INTRODUCTION:

Diarrhoea is a condition characterized by a variation in the bowel movement¹. It is one of the major health issues in developing countries. In children’s, it is most common to cause severe dehydration and sometime may lead to death^2,3. Its mortality index in children’s of less than 5 years of age has been crossing 5 million each year globally⁴.

Alteration of bowel movement and intestinal transit time is associated with various factors including psychological factor, anxiety or even some chemical substances are also involved⁵.

Available conventional medicines are not completely safe for any therapy. Now a day’s herbal medicines are widely utilized with greater importance to treat various ailments by considering its safety and efficacy⁶. So it is very crucial to identify and investigate available natural drugs for treating diarrhoea⁷.

Achyranthes aspera Linn, is widely known as ‘rough chaff tree’ in English and ‘Chirchira’ in Hindi. It belongs to a family Amaranthaceae. It grows everywhere as a wasteland herb⁸,⁹. It is spreading universally including tropical Asia and India as a weed¹⁰. Traditionally different parts of this plant were used to treat various ailments like skin rash, cough, nasal infection, fever, asthma, fistula, piles, snake bites, chronic malaria^11,12, stomach pain¹³, dysentery, gonorrhoea¹⁴, Cholecystitis¹⁵ and many more. Various Pharmacological activities like antidiabetic, spermicidal, anti-inflammatory, antitumor, antimicrobial, immunomodulatory, nephroprotective, antioxidant, wound healing, antihypertensive and many others were reported in Achyranthus aspera¹⁶. However, an antidiarrhoeal property of Achyranthus aspera has not been reported systematically. Hence in the present study, attempts were made to evaluate the antidiarrhoeal activity of the whole plant of Achyranthus aspera, by extracting it with various solvents like petroleum ether, dichloromethane and ethanol.

MATERIALS AND METHODS:

Extraction:

Achyranthes aspera plant was collected from B. G Nagara, Nagamangala Taluk. The dried powder was extracted by soxhlet apparatus. Extracting solvents were chosen based on their polarity that is petroleum ether, dichloromethane and ethanol.

Experimental Animals:

Albino mice and rats weighing approximately around 150 to 160g and 20 to 25g respectively were acquired from Sri Raghavendra Enterprises, Bangalore and kept under standard environmental provisions. Animals were maintained with free access to food and water. Institutional Animal Ethical Committee approval with ethical clearance No. SACCP-IAEC/26/2015 was taken for the study.

Acute toxicity studies:

The acute toxicity study of all the extracts of the Achyranthes aspera was performed using albino mice as per OECD guidelines 425. Survival of animals was observed for 48 hours and 14 days after different dose administration of the extract. The Lethal Dose₅₀value was calculated.

Phytochemical Screening:

Presence of Phytoconstituents was confirmed by following standard procedures given by Kokateand Khandelwal with some modification^17,18,19.

Castor oil-induced diarrhea:

Eight groups of Albino rats with six rats in each group were kept fasting for 24 hours then used for the study. The first group was served as control; the second group had received 3mg/kg of loperamide per oral route (standard drug). Third to eight group of the animal were treated with all solvent extracts of Achyranthes aspera at a dose of 200 and 400mg/kg body weight.

After 30 minutes of the above treatment, each rat received 1ml castor oil then housed separately. The dropping number and weight of stools were observed for 4 hours. Percentage protection of diarrhoeal episodes was calculated by a formula^20,21.

Percentage of protection (%) = A-B/A

Where

‘A’ is the total weight of stools of control animals.

‘B’ is the total weight of stools of extracts treated animals.

The data of stools weight are expressed as mean ± SEM.

Gastrointestinal Motility test:

Pazhani G.P. et.al. depicted method was followed here²². Albino rats were divided into 8 groups of 6 rats each fasted for 24 hours with free access to water were used for the evaluation. First group was served as control (0.2 ml of vehicle p. o.), Second group was treated with standard drug Atropine sulphate 5mg/kg orally. Group third, fourth, fifth, sixth, seventh and eight were treated with all the extracts of Achyranthes aspera at a dose of 200mg/kg and 400mg/kg bodyweight respectively.

