ADAPTOGENIC ACTIVITY OF MYXOPYRUM SMILACIFOLIUM BLUME

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1 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Samu et al. SJIF Impact Factor Volume 6, Issue 7, Research Article ISSN ADAPTOGENIC ACTIVITY OF MYXOPYRUM SMILACIFOLIUM BLUME E.N. Siju and Jolly Samu* Department of Pharmacology, Academy of Pharmaceutical Science, Pariyaram Medical College, Kannur , Kerala, India. Article Received on 07 May 2017, Revised on 28 May l 2017, Accepted on 18 June 2017, DOI: /wjpps *Corresponding Author Jolly Samu Department of Pharmacology, Academy of Pharmaceutical Science, Pariyaram Medical College, Kannur , Kerala, India. ABSTRACT Ethanol and aqueous extracts of Myxopyrum smilacifolium leaves were screened for adaptogenic activity using anoxia stress tolerance model and swimming endurance model. The effect was assessed by swimming survival time and anoxia tolerance time, estimation of various biochemical parameters in swimming endurance like glucose, cholesterol, triglycerides and blood urea nitrogen and morphological parameters like weight of organ such as liver, spleen, adrenal gland, at dose of 200mg/kg, 400mg/kg body weight per oral. Blood cell count and the differential count also determined. The extracts showed significantly increases the swimming endurance time and anoxia tolerance time. The extracts showed significant decrease in biochemical parameters, organ weights and increases the weight of spleen. Thus the obtained results revealed that the Myxopyrum smilacifolium may have a significant adaptogenic activity. KEYWORDS: LD 50 ; Adaptogen; Anoxia stress tolerance; Swimming endurance; Myxopyrum smilacifolium. 1. INTRODUCTION Stress is defined as a state of threatened homeostasis or disharmony and is counteracted by a complex repertoire of physiologic and behavioral responses that re-establish homeostasis. [1] Commonly seen in stress related symptoms are problems with sleep, irritability, fatigue, lethargy, depression and anxiety. From the birth of an infant to the end of life, stresses continuously hammer at the harmony and homeostasis of life. Although the exact role of stress in human diseases is not known, it is clear that stress can lead to particular diseases by Vol 6, Issue 7,

2 temporarily inhibiting certain components of the immune system. Stress related disorders include gastritis, irritable bowel syndrome, ulcerative colitis, hypertension, asthma, migraine, headache, rheumatoid arthritis, anxiety and depression. [2] People under stress are at a greater risk of developing chronic disease or dying prematurely. Adaptogens were defined as substances derived from plant origin that enhance the state of nonspecific resistance in stress, a physiological condition that is linked to various disorders of the neuro endocrine immune system. [3] In 1969 Brekhman and Dardymov defined the general pharmacological properties of adaptogenic substances. These include adaptogen must reduce stress induced damage, thus presenting stress protective effects such as anti fatigue, antidepressant and restorative activities, the stimulating effect of adaptogens must be different from those of conventional CNS stimulants and anabolic and give rise to negative side effects such as drug withdrawal syndrome. Myxopyrum smilacifolium are rich in volatile oil tannins, saponins, terpenoids. But few studies are available on the pharmacological effects of plant. Myxopyrum smilacifolium Blume (Family-Oleaceae) is an important medicinal plant widely used in indigenous system of medicine in India. The leaves are acrid, astringent, sweet, anodyne, febrifuge, thermogenic, and tonic. These are useful in vitiated conditions of kapha, vata, cough, asthma, nostalgia, rheumatism, consumption, otopathy, fever, neuropathy, cuts and wounds. [4,5] Earlier the plant has been studied for its antimicrobial [5], wound healing [6] and antiinflammatory activity. [7] In the present study ethanol and aqueous extracts of Myxopyrum smilacifolium was evaluated for its effects on adaptogenic activity. 2. MATERIALS AND METHODS 2.1. Collection of plant material The leaves of Myxopyrum Smilacifolium Blume were collected from Agricultural University, Odakkali, Kerala, India in the month of September 2013 and authenticated by Dr. Harikrishnan E, Assistant professor, Department of Botany, Payyanur College, Kannur, Kerala. The leaves were dried in shade at room temperature. The dried leaves of Myxopyrum smilacifolium were pulverized in a mechanical grinder to obtain powder. Vol 6, Issue 7,

