of Health Guide for Care and Use of the laboratory animals, and were approved by the Local Bioethics Committee (School of Pharmacy, Fudan University, China; document number: SYXK2007-002). Every effort was made to minimize the number and suffering of the animals used. 2.5. Drugs Acetic acid, formaldehyde, acetylsalicylic acid (ASA) and Tween-80 were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China), morphine hydrochloride from Shenyang First Pharmaceutical Company (Shenyang, China), and fluoxetine from Shanghai Zhongxi Pharmaceutical Co., Ltd (Shanghai, China). 2% Tween-80 suspension in saline was used as vehicle in the pharmacological tests. 2.6. Pharmacological tests 2.6.1. Forced swimming test (FST) A modification method of the FST [17] was carried out. Briefly, male mice were individually forced to swim for 15 min in a glass cylinder (height: 20 cm, diameter: 14 cm), filled with 10 cm high water at 25 °C ± 1 °C, which is a pretest. Twenty-three hours later, male mice were treated orally with EAE or amide alkaloids. One hour later, mice were placed in the cylinders again for a 6 min test in the same system depicted above. The immobility time during the final 4 min was recorded by competent observers manually. Immobility time was defined as the time spent by the mouse floating in the water without struggling, and making only those movements necessary to keep its head above the water. Fluoxetine was used as positive control drug. For EAE, the male mice were divided into five different groups (n = 10 per group), i.e., control (vehicle) group, F. (fluoxetine) 50 mg/kg group, EAE 30 mg/kg group, EAE 60 mg/kg group, EAE 120 mg/kg group. The amide alkaloids used in FST were all 20 mg/kg. There were also 10 male animals in each group. 2.6.2. Open field test The open field test method used in the present study was similar to that described previously by [9]. The apparatus was a square arena (diameter: 40 cm; height: 20 cm) with a light source of 120 lx, which was demarcated into 16 equal areas. The mice, when they finished FST, were placed in the center and their behavior was noted immediately and continued for 5 min. The score locomotion (number of line crossings) and rearing frequencies (number of times an animal stood on its hind legs) were recorded. 2.6.3. Acetic acid-induced writhing test According to previously described techniques [18,19], the acetic acid-induced writhing test was adopted. Mice (n = 10 per group) were treated orally 1 h before intraperitoneal injection of 1% acetic acid solution (10 ml/kg) with EAE at doses of 30, 60 and 120 mg/kg. The control group received only vehicle (10 ml/kg), and the reference group received
ASA (100 mg/kg). The number of writhes occurring between 5 and 25 min after acetic acid injection was recorded. 2.6.4. Formalin test The method used for formalin test was similar to that described by Hunskaar and Hole [20] with slight modifications. Mice (n = 10) were orally treated with EAE (30, 60 and 120 mg/kg, p.o.), morphine hydrochloride (7.5 mg/kg, s.c.) or 2% Tween-80. After 1 h, 20 μl of 2% formalin (v/v, in 0.9% saline) was injected subcutaneously into the plantar surface of the left hind paw of the mice. Immediately after, each mouse was placed into a glass cylinder provided with mirrors to enable a total panorama of the nociceptive behavior. The mice were observed for 60 min, the amount of time spent licking and biting the injected paw was recorded. The first 10 min is known as the first phase and the period during the following 50 min as the second phase. 2.7. Statistical analysis of data Data obtained were expressed as mean ± S.E.M. Statistical significance between groups was performed by the application of analysis of variance ANOVA followed by Bonferroni's test. P-values less than 0.05 (p b 0.05) were used as the significant level. 3. Results and discussion 3.1. Effects of EAE 3.1.1. Effect of EAE on immobility time in the mouse FST The forced swimming test, described originally by [17], is the most widely used model to screen new antidepression drugs. There are a wide variety of antidepressants and compounds with potential antidepressant-like activity to reduce the duration of immobility in the FST [17,21]. In the present study, we first investigated the antidepressant effects of ethyl acetate extract (EAE) .The results (Fig. 1) clearly showed that the treatment with doses of 60 and 120 mg/kg of EAE significantly and dose-dependently decreased the immobility time (p b 0.01 and p b 0.001). In order to detect any association of immobility in the FST with changes in motor activity, the activities of animals were tested in an open field test after FST. Fig. 2 summarizes the results. The treatment animals with 30, 60 mg/kg EAE
Fig. 1. Effects of the ethyl acetate extract (EAE) on the forced swimming test in mice. Results are expressed as mean ± S.E.M. (n = 10). **p b 0.01 and ***p b 0.001 compared with control. Control: vehicle, F: fluoxetine.
H. Xie et al. / Fitoterapia 82 (2011) 1086–1092
1089
FST seemed unlikely to be due to an increase in locomotive activity and the antidepressant-like effect of it would be specific.
Fig. 2. Effects of EAE on the open field for 5 min in mice. Results are expressed as mean ± S.E.M. (n = 10). **p b 0.01 compared with control. Control: vehicle.
Fig. 3. Effects of Laetispicine on acetic acid induced writhing test in mice. Data are expressed as mean ± S.E.M. (n = 10). ***p b 0.001 compared with control. Control: vehicle, A: acetylsalicylic acid.