Administered one ml of a charcoal meal orally (i.e. 3% charcoal (deactivated) in 10% of normal saline) after 30 minutes of the above treatment. Rats of all the groups were sacrificed after half an hour of a charcoal feed and the movement of charcoal feed in the intestine was measured. Percentage travelling and percentage inhibition were calculated by a formula

% Travelled = (A/B)*100.

% of Inhibition = {(B-A)/B}*100.

Where

‘A’ is the distance travelled by the charcoal meal,

‘B’ is the total length of the small intestine.

Prostaglandin-E₂ induced enteropooling:

A method of Murugesan T. et. al.²³ was followed here. Albino rats were divided into nine groups containing six animals in each group. Animals were deprived of food and water for 18 hour before the experiment. The first group was served as control. The second group was administered with 1ml of 5% ethanol in normal saline orally and considered as a positive control. The third group was served as standard and treated with 3 mg/kg Loperamide orally and group fourth to ninth received 200 and 400mg/kg body weight solvent extracts of Achyranthes aspera respectively.

After above treatment, Prostaglandin - E₂ (100µg/kg in 5% ethanol in normal saline) was administered orally to all the groups excluding the first group.

Half an hour later every animal were sacrificed, intestine was isolated and its fluid content was collected. Percentage inhibition of diarrhoea was calculated by a formula:

Percentage inhibition = 100 – (A/B)*100

Where

“A” is the volume of intestinal fluid in the extract treated animals

“B” is the volume of intestinal fluid in the animal group treated with prostaglandin E₂

Statistical Analysis:

Statistical analysis was done using ANOVA followed by Dunnett ‘t’ test.

RESULTS:

Phytochemical investigation exhibited the presence of saccharides, proteins, Tannins, Triterpenoids, Flavonoids, Steroids, Glycosides, Alkaloids in Petroleum ether extract. Proteins, Tannins, Triterpenoids, Flavonoids, steroids and Alkaloids in Dichloromethane extract. Proteins, Flavonoids, Steroids, Glycoside and Alkaloids in Ethyl alcohol extract.

Results of acute toxicity study showed no mortality or any toxicity up to the dose of 2000mg/kg body weight. Hence a dose of 200mg/kg and 400mg/kg were preferred for the study.

Castrol oil-induced diarrhea:

In this model, all the solvent extract showed significant inhibition of diarrhoeal episodes. Among all, percentage inhibition was more with the higher dose of ethanolic extract of Achyranthes aspera as shown in table 1.

Charcoal meal test model:

Here all the extracts and standard drug had diminished the movement of charcoal in the gastrointestinal tract by decreasing the distance travelled by the charcoal meal. The potency is in the order of atropine sulphate (standard drug), Petroleum ether extract lower dose, ethanolic extract higher dose, Petroleum ether extract higher dose, Dichloromethane extract higher and lower dose, ethanolic extract lower dose respectively as shown in table 2.

Prostaglandin-E₂ induced enteropooling model:

In Prostaglandin-E₂ induced enteropooling model, the volume of intestinal fluid was reduced by standard drug and all the extract as compared to the positive control. The percentage inhibition of diarrhoeal episodes was more in lower doses of all the extract as compared to higher doses. Details are shown in table 3.

Table 1: Anti-diarroeal activity of various extracts of Achyranthes aspera by castor oil induced diarhoeal model

Group	Treatment	Mean weight of stools± SEM after 4 h (g)	% Inhibition
1	Control	3.82 ± 0.2731	-
2	Standard	0.205 ± 0.257**	94.63
3	PEAA 400	0.41 ± 0.05596**	89.26
4	PEAA 200	1.52 ± 0.03103**	60.20
5	DCMAA 400	0.99 ± 0.07929**	74.08
6	DCMAA 200	0.88 ± 0.02512**	76.96
7	EEAA 400	0.23 ± 0.03731**	93.97
8	EEAA 200	0.46 ± 0.04551**	87.95

Values are expressed as mean ± SEM (n = 6). ***P<0.001, **P< 0.01 and *P< 0.05

PEAA 400: Petroleum ether extracts of Achyranthes aspera (400 mg), PEAA 200: Petroleum ether extracts of Achyranthes aspera (200 mg), DCMAA 400: Dichloromethane extracts of Achyranthes aspera (400 mg), DCMAA 200: Dichloromethane extracts of Achyranthes aspera (200 mg), EEAA 400: Ethanolic extracts of Achyranthes aspera (400 mg), EEAA 200: Ethanolic extracts of Achyranthes aspera (200 mg).

