Evaluation of Adaptogenic Activity of Moringa oleifera Lam.

 

Shameer Pasha*, Mohamed Khaleel and Sukhen Som

M.M.U College of Pharmacy, K. K. Doddi, Dist- Ramanagara- 571511, Karnataka (India)

ABSTRACT:

Moringa oleifera Lam. (Moringaceae) is an medicinal plant used in the treatment of many clinical conditions in India. Its adaptogenic activity have been investigated in this study using albino mice and rats. The ethanolic extract of Moringa oleifera leaves was prepared and subjected to preliminary qualitative phytochemical screening. Glycosides, phytosterols, tannins and amino acids were found to be present. Acute toxicity studies were carried out in albino mice. The ethanolic extract did not show the lethal effect upto the doses of 2000mg/ kg body weight with no signs of abnormalities or any mortality observed for 14 days period under observation after single dose of drug administration. The parameters studied were swimming endurance, cold-restraint induced ulcer, and effect of cold-restraint stress on adrenocortical activity and weights of organs like liver, spleen, adrenal glands. Geriforte was used as a standard adaptogenic drug, which is a commercial preparation of Himalaya Drugs Company. The results indicate that pretreatment with ethanolic extract of leaves of Moringa oleifera exhibited significant adaptogenic activity at the tested doses of 200 mg/ kg and 400 mg/ kg body weight. On the basis of results, it was concluded that Moringa oleifera possess adptogenic activity.

 

KEYWORDS: Adaptogenic, cold-restraint stress, Moringa oleifera, swimming endurance. 

 

INTRODUCTION

Dr. Hans Seyle defined stress as the sum of all nonspecific responses of the body to any external stimuli acting up on it1. Homeostatic mechanism attempts to counteract stress. When they are successful, the internal environment remains within normal physiological limits. If stress is extreme, unusual or long lasting, the normal mechanisms may not be enough2. Some medicinal plants have been demonstrated to induce a state of non-specific increase of resistance in experimental animals as well as human beings3. Active phytochemical constituents present in these plants may be acting as anti-stress agents by inducing resistance against aversive stimuli, impart immunity, give protection against disease, postpone ageing, improve vigor, vitality, longevity and so the drugs are adaptogens4. Studies indicate that adaptogens not only help the body to cope up with stress, but can enhance general health and performance5. In traditional systems of medicine various plants have been used to promote the in vivo anti-stress activity. It was considered worthwhile to validate the use of one such plant Moringa oleifera.

 

Moringa oleifera Lam. belongs to the family Moringaceae. A native of the Himalayan regions of India, it is now grown in Africa, Arabia, Southeast Asia, the Pacific Islands, the Caribean Islands and South America.


Leaves, flowers, fruits and bark of the plant have been used in traditional medicine6, 7. Several reports have been appeared on the antimicrobial7- 10, antihypertensive11, antitumor12, antiulcer13, anti-inflammatory and hepatoprotective14 activities of M. oleifera plant parts. Traditionally this plant is used as antispasmodic, stimulant, expectorant, anthelmintic, aphrodisiac, laxative and diuretic15.

 

Some of the important phytochemical constituents reported with Moringa oleifera were various amino acids, fatty acids, vitamins, nutrient elements, isothiocyanates, carbamates, thiocarbamates and polyphenols6-8, 11.

 

MATERIALS AND METHODS:

Plant material:

The leaves of Moringa oleifera were collected from the fields around the campus in the month of July- August. The collected material was authenticated by Botanist. The voucher specimen was deposited in the laboratory for future reference.

 

Extraction:

The fresh leaves were shade dried, pulverized and extracted with 95% ethanol in a soxhlet apparatus. The extract was concentrated under reduced pressure at 400C and dried over anhydrous sodium sulphate. The yield of the concentrated crude alcoholic extracts of leaves was 6.3%. A suspension of extract in 1% w/v gum acacia was prepared before oral administration to the animal for pharmacological studies.