showed no differences compared with control animals. Interestingly, the 120 mg/kg EAE group can decrease the score locomotion and rearing frequencies. There was significant difference compared with the control (p b 0.01). That suggested that the antidepressant actions of EAE in the mouse
3.1.2. Antinociceptive effect of EAE Previous studies [22] have suggested that the positive results obtained with antidepressants in nociceptive tests. And the P. laetispicum was usually used for analgesic. Then, we evaluated the antinociceptive effect of EAE using two models: acetic acid-induced writhing test and formalin test. The acetic acid-induced writhing test has long been used as a screening tool for the assessment of analgesic or antiinflammatory properties [18,19]. In the present study, the effect of EAE (120 mg/kg, 64.51%) was comparable to that of ASA (100 mg/kg, 61.64%) (Fig. 3), and there was a significant reduction (p b 0.001) in comparison to the control (Fig. 3). It is suggested that EAE at doses of 120 mg/kg had powerful antinociceptive effect. The results of formalin test were showing in Fig. 4, morphine hydrochloride (7.5 mg/kg) the reference drugs, significantly suppressed the formalin response in the first phases by 50.40%, and second phases by 57.12%, respectively. For EAE, during the first phase (0–10 min), only at doses of 120 mg/kg significantly reduced the licking activity by 32.75% (p b 0.05). In regard to the second phase (10–60 min), EAE at all given doses had a respectively significant effect on the diminished licking time by 42.24%, 32.37%, 44.60% (p b 0.05), as compared with the control group. The acetic acid-induced writhing test suggested that the abdominal writhing induced by acetic acid involves the production and release of arachidonic acid metabolites via cycloxygenase (COX) and prostaglandin biosynthesis; an increase in the peritoneal fluid levels of PGE1, PGF2 and lipooxygenase products; release of sympathetic nervous system mediators; and it might also cause the release of cytokines, such as TNF-α, interleukin-1β, and interleukin-8, by resident peritoneal macrophages and mast cells [20,23,24]. The formalin pain test is a very useful model of clinical pain in which the first phase seems to be due to direct chemical stimulation of nociceptors, whereas the second phase is dependent on peripheral inflammation and changes in
Fig. 4. Effects of EAE on formalin test in mice. Data are expressed as mean ±S.E.M. (n = 10). *p b 0.05, **p b 0.01 and ***p b 0.001 compared with control. Control: vehicle, M: morphine.
1090
H. Xie et al. / Fitoterapia 82 (2011) 1086–1092
Fig. 5. HPLC chromatogram of the ethyl acetate extract from P. laetispicum. Peak markers 1: Chingchingenamide, 2: Leatispiamide A, 3: Laetispiamide B, 4: Laetispicine.
central processing [25]. Drugs which act mainly centrally, such as narcotic analgesics, inhibit both phases of pain in this model while peripherally acting drugs, such as aspirin or indomethacin, only inhibit the second phase [26].
That suggested EAE at doses of 30 mg/kg and 60 mg/kg only possess peripheral antinociceptive effect, at doses of 120 mg/kg the antinociceptive effect may due to direct result of stimulation of nociceptors and inflammation occurring following the release of serotonin, histamine, bradykinnin and prostaglandins.
3.2. Chemical analysis of the ethyl acetate extract prepared from P. laetispicum
Fig. 6. Chemical structures of compounds Chingchingenamide (1), Leatispiamide A (2), Leatispiamide B (3), Laetispicine (4) and Guineensine (5).
In order to expound the active ingredients of EAE, a chromatogram of the ethyl acetate extract was obtained, according to the HPLC conditions described above (Fig. 5). Four amide alkaloids were identified as the major components of this extract. These are Chingchingenamide (1), Leatispiamide A (2), Leatispiamide B (3) and Laetispicine (4). Their molecular structures are shown in Fig. 6. The calibration curves of these four amide alkaloids showed good linear regression with high correlation coefficients (r N 0.9999) within the ranges tested, as shown in Table 1. The precision was below 3% (RSD) (Table 1). The recoveries for all the analytes were between 98.43% and 101.23%, RSDs were between 0.47% and 0.97%. That means the method is precise, sensitive and shows a high accuracy for the simultaneous quantification of the four amide alkaloids. The content of these four amide alkaloids in EAE was calculated by this method. As showing in Table 1, Leatispiamide A (2) and Laetispicine (4) were major compounds of it.
Table 1 Quantitative analysis of major components of EAE by HPLC. Analytes
Calibration curves (n = 7)
Correlation coefficients
Linearity range (mg/L)
Precision RSD
Content/EAE (mg/g)
Ch La A La B L
y = 62226x + 51699 y = 59231x + 40222 y = 138765x − 66610 y = 57300x + 7964.7
r = 0.9999 r = 0.9999 r = 1.000 r = 1.000
1.87–187.20 2.13–213.20 1.90–190.00 2.86–286.00
0.35% 0.94% 0.43% 1.47%
1.24 12.68 1.25 9.64
Ch: Chingchingenamide, La A: Leatispiamide A, La B: Leatispiamide B, L: Laetispicine.
H. Xie et al. / Fitoterapia 82 (2011) 1086–1092
1091
3.3. Structure of amide alkaloid with antidepressant-like effect of EAE Laetispicine (4) from P. laetispicum has been proven to possess antidepressant activities [12]. Its pharmacokinetic characteristics in plasma and brain distribution in rats also have been examined [27]. In this paper, the antidepressant effects of four major amide alkaloids of EAE, Chingchingenamide (1), Leatispiamide A (2), Leatispiamide B (3), Laetispicine (4) and Guineensine (5) (Fig. 6), another amide alkaloid extracted from P. boehmeriaefolium (Miq.)C. DC. var. tomkinense C.DC., were evaluated and the result was showed in Fig. 7. Only Leatispiamide A (2) and Laetispicine (4) significantly decreased the immobility time by 29.13% and 32.58% in FST. An open field test also was used to detect any changes in motor activity of animals. The results were shown in Fig. 8. There were no significant differences compared with control animals. That suggested that the antidepressant-like effect of Leatispiamide A (2) would be specific either, similar with Laetispicine (4). The view of the structures of these five amide alkaloids was similar, except some double bond locations in the carbon chain. The difference among them was analyzed. Chingchingenamide (1), Leatispiamide A (2), Laetispicine (4) and Guineensine (5) all have conjugated double bond located at 2–3 and 4–5. Among them Leatispiamide A (2) and Laetispicine (4) have isolated double bond from benzene ring, correspondingly Gu has a conjugated double bond with benzene ring and Chingchingenamide (1) has no other double bond in carbon chain. Leatispiamide B (3) also has isolated double bond from benzene ring, same as Leatispiamide A (2) and Laetispicine (4), but it has no conjugated double bond at 2–3 and 4–5. The result of pharmacological tests revealed that isolated double bond from benzene ring and conjugated double bond located at 2–3 and 4–5 are necessary for its antidepressant activities. 4. Conclusion The present study demonstrated that the ethyl acetate extract (EAE) from P. laetispicum had significantly antidepressant-like effect which was not due to an increase in locomotive activity. Its effective dosage was 60 mg/kg. The
Fig. 7. Effects of major amide alkaloids on the forced swimming test in mice. 4: Laetispicine, 2: Leatispiamide A, 3: Leatispiamide B, 1: Chingchingenamide, 5: Guineensine.