Table 2: Anti-diarroeal activity of various extracts of Achyranthes aspera by charcoal meal test model.

Group	Treatment	Mean total length ± SEM (cm)	Mean distance traveled ± SEM (cm)	Mean percentage movement of charcoal ± SEM (cm)	Percentage Inhibition
1	Control	88.66	76.83	91.22 ± 1.75	-
2	Standard	78.83	34.33	30.63 ± 1.96**	56.44
3	PEAA 400	76.66	55.83	72.34 ± 2.08**	27.17
4	PEAA 200	85	47.83	57.76 ± 4.45**	43.72
5	DCMAA 400	85.66	62	72.49 ± 2.23**	27.62
6	DCMAA 200	81.83	62.33	76.13 ± 2.98**	23.82
7	EEAA 400	85.83	49.83	58.48 ± 3.38**	41.94
8	EEAA 200	77.66	63	81.46 ± 2.35^ns	18.87

Values are expressed as mean ± SEM (n = 6). **P< 0.01 and *P< 0.05

Table 3: Anti-diarroeal activity of various extracts of Achyranthes aspera by prostaglanding-e₂ induced enteropooling model.

Group	Treatment	Mean volume of intestinal fluid ± SEM (ml)	Percentage Inhibition
1	Control	0.95±0.1586*	-
2	Positive Control	1.45±0.08851	-
3	Standard	0.58±0.1897**	60.00
4	PEAA 400	0.9±0.04472**	37.93
5	PEAA 200	0.8±0.07303**	44.83
6	DCMAA 400	0.86±0.08433**	40.69
7	DCMAA 200	0.7±0.1000**	51.72
8	EEAA 400	0.96±0.09545*	33.79
9	EEAA 200	0.93±0.04216**	35.86

DISCUSSION:

Diarrhoea usually occurs due to fluid imbalance where there is an alteration of absorption and secretion in the intestine. This leads to huge loss of fluid in the stools. Castor oil undergoes hydrolysis and induces diarrhoea by releasing ricinoleic acid which in turn alters water and electrolyte transportation and stimulates peristalsis in the intestine^{24, 25}. Ricinoleic acid activates adenylate cyclase²⁶, also boosts prostaglandin production and platelet factor activation²⁷. This raises intestinal fluid content and induces diarrhoea²⁸. Acetylcholine, a cholinergic agonist and Atropine, a cholinergic antagonist are involved in increasing and decreasing gastrointestinal motility respectively²⁹. The anti-diarrhoeal property of Achyranthes aspera may be due to its antisecretory mechanism which was proven by reducing weight and frequency of defecation in castor oil-diarrhoeal model, by reducing the motility of charcoal in gastrointestinal motility test and by diminishing the quantity of intestinal fluid in the prostaglandin-E2 diarrhoeal model. The particular Phytoconstituents of Achyranthes aspera for showing antidiarrhoeal potential is yet to be characterized. However tannins and flavonoids have been reported for its antidiarrhoeal property by reducing intestinal motility. Achyranthes aspera is also rich in tannins and flavonoids. Hence the antidiarrhoeal potential of Achyranthes aspera might be due to the presence of such phytoconstituents^30-34.

The findings of this research proved significant antidiarrhoeal property of Achyranthes aspera which could be due to the presence of some active pharmacological components. Thus this plant can be used in the suitable form as an alternative to available synthetic antidiarrhoeal medicines which are not safe completely. Hence adverse effects of synthetic drugs can be minimized. This research opened a new arena for the researchers to contribute further research to isolate and purify the molecules responsible for the antidiarrhoeal activity, explore its mechanism of action and prepare suitable formulation for administration.

ACKNOWLEDGEMENT:

The authors would like to extend a heartfelt thanks to the Principal and management of Sri Adichunchanagiri College of Pharmacy for facilitating this research.

CONFLICT OF INTEREST:

Declared None.

