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Journal of Ethnopharmacology 127 (2010) 521–527

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l homepage: www.e lsev ier .com/ locate / je thpharm

he antinociceptive activity of Polygonatum verticillatum rhizomes inain models

aroon Khana, Muhammad Saeeda,∗, Anwal-Ul-Hassan Gilanib, Murad Ali Khanc,hsana Dard, Inamullah Khana

Department of Pharmacy, University of Peshawar, Peshawar 25120, PakistanDepartment of Biological and Biomedical Sciences, Aga Khan University, Stadium Road, P.O. Box 3500, Karachi 74800, PakistanDepartment of Chemistry, Kohat University of Science and Technology, Kohat, PakistanInternational Centre for Chemical Sciences, HEJ Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan

r t i c l e i n f o

rticle history:eceived 14 May 2009eceived in revised form0 September 2009ccepted 5 October 2009vailable online 22 October 2009

eywords:olygonatum verticillatumiliaceaentinociceptiveiureticaponinslkaloids

a b s t r a c t

Aim of the study: The current study was designed to establish the pharmacological rationale for thetraditional use of the rhizomes of Polygonatum verticillatum in the treatment of painful conditions and asa plant diuretic.Materials and methods: The crude methanolic extract of the rhizomes of Polygonatum verticillatum (PR)was tested in various established pain models in rodents at 50, 100 and 200 mg/kg i.p. while the diureticactivity was assessed at 300 and 600 mg/kg p.o. in rats.Results: PR demonstrated significant reduction (14–72%) in the number of writhes induced by acetic acidin a dose-dependent manner. When nociceptive threshold was measured in the formalin test, PR stronglyattenuated the formalin-induced flinching behaviour in both phases (6–30% in first phase while 12–72%in second phase). Central involvement in the analgesic profile of PR was confirmed by the hot plate test, inwhich PR elicited a significant (P < 0.01) analgesic activity by increasing latency time. However, an opioidreceptor antagonist, naloxone (2 mg/kg s.c.) strongly antagonized the antinociceptive activity of PR. As

a plant diuretic, PR showed mild but statistically insignificant diuretic activity at 300 mg/kg. The crudeextract and solvent fractions of the plant contained reasonable quantity of total saponin and alkaloidcontents.Conclusions: The mechanisms underlying the analgesic action of PR shows that the opioid dependantcentral mediation has synergistic effect by enforcing the peripheral analgesic effects. Interestingly, ourfindings not only substantiated the folk use of the plant as an analgesic but also reported for the first time in the whole genus.

. Introduction

Polygonatum (King Solomon’s-seal, Solomon’s Seal) is a genus ofbout 57 species belongs to family Liliaceae or Convallariaceae. Its widely distributed in East Asia, mainly China and Japan where0 species of Polygonatum are found (Tamura, 1993; Monika et al.,006). Solomon’s seal has been used for thousands of years in herbaledicine. The rhizomes are antiperiodic, antitussive, cardiotonic,

emulcent, diuretic, energizer, hypoglycemic, sedative, tonic and

re used in the treatment of dry coughs and pulmonary problems,ncluding tuberculosis (Jiang, 1977, 1986; Flora of China, 1980). Thentibacterial and antifungal activity of the Polygonatum has beeneported (Alluri et al., 2006). Polygonatum reduced blood sugar level

∗ Corresponding author. Tel.: +92 919216750.E-mail address: saeedrph2000@yahoo.com (M. Saeed).

378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2009.10.003

© 2009 Elsevier Ireland Ltd. All rights reserved.

by different mechanisms including �-glucosidase inhibition andincreased insulin sensitivity (Li et al., 2004; Hong et al., 2008). Thecytotoxic activity with human MCF-7 breast cancer cells of varioussteroidal saponins isolated from the Rhizomes of Polygonatum isalso on the record (Mi-Jeong et al., 2006).

Polygonatum verticillatum [L.] All. (Nooreallam) is a perennialrhizomatous herb. The rhizomes are usually shortly branched and0.7–1.5 cm thick. Stem is usually erect and the leaves are four toeight in a whorl; the flowers are hermaphrodite. The syrup of freshrhizome is used in the treatment of pain, pyrexia, burning sensa-tion and for phthisis (Amrit, 2006). It is also used in combinationwith other herbs to promote urine discharge (diuretic) and removes

painful urine (Ballabh et al., 2008). Other ethnobotanical uses ofthe plant include as emollient, aphrodisiac, vitiated condition ofpitta and vata, appetizer and tonic, galactagogue (increases milkrelease), weakness (Ghayur, 2004). It is also used as a substituteof Polygonatum cirrhifolium (Parveen et al., 2004). Keeping in view

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22 H. Khan et al. / Journal of Ethn

he ethnobotanical use of the plant in the treatment of painfulonditions, the current study was designed to provide scientificackground to this claim.

