AIM- The main of study is to evaluate the In-vitro anti-inflammatory activity of different extracts of flowers of Tridex procumbens. Material &amp; Methods-Successive solvent extraction method was followed for extraction by using different solvents i.e. petroleum ether, chloroform, methanol, butanol and water. Preliminary phytochemical screening method was performed for the presence of different phytoconstituents. For the evaluation of anti-inflammatory activity, protein denaturation and proteinase inhibitory method were performed. Diclofenac sodium was used as standard. Result- Among all the extracts, methanolic extract of Tridex procumbens showed 80.23% inhibition. The petroleum ether, chloroform, n-butanolic and water extract had shown the 12.23, 23.33, 20.45 and 36.20% inhibition respectively at 500 µg/ml concentration. The Diclofenac sodium showed 83.28 % inhibition against denaturation of protein. The different extracts were evaluated by using this method. Methanolic extract of Tridex procumbens showed 74.50% inhibition at 500 µg/ml. The petroleum ether, chloroform, n-butanolic and water extracts had shown the 30.16%, 41.22%, 43.28% and 31.97% inhibition respectively at 500 µg/ml. The Diclofenac sodium showed 79.21% inhibition against proteinase inhibitory activity at 100 µg/ml. Conclusion- Besides from the obvious therapeutic importance, methanolic extract would be useful in understanding the mechanism of diseases with higher levels of cellular and molecular level. These components could serve as lead molecules for development of prospective anti-arthritic agents. Further detailed studies are required to isolate the active phytoconstituents from methanolic extract which is responsible for anti-arthritic activity.
Keywords: Protein denaturation model, proteinase inhibitory method, methanolic extract, anti-inflammatory activity, Tridex procumben

Giri, Sanjay

Jain, Neetesh K

Shaikh, Anis

Journal of Drug Delivery and Therapeutics (JDDT)

Giri et al                                                                                                       Journal of Drug Delivery & Therapeutics. 2018; 8(6-A):111-115  ISSN: 2250-1177                                                                                  [111]                                                                                 CODEN (USA): JDDTAO Available online on 10.01.2019 at http://jddtonline.info Journal of Drug Delivery and Therapeutics Open Access to Pharmaceutical and Medical Research © 2011-18, publisher and licensee JDDT, This is an Open Access article which permits unrestricted non-commercial use, provided the original work is properly cited Open  Access                                                                                                                                                     Research Article In-vitro anti-inflammatory activity of different extracts of flowers of Tridex procumbens Sanjay Giri 1, Neetesh K Jain1, Anis Shaikh2 1Department of Pharmacology, Oriental University, Indore (M.P.)-India 2Institute of Pharmacy, Vikram University, Ujjain (M.P.)-India  ABSTRACT AIM- The main of study is to evaluate the In-vitro anti-inflammatory activity of different extracts of flowers of Tridex procumbens. Material & Methods-Successive solvent extraction method was followed for extraction by using different solvents i.e. petroleum ether, chloroform, methanol, butanol and water. Preliminary phytochemical screening method was performed for the presence of different phytoconstituents. For the evaluation of anti-inflammatory activity, protein denaturation and proteinase inhibitory method were performed. Diclofenac sodium was used as standard. Result- Among all the extracts, methanolic extract of Tridex procumbens showed 80.23% inhibition. The petroleum ether, chloroform, n-butanolic and water extract had shown the 12.23, 23.33, 20.45 and 36.20% inhibition respectively at 500 µg/ml concentration. The Diclofenac sodium showed 83.28 % inhibition against denaturation of protein. The different extracts were evaluated by using this method. Methanolic extract of Tridex procumbens showed 74.50% inhibition at 500 µg/ml. The petroleum ether, chloroform, n-butanolic and water extracts had shown the 30.16%, 41.22%, 43.28% and 31.97% inhibition respectively at 500 µg/ml. The Diclofenac sodium showed 79.21% inhibition against proteinase inhibitory activity at 100 µg/ml. Conclusion- Besides from the obvious therapeutic importance, methanolic extract would be useful in understanding the mechanism of diseases with higher levels of cellular and molecular level. These components could serve as lead molecules for development of prospective anti-arthritic agents. Further detailed studies are required to isolate the active phytoconstituents from methanolic extract which is responsible for anti-arthritic activity.  