International Journal of Pharma Research & Review, Feb 2015; 4(2):1-8 ISSN: 2278-6074 Research Article Anti-inflammatory and Analgesic Activity of Methanolic Extract of Medicinal Plant Rhodiola rosea l. Rhizomes Avijit Chatterjee1, Biswarup Sen2, Sumanta Das2, *Tapan Kumar Chatterjee2 1. Dr. B. C. Roy College of Pharmacy & AHS, Bidhan Nagar, Durgapur- 713206, West Bengal, India. 2. Division of Pharmacology, Department of Pharmaceutical Technology, Jadavpur University, Kolkata-32, India. ABSTRACT Rhodiola rosea L. (Crassulaceae) have been used as traditional medicines that can increase someone’s physical strength, work productivity, longevity and resistance to high altitude sickness, fatigue, depression, anaemia, gastrointestinal ailments, infections, and nervous system disorders. The objective of this study was to evaluate the anti-inflammatory and analgesic activities from the methanolic extract of the rhizomes of Rhodiola rosea. Crude methanolic extract of the herb Rhodiola rosea were prepared and analyzed for its pharmacological activity. Swiss albino mice are used for acute toxicity study and also analgesic property of the extract by using Tail flick, Acetic acid induced writhing reflex and tail immersion method. Male Wister rat model are used to detect anti-inflammatory activity of the extract using carrageenan induced rat paw edema. After experiment statistical analysis like one way anova (nonparametric), Dunnet’s test was done. Results are plotted in graph and from this the effective activity of the plant is determined. The orally administered methanolic extract of Rhodiola rosea demonstrated a significant analgesic and anti-inflammatory in animal model. The findings in the study suggest that the methanolic extract of the herb Rhodiola rosea possesses analgesic and anti-inflammatory activities. This results may prove the fact that the herb may be used as analgesic and anti-inflammatory along with its adaptogenic properties. Keywords: Acute toxicity study, analgesic, anti-inflammatory activity, Rhodiola rosea Received 16 Dec 2014 Received in revised form 28 Jan 2015 Accepted 30 Jan 2015 *Address for correspondence: Dr. Tapan Kumar Chatterjee, Division of Pharmacology, Department of Pharmaceutical Technology, Jadavpur University, Kolkata-32, India. E-mail: crctkc@gmail.com, tkchatterjee_81@rediffmail.com _________________________________________________________________________________________________________________________ INTRODUCTION Rhodiola rosea, also known as “golden root” or “roseroot” belongs to the plant family Crassulaceae. For past few centuries, rhizomes of this plant has been used in the traditional medicine of Russia, Scandinavia, and other countries. In the rhizome it stores the secondary metabolites that have been shown to possess different medical activities and valuable adaptogenic effects. Traditional folk medicinal system used R. rosea to increase physical endurance, work productivity, longevity, and resistance to high altitude sickness, fatigue, depression, anaemia, impotence, gastrointestinal ailments, infections, and nervous system disorders [1]. Tapan Kumar Chatterjee et.al, IJPRR 2015; 4(2) Figure 1: Rhodiola rosea plant Inflammation was described two thousand years ago by Celsus by the four Latin words: Rubor, calor, tumor and dolor. Inflammation has different phases. The first phase is 1 International Journal of Pharma Research & Review, Feb 2015; 4(2):1-8 caused by an increase of vascular permeability resulting in exudation of fluid from the blood into the interstitial space, the second one by infiltration of leukocytes from the blood into the tissues and the third one by granuloma formation. Accordingly, antiinflammatory tests have to be divided into those measuring acute inflammation, subacute inflammation and chronic repair processes. In some cases, the screenings directed to test compounds for local application [2]. Analgesia is an ill-defined, unpleasant sensation, usually evoked by an external and internal noxious stimulus. Analgesics are drugs that selectively relieve pain by acting in the CNS or on peripheral pain mechanisms, without significantly altering consciousness. Analgesics relieve pain, without affecting its cause. Analgesics are divided into two groups, opioid analgesic