. Materials and methods

.1. Plant material

Polygonatum verticillatum All was collected from District SwatGabral and Miandam valleys), N.W.F.P, Pakistan, in July–August007. The botanical identity of the plant material was done byhe Taxonomy Department of PCSIR Laboratories Peshawar andspecimen with catalogue No.: 9970 (PES) was submitted to theerbarium of PCSIR Laboratories Peshawar.

.2. Plant extraction and fractionation

The air dried rhizomes of the Polygonatum verticillatum (8 kg)as ground to fine powder and extracted by maceration withethanol at room temperature for 14 days with occasional shaking

Khan et al., 2007, 2009). The methanol soluble residue was filteredff and concentrated under vacuum at low temperature (40 ◦C)sing rotary evaporator, yielded a dark greenish semisolid mate-ial (2.2 kg, 27.50%, w/w). The crude methanol extract (1.6 kg) wasissolved in distilled water and sequentially fractionated with hex-ne, chloroform, ethyl acetate and butanol, yielding hexane (258 g,6.13%, w/w), chloroform (219 g, 13.69%, w/w), ethyl acetate (226 g,4.13% w/w), butanol (265 g, 16.56%, w/w), and aqueous (501 g,1.31%, w/w). These fractions were then screened for various phar-acological and phytochemical analysis.

.3. Animals

Swiss albino mice (20–25 g) and Wistar rats (210–270 g) ofither sex were used in various experimental models. Animals wereoused 10 per cage and were fed laboratory diet ad libitum andllowed free access to drinking water under standard environmen-al condition of temperature (25 ◦C), relative humidity 60 ± 10% andight/dark cycles (12/12 h). Experiments were performed accordingo ethical principles established in 1979 for laboratory animals athe service of mankind Lyons, France.

.4. Drugs and reagents

The chemicals used in this study include: aspirin (Reckitt andolman, Pakistan), acetic acid, formalin, morphine sulphate, nalox-ne hydrochloride, sodium chloride hydrochlorothiazide (Sigmahemicals company, St. Louis, USA) Sterile normal saline was useds control in all studies and the crude methanol extract (PR) usedn various studies were prepared in normal saline.

.5. Acute toxicity studies

The acute toxicity test for PR was carried out to evaluate anyossible toxicity. Swiss albino mice (n = 6) of either sex were testedy administering different doses of PR by increasing or decreasinghe dose, according to the response of animal (Bruce, 1985). Theosing patron was 500, 1000 and 2000 mg/kg p.o., while the controlroup received only the normal saline. All the groups were observedor any gross effect or mortality during 24 h.

.6. Antinociceptive activity

.6.1. Visceral pain model (acetic acid-induced abdominalonstriction)

The peripheral nociceptive activity was investigated by usinghe acetic acid-induced abdominal constriction test (Koster et al.,

acology 127 (2010) 521–527

1959; Adzu et al., 2001). Briefly, the prescreened animals weredivided into five groups (n = 6). The writhes were induced byintraperitoneal injection of 1.0% acetic acid (v/v, 0.1 ml/10 g bodyweight). Group I was used as control, received normal saline(10 ml/kg, i.p.); groups II, III and IV were treated with PR (50,100 and 200 mg/kg, i.p.), respectively; group V received aspirin(100 mg/kg i.p.), as a standard drug. The number of muscular con-tractions was counted over a period of 20 min after acetic acidinjection. The number of writhes in each treated group was com-pared with control (saline treated group) and the percent inhibitionof the writhes was calculated.

2.6.2. Formalin testThe method used in our study for the assessment of formalin-

induced flinching behaviour in normal rats was describedpreviously (Dubuisson and Dennis, 1977; Tjolsen et al., 1992). Inthis method, 0.05 ml of formalin (2.5% formaldehyde) was injectedinto the plantar surface of the right hind paw, 30 min after treatingthe animals with the extracts (50, 100 and 200 mg/kg i.p.). Noci-ceptive behaviour was quantified as rat walking or can stand oninjected paw; paw partially elevated; total elevation of injectedpaw, injected paw licking or biting. Formalin injection induced astereotyped response characterized by two well distinct phases;phase I started almost immediately and was short lasting (0–5 min)followed, by prolonged tonic phase II lasting (15–30 min). Mor-phine (3 mg/kg s.c.) was used as a standard drug.