 

Experimental animal:

Adult swiss albino mice (22 to 26 gm) and adult wistar rats (160- 200 gm) of either sex procured from Venkateshwara Enterprises, Bangalore, were housed in a clean polypropylene cage under standard conditions of temperature (25 ± 20C), humidity (55 ± 2%) and light (dark/ light- 12/12 hours cycle). They allowed to take standard laboratory feed and water ad libitum.

 

All the animals were acclimatized for seven days to the laboratory environment. All animal experiments were carried out according to the guidelines and approval of the Animal Ethics Committee (Regd. No 131/99/CPCSEA).

 

Chemicals:

Geriforte tablet (Himalaya Drugs) was used as a standard adaptogenic drug which is a multiconstituent ayurvedic drug with 35 herbal and natural constituents like Whithania somnifera, Asparagus racemosus, Glycyrrhiza glabra, Shilajeet, Chichorium intybus, Myristica fragrans, Caesalpinia digyna etc.

All other chemicals used in the study were of analytical grade.

Phytochemical screening:

Preliminary phytochemical screening was performed as per standardized procedure 16 and various phytoconstituents present in the extract were identified.

Acute toxicity:

The acute toxicity study was carried out as per guideline set by Organization for Economic Co-operation and Development (OECD guideline no. 425) received from CPCSEA. Mortality within 48 hours was recorded in overnight fasted albino mice. The animals were observed for a further 14 days for any signs for delayed toxicity.

The ethanol extract has good margin of safety and did not show the lethal effects on the albino mice upto the doses of 2000 mg/ Kg. Hence 200 mg/ Kg and 400 mg/ Kg body weight of extract were fixed at ED50.

 

Evaluation of adaptogenic activity

In the present study the effect of ethanolic extract of M. oleifera on swimming endurance and cold restraint stress induced ulcer, adrenocortical activity, weights of organs was evaluated.

 

Swimming Endurance test:

Albino mice of either sex were divided into following groups of six animals each.

Group I (stress control): Animals received only 1% w/v Acacia as vehicle.

 

Group II (test 1 and stress): Animals received Moringa oleifera extract (200 mg/ Kg body weight)

 

Group III (test 2 and stress): Animals received Moringa oleifera extract (400 mg/ Kg body weight)

 

Group IV (standard and stress): Animals received Geriforte (100 mg/ Kg body weight)

 

Vehicle, extract and Geriforte were given to mice once daily for a period of 10 days orally. On tenth day all the mice were subjected to swimming stress by keeping them in cylindrical vessels (30 cm x 25 cm) containing water at room temperature. The mice were allowed to swim till they got exhausted. The end point was taken when the animals drowned and death occurred 17.

 

Swimming survival time for each animal was noted and mean swimming survival time for each group was calculated. The data obtained were subjected to statistical analysis.

 

Cold- Restraint stress induced Ulcers (CRU):

Albino rats of either sex were divided into following groups of six animals each.

Group I (stress control): Animals received only 1% w/v acacia as vehicle.

Group II (test 1 and stress): Animals received Moringa oleifera extract (200 mg/ Kg body weight)

Group III (test 2 and stress): Animals received Moringa oleifera extract (400 mg/ Kg body weight)

Group IV (standard and stress): Animals received omeprazole (20 mg/ Kg body weight)

 

Vehicle, extract and omeprazole were given to rats once daily for a period of 10 days orally. Rats were deprived of food for about 18 hours before the experiment. Rats were subjected to cold and restraint stress for 2 hours by tying them to the wooden plank and were placed at 4- 6 C 18. Two hour later, the animals were sacrificed by cervical dislocation. The stomach was then excised and cut along the greater curvature. The stomachs were washed carefully with 5 ml of 0.9% sodium chloride, rinsed under a slow stream of water and pinned flat on corkboard for ulcer scoring. The stomachs were examined with magnifying lens for the degree of ulceration 19. The ulcerogenic indices were scored 17. The following arbitary scoring system was used to grade the incidence and severity of the lesions.

 

     I.        No ulcerogenic changes = 0

   II.        Shedding of epithelium = 10

 III.        Petechial and frank haemorrhages = 20

 IV.        One or two ulcers = 30

   V.        More than two ulcers = 40

 VI.        Perforated ulcers = 50

 

Ulcer index (UI) is calculated from the scoring described as follows.