3 2.2. Preparation of extract [8] Ethanol Extract The shade dried powered leaves (500gm) were exhaustively extracted with 95% ethanol using a soxhlet apparatus. The ethanol was concentrated in vaccum. The percentage yield of extract is 3.75%. Aqueous extract The aqueous extract was prepared using fresh powder by maceration process. 100gm of the powdered drug was taken in a 2000ml conical flask with 500ml of distilled water and 10ml chloroform is added as preservative. It was extracted up to 7 days with stirring. After 7 days of the extract was filtered through the muslin cloth and the marc was is discarded and airtight container in its filtrate dried under hot air oven at 45 o C to semisolid mass. These were stored in refrigerator below 10 0 C. The percentage yield of extract was found to be 5% EXPERIMENTAL ANIMALS Albino mice (20-25g) and Wistar rats ( g) of either sex were used for the study. The animals were fasted overnight prior to the experiment. The animals were maintained under standard 12 hr light/dark cycle throughout the study. The study protocol was approved by IAEC (No: APSC/CPCSEA/-06/IAEC/2013). [9] 3. Determination of LD 50 The acute toxicity of aqueous and ethanol extracts of Myxopyrum smilacifolium were determined by using albino mice of either sex (20-30 gm), those maintained under standard conditions. The animals were fasted overnight prior to the experiment. The study was performed according to the acute toxic class method (as per CPCSEA/OECD GUIDELINES). The toxicity study carried out as per the guidelines of 423. The study protocol was approved by IAEC (No: APSC/CPCSEA/-06/IAEC/2013). The animals were selected and grouped, three animals per group. Animal should fasting prior to dosing (with mice, food but not water should be withheld for 3-4 hours). Following the period of fasting, the animal should be weighted and the test substance administered. Test substance was administered in a single dose by the oral route. A single administration of starting dose of 2000mg/kg body weight p.o. of the extract was administered to three female mice. After the extract has been administered food may be withheld for a further 1-2 hours in mice. Where a dose administered in fractions over a period it may be necessary to provide the Vol 6, Issue 7,

4 animals with food and water depending on the length of the period. They were noted individually after dosing at least once during the first 30 minutes, periodically during the first 24 hours, with special attention given during the first four hours, and daily thereafter for a total of 14 days. The mice were observed for three days to evaluate considerable changes in body weight and other sign of toxicity. Then the experiment was repeated again with same dose level, 2000 mg/kg body weight p.o. of plant extract for 7 more days and observed for fourteen days. [9] 4. Anoxia stress tolerance in mice All the body functions, including cellular respiration depends on the oxygen supply. Any lack of vital element will play havoc on all body mechanisms and increase in adaption during stress by any drug could be considered as its major anti stress effect. [10] During stress, adaptogens are capable of increasing succinate dehydrogenase (SDH) in the brain. The enzyme (SDH) is responsible for utilization and conservation of energy in the cellular system of the organism, which helps in adaptive process during stress. [11] Hermetic vessel of 2-litre air capacity was made airtight at the start of the experiments and used for the study. The animals were divided into seven groups of six mice each. [12] Each animal was kept in the hermetic vessel and the time was noted. The time duration from the entry of the animals to the hermetic vessel and the appearance of the first convulsion was noted and a delay of even one minute in removal of the animals may lead to death of the animal. After one week of drug treatment the animals were once again exposed to the anoxia stress. Depending on their capacity of tolerance, the animals were observed for the 2 nd and 3 rd week with the same treatment and the change in time duration of anoxia tolerance was noted. 5. Swimming endurance test in Rats Rats when forced to swim in a restricted space from which they cannot become immobile after an initial period of vigorous activity. It has been suggested that the observed immobility signifies behavioral despair resembling a state of mental depression and has been used to screen antidepressant. [13] It is now recognized that this behavioral depression is fairly a common consequence of stress. It is also evident that the animal s ability to cope with the stress largely influenced by the neurochemical consequence of stress. [14] Thus exposure of rats to inescapable and severe stress leads to depletion of central non adrenaline and serotonin, postulated to be the cause of endogenous depression. [15,16] Stress was exerted by keeping rats in glass tank ( cm, 4 inch thickness) filled with water to a height of Vol 6, Issue 7,