and non-opioid analgesic [3]. The use of herbal medicines worldwide has provided an excellent opportunity to India to look for therapeutic lead compounds from our ancient system of therapy, i.e. Ayurveda, which can be utilized for development of new drug [4]. Epidemiological evidence suggests that dietary factors play an important role in human health and in the treatment of certain chronic diseases including cancer [5].This study can open a new phase of treatment of pain and inflammation with the extract of this rhizomes. MATERIALS AND METHODS (i) Animals Male Wistar rats weighing 218.7±3.0 (range 180–250g) were used for this study. They were bred and housed in the animal house, Department of Pharmaceutical technology, Jadavpur University. The animal house was well ventilated; the rats were fed with mouse cubes and had water ad libitum. The animals were randomly divided into groups comprising five rats each. Male Swiss Albino mice (20-25 g) were obtained from animal house Department of Pharmaceutical technology, Jadavpur University. The animals were housed under standard environmental condition (25°C, 12 h light and 12 h dark cycle) and fed with standard diet, water ad libitum [6]. The experiments were performed with subject to minimum pain to the experimenting animals. All the ethical Tapan Kumar Chatterjee et.al, IJPRR 2015; 4(2) ISSN: 2278-6074 considerations have been followed. The research was conducted in accordance with the ethical rules on animal experimentation, approved by Ethical committee, Department of Pharmaceutical technology, Jadavpur University Approval No:147/1999/CPCSEA). (ii) Plant materials The rhizomes (Rhodiola rosea) used for this study were collected in August 2013 from the Pharmaceutical Technology Department, Jadavpur university.This is to be identified and authenticated by The Botanical Survey of India, Botanical Gardens, Howrah, and West Bengal. Rhizomes were cut on sites 5–10 cm long, splinted and dried in drying case at 40– 45oC. The dried rhizomes were reduced to powdery form and 150g of the powdered sample was extracted with 2.5 Litre of methanol (analytical grade) for 72hrs [7]. The macerated mixture was filtered and evaporated in a carefully regulated water bath maintained at 40-45◦C to yield a brownish solid extract weighing 8g. The extract was stored in a refrigerator at 4◦C. For the pharmacological tests, the extract was dissolved in 0.1% Na-CMC in normal saline solution to prepare 50 mg/kg and 200 mg/kg concentrations. (iii) Drugs and Chemicals Indomethacin, carrageenan and DiclofenacNa were purchased from Sigma-Aldrich, Germany. Acetic acid was purchased from Merck, Germany. Morphine Sulfate was purchased from Troikaa Pharmaceutical Ltd, Gujarat, India. Figure 2: Extracted rhizome powder Figure 3: Rhizome of Rhodiola rosea 2 International Journal of Pharma Research & Review, Feb 2015; 4(2):1-8 1. Acute Oral Toxicity Study Acute oral toxicity study was performed as per OECD (Organization of Economic Cooperation and Development) 423 guideline (Acute toxic class method).Healthy female (should be nulliparous and non-pregnant) albino Wistar rats were randomly divided into 6 groups with 6 animals in each group. The animals were kept fasting overnight providing only water, after which the methanolic extract of Rhodiola rosea L .rhizomes was administered orally with increasing doses (50, 100, 500, 1000 and 2000 mg/kg) by intragastric tube to determine the safe doses. The animals were noticed continuously for 1 h, then frequently for 4 h and later at the end of 24 h for general behavioral, neurological and autonomic profile. Further, one group was administered high dose of Rhodiola rosea L. rhizomes extract orally once daily for 15 days and observed for any lethality [8]. 