Keywords: Protein denaturation model, proteinase inhibitory method, methanolic extract, anti-inflammatory activity, Tridex procumbens   Article Info: Received 15 Oct 2018;     Review Completed 25 Dec 2018;     Accepted 28 Dec 2018;     Available online 10 Jan 2019 Cite this article as:  Giri S, Jain NK, Shaikh A, In-vitro anti-inflammatory activity of different extracts of flowers of Tridex procumbens, Journal of Drug Delivery and Therapeutics. 2018; 8(6-A):111-115 *Address for Correspondence:  Sanjay Giri, Department of Pharmacology, Oriental University, Indore (M.P.)-India   INTRODUCTION Inflammation is usual and essential protective response to the harmful stimuli such as infectious agents, antigen-antibody reactions, thermal, chemical, physical agents, and ischemia (Goldyne et al., 1984). It is caused by a assortment of stimuli, including physical damage, UV irradiation, microbial attack, and immune reactions. The classical facial appearance of inflammation is redness, warmth, swelling, and pain. Inflammation cascades can lead to the expansion of diseases such as chronic asthma, arthritis, multiple sclerosis, inflammatory bowel disease, and psoriasis. Many of these diseases are incapacitating and are flattering increasingly common in our ageing society1. Immunosuppressive DMARDs to produce some or the other side effects connected with suppression of immunity. On prolonged use, these drugs can cause renal complications, renal insufficiency, and hyperkalemia particularly in CHF, cirrhotic and ascetic at higher doses in patients2,3. Traditionally herbal plants were used equally outwardly and inside for treating inflammatory circumstances such as arthritis. The confident persuade of herbal medicine in alternating pathophysiology of arthritis has resulted in a substantial increase in their use as a treatment for arthritis4. In the absence of any scientific confirmation for anti-arthritic activity in inflammatory conditions of Tridex procumbens, there is a need in scientifically establishing the anti arthritic activity of different bioactive extracts with fewer side effects in assessment with existing synthetic drugs.   MATERIAL & METHODS Plant Materials The flowers of Tridex procumbens were collected from the college premises and from nursery.  Giri et al                                                                                                       Journal of Drug Delivery & Therapeutics. 2018; 8(6-A):111-115  ISSN: 2250-1177                                                                                  [112]                                                                                 CODEN (USA): JDDTAO Authentification of Plant Materials Flowers of plant were taxonomically identified by Dr. Anurag Titov, Professor, Department of Botany, Govt. Madhav  Sciences, PG College, Dewas Road, Ujjain and herbarium specimen was submitted in Department of Botany for future references. Methods Preparation of Total Crude Extract Flowers of plant were dried under shade and subjected to coarse powder for extraction process. Accurately weighed (200 gm) quantity of flowers of Tridex procumbens were extracted using petroleum ether, chloroform, methanol, butanol and finally water by soxhlet apparatus for 72 hr. The all extracts were dried under the reduced pressure to get crude extracts. After drying, the respective extracts were weighed and percent yield was determined5. Preliminary Phytochemical Tests Qualitative chemical tests of all extracts were subjected to various chemical tests to detect various presences of various phytoconstituents5,6. Evaluation of in-vitro anti-arthritic activity   Inhibition of protein denaturation method: All the extracts (pet ether, chloroform, methanol, butanol and water) of Tridex procumbens were evaluated by this method. The following procedure was followed for evaluating the percent of inhibition of protein denaturation.  