5 25cm. Extract was given to a rats for 7 days, the rats were allowed to swim till exhausted in separate tanks filled with water. The end point was taken when the animal starts drowning and swimming time for each animal was noted. The mean swimming time for each group was calculated on 8 th day blood was collected by cardiac puncture to estimate biochemical parameters like blood glucose, tri glycosides, cholesterol, BUN and blood cell count (RBC,WBC,DLC). The weight of organ such as liver, adrenals, spleen was recorded after washing with alcohol. [17,18] 6. Statistical analysis Values are presented a mean ±SD of 6 animals in each group. All data are statistically evaluated by ANOVA. P value of 0.05 and 0.01 were considered as statistically significant. 7. RESULTS 7.1. Determination of LD 50 In the present study, ethanol and aqueous extracts of leaves of Myxopyrum smilacifolium were subjected for toxicity study. Acute toxicity studies of both extracts were determined in mice by following OECD (423 guideline). The ethanol and aqueous extracts of Myxopyrum smilacifolium were found to be safe up to 2000 mg/kg body weight by oral route. After 24 hours, animals were found well tolerated. There was no mortality and sign of toxicity after 14 days. From this LD50, 1/10, 1/5 doses of LD50 was selected to study adaptogenic activity as metabolism of drugs in animals is 5-10 times more than human individuals. So the doses selected for the present studies are Ethanol extract (lower dose) -200mg/kg Ethanol extract (higher dose) 400mg/kg Aqueous extract (lower dose) 200mg/kg Aqueous extract (higher dose) 400mg/kg The mice and rats were selected for the assessment of adaptogenic activity. 7.2.Anoxia stress tolerance Table No. 1: Effect of Myxopyrum smilacifolium on anoxia stress tolerance in mice Sl. No Groups Mean duration of tolerance in min Mean ±SEM after treatment 7 th day 14 th day 21 st day 1 Normal control Stress control 14.24± ± ± Withania somnifera standard 18.48±2.82** 18.90±1.04** 19.40±2.82** Vol 6, Issue 7,

6 (100mg/kg) Ethanol extract of Myxopyrum smilacifolium (200mg/kg) Ethanol extract of Myxopyrum smilacifolium (400mg/kg) Aqueous extract of Myxopyrum smilacifolium (200mg/kg) Aqueous extract of Myxopyrum smilacifolium (400mg/kg) 15.68±2.26** 15.78±1.86** 15.96±1.94** 16.76±3.24** 16.84±2.26** 16.96±3.16** 14.86±1.82** 14.89±2.26** 14.92±2.18** 15.24±2.26** 15.36±1.86** 15.42±2.26** The values are expressed as ±SEM, n=6, *P<0.05, **P<0.01 as compared to stress control The result obtained from anoxia stress tolerance was expressed as mean±sem. Significantly increases in anoxia stress tolerance with ethanol and aqueous extracts but more with ethanol extract at a dose of 400mg/kg at 1 st, 2 nd and 3 rd week of the experimental study and significantly increasing anoxia tolerance time as compared with control. The standard Withania somnifera showed increases in anoxia stress tolerance as compared to control. Results are shown in Table No. 1. Table No: 2 Effect of Myxopyrum smilacifolium on swimming endurance test in rats Sl. No Groups Swimming endurance test in Rats in min Mean ±SEM after treatment 1 Normal control - 2 Stress control 23.5± Withania somnifera standard (100mg/kg) 30.52±2.32** 4 Ethanol extract of Myxopyrum smilacifolium (200mg/kg) 31.42±1.82** 5 Ethanol extract of Myxopyrum smilacifolium (400mg/kg) 33.42±1.42** 6 Aqueous extract of Myxopyrum smilacifolium (200mg/kg) 34.62±2.56** 7 Aqueous extract of Myxopyrum smilacifolium (400mg/kg) 35.46±1.62** The values are expressed as ±SEM, n=6, *P<0.05, **P<0.01 as compared to stress control. The animals when forced to swim in water eventually assumes a characteristic immobile posture, devoid of activity. The appearance of immobility reflects a state of tiredness, reduced stamina with the end point being the moment when the rats cannot swim further and starts drowning. Animals were pretreated with Myxopyrum smilacifolium and Withania somnifera for 7 days and subjected to swimming endurance on 8 th day. Withania somnifera and Myxopyrum smilacifolium showed significant increases in swimming endurance time when compared with control group. Results are in Table No. 2. Immobilization stress caused Vol 6, Issue 7,