Fig. 8. Effects of Laetispicine and Leatispiamide A on the open field for 5 min in mice. Results are expressed as mean ± S.E.M. (n = 10). Control: vehicle, 4: Laetispicine, 2: Leatispiamide A.
effective components of EAE were Leatispiamide A (2) and Laetispicine (4). The isolated double bond from benzene ring and conjugated double bond located at 2–3 and 4–5 in the molecular structure were necessary for its antidepressant activities. The ethyl acetate extract (EAE) also presented antinociceptive effect, in our studies. At lower doses (30 mg/kg) the antinociceptive effect was likely mediated via peripheral inflammation and changes in central processing, and at higher doses (120 mg/kg) that was due to both central and peripheral pathways. The possible mechanisms deserve further attention.
Acknowledgments The study was supported and funded by the Ministry of Science and Technology of the People's Republic of China (no. 2009zx09103-075), Science Committee of Shanghai, China (no. 08DZ1971203), and Pudong New District of Shanghai, China (no. PW2008A-7).
References [1] Kessler RC, Berglund P, Demler O, Jin R, Koretz D, Merikangas KR, et al. The epidemiology of major depressive disorder: results from the National Comorbidity Survey Replication (NCS-R). JAMA 2003;289: 3095–105. [2] Nemeroff CB. The burden of severe depression: a review of diagnostic challenges and treatment alternatives. J Psychiatr Res 2007;41: 189–206. [3] Patten SB. Major depression prevalence is very high, but the syndrome is a poor proxy for community populations' clinical treatment needs. Can J Psychiatry 2008;53:411–9. [4] Gaudiano BA, Young D, Chelminski I, Zimmerman M. Depressive symptom profiles and severity patterns in outpatients with psychotic vs nonpsychotic major depression. Compr Psychiatry 2008;49:421–9. [5] Kennedy SH. A review of antidepressant treatments today. Eur Neuropsychopharmacol 2006;16:619–23. [6] Linde K, Berner MM, Kriston L. St John's wort for major depression. Cochrane Database Syst Rev 2008;8:CD000448. [7] Baum SS, Hill R, Rommelspacher H. Effect of kava extract and individual kavapyrones on neurotransmitter levels in the nucleus accumbens of rats. Prog Neuropsychopharmacol Biol Psychiatry 1998;22:1105–20. [8] Dang HX, Chen Y, Liu XM, Wang Q, Wang LW, Jia W, et al. Antidepressant effects of ginseng total saponins in the forced swimming test and chronic mild stress models of depression. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:1417–24. [9] Kim SH, Han J, Seog DH, Chung JY, Kim N, Park YH, et al. Antidepressant effect of Chaihu-Shugan-San extract and its constituents in rat models of depression. Life Sci 2005;76:1297–306.
1092
H. Xie et al. / Fitoterapia 82 (2011) 1086–1092
[10] Zhonghua Herbals Editorial Committee. Zhonghua Herbals. Shanghai: Shanghai Science Technique Press; 1999. p. 433–4. [11] Pan SL, Xie J, Qian FG, Wang J, Shao YC. Antidepressant amidesfrom Piper laetispicum C. DC. Acta Pharmacol Sin 2005;40:355–7. [12] Yao CY, Wang J, Dong D, Qian FG, Xie J, Pan SL. Laetispicine, an amide alkaloid from Piper laetispicum, presents antidepressant and antinociceptive effects in mice. Phytomedicine 2009;16:823–9. [13] Parmar VS, Sinha R, Shakil NA, Tyagi OD, Boll PM, Wengel A. An insecticidal amide from Piper falconerl. Indian J Chem 1993;32B:392–3. [14] Fang J, Qian FG, Xie J. Studies on the chemical constituents of root and stem of Piper laetispicum C. DC. Chin Tradit Herb Drugs 2006:1882–901 (Supp l). [15] Fang J, Xie J, Shou YC, Qian FG. Studies on the chemical constituents of root and stem of Piper laetispicum C. DC. (II). Chin Tradit Herb Drugs 2007;38:1289–92. [16] Gupta OP, Dhar KL, Atal CK. Structure of new amide from Piper officinarum. Phytochemistry 1976;15:425. [17] Porsolt RD, Le Pichon M, Jalfre M. Depression: a new animal model sensitive to antidepressant treatments. Nature 1977;266:730–2. [18] Koster R, Anderson M, Beer EJ. Acetic acid analgesic screening. Fed Proc 1959;18:412. [19] Duarte IDG, Nakamura M, Ferreira SH, Braz J. Participation of the sympathetic in acetic acid-induced writhing in mice. Med Biol Res 1988;21:41.