2. Anti-inflammatory activity 2.1. Carrageenan-induced rat hind paw edema The anti-inflammatory activity of the methanolic extract of Rhodiola rosea L. was assessed by the carrageenan-induced right hind paw edema method in Male Wister rats. Acute inflammation was produced by subplanar injection of 0.1 ml of 1% suspension of carrageenan in normal saline, in the right hind paw of the rats 1 h after the oral administration of test materials. The paw volume was measured by plethysmometer at 0h, 1hr, 1.5hr, 2hrand 3hr after the carrageenan injection .The extract was administered at 50 and 200 mg/kg body weight orally. Diclofenac at a dose of 25 mg/kg body weight was used as standard anti-inflammatory agent. The negative control group received 0.1% Na-CMC in saline solution [9].The anti-inflammatory effect of the extract was calculated by the following equation: Anti-inflammatory activity (%) inhibition = ×100 Where, C= Mean paw volume of control, D= Mean paw volume of test. 3. Analgesic activity screening 3.1 Radiant heat tail-flick method Tapan Kumar Chatterjee et.al, IJPRR 2015; 4(2) ISSN: 2278-6074 The central analgesic activity of the root extract was studied by measuring druginduced changes in the sensitivity of the prescreened (Basal reaction time: 3-5 sec) mice to heat stress applied to their tails by using a Medicraft Analgesiometer. The current intensity passing through the naked nichrome wire was maintained at 5 ampere. The distance between the heat source and the tail skin was 1.5 cm and cut-off reaction time was fixed at 10 sec to avoid any tissue damage. Morphine was used to compare the analgesic effect of the plant extract. The extract was orally administered at 50 and 200 mg/kg body weight. Morphine was administered subcutaneously at a dose of 5mg/kg body weight [10]. 3.2 Acetic acid induced writhing reflex The peripheral analgesic activity of methanolic extract of Rhodiola rosea L. rhizomes was measured by the acetic acid induced writhing test in mice The abdominal writhing was induced by intra-peritoneal injection of acetic acid solution (0.9%) at a dose of 0.1 ml/10 g of body weight to each mouse. Indomethacin at oral dose of 25 mg/kg was used as standard analgesic agent. The extract was administered at 50and 200 mg/kg body weight. The extract, standard drug and control (normal saline solution, 1 ml/kg) were orally administered 30 min prior to the injection of acetic acid. The number of writhing was calculated for 10 min after the application of acetic acid [11]. % inhibition of pain response = ×100 [C=control, T=test] 3.3 Tail immersion method Tail immersion test was used to assess the analgesic activity of Rhodiola rosea L. In this method six rats per group were used. Tail immersion method involved immersing the extreme 3cm of the rat’s tail in a water bath containing water at a temperature of (55.0±0.5) °C. After immersing within a few minutes, the rat reacted by withdrawing the tail. The reaction time was noted on a stopwatch. Each animal served as its own control and two readings were obtained for the control at 0 and 10min interval. The average of the two values was the initial reaction time. The test groups were given methanolic extract of Rhodiola rosea L. (50 mg/kg and 200 mg/kg, p.o.). Morphine (5 mg/kg, S.C.) 3 International Journal of Pharma Research & Review, Feb 2015; 4(2):1-8 were given for standard group, and 1ml/kg saline solution for control group (p.o.). The reaction time of the test groups was taken at 30 min, 1.0 h, 2.0h and 3.0h after a latency period of 30 min following the administration of the tests substances. The cut off time, i.e. time of no response was put at 120 seconds. The reaction time was measured and calculated [12]. 4. Statistical analysis Data were analyzed by one-way ANOVA followed by Dunnett’s test and p value of 0.05 was considered statistically significant. RESULT 1. Anti-inflammatory activity ISSN: 2278-6074 The anti-inflammatory activity of the plant extract was measured at a dose of 50 and 200 mg/kg body weight against acute paw edema induced by carrageenan. A strong inhibition of the paw edema was observed with the different doses of the extract and with Diclofenac (standard drug). The two doses tested (50 and 200 mg/kg) produced significant (p<0.05) anti-inflammatory activity and reduced the paw by 50.99% and 64.24% respectively, whereas Diclofenac caused 79.47% reduction when used as a standard drug. Result of anti-inflammatory activity is written on (Table 1) and plotted in (Fig. 1). Table 1: Anti-inflammatory activity of Rhodiola rosea extracts in Rats Treatment group Dose mg/kg % Inhibition Paw volume (mm³) 0 min 30 min 60 min 120 min 240 min Control 0.9% 0.79±0.03 1.35±0.15 1.72±0.11 1.80±0.11 1.51±0.03 Diclofenac 25mg/kg 0.75±0.09 0.88±0.11 0.77±0.15 0.58±0.16 