               UI = UN + US + UP  x 10-1

Where UN = average number of ulcers per animal

US = average of severity scores

UP = percentage of animals with ulcers

Percentage protection index is calculated as follows

 

% protection index = C – T x 100

                                        C

Where, C = ulcer index in control group

T = ulcer index in treated group

 

Effect of Moringa oleifera on Adrenocortical activity and organ weights in cold-restraint stress model

Albino rats of either sex were divided into following groups of six animals each.

Group I (control): Animals received saline

Group II (stress control): Animals received only 1% w/v acacia as vehicle

Group III (test and stress): Animals received Moringa oleifera extract (400 mg/ Kg body weight)

Group IV (standard and stress): Animal received Geriforte (100 mg/ Kg body weight)

 

Vehicle, extract and Geriforte were given to rats once daily for a period of 10 days orally. On 10th day animals from group 2, group 3 and group 4 were subjected to cold and restraint stress for 2 hours by tying them to the wooden plank and were placed at 4-60C.

 

Animals from all the groups were anaesthetized using Ether to avoid further stress and blood samples were collected from the jugular vein in heparinised syringes for the estimation of plasma corticosterone 20.

 

All the animals were sacrificed by cervical dislocation to isolate adrenal glands, liver and spleen. The organs were washed with alcohol and individual weights were recorded with respect to their body weight i.e. 100 gm.

 

Statistical analysis:

The resulted values of all the studies were expressed as mean ± SEM (n = 6) and analysed by one-way ANOVA followed by Dunnet’s test for individual comparison of groups with control.

 

RESULTS:

Preliminary phytochemical screening revealed the presence of glycosides, phytosterols, tannins and amino acids.

 

The animals did not show significant autonomical and behavioural changes observed for 14 days, thereby showing the safety of Moringa oleifera.

 

Swimming endurance test: As shown in table-1 the survival time of swimming mice was significantly (p< 0.01) increased in the animals treated with extract at both the tested doses of 200 and 400 mg/ Kg body weight as compared to the untreated animals. The extract effects were at par with that of the standard drug.

 

Cold- restraint stress induced ulcers: Control group rats exhibited ulcers, Frank haemorrhages and perforated ulcers. The ethanolic extract of Moringa oleifera at the tested doses of 200 and 400 mg/ Kg body weight showed protection index of 59.02 and 71.65 respectively, whereas omeprazole showed protection index of 87.52 at a dose of 20 mg/ Kg body weight (Fig- 1). So group of rats pre-treated with Moringa oleifera extract for 10 days exhibited a significant (p< 0.01) protective effect on ulceration induced by cold-restraint stress and was comparable to the standard drug.

 

Fig 1: Percentage protection of ulcer index

 

Effect of ethanolic extract of M. oleifera and omeprazole at doses of 200, 400 and 20 mg/ kg body weight respectively, on percentage protection of ulcer index in cold-restraint stress model. p<0.01 when compared to control (n = 6 in each group).

Table1: Effect of Moringa oleifera extract on swimming endurance test in mice

Group

Treatment

Dose

Swimming survival time (min.) Mean ± SEM

I (stress control)

Vehicle (1% w/v Acacia)

0.25 ml

387.00 ± 28.30

II(test 1 and stress)

M.oleifera extract

200mg/kg body weight

464.11± 23.40 *

III(test 2 and stress)

M.oleifera extract

400mg/kg body weight

481.17 ± 11.55 *

IV(standard and stress)

Geriforte

100mg/kg body weight

493.88 ± 14.04 *

* p<0.05 when compared with stress control

 

 

Table 2: Effect of Moringa oleifera extract on Adrenocorftical activity and organ weights in cold- restraint stress model.

