Studies on the Anti-inflammatory potential of selected medicinal plants in vitro

 

K. Kousalya¹, T. Priya¹, P. Venkatalakshmi^1,2

¹PG Research Department of Biochemistry, STET Women’s College, Mannargudi

²Department of Biochemistry, PSG College of Arts and Science, Coimbatore

 

ABSTRACT:

Chronic inflammation is the underlying factor in causing multitude of human ailments ranging from arthritis to cancer. Amidst the emerging synthetic drugs, people started re-evaluating traditional plant based medicines because of the efficacy and safety of herbal medicine. Hence in the present study, efforts are made to validate the anti-inflammatory potential of four medicinal plants using in-vitro procedures. Ten different concentrations of aqueous extracts of selected medicinal plants ranging from 0.01-10mg/ml were taken. At a concentration of 10 mg/ml Erythrina indica has shown 74.15% of protein denaturation inhibition followed by Mukia maderaspatana (53%). Cardiospermum halicacabum has shown only 42.40% activity, whereas Delonix elata has shown only 14% activity. In the case of proteinase inhibition assay, Mukia maderaspatana at a concentration of 10 mg/ml has exhibited 74% inhibition of proteinase followed by Cardiospermum halicacabum 58%, Erythrina indica 38% and Delonix elata 30%. Among the four plants selected, except Delonix elata all the other three plants have revealed remarkable anti-inflammatory activity.

 

 

 

INTRODUCTION:

Inflammation is a normal protective response to inactivate and destroy the invading organism to remove the irritant and set the stage for tissue repair. It is triggered by the release of chemical mediators from injured tissue and migrating cells¹.The commonly used drugs for management of inflammatory conditions are non-steroidal anti-inflammatory drugs, which have several adverse effects especially gastric irritation leading to formation of gastric ulcers². Natural products contribute significantly towards the development of modern medicine. Recently traditional medicine is being re-evaluated worldwide due to the perceived efficacy, low incidence of serious adverse effects, and cost. The rich wealth of plant kingdom can represent a novel source of phytochemicals with significant anti-inflammatory activity.

 

Several researches carried out on medicinal plants across the globe, ascertain their anti-inflammatory efficacy^3,4,5,6,7. Cardiospermum halicacabum L. belonging to the family Sapindaceae is an herbaceous plant. This herb acts as a diaphoretic, diuretic, emetic, laxative, refrigerant, stomachic and sudorific and has antibacterial⁸, anti-diarrheal⁹, antioxidant activities¹⁰, suppresses TNF production¹¹, exhibits anticancer¹², vasodepressant ¹³effect. This herb is also useful in curing rheumatism, severe bronchitis and firmness of the appendages and snakebite^14,15,16,17 Antiulcer¹², analgesic¹⁸, antiparasitic, antimalarial¹⁹, antifilarial²⁰ and antipyretic role were also accounted for this herb²¹.

 

Erythrina indica belongs to the family Fabaceae is a shrub with prickly stems: it is a wild growing forest plant in India. Each part of the plant is used to treat various diseases in India. The different extracts of Erythrina indica showed anticonvulsant activity²², antibacterial activity²³, antimicrobial activity²⁴, diuretic activity²⁵ etc. Mukia maderaspatana (L.) M. Roem. (Family-Cucurbitaceae) grows common in village hedges and other open habitats and in the disturbed sites of the semi-evergreen and deciduous forests. Different parts of the plant are being utilized for the health-care needs of human beings and livestock. The leaves and tender shoots are also frequently consumed as a part of the South Indian cuisine. Preclinical animal experiments have substantiated a number of traditional and folkloric medicinal claims, including hypotensive²⁶, hepatoprotective²⁷, hypoglycaemic²⁸, immunomodulatory²⁹, antimicrobial³⁰ characteristics of the plant extract. Encouraging results have emerged from clinical investigations also as a chemoprotective plant in subjects suffering from hypertension, diabetes, dyslipidemia and rheumatoid arthritis.