REFERENCES:

1. Guerrant RL, Van Gilder T, Steiner TS, Theilman MN, Slutsker L. Pratice guidelines for the management of infectious diarrhea. The Journal of Infectious Disease. 2001; 32: 331-351.

2. Shoba FG, Thomas M. Study of antidiarrhoeal activity of four medicinal plants in castor oil induced diarrhoea. Journal of Ethnopharmacology. 2001; 76: 73-6.

3. Yeshwante SB, Juvekar AR, Pimprikar RB, Kakade RT, Tabrej M, Kale MK, Firke SD. Anti-diarrheal activity of methanolic and aqueous extracts of Dillenia indica L. Research Journal of Pharmacology and Pharmacodynamics. 2009; 1(3): 140-142.

4. Snyder JD, Merson MH. The magnitude of the global problem of acute diarrhea disease. A review of active surveillance of data. Bull WHO. 1982; 60: 605-613.

5. Paul S, Saha D. Evaluation of antimotility effect of Alpinia conchigera Griff on charcoal induced gastrointestinal motility in mice. Asian Journal of Pharmaceutical Research. 2012; 2(2): 71-74.

6. Hasan S. Pharmacological and medicinal uses of Achyranthes aspera. International Journal of Science, Environment and Technology. 2014; 1(3): 123-129.

7. Chopra RN, Nayar SL, Chopra IC. Glossary of Indian medicinal plants. New Delhi. Council of Scientific and Industrial Research. 1956.

8. Dwivedi, Sumeet, Dubey, Raghvendra, Mehta, Kushagra. Achyranthes aspera Linn. (Chirchira): A Magic Herb in Folk Medicine. Ethnobotanical Leaflets. 2008; 12: 670-676.

9. Sarma D, Kumar DR, Nagajyothi D, Prabhavathi V, Bai MS. A Review on Achyranthes aspera. Research Journal of Pharmacognosy and Phytochemistry. 2014; 6(2): 66-69.

10. Dey A. Achyranthes aspera L: Phytochemical and Pharmacological aspects. International. Journal of Pharmaceutical Sciences. 2011; 2(9).

11. Selvanayagam ZE, Gnanavendan SG, Balakrishnan K, Rao RB. Antisnake venom botanicals from Ethnomedicine. Journal of Herbs, Spices and Medicinal Plants. 1994; (2): 45-100.

12. Warke PD, Kale MK. Phytopharmacognostic Study of Leaves of Achyranthes aspera Linn. Research Journal of Pharmacy and Technology. 2015; 8(2): 212-5.

13. Ghani A. Medicinal plant of Bangladesh with chemical constituents and uses. Asiatic Society of Bangladesh, Dhaka. 2003; 2: 71-72.

14. Aziz A, Rahman M, Mondal AK, Muslim T, Rahman A, Quader A. 3-Acetoxy-6-benzoyloxyapangamide from Achyranthes aspera, Dhaka Universal Journal of Pharmaceutical Sciences. 2005; 4(2): 113-116.

15. Sujitha K, Sri AP, Rao PM, Lal M, Srinivasarao K, Sree VK. Preliminary screening of Syzygium cumini and Achyranthes aspera for their anthelmintic activity. Research Journal of Pharmacognosy and Phytochemistry. 2010; 2(6): 445-449.

16. Rahman HM, Bashir S, Mandukhail SR, Huda S, Gilani AH. Pharmacological Evaluation of Gut Modulatory and Bronchodilator Activities of Achyranthes aspera Linn. Phytotherapy Research. 2017; 31(11):1776-85.

17. Khandelwal. Practical Pharmacognosy. Nirali Publication. 1995; pp. 140-3.

18. Kokate CK, Purohit AP and Gokhle SB. Practical Pharmacognosy. Nirali Prakashan. 2005; 4: pp. 108-11.

19. Datir SB, Nirmal SA, Ganjare AB, Bhawar SB, Patil MJ. Antioxidant activity of the aerial parts of the Achyranthes aspera Var. Porphyristachya (Wall. Ex Moq.) Hook. F. Research Journal of Pharmacognosy and Phytochemistry. 2009; 1(3): 220-223.