[20] Hunskaar S, Hole K. The formalin test in mice—dissociation between inflammatory a noninflammatory. Pain 1987;30:103–14. [21] Cryan JF, Markou A, Lucki I. Assessing anti-depressant activity in rodents: recent developments and future needs. Trends Pharmacol Sci 2002;23:238–45. [22] Eschalier A, Ardid D, Coudore F. Pharmacological studies of the analgesic effect of antidepressants. Clin Neuropharmacol 1992;15:373–4. [23] Guzzo LS, Saude-Guimaraes DA, Silva ACA, Lombardi JA, Guimaraes HN, Grabe-Guimaraes A. Antinociceptive and anti-inflammatory activities of ethanolic extracts of Lychnophora species. J Ethnopharmacol 2008;116:116–20. [24] Elisabetsky E, Amador TA, Albuquerque RR, Nunes DS, Carvalho ACT. Analgesic activity of Psychotria colorata (wild ex Ret S) Muell. Arg Alkaloids J Ethnopharmacol 1995;48:77–83. [25] Tjolsen A, Berge OG, Hunskaar S, Rosland JH, Hole K. The formalin test: an evaluation of the method. Pain 1992;51:5–17. [26] Santos ARS, Filho VC, Niero R, Viana AM, Moreno FN, Campos MM, et al. Analgesic effects of callus-culture extracts from selected species of Phyllanthus in mice. J Pharm Pharmacol 1994;46:755–9. [27] Wang Y, Xie H, Pan SL. Pharmacokinetics of Laetispicine and its brain distribution in Rats. Am J Chin Med 2010;38:895–907.
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Studies on antidepressant and antinociceptive effects of ethyl acetate extract from Piper laetispicum and structure–activity relationship of its amide alkaloids Hui Xie a, 1, Ma-cheng Yan b, 1, Di Jin c, Jia-jia Liu d, Min Yu c, Dong Dong a, Cheng-cheng Cai a, Sheng-Li Pan a,⁎ a b c d
School of Pharmacy, Fudan University, Shanghai, 201203, China Pharmaceutical Preparation Section, Punan Hospital, Pudong New District, Shanghai, 200127, China Department of Pharmacognosy, College of Pharmacy, Jiamusi University, Jiamusi, 154007, China Academy of Chinese Medical, Yunnan University of Traditional Chinese Medicine, Kunming, 650500, China
a r t i c l e
i n f o
Article history: Received 12 April 2011 Received in revised form 8 July 2011 Accepted 8 July 2011 Available online 23 July 2011 Keywords: Piper laetispicum Ethyl acetate extract Antidepressant Antinociceptive Amide alkaloid Structure–activity relationship
a b s t r a c t Piper laetispicum C.DC. (Piperaceae), is an endemic climbing, glabrous plant distributed in the southern part of China. A novel alkaloid amide, Laetispicine, from it has been proven to possess antidepressant activity. In this present study, antidepressant and antinociceptive effects of the ethyl acetate extract (EAE) of P. laetispicum have been studied in forced swimming, open field, acetic acid writhing and formalin tests in KM mice. A significantly antidepressant-like effect was showing at doses of higher than 60 mg/kg, which was not due to an increase in locomotive activity. The EAE also presented an analgesic effect, in our studies. At lower doses (30 mg/kg) the antinociceptive effect was likely mediated via peripheral inflammation and changes in central processing, and at higher doses (120 mg/kg) that was due to both central and peripheral pathways. We also quantitatively analyzed the major components of EAE by HPLC and approached the structure–activity relationship between structure of amide alkaloids and its antidepressant activities. The antidepressant effective components of EAE might be Leatispiamide A and Laetispicine. In their molecular structures, the isolated double bond from benzene ring and conjugated double bond located at 2–3 and 4–5 were necessary for its antidepressant activity. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Depression, with a lifetime incident of 15–25% [1–3], has been ranked by WHO as one of the most burdensome diseases in the world. Its symptoms are intense feelings of sadness, hopelessness, and despair; the inability to experience pleasure in usual activities; changes in sleep patterns and appetite; loss of energy; and suicidal thoughts [4]. Although, numerous antidepressants are clinically prescribed, including tricyclic antidepressants (TCAs), selective serotonin reuptake inhibi⁎ Corresponding author. Tel.: + 86 21 51980137. E-mail address: [email protected] (S.-L. Pan). 1 The authors contributed equally to the paper. 0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2011.07.006
tors (SSRIs), serotonin-noradrenergic reuptake inhibitors (SNRIs), and other atypical antidepressant drugs-monoamine oxidase inhibitors (MAOIs) and so on [2], the heterogeneity of clinical responses and side-effects were noticed and have become the major problems, such as sedation, apathy and fatigue, sleep disturbance, cognitive impairment, sexual dysfunction, etc. [5]. Concurrently, a lot of herbal medicines have been researched as alternative therapies for depression, such as Hypericum perforatum L. (St. John's wort), Nelumbo nucifera Gaertn, Ginkgo biloba L., Panax ginseng C.A. Meyer, Piper methysticum Forst and Chaihu-Shugan-San etc.[6–9]. Piper laetispicum C.DC. (Piperaceae) is an endemic climbing, glabrous plant distributed in the southern part of
H. Xie et al. / Fitoterapia 82 (2011) 1086–1092
China, which was usually used for invigorating circulation and reducing stasis, detumescence and analgesia, and is popularly known as Xiao Chang-feng, Shan Hu-jiao, and Ye Hu-jiao in folk terms [10]. Previous studies of our group demonstrated that Laetispicine (N-isobutyl-(3,4-methylendioxyphenyl)-2E, 4E, 9E-undecatrienoamide), a novel amide alkaloid from P. laetispicum, presents antidepressant activity [11,12]. In this paper, pharmacological effects of the ethyl acetate extract of P. laetispicum have been researched, major components from the ethyl acetate extract have been examined by HPLC and the relationship between structure of amide alkaloids and its activities has been discussed. Those were designed to clue a novel alternative antidepressant. 