2.6.3. Thermal nociception (hot plate test)In thermal nociception (hot plate test) mice were screened by

placing them on a hot metal plate maintained at 50 ± 0.05 ◦C (Daret al., 2005). The mice were treated either with vehicle (10 ml/kgi.p.), PR (50, 100 and 200 mg/kg i.p) or morphine (10 mg/kg s.c.)an opioid analgesic as a standard drug. Thermal nociception wasestimated by measuring withdrawal response latency in the formof jumping, withdrawal of the paws or the licking of the paws.In the pretreatment session, mice were tested on two separatedoccasions, each 30 min apart and then only those mice wereselected for the study, which responded within 15 s and whichshowed comparatively similar results. The response latencies wererecorded at 0, 30, 60, 90 and 120 min with a cut off period of30 s to avoid damage to the paw in the absence of response. Inorder to investigate the participation of the opioid system in theantinociceptive effect of PR, naloxone hydrochloride (2 mg/kg s.c.),a non-selective opioid receptor antagonist was injected, 15 minprior to the administration of test samples, as explained above andthe hot plate latencies were sequentially measured at 0, 30, 60, 90and 120 min.

2.7. Diuretic activity

The diuretic activity of PR was determined by method previouslyestablished (Jabeen et al., 2009). Male Albino rats were divided into four groups (n = 6). The animals were fasted for 24 h and werefed laboratory diet ad libitum and allowed free access to drink-ing water. On the day of experiment, the animals of group I wastreated with saline (15 ml/kg p.o) and this group served as con-trol. Similarly, the animals of group II, III and IV were administeredhydrochlorothiazide (10 mg/kg p.o) as standard drug, PR (300 and600 mg/kg p.o), respectively. The extract was dissolved in saline and

treatment, the animals were placed in metabolic cages (1 animalin each metabolic cage). Urine was collected in graduated cylin-ders and its volume was recorded at 2, 3 and 6 h. Cumulative urineexcretion was calculated in relation to body weight and expressedas ml/100 g body weight.

pharmacology 127 (2010) 521–527 523

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Fig. 1. Effect of intraperitoneal administration of PR (50, 100, 200 mg/kg) in acetic

60, 90 and 120 min after the administration of vehicle, PR and stan-dard drug morphine. The peak protection against thermal pain wasachieved at 90 min of PR administration compared to vehicle. Theinvolvement of opioid receptor was assessed by the administration

H. Khan et al. / Journal of Ethno

.8. Phytochemical analysis

.8.1. Total saponin contentsSaponin contents were determined for the crude extract

nd solvent fractions according to the method of Tomowa andjulemetowa (1978). Briefly, 2 g of test samples were taken in aeaker and 50 ml of petroleum ether was added and heated gen-ly on water-bath to 40 ◦C for 5 min with regular shaking. Theetroleum ether was filtered and repeated the operation twice withurther 50 ml of pet ether. The marc obtained was extracted with× 60 ml of methanol on gentle heating. The methanol layer wasoncentrated to approximately 25 ml on water-bath and 150 mlf dry acetone was added to precipitate the saponins, which wasollowed by filtration and drying in oven at 100 ◦C for constanteight.

.8.2. Total alkaloid contentsThe total alkaloid contents of PR and subsequent solvent frac-

ions of Polygonatum verticillatum was estimated by using methodeveloped previously (Obadoni and Ochuko, 2001). Briefly, 2 g ofach was defatted by extraction with petroleum ether, heated gen-ly on water-bath to 40 ◦C for 5 min with regular shaking. The

arc obtained was acidified with 100 ml of 20% acetic acid inthanol and allowed to extract for 4 h. The resulting solution wasltered, concentrated and then basified with concentrated ammo-ium hydroxide to pH 9 followed by precipitation. The final weightf precipitated mass represented the total alkaloid contents.

.9. Statistical analysis

Results are presented as the mean ± S.E.M. for at least six ani-als per group. The statistical analysis of the results was carried out

sing one-way ANOVA followed by Dunnet’s multiple comparisons.he criterion for statistical significance was P < 0.05.