 

Delonix elata (L) gamble commonly known as "white gulmohur" in English and is a reputed folklore remedy for arthritic disorders in many parts of India. The leaf extract is an anti-inflammatory agent³¹. Root decoction of this plant is consumed for abdominal pains. Leaves are reported to be used in scorpion bite treatments. Hence in the present study, initiatives are taken to evaluate the in vitro anti-inflammatory activity of Cardiospermum halicacabum, Erythrina indica, Mukia maderaspatana and Delonix elata.

 

MATERIALS AND METHODS:

Collection of plant material and Preparation of the extract:

The leaves of Cardiospermum halicacabum, Erythrina indica, Delonix elata and Mukia maderaspatna were collected from Mannargudi, Tamil Nadu. Plant materials were identified and authenticated by Dr. P. Brindha, Associate Dean and Coordinator, Centre for Advanced Research in Indian System of Medicine, SASTRA University, Thirumalaisamudhram, and Tamil Nadu. The collected materials were cleaned, shade dried and coarsely powdered.10g of powdered samples were extracted with 100ml of distilled water separately under shaking condition (500rpm) for 8 h and filtered. The filtrates were collected and dried in rotovapor and then the dried extracts were re-suspended in water at 10 mg/ml ratio and used for further analysis.

 

Preliminary phytochemical screening:

It involves testing of plant powder for the presence of different classes of compounds and to determine their chemical profile. The methods used for detection of various phytochemicals were done as per standard texts^{32, 33, 34}.

 

Inhibition of protein denaturation:

The protein denaturation assay was conducted by taking 50 µl of different concentrations of aqueous extract (10.0, 5.0, 2.50, 1.25, 0.62, 0.31, 0.15, 0.07, 0.03 and 0.01 mg/ml) with 450 µl of 5% bovine serum albumin (BSA). The pH of the reaction mixture was adjusted to 6.3 using 1 N HCl and vortexed and then incubated at 37⁰ C for 20 min. The mixture was further incubated at 57⁰ C for 3 min and thereafter allowed to cool at room temperature. Then 2.5 ml of phosphate buffered saline (pH 6.3) was added to each tube and the turbidity was measured at 660 nm. Distilled water was added instead of extract in blank whereas for control, water was used instead of BSA. The percentage of inhibition of protein denaturation was calculated using the formula, Inhibition of protein denaturation (%) = (100 – (Abs. Test - Abs. Control)/Abs. Blank)³⁵.

 

Protease inhibition activity:

The protease inhibition activity was measured by taking 1 ml of trypsin enzyme (0.06 mg in 1 ml 25 mM Tris-HCl buffer, pH 7.4) with 1 ml of different concentrations of water extract (10.0, 5.0, 2.50, 1.25, 0.62, 0.31, 0.15, 0.07, 0.03 and 0.01 mg/ml). The reaction mixture was incubated at 37⁰ C for 5 min followed by the addition of 1 ml of 0.8% casein. The mixture was further incubated at 57⁰ C for 20 min and thereafter the reaction was terminated by adding 2 ml of 70% perchloric acid. The cloudy suspension was centrifuged at 2000 rpm and the absorbance was measured at 280 nm. Distilled water was added instead of extract in blank whereas for control water was used instead of casein. The percentage of protease inhibition was calculated using the formula, Protease inhibition (%) = (100 – (Abs. Test - Abs. Control)/Abs. Blank)³⁶.

 

RESULTS AND DISCUSSION:

Preliminary phytochemical analysis of the aqueous extracts of Cardiospermum halicacabum revealed the presence of Carbohydrates, Tannins, Saponins, Flavonoids, Alkaloids, glycosides, Terpenoids, Steroids and Protein. Aqueous extract of Erythrina indica revealed the presence of Carbohydrates, Tannins, Saponins, Flavonoids, Alkaloids, glycosides, Terpenoids, phenol and Steroids. Aqueous extract of Mukia maderaspatana revealed the presence of Carbohydrates, Tannins, Saponins, Flavonoids, Alkaloids, Terpenoids, Phenols and Protein. Aqueous extract of Delonix elata revealed the presence of Flavanoids, Carbohydrates, Protein, Alkaloids, Glycosides, Phenol, Saponins, Tannins, glycosides and steroid (Table 1).