2. Materials and methods 2.1. Plant material and preparation of extract The stems of P. laetispicum were collected in 2006, from Hainan Province, China. The plant was identified by Prof. Sheng-li Pan, School of Pharmacy, Fudan University, where a voucher specimen (no. 060812) of the plant material has been deposited for further reference. Dried stems of P. laetispicum (10 kg) were powdered and percolated with 95% EtOH, and the solution was concentrated under reduced pressure to give crude extract (760 g). The crude extract was subsequently suspended in three volumes of water saturated ethyl acetate (M/V), stored for 30 min, and the upper phase was removed into a new tube. This treatment was repeated twice. All of the upper phase were pooled together and concentrated under reduced pressure. With a yield of 44.19%, the ethyl acetate extract (EAE) was obtained, which was used in activity tests and the isolation of its major amide alkaloids. The EAE was kept in a refrigerator at + 4 °C, and suspended in a vehicle (2% Tween-80 suspension in saline) using ultrasonic cleaner under 60 °C, while any pharmacological screening were taken. All dosages were expressed as milligrams of it per kilogram body weight. 2.2. Isolation of major amide alkaloids The EAE was chromatographed on a silica gel column by a gradient elution using mixtures of petroleum ether and acetone to afford six fractions. Fraction 2, 3, 4 was subsequently separated by repeated chromatography on silica gel column by chloroform: methanol (1:1). Finally, preparative liquid chromatography was used for isolation of Chingchingenamide (1), Leatispiamide A (2), Leatispiamide B (3) and Laetispicine (4). The solvent system was composed of A (methanol) and B (water), with an elution 85(A):15(B), V/V. Guineensine (5) was extracted and isolated from another plant of Genus Piper L., Piper boehmeriaefolium (Miq.)C. DC. var. tomkinense C.DC. The structure of these five compounds was fully characterized by UV, IR, NMR and MS [11,13–16]. The obtained amide alkaloids were kept in a refrigerator at + 4 °C, and suspended in a vehicle (2% Tween-80 suspension in saline) using ultrasonic cleaner under 60 °C, when used in any pharmacological screening. All dosages
1087
were expressed as milligrams of the respective drugs per kilogram body weight. 2.3. Quantitative analysis of major components of ethyl acetate extract by HPLC 2.3.1. Chromatographic instrumentation and conditions A liquid chr7omatograph 105 system (LC-10AT, Shimadzu), equipped with an UV–Vis detector (SPD-10A, Shimadzu) was used. The samples were separated on a Luna ODS column (250 × 4.6 mm, 5 μm, Phenomenex). Data analyses were carried out using ChemStation software (HW2000, Dianpu Co., Ltd, China).The solvent system was composed of A (methanol) and B (water), with an elution 77(A):23(B), V/V. The flow rate was 1 ml/min. Injection volume was 20 μl and the detection was conducted using the wavelength of 259 nm. 2.3.2. Standard solution A methanolic stock solution 5 ml containing Chingchingenamide (1) 9.36 mg, Leatispiamide A (2) 10.66 mg, Leatispiamide B (3) 4.75 mg and Laetispicine (4) 14.30 mg was prepared and stored out of light at 4 °C until required for analysis. 2.3.3. Calibration curve Calibration curve of each compound was constructed by plotting the peak area against the concentration. Each calibration curve contained seven different concentrations and was performed in triplicate. For the constituents detected by UV detection, their regression equations were calculated in the form of y = bx + a, where y and x were the peak area and sample concentration, respectively. 2.3.4. Precision Instrument precision was evaluated by carrying out intraday assay. Briefly, the mixed standard solution was continuously injected six times in 1 day. 2.3.5. Recovery To evaluate accuracy, recovery tests were carried out. The powders of P. laetispicum were spiked with the mixed standard solutions at high, middle and low concentration levels. Triplicate experiments at each level were performed. The total amount of each compound was calculated from the corresponding calibration curve, the detected amount of each compound was calculated by subtracting the amount of each compound contained in the herb from the total amount of each compound after spiking. The recovery of each compound was obtained using the following equation: Recovery (%) = (Amount detected) / (Amount spiked) × 100%. 2.4. Animals and grouping Adult KM mice were purchased from the Department of Experimental Animal Center of Fudan University, and were housed under a normal 12 h light/dark cycle with lights on at 07:00 a.m. The mice had free access to tap water and food. Ambient temperature and relative humidity were maintained at 22–25 °C and 55 ± 10%, respectively. Prior to the test procedure, mice were acclimatized to the laboratory for at
1088
H. Xie et al. / Fitoterapia 82 (2011) 1086–1092
least 5 days. All behavioral tests were conducted during the light cycle. The experiment procedures were conducted in compliance with the National Institutes of Health Guide for Care and Use of the laboratory animals, and were approved by the Local Bioethics Committee (School of Pharmacy, Fudan University, China; document number: SYXK2007-002). Every effort was made to minimize the number and suffering of the animals used. 2.5. Drugs Acetic acid, formaldehyde, acetylsalicylic acid (ASA) and Tween-80 were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China), morphine hydrochloride from Shenyang First Pharmaceutical Company (Shenyang, China), and fluoxetine from Shanghai Zhongxi Pharmaceutical Co., Ltd (Shanghai, China). 2% Tween-80 suspension in saline was used as vehicle in the pharmacological tests. 2.6. Pharmacological tests 2.6.1. Forced swimming test (FST) A modification method of the FST [17] was carried out. Briefly, male mice were individually forced to swim for 15 min in a glass cylinder (height: 20 cm, diameter: 14 cm), filled with 10 cm high water at 25 °C ± 1 °C, which is a pretest. Twenty-three hours later, male mice were treated orally with EAE or amide alkaloids. One hour later, mice were placed in the cylinders again for a 6 min test in the same system depicted above. The immobility time during the final 4 min was recorded by competent observers manually. Immobility time was defined as the time spent by the mouse floating in the water without struggling, and making only those movements necessary to keep its head above the water. Fluoxetine was used as positive control drug. For EAE, the male mice were divided into five different groups (n = 10 per group), i.e., control (vehicle) group, F. (fluoxetine) 50 mg/kg group, EAE 30 mg/kg group, EAE 60 mg/kg group, EAE 120 mg/kg group. The amide alkaloids used in FST were all 20 mg/kg. There were also 10 male animals in each group. 2.6.2. Open field test The open field test method used in the present study was similar to that described previously by [9]. The apparatus was a square arena (diameter: 40 cm; height: 20 cm) with a light source of 120 lx, which was demarcated into 16 equal areas. The mice, when they finished FST, were placed in the center and their behavior was noted immediately and continued for 5 min. The score locomotion (number of line crossings) and rearing frequencies (number of times an animal stood on its hind legs) were recorded. 2.6.3. Acetic acid-induced writhing test According to previously described techniques [18,19], the acetic acid-induced writhing test was adopted. Mice (n = 10 per group) were treated orally 1 h before intraperitoneal injection of 1% acetic acid solution (10 ml/kg) with EAE at doses of 30, 60 and 120 mg/kg. The control group received only vehicle (10 ml/kg), and the reference group received