. Results

.1. Acute toxicity

No gross behaviour effect or mortality was observed during thessessment time (24 h), therefore concluded to be safe up to 2 g/kg.o.

.2. Effect of acetic acid-induced abdominal constriction

PR demonstrated a marked and significant (P < 0.01) reduction15–72%) in the number of writhes induced by acetic acid. As shownn Figs. 1 and 2, the analgesic activity at all tested doses (50, 100nd 200 mg/kg i.p.) indicated a dose-dependent relationship inhe inhibition of writhing reflux and the analgesic response of PRt 200 mg/kg was quite similar to standard drug aspirin (77% at00 mg/kg i.p.).

.3. Effect of formalin-induced pain

The formalin-induced flinching behaviour was strongly atten-ated by PR in both phases, as compared to control. As shown inFig. 3A and B), the pain relieving potential of the PR was morerominent and significant (P < 0.01) in the first phase of injectedormalin as compared to second phase. The antinociceptive effectas found dose-dependent in both phases. Morphine (5 mg/kg s.c.)

ffered marked and significant (P < 0.01) reduction in pain inducedy formalin in both the phases.

acid-induced writhing in mice. Values are reported as mean ± S.E.M. for group of sixanimals. The data were analyzed by ANOVA followed by Dunnett’s test. Asterisksindicated statistically significant values from control. *P < 0.05, **P < 0.01.

3.4. Effect of thermal nociception

As presented in Table 1, PR elicited marked analgesic activity inthe thermal nociceptive test at 50, 100 and 200 mg/kg. The responsewas showed by the increase in latency time in seconds. The increasein latency time was dose-dependent which was measured at 0, 30,

Fig. 2. Inhibition of writhes in percentage of intraperitoneal administration of PR(50, 100, 200 mg/kg) in acetic acid-induced writhing in test in mice. Data is expressedas mean ± S.E.M. (n = 6).

524 H. Khan et al. / Journal of Ethnopharmacology 127 (2010) 521–527

Table 1Effect of the PR on the hot plate (thermal stimuli) test with and without Naloxone in mice at 50, 100 and 200 mg/kg, i.p.

Group Dose mg/kg Latency time (mean ± S.E.M.)

0 min 30 min 60 min 90 min 120 min

Saline 10 mL/kg 7.10 ± 0.49 7.20 ± 0.13 7.35 ± 0.20 7.40 ± 0.42 7.60 ± 0.49PR 50 7.20 ± 0.49 7.35 ± 0.15 7.65 ± 0.15 8.00 ± 0.44 8.00 ± 0.44

100 7.20 ± 0.35 7.40 ± 0.17 8.50 ± 0.44 9.70 ± 0.89 9.10 ± 0.89200 7.00 ± 0.44 7.40 ± 0.13 11.50 ± 0.44** 13.70 ± 0.84** 12.90 ± 0.84**

Morphine 10 7.10 ± 0.40 7.90 ± 0.40 12.70 ± 0.17** 16.10 ± 0.89** 14.36 ± 0.93**With naloxonePR 50 7.30 ± 0.44 7.30 ± 0.58 7.60 ± 0.26 7.50 ± 0.44 7.50 ± 0.89

100 7.40 ± 0.17 7.30 ± 0.31 8.20 ± 0.44 8.10 ± 0.53 8.15 ± 0.51200 7.40 ± 0.13 7.10 ± 0.89 10.00 ± 0.89* 9.26 ± 0.51** 9.40 ± 1.11*

Morphine 10 7.60 ± 0.26 7.10 ± 0.58 7.10 ± 0.40*** 6.85 ± 0.60*** 6.85 ± 0.89***

Without naloxone, data was compared with control (Saline 10 ml/kg). With naloxone (2were compared with their respective test substances in the absence of naloxone. Valueby ANOVA followed by Dunnett’s test. Asterisks indicated statistically significant valuescomparison between 2 groups.