0.31±0.05* 79.47 R. rosea L. 50mg/kg 0.81±0.10 1.44±0.13 1.38±0.17 0.86±0.150 0.74±0.21* 50.99 extract R. rosea L. 200mg/kg 0.76±0.11 0.94±0.11 0.85±0.15 0.70±0.14 0.74±0.21* 64.24 extract The data represent the Mean±SEM (n=8). *: P<0.05, compared to corresponding control. Control: 0.9% CMC (p.o); Standard drug: Diclofenac (p.o.); Extract: Methanolic extract of R. rosea (50 and 200mg/kg) (p.o). Dose response comparision by carragennan induced rat paw edeoma method Inhibition 1.5 control Diclofenac Drug 50 Drug 200 1.0 0.5 0.0 control Di cl ofenac Drug 50 Drug 200 Dose Figure 4: Dose response comparison graph for R.rosea extracts by Carrageenan induced rat paw edema method 2. Radiant heat tail-flick method In the radiant heat tail-flick method, the Rhodiola extract prolonged the heat stress tolerance capacity of the mice. In radiant heat tail-flick test, the extract produced 29.50% and 62.68% (p <0.05 significant) Tapan Kumar Chatterjee et.al, IJPRR 2015; 4(2) increase in analgesic activity after oral doses of 50 and 200 mg/kg body weight respectively. Morphine caused 74.650 % (p<0.01) increase in analgesic activity when used as a standard drug at 5 mg/kg body weight. Result of radiant heat tail flick 4 International Journal of Pharma Research & Review, Feb 2015; 4(2):1-8 ISSN: 2278-6074 method is written on (Table 2) and plotted in (Fig. 2). Table 2: Analgesic Activity of Rhodiola rosea extracts by Tail-Flick Method in Mice Treatment Group Flicking response of tail Before treatment After treatment Control Morphine(5mg/kg) R. rosea ext(50mg/kg) R. rosea ext(200mg/kg) Difference % Increase in analgesic activity 3.500±0.1890 3.650±0.2053 3.400±0.2179 3.531±0.05984 6.375±0.6847** 4.403±0.3021 0.031 2.725 1.003 27.25% 10.03% 3.650±0.1309 5.938±0.5984* 2.288 22.88% Data presented as mean ± SEM, n = 8 for all groups. **P<0.01(Morphine)*P<0.05.P value <0.05 taken as significant Dose response comparision by using Tail flick method Analgesic response 7.5 Control Morphine Drug 50 Drug 200 5.0 2.5 0.0 Control Morphine Drug 50 Drug 200 Dose Figure 5: Dose response comparison of different dose of R. rosea extract by radiant heat tail flick method 3. Acetic acid induced writhing method when compared to control untreated groups. The methanolic extract of the plant Rhodiola The two doses tested (50 and 200 mg/kg) rosea L. rhizomes at the doses of 50 and 200 produced significant (p<0.01) analgesic mg/kg body weight and indomethacin 25 activity. Result of acetic acid induced mg/kg body weight induced respectively writhing reflex is written on table-3 and significant (p<0.01) 22.67%, 49.38% and plotted in (Fig. 3). 62.76% decrease in the number of writhes Table 3: Analgesic Activity of Rhodiola rosea by Acetic Acid Induced Writhing Response in Mice Treatments Dose (mg/kg) No of abdominal writhing % Inhibition Control 1ml/kg 0.88±1.995 Indomethacin 25mg/kg 11.50±0.8018** 62.76 R.rosea extract 50mg/kg 23.88±1.420** 22.67 R.rosea extract 200mg/kg 15.63±1.068** 49.38 Data presented as mean ± SEM, n = 8 for all groups.*= p< 0.01 (significant) when last three groups are compared to control in Dunnett’s multiple comparison test. 4. Tail immersion method The methanolic extract of the plant Rhodiola rosea L. rhizomes at the doses of 50 and 200 Tapan Kumar Chatterjee et.al, IJPRR 2015; 4(2) mg/kg body weight and Morphine 5 mg/kg body weight induced a significant (p<0.05) decrease in the reduction of painful 5 International Journal of Pharma Research & Review, Feb 2015; 4(2):1-8 sensation when compared to control untreated groups in tail immersion method. The 200 mg/kg dose produced significant (p<0.05) analgesic activity and reduced the painful sensation by 44.59%, whereas morphine caused 52.87% reduction when used as a reference drug. There was a ISSN: 2278-6074 significant reduction of painful sensation due to tail immersion in warm water, after a latency period of 0.5 h following oral administration of the plant extract at the dose of 200 mg/kg. Result of tail immersion is written on table-4 and plotted in (Fig. 4). Dose response comparision by using Acetic acid induced writhing reflex method Inhibition 40 Control standard Drug 50 Drug 200 30 20 10 0 Control standard Drug 50 Drug 200 Dose Figure 6: Dose response comparison of R.rosea extracts by using