 

Table 1: Preliminary phytochemical screening of selected plants

Phytochemicals	AECH	AEEI	AEMM	AEDE
Carbohydrates	+	+	+	+
Tannins	+	+	+	+
Saponins	+	+	+	+
Flavonoids	+	+	+	+
Alkaloids	+	+	+	+
Glycosides	+	+	-	+
Terpenoids	+	+	+	+
Phenol	-	+	+	+
Steroids	+	+	-	+
Protein	+	-	+	+

AECH: Aqueous extract of Cardiospermum halicacabum; AEEI: Aqueous extract of Erythrina indica

AEMM: Aqueous extract of Mukia maderaspatana; AEDE: Aqueous extract of Delonix elata

There are certain problems in using animals in experimental pharmacological research, such as ethical issues and the lack of rationale for their use when other suitable methods are available or could be investigated³⁷. In the present study, methods such as proteinase inhibitory activity and Inhibition of protein denaturation bioassay were selected for in vitro assessment of anti-inflammatory property of Cardiospermum halicacabum, Erythrina indica, Mukia maderaspatana and Delonix elata.

 

Protein denaturation is a process in which proteins loss their tertiary structure and secondary structure by physical or chemical factors. Most biological proteins loss their biological function when denaturated. Denaturation of tissue proteins is one of the well-documented causes of inflammatory and arthritic disease. Production of auto antigens in certain arthritic disease may be due to denaturation of protein in            vivo^38,39. Agent that prevents protein denaturation therefore, would be worthwhile for anti-inflammatory drug development. Hence the ability of a plant extract to inhibit protein denaturation can be studied to assess the anti-inflammatory activity of the extract. In the present study, at a concentration of 10 mg/ml Erythrina indica has shown 74.15% of protein denaturation inhibition and Mukia maderaspatana has shown 53% of protein denaturation inhibition. Cardiospermum halicacabum has shown only 42.40% activity where as Delonix elata has shown only 14% activity. Among the selected plants, Erythrina indica has shown greater potential than the other plants (Table 2).

 

Table 2: Protein denaturation inhibition activity of the selected medicinal plants

Concentration of the extract (mg/ml)	AEEI	AECH	AEMM	AEDE
10.00	74.15 ± 6.26	42.40 ± 3.37	53.98 ± 3.70	14.34 ± 1.02
05.00	65.83 ± 2.71	41.90 ± 3.81	52.17 ± 4.26	12.30 ± 1.85
02.50	51.53 ± 3.80	37.76 ± 2.01	48.57 ± 3.11	11.69 ± 1.58
01.25	45.02 ± 3.01	32.96 ± 2.08	42.96 ± 4.26	08.59 ± 0.56
00.62	37.71 ± 2.53	29.74 ± 2.52	39.54 ± 3.01	06.24 ± 0.41
00.31	30.73 ± 2.68	26.06 ± 1.78	31.08 ± 2.15	04.82 ± 0.56
00.15	24.22 ± 2.08	21.27 ± 1.45	27.23 ± 2.97	02.81 ± 0.70
00.07	18.42 ± 1.13	19.45 ± 1.61	24.36 ± 1.26	01.80 ± 0.85
00.03	15.92 ± 1.64	16.40 ± 1.68	20.53 ± 1.51	01.77 ± 0.56
00.01	12.91 ± 1.01	13.02 ± 1.33	18.31 ± 1.26	00.55 ± 0.20

AEEI: Aqueous extract of Erythrina indica; AECH: Aqueous extract of Cardiospermum halicacabum

AEMM: Aqueous extract of Mukia maderaspatana; AEDE: Aqueous extract of Delonix elata

 