20. Awouters F, Niemegeers CJ, Lenaerts FM, Janssen PA. Delay of castor oil diarrhoea in rats: a new way to evaluate inhibitors of prostaglandin biosynthesis. Journal of Pharmacy and Pharmacology. 1978; 30(1): 41-5.

21. Anowi CF, Umanah U, Emezie AU, Utoh-Nedosa AU. Anti-diarrhoeal, antispasmodic and phytochemical properties of ethanol extract of the leaves of Ficus exasperate. Asian Journal of Research in Pharmaceutical Science. 2012; 2(1): 26-32.

22. Pazhani GP, Subramanian N, Arunachalam G, Hemalatha S, Ravichandran V. Anti-diarrhoeal potential of Elephanthus scaber L leaf extract. Indian Drug. 2001; 38(5): 269-271.

23. Murugesan T, Ghosh L, Mukherjee K, Das J, Pal M, Saha BP. Evaluation of antidiarrhoeal profile of Jussiaea suffruticosa Linn. extract in rats. Phytotherapy Research. 2000; 14(5): 381-3.

24. Iwao I, Terada Y. On the mechanism of diarrhea due to castor oil. The Japanese Journal of Pharmacology. 1962; 12(2): 137-45.

25. Ammon HV, Thomas PJ, Phillips SF. Effects of oleic and ricinoleic acids on net jejunal water and electrolyte movement, Perfusion studies in man. The Journal of Clinical Investigation. 1974; 53(2): 374-9.

26. Naidu BB, Palaksha MN, Ravishankar K, Naveen M, Basha N. Evaluation of antidiarrhoeal and antiulcer activities of hydroalcoholic extract of Luffa aegyptiaca fruits. Research Journal of Pharmacology and Pharmacodynamics. 2014; 6(4): 184-189.

27. Maiti A, Dewanjee S, Mandal SC. In vivo evaluation of antidiarrhoeal activity of the seed of Swietenia macrophylla King (Meliaceae). Tropical Journal of Pharmaceutical Research. 2007; 6(2): 711-6.

28. Ooms LA, Degryse AD, Janssen PA. Mechanisms of action of loperamide. Scandinavian Journal of Gastroenterology, Supplement. 1984; 96: 145-55.

29. Paul S, Saha D. Evaluation of antimotility effect of Plumbago indica (L.) on charcoal induced gastrointestinal motility in mice. Asian Journal of Research in Pharmaceutical Science. 2012; 2(3): 95-97.

30. Otshudi AL, Vercruysse A, Foriers A. Contribution to the ethnobotanical, phytochemical and pharmacological studies of traditionally used medicinal plants in the treatment of dysentery and diarrhoea in Lomela area, Democratic Republic of Congo (DRC). Journal of Ethnopharmacology. 2000; 71(3): 411-23.

31. Venkatesan N, Thiyagarajan V, Narayanan S, Arul A, Raja S, Kumar SV, Rajarajan T, Perianayagam JB. Anti-diarrhoeal potential of Asparagus racemosus wild root extracts in laboratory animals. Journal of Pharmacy and Pharmaceutical Sciences. 2005; 8(1): 39-46.

32. Di Carlo G, Autore G, Izzo AA, Maiolino P, Mascolo N, Viola P, Diurno MV, Capasso F. Inhibition of intestinal motility and secretion by flavonoids in mice and rats: structure‐activity relationships. Journal of Pharmacy and Pharmacology. 1993; 45(12): 1054-9.

33. Gavez J, Crespo ME, Jimenez J, Suarez A, Zarzuelo A. Anti-diarrhoeic activity of quercitin in mice and rats. Journal of Pharmacy and Pharmacology. 1993; 45: 1054-1059.

34. Rao VS, Santos FA, Sobreira TT, Souza MF, Melo CL, Silveira ER. Investigations on the gastroprotective and antidiarrhoeal properties of ternatin, a tetramethoxyflavone from Egletes viscose. Planta Medica. 1997; 63(02): 146-9.

Received on 19.06.2020 Modified on 24.08.2020

Research J. Pharm. and Tech. 2021; 14(8):4173-4177.

DOI: 10.52711/0974-360X.2021.00722