7 marked increase in serum glucose, cholesterol, triglycerides and BUN in rats. The stress induced elevated levels of biochemical parameters were significantly reversed by Myxopyrum smilacifolium leaves extracts. The results are given in Table No 3,5. Myxopyrum smilacifolium leaves extracts at a doses of 200, 400mg/kg offered significant protection against change in the weight of liver, adrenal gland and spleen. The results are presented in Table No Vol 6, Issue 7,

8 Table No. 3: Effect of Myxopyrum smilacifolium on blood cell count in swimming stress induced rats Sl. No. parameters RBC count (million) DLC Polymorphs Eosinophils Lymphocytes Monocytes No of cells/ cumm WBC count 1. Group1 6.28± ± ± ± ± ± Group2 9.58± ± ± ± ± ± Group3 6.49±0.01** 5672±582** 1649±3.181** ±3.480** 5104±10.08** 8.35±0.284** 4. Group4 7.65±0.327* 6001±384.01* 1981±52.47* ±5.26* 5848±139.98* 8.55±0.282* 5. Group5 7.05±0.245** 5910±287** 1811±49.21** ±5.23** 5498±139.98* 8.18±0.307* 6. Group6 8.25±0.312* 6627±405.1* 2615±179.52* ±14.57* 5920±179.52* 8.62±0.307* 7. Group7 7.89±0.199* 6347±416.0** 2578±77.6** ±14.28** 5687±170.55* 8.32±0.223** The values are expressed as ±SEM, n=6, *P<0.05, **P<0.01 as compared to normal control. Table No. 4: Effect of Myxopyrum smilacifolium on organ weights in swimming stress induced rats. Sl. No Groups Liver Adrenal gland Spleen (Mean ±SEM) gms/100gms 1 Group ± ± ± Group ± ± ± Group ±1.91** 0.021±3.256** 0.482±0.44** 4 Group ±1.39* 0.031±0.01* 0.351±0.21* 5 Group ±0.78* 0.025±0.44* 0.435±0.02* 6 Group ±0.71* 0.030±0.788* 0.398±0.05* 7 Group ±0.78* 0.027±1.37** 0.441±0.06** The values are expressed as ±SEM, n=6, *P<0.05, **P<0.01 as compared to normal control. Vol 6, Issue 7,

9 Table No. 5: Effect of Myxopyrum smilacifolium on biochemical parameters in swimming stress induced rats Sl No. Groups Glucose Cholesterol Triglyerides BUN 1. Group ± ± ± ± Group ± ± ± ± Group ±0.38** 56.32±036** 74.28±0.44** 29.24±0.003** 4. Group ±0.44** 64.03±0.46* 84.38±0.32* 33.66±0.002* 5. Group ±0.34** 61.20±0.32** 82.04±0.46** 30.54±0.72** 6. Group ±0.46** 69.04±0.44* 89.42±0.28* 38.06±0.44* 7. Group ±0.46** 60.06±0.42** 85.32±0.34** 35.64±0.032* The values are expressed as ±SEM, n=6, *P<0.05, **P<0.01 as compared to positive control. 7. DISCUSSION Adaptogens compared to synthetic drugs that are usually concentrated, single substances, plant extracts have a complex synergic action that has made the scientific investigation of adaptogen remedies a precipitous and tortuous affair. Triterpenes also include phytosterols and phytoecdysteroids, both of which are thought to have adaptogenic roles in mammals and in humans. Triterpens are structurally similar to the corticoids which are stress hormone involved in the inactivation of the stress system and in protecting the organism from over reaction in response to stressors. [19] In addition, there are reports that polyphenolic compounds like flavanoids and tannins are useful as antioxidants. Therefore, many researchers are attempting to screen the herbs containing polyphenolic compounds for antistress properties. The leaves of Myxopyrum smilacifolium Blume. were collected and extracts were prepared by different solvents. The prepared extracts were subjected to preliminary phytochemical studies. The phytochemical studies of petrolium ether, chloroform, methanol extract revealed the presence of triterpenoids and steroids. Ethanol contains carbohydrates, flavanoids, glycosides, saponins, steroids, tannins and terpenoids. Aqueous extracts contains alkaloids, carbohydrates, saponins, flavanoids and tannins. The aqueous and ethanol extracts rich in high poly phenolic content which may be responsible for the observed adaptogenic activity. Triterpenoids, tannins, volatile oil, ursolic acid are previously reported from Myxopyrum smilacifolium. Thus gave strong evidence to adaptogenic, activity. The ethanol and aqueous extracts of Myxopyrum smilacifolium inhibited accumulation of free radicals will put the body in a state of oxidative stress and bring injury to the body by attacking large molecules and cell organs. Several studies have shown that exogenous dietary antioxidants can decrease the contribution of exercise induced oxidative stress and improve the animal s physiological condition. In past few decades, health Vol 6, Issue 7,