Fig. 3. Effect of intraperitoneal administration of PR (50,100 and 200 mg/kg) informalin-induced flinching behaviour in rats, in the 1st phase (A) and 2nd phase(B) of formalin test. Values are reported as mean ± S.E.M. for group of six animals.The data was analyzed by ANOVA followed by Dunnett’s test. Asterisks indicatedstatistically significant values from control. *P < 0.05, **P < 0.01.

mg/kg s.c.), was administered 15 min prior to extract or morphine. All the resultss are reported as mean ± S.E.M. for group of six animals. The data were analyzedfrom control. *P < 0.05, **P < 0.01, ***P < 0.001. The independent t-test was used for

of naloxone (2 mg/kg) subcutaneously, 15 min prior to the admin-istration of test samples. The administration of naloxone causedsignificant (P < 0.01) attenuation of antinociceptive activity of PR,as shown in Table 1, establishing the participation of opioid recep-tor in the central analgesic activity. Moreover, morphine inducedanalgesia was completely abolished by the presence of naloxone.

3.5. Effect of diuretic activity

The diuretic activity of PR was evaluated at 300 and 600 mg/kg, ispresented in Fig. 4. The data indicating that PR exhibited mild butstatistically insignificant diuretic activity at 300 mg/kg p.o. com-pared to control. Surprisingly, the diuretic activity was markedlydecreased at the dose of 600 mg/kg as compared to 300 mg/kg andwas quite similar to normal saline (Fig. 5).

3.6. Effect of total saponins and alkaloids content

The crude extract and subsequent solvent fractions of PR pos-sessed considerable concentration of total saponins and alkaloidsas shown in Figs. 6 and 7.

Fig. 4. Protection (%) of PR (50, 100 and 200 mg/kg i.p.) in the both phases offormalin-induced pain in rats. Symbols represent mean ± S.E.M. (n = 6).

H. Khan et al. / Journal of Ethnopharmacology 127 (2010) 521–527 525

Fig. 5. Effect of oral administration of PR (300 and 600 mg/kg) in rats as urine vol-ume per 100 kg body weight after 6 h. Hydrochlorothiazide (HCT; 10 mg/kg o.p.)wTs

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Fig. 7. Total alkaloids contents in crude extract and subsequent solvent fractions of

as standard drug. Values are reported as mean ± S.E.M. for group of six animals.he data were analyzed by ANOVA followed by Dunnett’s test. Asterisks indicatedtatistically significant values from control. *P < 0.05, **P < 0.01.

. Discussion

Pain management for patients with chronic pain is a difficultask because of the risks associated with toxicity due to the drugntervention (Sam, 2008). Non-steroidal anti-inflammatory drugsNSAIDs) are the most widely prescribed medications for the man-gement of painful conditions, but they are frequently causing

astrointestinal damage (Fiorucci et al., 2001). Ethnobotanicals toreat diseases is a therapeutic modality, which has stood the testf time for the treatment of various ailments (Gilani and Atta-ur-ahman, 2005). To explore new effective and safe analgesic for the

ig. 6. Total saponins contents in crude extract and subsequent solvent fractions ofhizomes of Polygonatum verticillatum All. PR-1: crude methanol extract, PR-2: hex-ne fraction, PR-3: chloroform fraction, PR-4: ethyl acetate fraction, PR-5: butanolraction, PR-6: aqueous fraction. Values are expressed as mean ± S.E.M. (n = 3).

rhizomes of Polygonatum verticillatum All. PR-1: crude methanol extract, PR-2: hex-ane fraction, PR-3: chloroform fraction, PR-4: ethyl acetate fraction, PR-5: butanolfraction, PR-6: aqueous fraction. Values are expressed as mean ± S.E.M. (n = 3).

management of different painful conditions, the crude methanolextract (PR) was screened in various pain models, along withdiuretic activity.

PR showed marked antinociceptive activity in various pain mod-els including visceral pain model, formalin test and hot platetest. Acetic acid-induced writhing is a highly sensitive and use-ful test for analgesic drug development especially peripherallyacting analgesics. Acetic acid induces pain by liberating endoge-nous substances (bradykinin, serotonin, histamine, substance P)(Tsung-Chun et al., 2007). Hyperalgesia induced by the injectionof acetic acid is characterized by contraction of the abdominalmuscle accompanied by an extension of the forelimbs and bodyelongation. These peripheral nociceptive fibers are sensitive to bothnarcotics analgesic (morphine) and non-steroid anti-inflammatorydrugs like aspirin (Wibool et al., 2008). In our observation, PR sig-nificantly (P < 0.01) reduced the abdominal constriction responseinduced by the acetic acid in a dose-dependent manner. There-fore, one possible mechanism of antinociceptive activity of PRcould be due to the blockade of the effect or the release ofendogenous substances (arachidonic acid metabolites) that excitepain nerve endings by the pharmacologically active principles ofPR.

