Analgesic, Anti-inflammatory and Antipyretic Activity of Rotula aquatica Lour Leave

 

P. Agrawal¹*, K. Mruthunjaya³, K. Goyal¹, D. Ahuja¹, M.K. Gupta²

¹Research Scholar, Jayoti Vidyapeeth Women's University, Jaipur Rajasthan, India.

²Saint Soldier College of Pharmacy, Tonk, Rajasthan, India.

³J.S.S College of Pharmacy, J.S.S University, Mysore, Karnataka, India.

 

ABSTRACT:

The present study was aimed to evaluate analgesic, anti-inflammatory and anti pyretic activity of methanolic extract and aqueous extract of leave of Rotula aquatica L. in mice and rats. The methanolic extract of R. aquatica L. leave at a dose of 200mg/kg body weight has shown significant analgesic, antipyretic and anti-inflammatory activity as compared to aqueous extract. The result of hot plate method indicated that the total methanolic extract showed significant increase (P<0.01) in reaction time at a 3,4and 6 hours comparable to the reference drug Pentazocin but lesser (P<0.05) after 2hr. The tail immersion and hot plate tests revealed that plant has high analgesic activity. Both tests showed highest degree of analgesia in methanolic extract compared to aqueous extract. The total methanolic extract of R.aquatica L. leave at the a dose of 200mg/kg body weight has shown significant (p<0.01) antipyretic activity as compared to aqueous extract, methanolic extract also found to have significant result in Carrageenin-induced paw oedema; suspect edmodeo faction may be due to possible inhibition of lipooxygenase pathway.

 

 

INTRODUCTION:

The problem of uncontrolled pain led early human to seek remedies from any materials that they could lay their hand on. In recent times, focus on plant research has increase and non-steroidal anti-inflammatory drugs constitute one of the most widely used classes of drugs. Herbal drugs are being proved as effective as synthetic drugs with lesser side effects. Herbal medicines are in line with nature, with less hazardous reaction¹.

 

The leave of Rotula aquatic is also called as pashanbed, belonging to family Boraginaceae. It is widely distributed in India from Kumaun to Assam and western to southern India² One such plant is Rotula aquatic (Boraginaceae), which is used as anti-inflammatory, diuretic, and laxative, in hemorrhoids, renal and vesical calculi, diabetes and venereal diseases.

 

The plant contains Baunerol, steroid, alkaloid and rhabdiol. The medicinal value so plant lie in their component Phytochemical such as alkaloids, flavonoids, phenolic compounds and other nutrients like as amino acid and proteins.3 The plant is a mandatory component of many ayurvedic drug preparation sand is an important traditional medicine for kidney and bladder stones. The Rotula aquatica tuber is astringent, bitter, diuretic and also use ful in treating coughs, heart diseases, dysuria, blood disorders, fever, poisonings, ulcers and uterine diseases4. Rotula aquatica decoctions are both diuretic and laxative and are used to treat bladder stones and sexually transmitted diseases5. On the basis of traditional system of medicine there is no scientific evidence on this anti-inflammatory, antipyretic and analgesic activity so courmain is to evaluate this activity in various experimental models.

 

MATERIALS AND METHODS:

Plant Materials:

Leave of R. aquatic L. was collected from local areas Manipal, Karnataka. The taxonomical identification of the plant was done by Dr. Gajendra Pal Singh, Department of Botany, University of Rajasthan, Jaipur, and voucher specimens were deposited at the herbarium, Department of Botany, University of Rajasthan, Jaipur (Specimen no. RUBL51008). Leave was dried under shade, coarsely powdered and stored in air tight container for further use.

 

Preparation of Extract:

The powdered leave was Soxhlet-extracted with methanol. The extract, on removal of solvent in vacuum, gave green semisolid residue. The leave of R. aquatica L. was shade dried at room temperature, pulverized, and 100g of coarse powder was macerate exhaustively with water then being kept for 5 days in tightly sealed vessels at room temperature, protected from sunlight and shaken several times daily and add preservative (2% chloroform). Concentrate extract by distilling off the solvent and then evaporating to dryness on water–bath, gave light green semi solid residue.^6-7

 

Phytochemical Screening:

Preliminary Phytochemical investigation was carried out for extracts. Presence of alkaloids was determined by Mayer’s,  Dragendorf’s, Wagner and Hager’s test, Flavonoids by Shinoda, Ferric chloride and lead acetate tests, Saponins by Foam and haemolysis test and sterols by Salkowaski and Libermann and Burchardstests⁸.