Neutrophils are known to be a rich source of serine protease and are localized at lysosomes. It was previously reported that leukocyte protease play an important role in the development of tissue damage during inflammatory reaction and significant level of protection was provided by protease inhibition. Neutrophils are important source of protease in their lysosomal granules. Proteinase has been implicated in arthritic reaction. It was already reported that leukocyte protease plays important role in the development of tissue damage during inflammatory reaction and significant level of protection was provided by protease inhibitors. Recent studies have shown that many flavonoid and related polyphenols contribute significantly to the anti-inflammatory activity of many plants⁴⁰. In the present study, at a concentration of 10 mg/ml of Erythrina indica has shown 33.98% of proteinase inhibitory activity and Cardiospermum halicacabum has shown 57.88% activity. Mukia maderaspatana has shown 73% of protease inhibition where as Delonix elata has shown only 30% activity. Among the plants selected, Mukia maderaspatana has shown greater potential to inhibit protease than the other plants (Table 3).

 

Table 3: Protease inhibition potential of the selected medicinal plants

Concentration of the extract (mg/ml)	AEEI	AECH	AEMM	AEDE
10.00	33.98 ± 1.70	57.83 ± 3.41	73.98 ± 4.70	30.36 ± 2.01
05.00	32.17 ± 3.26	50.00 ± 4.43	71.17 ± 5.26	28.55 ± 2.85
02.50	29.57 ± 2.11	44.38 ± 3.68	67.57 ± 5.11	22.35 ± 1.85
01.25	25.96 ± 2.26	35.54 ± 2.13	60.96 ± 4.26	19.34 ± 1.70
00.62	22.54 ± 1.01	28.31 ± 2.43	55.54 ± 3.01	15.13 ± 1.25
00.31	18.08 ± 1.15	24.10 ± 1.85	51.08 ± 2.15	11.12 ± 1.56
00.15	17.23 ± 1.97	21.08 ± 1.28	47.23 ± 3.97	09.30 ± 0.85
00.07	14.36 ± 1.26	16.27 ± 1.43	40.36 ± 2.26	08.05 ± 0.62
00.03	12.53 ± 1.51	12.05 ± 0.85	36.53 ± 2.51	06.42 ± 0.40
00.01	08.31 ± 1.26	05.42 ± 0.43	31.31 ± 1.26	04.96 ± 0.61

 

CONCLUSION:

From the data of the results obtained, it could be concluded that among the four plants selected, Mukia maderaspatana and Erythrina indica have shown remarkable anti-inflammatory properties than the other two plants. Presence of phytochemicals might have been responsible for the biological activity of the selected plants.

 

ACKNOWLEDGEMENT:

Authors place on record their sincere gratitude to the management, S.T.E.T. Women’s College, Mannargudi.

 

REFERENCES:

1.      Chatterjee P, Chandra S, Dey P, Bhattacharya S. Evaluation of anti-inflammatory effects of green tea and black tea: A comparative in vitro study. J Adv Pharm Tech Res 2012; 3:136-8.

2.      Tripathi KD. Essentials of Medical Pharmacology. 6th Ed. New Delhi: Jaypee Brother’s Medical Publishers (P) Ltd.; 2008.

3.      Sivaranjani C, Venkatalakshmi P, and Brindha P. In vitro anti-inflammatory and antioxidant activity on fruits of Terminalia catappa L. Res. J. Pharm. Tech., 2015; 8(10): 1409-1411.

4.      Padmanabhan P, and Jangle SN. Evaluation of in vitro Antioxidant activity of Herbal preparation. A combination of four medicinal plants. International Journal Basic and Applied Medicinal Science. 2012; 2(1): 109-116.

5.      Venkatalakshmi P, Brindha P, and Vadivel V, 2014. In vitro antioxidant and anti-inflammatory studies on fruits of Terminalia catappa. Int J Phytomed. 2015; 7 (3): 246-253.

6.      Divya, RS, Venkatalakshmi P, Vadivel, V, and Brindha P. In vitro studies on the biological activities of flowers of banana (Musa paradisiaca L.,). Der Pharmacia Letter. 2016; 8(10): 238-246.