10 scholars and athletic ability, postpone fatigue and accelerate the elimination of fatigue in human beings, but also have few side effects. The physical stress caused significant fatigue and motor coordination. The significant reduction in antifatigue effect demonstrates the efficacy of Myxopyurm smilacifolium as an effective adaptogenic agent. Before screening the pharmacological activities the ethanol and aqueous extract were subjected to acute toxicity studies. The acute toxicity studies were carried out in accordance to OECD guidelines. The ethanol and aqueous extract of Myxopyrum smilacifolium were found to be safe and no mortality was seen at the dose of 2000 mg/kg. Hence 2000 mg/kg was fixed as LD 50, so those 200mg/kg(lower dose) and 400mg/kg (higher dose) were selected for the further study. In the present investigation ethanol and aqueous extracts of Myxopyrum smilacifolium Blume. have evaluated for the antistress activity against different types of stress viz. anoxia and swimming endurance models. Withania somnifera was taken as a reference standard in the present study. In case of anoxia stress tolerance test the ethanol extract of Myxopyrum smilacifolium at a dose 400mg/kg exhibited significant antistress activity as indicated by increase in duration of anoxia stress tolerance time. The test drug also produced mild increase in anoxia tolerance at a dose of 200mg/kg. The aqueous extract at a dose of 200mg/kg, 400mg/kg has shown significant increasing tolerance stress time in 7 th, 14 th and 21 st day as compared to the control. The extract of Myxopyrum smilacifolium pocessess adaptogenic activity as its significantly increased in swimming time. Swimming endurance stress resulted in significant increase in adrenal gland weight and liver weight with concomitant decrease in spleen weight in stress control group, which has significantly reverted by Myxopyrum smilacifolium Blume. at a doses of 200 mg/kg and 400mg/kg. Stress induced elevated blood cell counts of RBC and DLC i.e polymorphs, eosinophils, monocytes and lymphocytes have been significantly reduced by the extracts in dose dependent manner. The mechanism by which stress rises serum cholesterol is likely to be related to the enhanced activity of hypothalamo- hypophysial axis (HPA) resulting in liberation of cathecholamines and corticosteroids. This could lead to increase in blood cholesterol level since epinephrine is known to mobilize lipids from adipose tissue. The effect of stress on serum triglycerides has been shown to be variable. The increase in release of catacholamines leads to elevated levels of glucose and BUN. In Vol 6, Issue 7,