Control solution (50 ml) consists of 2ml of Egg albumin (from fresh hen’s egg) and 28 ml of phosphate buffer (PBS, pH 6.4) and 20ml distilled water.  Standard drug (50 ml) consists of 2ml of Egg albumin and 28 ml of phosphate buffer and 20 ml of various concentrations of standard drug Diclofenac sodium (20, 40, 60, 80 & 100 µg/ml).  Test solution (50 ml) consists of 2ml of Egg albumin and 28 ml of phosphate buffer and 20 ml of various concentrations of petroleum ether, chloroform, methanol, n-butanol & aqueous extracts (100, 200, 300, 400, 500 µg/ml). All of the above reaction mixtures were adjusted to pH 6.4, using a small amount of 1N HCl. The samples were incubated at 37°C for 15 minutes and heated at 70°C for 5 minutes. After cooling, the absorbance of the above solutions was measured using UV- spectrophotometer at 660 nm. The percent inhibition of protein denaturation was calculated using the following formula7,8. Percent inhibition = (Vt/Vc-1) x 100) Where, Vt= absorbance of test sample, Vc= absorbance of control Proteinase inhibitory activity  Different extracts of Tridex procumbens were evaluated by this model. The following procedure was followed for evaluating the Proteinase Inhibitory Activity. Control solution (50 ml) 20 ml consists of 0.06 mg Trypsin, 10 ml 20mM Tris HCl buffer (pH 7.4) and 10 ml distilled water. Standard drug (50 ml) 20 ml consists of 0.06 mg Trypsin, 10 ml 20mM Tris HCl buffer (pH 7.4) and 10 ml of various concentrations of standard drug Diclofenac sodium. (20, 40, 60, 80 & 100 µg/ml). Test solution (50 ml) 20 ml consists of  0.06 mg Trypsin, 10 ml 20mM Tris HCl buffer (pH 7.4) and 10 ml of various concentrations of petroleum ether, chloroform, methanol, n-butanol & aqueous extracts, obtained from Tridex procumbens (100, 200, 300, 400, 500 µg/ml). All the reactions were incubated at 37o C for 5 minutes and then 10 ml of 0.8% (w/v) casein was added. Again the samples were incubated for an additional 20 min and 10 ml of 70% perchloric acid was added to arrest the reaction. Cloudy suspension was centrifuged and the absorbance of the supernatant was read at 210 nm against buffer as blank. The experiment was performed as triplicate. The percent inhibition of proteinase inhibitory activity was calculated9.  Percent inhibition =  (Abs Control – Abs Sample) x 100 / Abs control. RESULTS Extractive Value Determination Dried flowers of Tridex procumbens were extracted using pet ether, chloroform, methanol, butanol and finally water by soxhlet apparatus. The percent yields were determined by using the following formula.                                         Weight of Extract   Percent yield = -------------------------------------- x 100                                 Weight of powder drug Taken   Table 1: Different extracts with their appearance and % yield (in gm) S. No. Extracts Color of dried extracts Consistency of dried extracts % Yield (W/W) 1 Pet ether extracts of Tridex procumbens Dark brown Sticky 8.52 % 2 Chloroform extracts of Tridex procumbens Dark Green Sticky 6.56 % 3 Methanolic extracts of Tridex procumbens Dark Yellowish Green Sticky 12.23 % 4 Butanolic extracts of Tridex procumbens Dark Yellowish Green Sticky 4.88% 5 Aqueous extracts of Tridex procumbens Dark Green Sticky 5.58 %     Giri et al                                                                                                       Journal of Drug Delivery & Therapeutics. 2018; 8(6-A):111-115  ISSN: 2250-1177                                                                                  [113]                                                                                 CODEN (USA): JDDTAO Preliminary Phytochemical Screening The preliminary phytochemical analysis revealed that different active constituent present in different extracts such as carbohydrates, proteins, amino acids, fat, oils, steroids, terpenoids, glycosides, alkaloids, tannins and other phenolics compounds. Evaluation of in-vitro anti-arthritic activity of Tridex procumbens  Protein Denaturation Methods Among all the extracts, methanolic extract of Tridex procumbens showed 80.23% inhibition. The petroleum ether, chloroform, n-butanolic and water extract had shown the 12.23, 23.33, 20.45 and 36.20% inhibition respectively at 500 µg/ml concentration. The Diclofenac sodium showed 83.28 % inhibition against denaturation of protein. The results are summarized in Table No. 2.   