7.      Karthika, T, Venkatalakshmi, P, Vadivel V, and Brindha P. In vitro Anti-Obesity, Antioxidant and Anti-Inflammatory Studies on the Selected Medicinal Plants. International Journal of Toxicological Research. 2016; 8(5):332-340.

8.      Raman N, Radha A, and Ravi M. Antibacterial activity of Cardiospermum halicacabum against human and plant pathogens. Indian Drugs. 1998; 35: 29–31.

9.      Rao V, Chandra PNK, and Shanta KSM. Pharmacological investigation of Cardiospermum halicacabum (Linn) in different animal models of diarrhoea. Indian J. Pharm., 2006; 38: 346-349.

10.   Kumaran A, and Karunakaran RJ. Antioxidant activities of the methanol extract of Cardiospermum halicacabum. J Pharm Biol. 2006; 44:145-151.

11.   Babu V, and Krishnakumari KCS. Cardiospermum halicacabum suppresses the production of TNF-alpha and nitric oxide by human peripheral blood mononuclear cells. Afr. J. Biomed. 2006; 9: 95–99.

12.   Sheeba MS, and Asha VV. Effect of Cardiospermum halicacabumon ethanol induced gastric ulcers in rats. J. Ethnopharmacol. 2006; 106:105–110

13.   Gopalakrishnan C, Dhannanjayan R, and Kameshwaran L. Studies on the pharmacological actions of Cardiospermum halicacabum. Indian J. Physiol. Pharmacol. 1976; 20: 203–208.

14.   Abdulla, P. In: Flora of West Pakistan. (Eds.): E. Nasir and S.I. Ali. Islamabad, Pakistan. 1973; pp. 39: 1-8.

15.   Nadkarni KM. Indian Materia Medica. Popular Book Depot, Bombay. 1976;271.

16.   Chopra RN, Nayar SL, and Chopra IC. Glossary of Indian Medicinal Plants, New Delhi; Council for Scientific Ind. Res. 1980; 51-55.

17.   Joshi SK, Dhstms BD, Bhatia CR, Singh RV, and Thakur RS. The Wealth of India Raw Materials. New Delhi; Council of Scientific Ind Res Pub. 1992; 3:270-271.

18.   Muthumani P, Meera R, Venkatraman S, Ganapathy S, and Devi P. Study of phytochemical, analgesic and anti ulcer activity of extracts of aerial parts of Cardiospermum halicacabum Linn., IJPSR.2010; 1(10):128-137.

19.   Waako PJ, Gumede B, Smith P and Folb PI. 2005. The in vitro and in vivo antimalarial activity of Cardiospermum halicacabum L. and Momordica foetida Schumch. Et Thonn. J. Ethnopharmacol. 2005; 99(1):137-143.

20.   Khunkitti W, Fujimaki Y, and Aoki Y. In vitro antifilarial activity of extracts of the medicinal plant Cardiospermum halicacabum against Brugia pahangi. J. Helminthol. 2000; 74(3): 241-246.

21.   Asha VV, and Pushpangadan P. 1999. Antipyretic activity of Cardiospermum halicacabum. Indian J. Exp. Biol.1999; 37(4): 411-414.

22.   Chinchawade AB, Deshmukh DB, Gaikwad DD, Grampurohit ND. Anticonvulsant activity of chloroform extract of bark and root of Erythrina variegata L. International Journal of Pharmaceutical and Clinical Research.2013; 5. 23-25.

23.   Tanaka H, Sato M, Fujiwara S, Hirata M, Etoh H and Takeuchi H. Antibacterial activity of isoflavonoids isolated from Erythrina variegata against methicillin-resistant Staphylococcus aureus. Lett. App. Microbio. 2002; 35: 494-498.

24.   Venkatesan D and Karrunakaran CM. Anti microbial activity of selected Indian Medicinal Plants. Journal of Phytology. 2010; 2(2): 44-48.