11 immobilization stress model, the test extracts reduced the levels of serum biochemical parameters in dose depend manner. Experimental studies have confirmed the adaptogenic properties of Myxopyrum smilacifolium and the effects are apparently due to presence of saponins, terpenoids and glycoside in the leaves. The adaptogenic activity of Myxopyrum smilacifolium so observed is attributed to the presence of flavanoids, saponins, glycosides and tannins. Literature survey indicates that flavanoids and tannins were reported to possess variety of pharmacological activities including adaptogenic activity. In the present investigation also preliminary phytochemical screening on ethanol and aqueous extracts of Myxopyrum smilacifolium gave positive tests for flavanoids and tannins that could be the reason for significant adaptogenic activity of test extracts. Analytical studies showed the presence of iridoid glycoside; may be the reason for its adaptogenic activity. Increased generation of oxidative free radicals or impaired antioxidant defense mechanism have been implicated in chronic stress induced perturbed homeostasis including inflammation, immunosuppression, diabetes mellitus, peptic ulceration and other stress related disease. The leaves of Myxopyrum smilacifolium have shown to exert significant antioxidant activity induced by scavenging enzymes namely superoxide dismutase (SOD), catalase (CA) and glutathione peroxidase (GPx). Thus at least part of observed adaptogenic (antistress) effects of ethanol and aqueous extracts of Myxopyrum smilacifolium may be due to antioxidant activity. 1. CONCLUSION Myxopyrum smilacifolium Blume. is an important medicinal plant widely distributed throughout Kerala, in evergreen forests. The phytochemical and pharmacological investigations of the leaves extracts of Myxopyrum smilacifolium have been represented here in this thesis. Preliminary phytochemical studies reveal that presence of poly phenolic compounds. Analytical studies showed the presence of iridoid glycoside; may be the reason for its adaptogenic activity. Ethanol and aqueous extracts of Myxopyrum smilacifolium showed good adaptogenic activity by anoxia stress tolerance and swimming endurance test. Myxopyrum smilacifolium leaves extracts at doses of 200mg/kg; 400mg/kg have shown significant activity, they possess the ability to prevent the alterations due to stress. The above results are promising and further dose studies would be helpful in substantiating the claim of adaptogenic action of the drug. The above activity may be due to controlling of hypothalamus pituitary adrenal axis and antioxidant properties. Thus the investigational works under taken Vol 6, Issue 7,

12 and furnished in this thesis deals with the thorough evaluation of phytochemical and pharmacological profiles of the leaves of Myxopyrum smilacifolium. Thus with all the above results it can be claimed and concluded that the leaves of Myxopyrum smilacifolium may have adaptogenic activity. 2. REFERENCES 1. Gerard J. Tortora and S.R. Grabowski, Principles of Anatomy and Physiology, twelfth ed., John Wiley & Sons Inc., United States of America, 2009; , , Virginia Hill, Theories of stress and its relationship to health, Health News Network, 3. C.K. Kokate, Text book of Pharmacognosy Twenty fourth ed., New delhi, 2003; P.S. Varier, Indian medical plants: a compendium of 500 species, Longman, Madras, 1995; D. Sundharmini, S.N. Ashalatha, Antimicrobial activity of triterpenoid fractions from Myxopyrum smilacifolium Blume, Ethnobot Leaflets. 2008; 12: S. Gopalakrishnan, R. Rajameena, Wound healing activity of the Ethanol extract of the leaves of Myxopyrum serratulum A.W. Hill in Rats, Int J Pharma Sci Rev Res., 2013; 22: Jolly Samu, E.N. Siju, M. Minil, G.R. Rajalakshmi, Invitro antiinflammatory activity of Myxopyrum smilacifolium Blume, World J. of Pharmacy and Pharm Sci., 2014; 3: W.C. Evan, Trease and Evans Pharmacognosy, fourteenth ed., Harcourt Brace and company, India, 1998; : OECD guidelines 423, for testing of chemicals. Acute oral toxicity- Acute Toxic class method. December S. Vinod Pawar, Shivakumar Hugar, Adaptogenic activity of Trigonella foenum graecum seeds in rodents exposed to anoxia and immobilization stress, Asian Pac J Trop Biomed, 2012; B.V.S. Lakshmi, M. Sudhakar, Screening of Psidium guajava leaf extracts for Antistress activity in different experimental animal models, Pharmacognosy Res., 2009; S.K. Battacharys, Muruganadam, Adaptogenic activity of Withania somnifera; an experimental study using a model of chronic stress, Pharmacology Biochemistry and Behavior, 2003; 75: Vol 6, Issue 7,

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