From a mechanistic point of view, the lack of specificity inacetic acid-induced writhing test suggesting the involvement ofdifferent nociceptive mechanisms in the reduction of muscularconstriction such as sympathetic system through the release ofbiogenic amines, cyclooxygenases and their metabolites inhibi-tion and through opioids receptors mechanisms (Andrade et al.,2007). This deficiency can be overcome by using other paradigms.The formalin test is used as a primary behavioural screen forthe assessment of the antinociceptive activity of compounds usedin moderate, long lasting clinical pain. In rats, the spontaneousnociceptive responses are typically characterized by lifting, bit-ing/licking and flinching of the hind paw after the injection offormalin into either the dorsal or plantar surface of the paw (Nisar

et al., 2008). Formalin test produced a distinct biphasic response.First neurogenic phase (0–5 min) occurs within seconds of formalininjection as a direct result of chemical stimulation of peripher-ally localized TRPA-1 containing nociceptors (McNamara et al.,

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26 H. Khan et al. / Journal of Ethn

007). The second, later tonic phase (25–30) occurs as a resultf increased primary afferent drive with subsequent sensitiza-ion of nociceptive spinal neuron. From a mechanistic point ofiew, different analgesics may act differently. Centrally acting drugsuch as opioid inhibit both phases equally but peripherally actingrugs, such as cyclooxygenase inhibitors (aspirin, indomethacinnd corticosteroids) (Morteza et al., 2004) inhibits only the latehase.

During our investigation in the formalin test, PR significantlyP < 0.01) attenuated the hyperalgesia produced by formalin injec-ion in both phases. The pain relieving effect was comparatively

ore promising in the late phase and was dose-dependent. There-ore, the antinociceptive activity of PR in formalin test is stronglyttributed to peripherally acting as well as centrally acting painediators. To confirm the participation of central system in the

ntinociceptive activity of PR, hot plate test was employed. Hotlate test is predominantly a spinal reflex and used to test supra-pinal analgesia in compounds. It is therefore, selective for centrallycting analgesic drugs, like morphine, while peripheral antinoci-eptive agents are found to be inactive on thermal stimulusYu-Feng et al., 2008).

PR exhibited marked inhibition on thermally induced hyper-lgesia. The PR possesses significant (P < 0.01) activity 200 mg/kgfter 60, 90 and 120 min of drug administration. Morphine10 mg/kg s.c.) being standard drug, showed more potent activitynd was increased with time up to 120 min. Therefore, the resultf our study showed the central mediation in the antinociceptivectivity of PR. For the assessment of opioid system involvementn the analgesic activity of PR, a non-selective opioid receptorntagonist, naloxone (2 mg/kg s.c.) was administered. The anal-esic activity of PR was significantly (P < 0.001) antagonized by thedministration of naloxone and it is therefore, confirmed the opioideceptors participation in the attenuation of thermal hyperalgesia.R and subsequent solvent fractions of the rhizomes of plant pos-essed responsible quantity of saponins and alkaloids (Figs. 6 and 7).he antinociceptive potential of saponins and alkaloids are wellocumented in literature (Dilipkumar et al., 2005; Kumar andemmani, 2002; Farouk et al., 2008), therefore, the saponins and/orlkaloids of PR could be the antinociceptive constituents. Moreover,o the best of our knowledge on the basis of available literature,olygonatum verticillatum is the first member of the genus Polygo-atum which demonstrated marked antinociceptive activity in theresent study.

In the traditional system of medicine, PR is used in combinationith other plant based diuretics for the enhancement of urine outut. Therefore, PR was also screened for the possible diuretic activ-

ty. In our assessment on the diuretic activity of PR, mild diureticctivity was computed at 300 mg/kg compared to saline. In con-rast, the urine discharge was similar to saline at 600 mg/kg. The

ild diuretic potential of the plant could be the reason for its usen polypharmacy for diuresis.

In conclusion, this study demonstrated that the opioid depen-ant central effect of the PR has synergistic effect by enforcing theeripheral analgesic effects and thus substantiated the traditionallaim of the plant in the treatment of painful conditions. However,he diuretic potential of the plant, as a mono-therapy, is not sup-orted by this study. For further clarification of mechanism and

nvolvement of specific components in the present study, bioas-ay directed isolation of pharmacologically active molecules of thelant is needed.

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