ASA (100 mg/kg). The number of writhes occurring between 5 and 25 min after acetic acid injection was recorded. 2.6.4. Formalin test The method used for formalin test was similar to that described by Hunskaar and Hole [20] with slight modifications. Mice (n = 10) were orally treated with EAE (30, 60 and 120 mg/kg, p.o.), morphine hydrochloride (7.5 mg/kg, s.c.) or 2% Tween-80. After 1 h, 20 μl of 2% formalin (v/v, in 0.9% saline) was injected subcutaneously into the plantar surface of the left hind paw of the mice. Immediately after, each mouse was placed into a glass cylinder provided with mirrors to enable a total panorama of the nociceptive behavior. The mice were observed for 60 min, the amount of time spent licking and biting the injected paw was recorded. The first 10 min is known as the first phase and the period during the following 50 min as the second phase. 2.7. Statistical analysis of data Data obtained were expressed as mean ± S.E.M. Statistical significance between groups was performed by the application of analysis of variance ANOVA followed by Bonferroni's test. P-values less than 0.05 (p b 0.05) were used as the significant level. 3. Results and discussion 3.1. Effects of EAE 3.1.1. Effect of EAE on immobility time in the mouse FST The forced swimming test, described originally by [17], is the most widely used model to screen new antidepression drugs. There are a wide variety of antidepressants and compounds with potential antidepressant-like activity to reduce the duration of immobility in the FST [17,21]. In the present study, we first investigated the antidepressant effects of ethyl acetate extract (EAE) .The results (Fig. 1) clearly showed that the treatment with doses of 60 and 120 mg/kg of EAE significantly and dose-dependently decreased the immobility time (p b 0.01 and p b 0.001). In order to detect any association of immobility in the FST with changes in motor activity, the activities of animals were tested in an open field test after FST. Fig. 2 summarizes the results. The treatment animals with 30, 60 mg/kg EAE
Fig. 1. Effects of the ethyl acetate extract (EAE) on the forced swimming test in mice. Results are expressed as mean ± S.E.M. (n = 10). **p b 0.01 and ***p b 0.001 compared with control. Control: vehicle, F: fluoxetine.
H. Xie et al. / Fitoterapia 82 (2011) 1086–1092
1089
FST seemed unlikely to be due to an increase in locomotive activity and the antidepressant-like effect of it would be specific.
Fig. 2. Effects of EAE on the open field for 5 min in mice. Results are expressed as mean ± S.E.M. (n = 10). **p b 0.01 compared with control. Control: vehicle.
Fig. 3. Effects of Laetispicine on acetic acid induced writhing test in mice. Data are expressed as mean ± S.E.M. (n = 10). ***p b 0.001 compared with control. Control: vehicle, A: acetylsalicylic acid.
showed no differences compared with control animals. Interestingly, the 120 mg/kg EAE group can decrease the score locomotion and rearing frequencies. There was significant difference compared with the control (p b 0.01). That suggested that the antidepressant actions of EAE in the mouse
3.1.2. Antinociceptive effect of EAE Previous studies [22] have suggested that the positive results obtained with antidepressants in nociceptive tests. And the P. laetispicum was usually used for analgesic. Then, we evaluated the antinociceptive effect of EAE using two models: acetic acid-induced writhing test and formalin test. The acetic acid-induced writhing test has long been used as a screening tool for the assessment of analgesic or antiinflammatory properties [18,19]. In the present study, the effect of EAE (120 mg/kg, 64.51%) was comparable to that of ASA (100 mg/kg, 61.64%) (Fig. 3), and there was a significant reduction (p b 0.001) in comparison to the control (Fig. 3). It is suggested that EAE at doses of 120 mg/kg had powerful antinociceptive effect. The results of formalin test were showing in Fig. 4, morphine hydrochloride (7.5 mg/kg) the reference drugs, significantly suppressed the formalin response in the first phases by 50.40%, and second phases by 57.12%, respectively. For EAE, during the first phase (0–10 min), only at doses of 120 mg/kg significantly reduced the licking activity by 32.75% (p b 0.05). In regard to the second phase (10–60 min), EAE at all given doses had a respectively significant effect on the diminished licking time by 42.24%, 32.37%, 44.60% (p b 0.05), as compared with the control group. The acetic acid-induced writhing test suggested that the abdominal writhing induced by acetic acid involves the production and release of arachidonic acid metabolites via cycloxygenase (COX) and prostaglandin biosynthesis; an increase in the peritoneal fluid levels of PGE1, PGF2 and lipooxygenase products; release of sympathetic nervous system mediators; and it might also cause the release of cytokines, such as TNF-α, interleukin-1β, and interleukin-8, by resident peritoneal macrophages and mast cells [20,23,24]. The formalin pain test is a very useful model of clinical pain in which the first phase seems to be due to direct chemical stimulation of nociceptors, whereas the second phase is dependent on peripheral inflammation and changes in
Fig. 4. Effects of EAE on formalin test in mice. Data are expressed as mean ±S.E.M. (n = 10). *p b 0.05, **p b 0.01 and ***p b 0.001 compared with control. Control: vehicle, M: morphine.