Group

Treatment

Dose

Plasma

corticosterone (µg/ dl)

Organs weight (wt/ 100 gm body weight)

Adrenals (mg)

Spleen (mg)

Liver (gm)

I (control)

Saline

0.25 ml

26.45 ± 0.84

11.45 ± 0.86

283. 908 ± 6.738

2.84 ± 0.03

II (stress control)

Vehicle (1% w/v acacia)

0.25 ml

51.67 ± 0.27 *

18.37 ± 0.24 *

259.841 ± 4.697

3.01 ± 0.46

III ( test and stress)

M. oleifera extract

400 mg/ Kg body weight

38.32 ± 0.26 *

13.62 ± 0.12 *

265.367 ± 3.567

2.99 ± 0.17

IV (standard and stress)

Geriforte

100 mg/ Kg body weight

34.20 ± 0.16 *

12.28 ± 0.15 *

270.020 ± 1.836

2.90 ± 0.21

Values are mean ± SEM, * p< 0.01 when compared with control.

 

 

 

Effect of Moringa oleifera on Adrenocortical activity and organ weights in cold- restraint stress model: As shown in table 2, stress controlled animals exhibited marked increase in plasma corticosterone as compared to control group. Pre-treatment with M. oleifera extract prevented the increase in plasma corticosterone due to cold-restraint stress significantly (p< 0.01). Stress control animals exhibited marked increase in the weights of liver, adrenal glands and decrease weight of spleen as compared to control group. Pre-treatment with M.oleifera extract at 400 mg/ Kg significantly (p< 0.01) prevented the increase in adrenal weight due to cold-restraint stress. Moringa oleifera extract at 400 mg/ Kg had not significantly prevented the variations in liver and spleen weight due to cold-restraint stress.

 

DISCUSSION:

Adaptogens are pharmacological agents that induce a state of non-specific increase of resistance of organism (SNIR) to aversive stimuli that threaten to perturb internal homeostasis3, 21. During 1958, medicinal substance causing SNIR were named as ADAPTOGENS22. Later on in 1961, such substances were called as “Athenktotropic” in the Anglo-American medicinal literature23. A rational approach was made to evaluate its anti-stress property. For this, various stressful situations were induced in animals. Greater swimming endurance has reported in mice when pre-treated with anti-stress agents24 and the test has been utilized to investigate the adaptogenic activity of different agents, based on the fact that swim endurance reflects physical endurance17. The swim endurance test results indicate clearly that the extract of Moringa oleifera have the properties whereby they increased the physical endurance as well as the overall performance in mice.

 

Stress is known to induce peptic ulcers and severity of ulceration is entirely dependent upon the intensity of the stress17.

 

Stress induced ulcers are caused by a number of factors, both physical and psychological25. The mechanism is still unclear but the Central Nervous System plays an important role in pathogenesis and prevention of restraint stress induced ulcers in rats17. Peripheral sympathetic activation plays an important role in induction of ulcers by restraint26. In the present study Moringa oleifera extract exhibited protective effect on ulceration induced by cold-restraint stress. Its antiulcerogenic effect may be due to its effects on neural pathways27 controlling acid secretion, thereby strengthening the animals physiological capabilities to decrease stress and hence ulcers.

 

Any type of stress, whether physical or neurogenic, will cause an immediate and marked increase in ACTH secretion by the anterior pituitary gland, followed within minutes by greatly increased adrenocortical secretion of cortisol28. Anti-stress drugs or the so called adaptogens, appear to have a corticosparing effect during experimental stress 3, 17. It is known that cold swimming stress increases plasma corticosterone level29. In the present investigation it was observed that Moringa oleifera extract prevented the rise in plasma corticosterone indicating the corticosteroid sparing effect which is in agreement with reported study 30.

 

Stress induces adrenomedullary response in man leading to increased production of corticotropic hormone that leads to increase in weight of adrenal gland, liver and reduction in spleen weight31. In the present study it was observed that Moringa oleifera extract prevented the increase in adrenal weight indicating anti-stress activity.

 

CONCLUSION:

The present study clearly indicate that Moringa oleifera could increase the non-specific resistance of the organism against various stressor thereby proving a promising adaptogen and further in depth studies are required to elucidate its mechanism of action.

 

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Received on 02.01.2010

Accepted on 24.03.2010     

© A&V Publication all right reserved

Research J. Pharmacology and Pharmacodynamics. 2(3): May-June 2010, 243-247