Table 2: Effect of different extracts on Protein Denaturation Method. S. No. Treatment Concentration µg/ml % Inhibition 1 Control ---- ----- 2 Petroleum ether Extract  100 8.61 200 9.44 300 10.42 400 11.41 500 12.23 3 Chloroform Extract 100 12.55 200 13.82 300 14.24 400 20.76 500 23.33 4 Methanolic Extract 100 30.72 200 37.41 300 42.58 400 60.99 500 80.23 5 n-butanolic  Extract 100 10.33 200 12.42 300 15.57 400 18.59 500 20.45 6 Aqueous Extract 100 13.73 200 18.22 300 20.33 400 30.45 500 36.20 7  Diclofenac Sodium 20  33.44 40 36.55 60 41.56 80 70.88 100 83.28  Proteinase inhibitory action: The different extracts were evaluated by using this method. Methanolic extract of Tridex procumbens showed 74.50% inhibition at 500 µg/ml. The petroleum ether, chloroform, n-butanolic and water extracts had shown the 30.16%, 41.22%, 43.28% and 31.97% inhibition respectively at 500 µg/ml. The Diclofenac sodium showed 79.21% inhibition against proteinase inhibitory activity at 100 µg/ml. The results are summarized in Table No 3.  Table 3: Effect of different extracts on proteinase inhibitory activity S. No. Treatment Concentration µg/ml % Inhibition  1 Control ---- ----- 2 Petroleum ether Extract  100 17.56 200 21.31 300 27.34 400 28.22 500 30.16 3 Chloroform Extract 100 24.51 200 35.55 300 38.76 Giri et al                                                                                                       Journal of Drug Delivery & Therapeutics. 2018; 8(6-A):111-115  ISSN: 2250-1177                                                                                  [114]                                                                                 CODEN (USA): JDDTAO 400 40.31 500 41.22 4 Methanolic Extract  100 35.64 200 39.88 300 47.98 400 59.64 500 74.50 5 n-butanolic  Extract 100 32.22 200 36.46 300 37.88 400 40.89 500 43.28 6 Aqueous Fraction 100 17.54 200 21.29 300 25.78 400 30.58 500 31.97 7 Diclofenac Sodium  20 33.82 40 35.44 60 44.55 80 60.51 100 79.21  DISCUSSION Rheumatoid arthritis is a unrelieved inflammatory disease which leads to the smash up of synovial membranes, cartilage and bone. Although etiology and pathogenesis of RA is poorly unspoken, pro-inflammatory cytokines are well thought-out to be one of the most imperative peacekeepers involved in the pathogenesis of RA (Mclnnes & Schett, 2007). Various cytokines i.e. Tumor necrosis factor (TNF-α) & Interleukin-1 (IL-1) is known to contribute a critical role in the pathogenic mechanisms of arthritis and is well recognized as another pro-inflammatory cytokine concerned in arthritis10.  The unbelievable development in the area of synthetic drugs throughout present era is accompanied by various undesirable side effects. Whereas plants still hold their own unique breathing space, with lesser side effects11.  Thus, in the present investigation, an effort was made to evaluate the anti-arthritic activity of Tridex procumbens on the basis of ayurveda and their traditional uses in a suitable experimental animal.  In the preliminary study, dried powders of flowers plant was extracted by using pet ether, chloroform, methanol, butanol and finally water. The extracts were dried and screened for the presence of various active constituents. The extracts showed the presence of alkaloids, terpenoids, flavonoids, glycosides, phenolic compounds, tannins, steroids and fatty acids. For the beginning assessment, all extracts were evaluated by in-vitro anti-arthritic activity i.e. protein denaturation inhibitory activity & proteinase inhibitory activity. The arthritic disease sequence was correlated with the weakness of the lysosomal membranes, denaturation of proteins & discharge of inflammatory mediators. Denaturation of proteins is a well known reason of inflammation. The preponderance of biological proteins misplaces their biological functions when denatured. Within body, denaturation of proteins causes production of auto-antigens in arthritic disorders12,13. Agents that can put off protein denaturation consequently would be meaningful for anti-arthritic activity.  