1090
H. Xie et al. / Fitoterapia 82 (2011) 1086–1092
Fig. 5. HPLC chromatogram of the ethyl acetate extract from P. laetispicum. Peak markers 1: Chingchingenamide, 2: Leatispiamide A, 3: Laetispiamide B, 4: Laetispicine.
central processing [25]. Drugs which act mainly centrally, such as narcotic analgesics, inhibit both phases of pain in this model while peripherally acting drugs, such as aspirin or indomethacin, only inhibit the second phase [26].
That suggested EAE at doses of 30 mg/kg and 60 mg/kg only possess peripheral antinociceptive effect, at doses of 120 mg/kg the antinociceptive effect may due to direct result of stimulation of nociceptors and inflammation occurring following the release of serotonin, histamine, bradykinnin and prostaglandins.
3.2. Chemical analysis of the ethyl acetate extract prepared from P. laetispicum
Fig. 6. Chemical structures of compounds Chingchingenamide (1), Leatispiamide A (2), Leatispiamide B (3), Laetispicine (4) and Guineensine (5).
In order to expound the active ingredients of EAE, a chromatogram of the ethyl acetate extract was obtained, according to the HPLC conditions described above (Fig. 5). Four amide alkaloids were identified as the major components of this extract. These are Chingchingenamide (1), Leatispiamide A (2), Leatispiamide B (3) and Laetispicine (4). Their molecular structures are shown in Fig. 6. The calibration curves of these four amide alkaloids showed good linear regression with high correlation coefficients (r N 0.9999) within the ranges tested, as shown in Table 1. The precision was below 3% (RSD) (Table 1). The recoveries for all the analytes were between 98.43% and 101.23%, RSDs were between 0.47% and 0.97%. That means the method is precise, sensitive and shows a high accuracy for the simultaneous quantification of the four amide alkaloids. The content of these four amide alkaloids in EAE was calculated by this method. As showing in Table 1, Leatispiamide A (2) and Laetispicine (4) were major compounds of it.
Table 1 Quantitative analysis of major components of EAE by HPLC. Analytes
Calibration curves (n = 7)
Correlation coefficients
Linearity range (mg/L)
Precision RSD
Content/EAE (mg/g)
Ch La A La B L
y = 62226x + 51699 y = 59231x + 40222 y = 138765x − 66610 y = 57300x + 7964.7
r = 0.9999 r = 0.9999 r = 1.000 r = 1.000
1.87–187.20 2.13–213.20 1.90–190.00 2.86–286.00
0.35% 0.94% 0.43% 1.47%
1.24 12.68 1.25 9.64
Ch: Chingchingenamide, La A: Leatispiamide A, La B: Leatispiamide B, L: Laetispicine.
H. Xie et al. / Fitoterapia 82 (2011) 1086–1092
1091
3.3. Structure of amide alkaloid with antidepressant-like effect of EAE Laetispicine (4) from P. laetispicum has been proven to possess antidepressant activities [12]. Its pharmacokinetic characteristics in plasma and brain distribution in rats also have been examined [27]. In this paper, the antidepressant effects of four major amide alkaloids of EAE, Chingchingenamide (1), Leatispiamide A (2), Leatispiamide B (3), Laetispicine (4) and Guineensine (5) (Fig. 6), another amide alkaloid extracted from P. boehmeriaefolium (Miq.)C. DC. var. tomkinense C.DC., were evaluated and the result was showed in Fig. 7. Only Leatispiamide A (2) and Laetispicine (4) significantly decreased the immobility time by 29.13% and 32.58% in FST. An open field test also was used to detect any changes in motor activity of animals. The results were shown in Fig. 8. There were no significant differences compared with control animals. That suggested that the antidepressant-like effect of Leatispiamide A (2) would be specific either, similar with Laetispicine (4). The view of the structures of these five amide alkaloids was similar, except some double bond locations in the carbon chain. The difference among them was analyzed. Chingchingenamide (1), Leatispiamide A (2), Laetispicine (4) and Guineensine (5) all have conjugated double bond located at 2–3 and 4–5. Among them Leatispiamide A (2) and Laetispicine (4) have isolated double bond from benzene ring, correspondingly Gu has a conjugated double bond with benzene ring and Chingchingenamide (1) has no other double bond in carbon chain. Leatispiamide B (3) also has isolated double bond from benzene ring, same as Leatispiamide A (2) and Laetispicine (4), but it has no conjugated double bond at 2–3 and 4–5. The result of pharmacological tests revealed that isolated double bond from benzene ring and conjugated double bond located at 2–3 and 4–5 are necessary for its antidepressant activities. 4. Conclusion The present study demonstrated that the ethyl acetate extract (EAE) from P. laetispicum had significantly antidepressant-like effect which was not due to an increase in locomotive activity. Its effective dosage was 60 mg/kg. The
Fig. 7. Effects of major amide alkaloids on the forced swimming test in mice. 4: Laetispicine, 2: Leatispiamide A, 3: Leatispiamide B, 1: Chingchingenamide, 5: Guineensine.