 

Animals Used:

Wister rats of either sex weighing 180-200g and Swiss mice weighing 18-28g were maintained under standard nutritional and environmental conditions throughout the experiment. The animals were of food for24 h before experimentation but allowed access to tap water throughout. Animal were divided into five (n=6) for each experimental model, control, standard, two extract.  Approval for the project was obtained by the institutional ethical committee, IAEC, Sri Balaji College of Pharmacy, Jaipur (Letter No. IAEC/SBCP/2020/22)

 

Toxicity Study9:

Leave of R. aquatic L. was tested in single doses in each experimental model as per following the OECD guideline no. 420 fixed dose method procedure, the safest dose of total methanolic extract and are 2000mg/kg body weight. The safe dose was found to be 2000mg/kg body weight; hence 1/10thof the dose was taken as effective dose which was found to be 200mg/kg body weight. Pentazocine 5mg/kg was used as the standard analgesic in hot- plate and Acetyl salicylic acid 640mg/kg p.o in tail immersion in mice. Paracetamol was used as standard drug (positive control) in anti-pyretic models in the dose of 150mg/kg and required quantity was dissolved in normal saline. In the anti-inflammatory model aspirin was used as the standard drug in a dose of 150mg/kg.

 

Assessment of Analgesic Activity^10-12

Hot Plate Method:

In the hot plate method albino mice (18-28) were divided into four groups each consisting of six animals. One group served as a control (received vehicle), second group served as a standard (received Pentazocine 5mg/kg) while the third group received the methanolic extract (As per b/w), and four group received the aqueous extract (As per b/w). The basal reaction time was noted before and 30, 60, 90 and120 minutes after the administration of the drugs.

 

Tail Immersion Method:

In the Tail immersion method albino rats (180-200g) were divided into four groups each consisting of six animals. One group served as a control (received vehicle), second group served as a standard (received Acetyl salicylic acid 640mg/kg p.o) while the third group received the methanolic extract (As per b/w), and four group received the aqueous extract (As per b/w). The time in second to withdraw the tail clearly out the water was taken as the reaction time.

 

Assessment of Anti-Pyretic Activity13

Induction of Yeast-induced Pyrexia:

Rats were divided into four groups of six each for this experiment. The normal body temperature of each rat was measured rectally

 

The results were analyzed by ANOVA followed by Dunnet's test ,(p-value 0.05 was taken as significant) at predetermined intervals and recorded. The rats were trained to remain quiet in a restraint cage. A thermister probe was inserted 3-4 cm deep into the rectum and fastened to the tail by adhesive tape. The temperature was measured on a thermometer. After measuring the basal rectal temperature, animals were given a subcutaneous injection of 10 ml/kg body wt. of 15% w/v yeast suspended in 0.5%w/v methyl cellulose solution. Rats were then returned to their housing cages. After19hof yeast injection, the animals were again restrained in individual cages for another recording of their rectal temperature as described above.

 

Table 1:Effect of Pentazocine, methanolic extract, and Aqueous extract of leaves of Rotula aquatica lour on Eddy’s Hot plate test in mice.

S. No	Treatment	Reaction time in seconds
		0 min	30 min.	60 min	90 min	120 min
1.	Control	2.83±0.3073	3.66±0.3333	3.66±0.3333	3.00±0.2582	3.00±0.2582
2.	Pentazocine (5mg/kg.s.c)	2.83±0.3073	3.66±0.2108	5.5±0.2236**	6.65±0.3333**	7.5±0.4282**
3.	RALML (200mg/kg,p.o)	2.66±0.2108	3.33±0.3333	3.16±0.1667	4.5±0.2236*	5.16±0.1667**
4.	RALAQ (200mg/kg,p.o)	2.66±0.2108	3.33±0.2108	3.5±0.2236	3.83±0.3073	4.83±0.1667*

The results were analyzed by ANOVA followed by Dunnet's test , p<0.05- Significant, p<0.01 Moderately significant, p<0.001 Highly significant

 

Table 2: Effect of Acetyl salicylic acid, methanolic extract, and Aqueous extract of leaves of  Rotula aquatic lour on Tail immersion test in rats