25.   Liu IX, Durham DG, and Richards ME. Vancomycin resistance reversal in Enterococci by flavonoids. Journal of Pharmacy and Pharmacology. 2001; 53: 129-132.

26.   Veeramani C, Aristatle B, Pushpavalli G, and Pugalendi KV. Effects of Melothria maderaspatana leaf extract on antioxidant status in sham-operated and uninephrectomized DOCA-salt hypertensive rats. Saudi J Biol Sci. 2011; 18 (1): 99-105.

27.   Wani VK, Dubey RD, Verma S, Sengottuvelu S and Sivakumar T. Antidiabetic activity of methanolic root extract of Mukia maderaspatana in Alloxan induced diabetic rats. Int J Pharm Technol Res. 2011; 3: 214-220.

28.   Balaraman AK, Singh J, Dash S, and Maity TK. Antihyperglycemic and hypolipidemic effects of Melothria maderaspatana and Coccinia indica in streptozotocin induced diabetes in rats, Saudi Pharmac J. 2010; 18 (3): 173-178.

29.   Thabrew MI, Gove CD, Hughes RD. McFarlane IG and Williams R. Protection against galactosamine and tert-butyl hydroperoxide induced hepatocyte damage by Melothria maderaspatana extract. Phytother Res. 1995; 9 (7): 513-517.

30.   Sasi Priya G, Radhika R, and Siddhuraju P. Antioxidant and antimicrobial activity of traditional Indian leafy vegetables: Mukia maderaspatana and Solanum trilobatum. Int J Pharm Pharm Sci. 2012; 4 (2), 513-521.

31.   Krishna RRV, Ganapathy P, Mallikarjuna R, and Ganga RB. Antiinflammatory activity of the leaves and bark of Delonix elata. Ancient Sci Life. 1997; 17(2): 141-143.

32.   Evans WC, and Trease. Pharmacognosy. 15^th edition, W.S Sauder company Ltd. London. 2002; 137-139, 230-240.

33.   Khandelwal KR. Practical pharmacognosy, Techniques and experiment Nirali Prakashan (Pune), 13^th edition. 2005.

34.   Kokate CK, Purohit AP, and Gokhale SB. A textbook of pharmacognosy, Nirali Prakashan., 2009;27-28.

35.   Osman NI, Sidik NJ, Awal A, Adam NAM, and Rezali NI. In vitro xanthine oxidase and albumin denaturation inhibition assay of Barringtonia racemosa L. and total phenolic content analysis for potential anti-inflammatory use in gouty arthritis. Journal of Intercultural Ethnopharmacology. 2016; 5 (4): 343-349.

36.   Leelaprakash G, and Mohan Dass S. In vitro anti-inflammatory activity of methanol extract of Enicostemma axillare. International Journal of Drug Development and Research. 2011; 3 (3): 189-196.

37.   Sangita Chandra, Priyanka Chatterjee, Protapaditya Dey, Sanjib Bhattacharya, et al. Evaluation of in vitro anti-inflammatory activity of coffee against the denaturation of protein. Asian Pacific Journal of Tropical Biomedicine, 2012; 178- 180.

38.   Opie EL. On the relation of necrosis and inflammation to denaturation of proteins. J. Exp. Med. 1962; 115: 597-608.

39.   Umapathy E, Ndebia EJ, Meeme A, Adam B, Menziwa P, Nkeh-Chungag BN, Iputo JE. An experimental evaluation of Albuca setosa aqueous extract on membrane stabilization, protein denaturation and white blood cell migration during acute inflammation. J. Med Plants Res. 2010; 4: 789-795.

40.   Govindappa M, Naga Sravya S, Poojashri MN, Sadananda TS, Chandrappa CP. Antimicrobial, antioxidant and in vitro anti-inflammatory activity of ethanol extract and active phytochemical screening of Wedelia trilobata (L.). Journal of Pharmacognosy and Phytotherapy, 2011; 3: 3-51.

 

 

 

Received on 02.07.2019           Modified on 10.09.2019

Accepted on 19.11.2019         © RJPT All right reserved