Acetic acid induced writhing reflex method Table 4: Analgesic activity of Rhodiola rosea extracts by tail immersion methods in mice Treatment Dose 0 min 30 min 1 hr. 2 hr. group (mg/kg) Control 1ml/kg 3.500±0.1890 3.750±0.1402 3.838±0.1700 3.875±0.1176 Morphine 5mg/kg 3.925±0.2210 6.275±0.1306 6.725±0.1612** 6.650±0.1535** R. rosea 50mg/kg 4.350±0.1711 4.825±0.1013 4.925±0.0839 4.575±0.1236 extract R. rosea 200mg/kg 3.925±0.2358 5.713±0.1481 5.900±0.1086* 5.888±0.1076* extract The data represent the mean±SEM (n=8). *: P<0.05, **: P<0.01, compared to corresponding Control: 1ml/kg (p.o); Standard drug: Morphine (s.c); Test drug: Methanolic extract of Rhodiola (p.o). Dose response comparision by using Tail immersion method Increase in analgesic activity 7.5 % Inhibition 52.87 5.17 44.59 control. rosea L. Control Morphine Drug 50 Drug 200 5.0 2.5 0.0 Control Morphi ne Drug 50 Drug 200 Dose Figure 7: Dose response comparison of R. rosea extracts by using tail immersion method Tapan Kumar Chatterjee et.al, IJPRR 2015; 4(2) 6 International Journal of Pharma Research & Review, Feb 2015; 4(2):1-8 DISCUSSION The acute toxicity study of the four different doses of Rhodiola rosea extract shows no lethality within 24 hrs and there are no behavioral changes take place within 14 days after administer 2000mg/kg dose of the extract. So we can conclude that the extracts are safe for further work. Inflammation has different phases the first phase is caused by an increase in vascular permeability, the second one by infiltrate of leucocytes and the third one by granuloma formation. We determined anti-inflammatory activity by using inhibition of carrageenan-induced inflammation which is one of the most feasible methods to screen antiinflammatory agents. The development of carrageenan-induced edema is bi-phasic; the first phase is attributed to the release of histamine, serotonin and kinins and the second phase is related to the release of prostaglandins and bradykinins [13-16]. We observed that R.rosea (50mg/kg) and R.rosea (200mg/kg) showed significant inhibition against carrageenan-induced paw edema in the dose dependent manner This response tendency of the extract in carrageenan induced paw edema revealed good peripheral anti-inflammatory properties of the methanolic extract. This antiinflammatory effect of R.rosea (50mg/kg) and R.rosea (200mg/kg) may be due to the presence of flavonoids. It has been reported that a number of flavonoids possess antiinflammatory [17] and analgesic [18] activities. Flavonoids are known to inhibit the enzyme prostaglandin synthetase, more specifically the endoperoxidase and reported to produce anti-inflammatory effect. Since, prostaglandins are also involved in the pain perception; inhibition of their synthesis might be the possible reason for the analgesic activity of the methanolic extract. The presence of flavonoid identified might be responsible for the analgesic and antiinflammatory activities in methanolic extract. CONCLUSION Thus, it is concluded that the methanolic extract of rhizomes of Rhodiola rosea produces significant analgesic and antiinflammatory activities in dose dependent manner. Flavonoids are known to inhibit the enzyme prostaglandin synthetase, more Tapan Kumar Chatterjee et.al, IJPRR 2015; 4(2) ISSN: 2278-6074 specifically the endoperoxidase and reported to produce anti-inflammatory effect. Since, prostaglandins are also involved in the pain perception; inhibition of their synthesis might be the possible reason for the analgesic activity of the methanolic extract. The presence of flavonoid identified might be responsible for the analgesic and antiinflammatory activities in methanolic extract. ACKNOWLEDGEMENT The entire work is supported by Dr. Tapan Kumar Chatterjee, Associate professor, Dept. of Pharmaceutical technology, Jadavpur University, Kolkata-700032, West Bengal, Mr Souvik Debnath, Dept. of Pharmaceutical technology, Jadavpur University, Kolkata700032, Mr.Saswata Banerjee. Dept. of Pharmaceutical technology, Jadavpur University, Kolkata-700032.We are very grateful to them for contribute towards the study by making substantial contributions to conception, design, acquisition of data, or analysis and interpretation of data. 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