Fig. 8. Effects of Laetispicine and Leatispiamide A on the open field for 5 min in mice. Results are expressed as mean ± S.E.M. (n = 10). Control: vehicle, 4: Laetispicine, 2: Leatispiamide A.
effective components of EAE were Leatispiamide A (2) and Laetispicine (4). The isolated double bond from benzene ring and conjugated double bond located at 2–3 and 4–5 in the molecular structure were necessary for its antidepressant activities. The ethyl acetate extract (EAE) also presented antinociceptive effect, in our studies. At lower doses (30 mg/kg) the antinociceptive effect was likely mediated via peripheral inflammation and changes in central processing, and at higher doses (120 mg/kg) that was due to both central and peripheral pathways. The possible mechanisms deserve further attention.
Acknowledgments The study was supported and funded by the Ministry of Science and Technology of the People's Republic of China (no. 2009zx09103-075), Science Committee of Shanghai, China (no. 08DZ1971203), and Pudong New District of Shanghai, China (no. PW2008A-7).
References [1] Kessler RC, Berglund P, Demler O, Jin R, Koretz D, Merikangas KR, et al. The epidemiology of major depressive disorder: results from the National Comorbidity Survey Replication (NCS-R). JAMA 2003;289: 3095–105. [2] Nemeroff CB. The burden of severe depression: a review of diagnostic challenges and treatment alternatives. J Psychiatr Res 2007;41: 189–206. [3] Patten SB. Major depression prevalence is very high, but the syndrome is a poor proxy for community populations' clinical treatment needs. Can J Psychiatry 2008;53:411–9. [4] Gaudiano BA, Young D, Chelminski I, Zimmerman M. Depressive symptom profiles and severity patterns in outpatients with psychotic vs nonpsychotic major depression. Compr Psychiatry 2008;49:421–9. [5] Kennedy SH. A review of antidepressant treatments today. Eur Neuropsychopharmacol 2006;16:619–23. [6] Linde K, Berner MM, Kriston L. St John's wort for major depression. Cochrane Database Syst Rev 2008;8:CD000448. [7] Baum SS, Hill R, Rommelspacher H. Effect of kava extract and individual kavapyrones on neurotransmitter levels in the nucleus accumbens of rats. Prog Neuropsychopharmacol Biol Psychiatry 1998;22:1105–20. [8] Dang HX, Chen Y, Liu XM, Wang Q, Wang LW, Jia W, et al. Antidepressant effects of ginseng total saponins in the forced swimming test and chronic mild stress models of depression. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:1417–24. [9] Kim SH, Han J, Seog DH, Chung JY, Kim N, Park YH, et al. Antidepressant effect of Chaihu-Shugan-San extract and its constituents in rat models of depression. Life Sci 2005;76:1297–306.
1092
H. Xie et al. / Fitoterapia 82 (2011) 1086–1092
[10] Zhonghua Herbals Editorial Committee. Zhonghua Herbals. Shanghai: Shanghai Science Technique Press; 1999. p. 433–4. [11] Pan SL, Xie J, Qian FG, Wang J, Shao YC. Antidepressant amidesfrom Piper laetispicum C. DC. Acta Pharmacol Sin 2005;40:355–7. [12] Yao CY, Wang J, Dong D, Qian FG, Xie J, Pan SL. Laetispicine, an amide alkaloid from Piper laetispicum, presents antidepressant and antinociceptive effects in mice. Phytomedicine 2009;16:823–9. [13] Parmar VS, Sinha R, Shakil NA, Tyagi OD, Boll PM, Wengel A. An insecticidal amide from Piper falconerl. Indian J Chem 1993;32B:392–3. [14] Fang J, Qian FG, Xie J. Studies on the chemical constituents of root and stem of Piper laetispicum C. DC. Chin Tradit Herb Drugs 2006:1882–901 (Supp l). [15] Fang J, Xie J, Shou YC, Qian FG. Studies on the chemical constituents of root and stem of Piper laetispicum C. DC. (II). Chin Tradit Herb Drugs 2007;38:1289–92. [16] Gupta OP, Dhar KL, Atal CK. Structure of new amide from Piper officinarum. Phytochemistry 1976;15:425. [17] Porsolt RD, Le Pichon M, Jalfre M. Depression: a new animal model sensitive to antidepressant treatments. Nature 1977;266:730–2. [18] Koster R, Anderson M, Beer EJ. Acetic acid analgesic screening. Fed Proc 1959;18:412. [19] Duarte IDG, Nakamura M, Ferreira SH, Braz J. Participation of the sympathetic in acetic acid-induced writhing in mice. Med Biol Res 1988;21:41.
[20] Hunskaar S, Hole K. The formalin test in mice—dissociation between inflammatory a noninflammatory. Pain 1987;30:103–14. [21] Cryan JF, Markou A, Lucki I. Assessing anti-depressant activity in rodents: recent developments and future needs. Trends Pharmacol Sci 2002;23:238–45. [22] Eschalier A, Ardid D, Coudore F. Pharmacological studies of the analgesic effect of antidepressants. Clin Neuropharmacol 1992;15:373–4. [23] Guzzo LS, Saude-Guimaraes DA, Silva ACA, Lombardi JA, Guimaraes HN, Grabe-Guimaraes A. Antinociceptive and anti-inflammatory activities of ethanolic extracts of Lychnophora species. J Ethnopharmacol 2008;116:116–20. [24] Elisabetsky E, Amador TA, Albuquerque RR, Nunes DS, Carvalho ACT. Analgesic activity of Psychotria colorata (wild ex Ret S) Muell. Arg Alkaloids J Ethnopharmacol 1995;48:77–83. [25] Tjolsen A, Berge OG, Hunskaar S, Rosland JH, Hole K. The formalin test: an evaluation of the method. Pain 1992;51:5–17. [26] Santos ARS, Filho VC, Niero R, Viana AM, Moreno FN, Campos MM, et al. Analgesic effects of callus-culture extracts from selected species of Phyllanthus in mice. J Pharm Pharmacol 1994;46:755–9. [27] Wang Y, Xie H, Pan SL. Pharmacokinetics of Laetispicine and its brain distribution in Rats. Am J Chin Med 2010;38:895–907.