S. No	Treatment	Response Time In Seconds
		0.0 min	30min	1st hr.	2nd hr.	3rd hr	4th hr.	6th hr.
1	Control	2.83± 0.1667	2.88± 0.1667	3.00± 0.2582	3.00± 0.2582	3.00± 0.2582	3.16± 0.3073	3.16± 0.1667
2	Aspirin (640mg/kg, p.o)	3.16± 0.3073	3.33± 0.2108	3.83± 0.3073	5.66± 0.3333**	6.83± 0.3073**	7.67± 0.2108**	8.00± 0.2582**
3	RALML (200 mg/kg, p.o)	3.33± 0.2108	2.66± 0.2108	3.5± 0.2236	3.83± 0.3073	4.5± 0.2236*	4.5± 0.2236*	4.83± 0.3073**
4	RALAQ (200 mg/kg, p.o)	3.16± 0.1667	2.83± 0.1667	3.33± 0.2108	3.5± 0.2236	4.00± 0.2582	4.2± 0.2108	4.40± 0.3073*

The results were analyzed by ANOVA followed by Dunnet's test (p-value 0.05 was taken as significant)

 

Table 3: Effect of Paracetamol, Methanolic and Aqueous extracts of leaves of Rotula aquatica lour on yeast-induced pyrexia

S.No.	Treatment	Initial temp (°C)	Temp. after 19 hr yeast admn.(°C)	Temp at different hr after treatment (°C)
				20 hr	21 hr	22 hr	23 hr
1	Control	37.18±0.0945	39.16±0.0667	37.30±0.5574	39.45±0.6708	39.51±0.0654	39.56±0.0843
2	Paracetamol 150 mg/b.w	37.33±0.0894	39.38±0.600	38.085±0.7188	38.18±0.1887	37.51±0.0794**	37.25±0.600**
7	RALML 200mg/b.w	37.43±0.0988	39.43±0.1256	38.65±0.0922	38.53±0.0922	38.21±0.01759	38.03±0.0182*
8	RALAQ 200mg/b.w	37.55±0.0763	39.5±0.1033	38.60±0.1202	38.43±0.0954	38.30±0.0816	38.23±0.0557

The results were analyzed by ANOVA followed by Dunnet's test (p-value 0.05 was taken as significant)

 

Table 4: Effect of Aspirin, Methnolic extract, and Aqueous extract of leaves of Rotula aquatic lour on paw edema in carrageenan paw edema model in rat

S. No	Treatment	(Paw size) Change in volume (ml) at h
		0 Hr.	1 Hr.	2 Hr.	3 Hr.	4 Hr.
1.	Control	0.84±0.0160	1.02±0.0275	1.16±0.0518	1.19±0.0393	1.21±0.0339
2.	Aspirin 150 mg/b.w	0.77±0.0315	0.74±0.0049	0.70±0.0079**	0.69±0.0060**	0.64±0.0085**
3.	RALML 200 mg/b.w	0.77±0.0083	0.81±0.0076	0.84±0.0060	0.85±0.0108	0.83±0.0102**
4.	RALAQ 200 mg/b.w	0.80±0.0080	0.81±0.0094	0.84±0.0095	0.86±0.0103	0.86±0.0080

 

Drug Administration:

After 19h of yeast injection, the methanolic and aqueous extracts were administered orally at doses of 200 mg/kg body wt. to two groups of animals, respectively. A similar volume (5ml/kg body wt.) of normal saline solution was administered orally to the control group. The fourth group of animals received the standard drug paracetamol (150 mg/kg body wt.) orally. Rats were restrained for recording of the irrectal temperature at the nineteenth hour, immediately before methanolic and aqueous extract, saline or paracetamol administration, and again at one- hour intervals up to the twenty-third hour after yeast injection.

 

Assessment of Anti- Inflammatory Activity ^{12, 14-15}

Carrageen in-induced Rat Paw Oedema The rats were divided into four groups, each groups consisting of six animals. Oedema was induced by sub plantar injection of 0.1mlof 1%    freshly repared suspension of Carrageen in into the right hind paw of each rat.The paw volume was measured before (Oh) and 1 h after the injection of Carrageenin using a Plethysmometer. The total alcoholic and aqueous extract bark in 2% Tween 80 solution (200 mg/kg) was administered orally to two groups of rats, 30 min before the injection of Carrageenin. The third and fourth group of rats received 2% aqueous. Tween 80 solution 10 ml/kg orally (control) and Aspirin 150mg/kg as a reference drug.

 

Statistical Analysis:

Values are expressed as mean±S.E.M Statistical significance was analyzed using one way ANOVA and p<0.05 was considered as statistically significant.

 

RESULTS:

The OECD guideline 420 fixed dose methods study showed that extract was safe at a dose of 2000 mg /kg body weight. The methanolic of R. aquatica L. root at a dose of 200 mg/kg body weight has shown significant analgesic, antipyretic and anti-inflammatory activity as compared to aqueous extract. The analgesic activity of root of R. aquatica L. was studied for its central activity. The result of hot plate indicated that the methanolic extracts haws a significant increase (P<0.01) in reaction time at 3 and 4 hours comparable to the reference drug Pentazocin but lesser (P<0.05) at 2hr. Aspirin leads to are life from inflammatory pain by suppressing the formation of pain inducing substances in the peripheral tissues, prostaglandins and bradykinin were suggested to play an important role in the pain process16. Therefore it is likely that R. aquatic L. root might suppress the formation of these substances. It has been widely accepted that Carrageen in-induced paw oedema model is applied for the evaluation of the anti oedemal effect of drugs. Recent investigation demonstrated that Carrageen in oedema is effectively decreased by lipooxygenase inhibitors. In the present study, total alcoholic extract significantly inhibited Carrageen in-induced paw oedema as compared; it may be due to possible inhibition of lipooxygenase pathway although such assumption obviously requires confirmation by further detailed experimentation17.The total alcoholic extract of R. aquatica L. root at the a dose of 200 mg/kg body weight has shown significant (p<0.01) antipyretic activity as compared to aqueous extract, it has shown significant fall in body temperature up to 4h following its admistraction. The antipyretic activity stared as early as 1 hrs. and the effect was maintained for 4h. The response was comparable to that antipyretic activity of Paracetamol a standard antipyretic drug. But aqueous extract did not showed significant activity as compared methanolic extract.

 

DISCUSSION:

A drug with anti‐inflammatory activity usually exhibit anti‐pyretic and analgesic properties. The best examples would be the non steroidal anti‐inflammatory drugs, which possess all three activities18. Inflammation is a defensive reaction of the local microcirculation to tissue injury a rising from cell damages due to mechanical trauma, chemical, physical and thermal injury, antigen antibody reactions and infections. The signs and symptoms of inflammation in clued redness, swelling, heat, pain and loss of function of the affected area. Pain is an unpleasant sensation that is a consequence of complex neuro chemical processes in the central and peripheral nervous systems. Non-steroidal anti-inflammatory drugs (NSAIDS) and opioids are used in management of mild to moderate and severe pains respectively. These drugs have serious limit at ions due to their side effects. Most of the drug used presently for the management of pain and inflammation possess some side and toxic effects. It is therefore, in evitable to search for new, less toxic and more effective anti-inflammatory and analgesic agents19. Fever may due to infection of the sequel soft tissue damage, inflammation, graft rejection, orother disease states. Antipyretic are agent which reduce the elevated body temperature. Regulation of body temperature requires a delicate balance between production and loss of heat, and the hypothalamus regulates the set point at which body temperature is maintained. In fever this set point elevates and a drug like paracetamol does not influence body temperature when it is elevated by the factors such as exercise or increase in ambient temperature. Yeast-induced fever is called pathogenic fever. Its etiology includes production of prostaglandins which set the thermoregulation center at a lower temperature20. In the present study, methanolic extract significantly inhibited Carrageen in-induced paw oedema as compared to aqueous extract. The tail immersion and hot plate test reveal that this plant has high analgesic activity. This is because some form of error may be introduced with the animal handing while the test is being elicited. Both test show highest degree of analgesia in methanolic extract compared to aqueous extract. methanolic extract of bark possesses a significant antipyretic effect in yeast provoked elevation of body temperature in rats as compared to aqueous extract but its effect less than that of paracetamol (standard drug). The result clearly indicates that the methanolic extract of R. aquatica L. leave in context of analgesic, antipyretic and anti- inflammatory activity. The detailed study is required in order to identify the actual active constituent from this drug.

 

ACKNOWLEDGMENTS:

We are thankful to (Dr.) Mini Arrawatia, Director- Research Jayoti Vidhyapeeth Women’s University Jaipur and Prof. (Dr.) Dharmendra Ahuja, Director, Department of Pharmacy, Jayoti Vidhyapeeth Women’s University, Jaipur for providing the facilities to carry out the research work. We also wish to extend our thanks to herbarium, Department of Botany, University of Rajasthan, Jaipur  (Rajasthan) for taxonomic identification of the plant. One of the authors, JVn Parul Agrawal is highly thankful to Dr. K. Goyal Jayoti Vidhyapeeth Women’s University, Jaipur and Dr. K. Mruthunjaya, HOD, Department of Pharmacognosy, JSS College of Pharmacy, JSS University, Mysore India for providing necessary facility during the research work.

 

REFERENCES AND NOTES:

1.      A.L. Udupa, U.P. Rathnakarand S. Udupa. Anti- inflammatory, anti-pytrtic and analgesic effect of Tamarindus indica. Indian Drugs. 44(6):466- 470(2007).

2.      Mamta K, Abhishek B, Rohit. Pharmacognostical evaluation on the root of Rotula aquatic lour. International Journal of Pharma and Bio Sciences. 2010;VI(2):01-04.

3.      Gilhotra U, Christina AJM .Effect of Rotula aquatic lour on ethylene – glycol induced urolithias is inrat. Int .J .Drug Devand Res. 2011;3(1)273-280

4.      Sivarajan, V. V, Indira Balachandran (1994). Ayurvedic Drugs and Their Plant Sources. New Delhi, India: Oxford and IBH Publishing Co. pp.358–359.

5.      Christiana, A.J.,  M. Priyamole, P. Moorthy (July– August 2002). Studies on the antilithic effect of Rotula aquatic Lo urin male Wistar rats. Methods and Findings in Experimental and Clinical Pharmacology. 24(6):357–359.

6.      Mukherjee P.K. Quality Control of Herbal Drugs: New Delhi, India, Business Horizone, 2002. Indian Pharmacopoeia. 1996. Vol II, Appendix 3.23, A47.

7.      C.K Kokate. Practical Pharmacognosy.Nirali Prakashan;1994.

8.      OECD [Organosation for Economic Co-opration and Development] 1992. Guideline 420: Acute oral toxicity–Fixed dose procedure, Paris: OECD.

9.      Acosta SL, Muro LV, Sacerio AL, Pena AR, Okwei SN. Analgesic Properties of Caprariabi flora leaves aqueous extract. Fitoterapia. 2002;74: 686-8.

10.   Udupa AL, Rathnakar UP, Udupa S. Anti- inflammatory, Anti-pyretic, Analgesic effect of Tamarindus indica. Indian drug, 2007; 44(6):466-0.

11.   Vogel GH, Drug Discovery and Evaluation. 2nd Ed, Springer-Verlag Berlin Heidelberg. Germany; 2002.

12.   Mandal SK, Mandal SC, Das AK, Tag H, Sur T. Antipyretic activity of Eupatorium adenophorum leaf extract. Ind. J. Nat. Prod 2005;21(1):6-8.

13.   B.V. Owyele, S.B.Olaleye, J.M. Okeand R.A. Elegbe. Anti-inflammatory and Analgesic activity of Notho spondiasstaudtii. Nigerian journal of Physiological Sciences 19(1-2):102- 105(2004)

14.   B.V. Owoyele, S.B. Olaleye, J. MO Keand R.A. Elegbe. Anti-inflammatory and Analgesic activity leaf extract of Land olphiao wariensis. Afr. J. Biomed. Res, 4:131-133(2001).

15.   Lalitha KG, Sethuraman MG, and Rajkapoor B. Analgesic activity of Sarco stem mabrevistigma. Indian Drug. 2002; 39(10):541-2

16.   S.M. Farook, W.C.Atlee, S. Kannan, S. Kumarand M.S. Davey. Assessment of Analgesic, Anti- Pyretic and Anti-inflammatory of Hydro- alcoholic fraction of Hemi desmus indicus root in experimental animals. Scholars Research Library, 3(1):448-453(2011)

17.   S.R Swain, B.N.Sinhaand P.N Murthy. Comparative evaluation of Antipyretic and Analgesic activity of Rungi are pens Needs and Rung iapectinata L. Asian Journal of Pharmaceutical and Clinical Research. 4(2): 103-106(2011).

18.   T.S. M. Saleem, S.D. Basha, G. Mahesh, P.V.S. Rami, N.S. Kumar and C.M. Chettv. Analgesic, Anti-Pyretic and Anti-inflammatory of Dietary Sesame Oil in Experimental Animal Models. Pharmacologia. 2(6):172-177(2011).

19.   K.K Hullatti and M.S.Sharada.Comparative Antipyretic activity of Patha: An Ayurvedic drug. Pharmacognosy Magazine. 11(3):173- 175(2007).

 

 

 

Accepted on 29.03.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(10):5503-5507.