Serine proteinase from inflammatory cells is worried in various inflammatory disorders such as arthritis and pulmonary emphysema. Neutrophils are known to be a wealthy starting place of serine proteinase and are confined to a small area at Lysosomes. Deficiency of protease inhibitors in transmission is the major risk factor for enlargement of inflammatory disorder14,15,16. In our examination, the different extracts were studied for protein denaturation as well as proteinase inhibitory activity. The methanolic extract was found to be effectual in inhibiting heat induced albumin denaturation & proteinase inhibition at different concentrations.  CONCLUSION This study was designed to find out and prove the claims put forth by the traditional and folk medicine of applicability of Tridex procumbens that have been used in crippling arthritis and frozen joints in Siddha and Ayurvedic system of medicine.  Besides from the obvious therapeutic importance, methanolic extract would be useful in understanding the mechanism of diseases with higher levels of cellular and molecular level. These components could serve as lead molecules for development of prospective anti-arthritic agents. Further detailed studies are required to isolate the active phytoconstituents from methanolic extract which is responsible for anti-arthritic activity.      Giri et al                                                                                                       Journal of Drug Delivery & Therapeutics. 2018; 8(6-A):111-115  ISSN: 2250-1177                                                                                  [115]                                                                                 CODEN (USA): JDDTAO REFERENCES  1. Brown JH, Mackey HK. Inhibition of heat-induced denaturation of serum proteins by mixtures of nonsteroidal anti-inflammatory agents and amino acids. Proc Soc Exp Biol Med 1968; 128:225. 2. Chandra S, Chatterjee P, Dey P, Bhattacharya S. Evalauation of anti-inflammatory effect of Ashwagandha: A preliminary study invitro. Pharmacog J 2012; 4(29):47-49. 3. Clive DM and Stoff JS. Renal syndromes associated with NSAIDs, N. Eng. J. Med., 1984; 144:2165-2166. 4. Das SN and Chatterjee S. Long term toxicity study of ART-400. Indian Indg Med 1995; 16(2):117-123. 5. Eric FM, Michelle L, Jurgen B. MIF: a new cytokine link between rheumatoid arthritis and atherosclerosis. Nature Reviews Drug Discovery 2006; 5(5):399–411. 6. Garella S and Matarese RA. Renal effects of PGs and clinical adverse effects of NSAIDs. Medicine., 1984; 63:165-181. 7. Goldyne ME, Burrish GF, Poubelle P and Borgeat P. Arachidonic acid metabolism among human mononuclear leukocytes. Lipoxygenaserelated pathways, J Biol Chem., 1984; 259:8815-8819. 8. Hiemstra PS. Novel roles of protease inhibitors in infection and inflammation. Biochemical Society Transactions 2002; 30(2):116-120 9. Kumar V, Cotran RS and Robbins SL (2001). Acute and Chronic inflammation. In Basic Pathology, 6th edition, Harcourt India Pvt. Ltd. Philadelphia, pp. 25-46. 10. McInnes IB. Rheumatoid arthritis. From bench to bedside. Rheumatic diseases Clinics of North America 2001; 27(2):373-387. 11. Mukherjee,Pulok. Quality control of Herbal Drug, Business Horion publishers. Page no 693. 2002 12. Patel D, Kaur G, Sawant MG, Deshmukh P. Herbal Medicine- A Natural cure to arthritis. Indian Journal of Natural Products & Resources 2013; 4(1):27-35. 13. Sakat S, Juvekar AR, Gambhire MN. In vitro antioxidant and inflammatory activity of methanol extract of Oxalis corniculata Linn. Int J Pharma and Pharm Sci 2010; 2(1):146-155. 14. Woolf AD and Pfleger B. Burden of major musculoskeletal conditions, Bull World Health Org., 2003; 81:646–656. 15. Kokate, C.K., 1996, Practical Pharmacognosy. Delhi, Vallabh Prakashan. 16. Khandelwal, K.R., 2006. Practical Pharmacognosy. Pune, Nirali Prakashan. 

In-vitro anti-inflammatory activity of different extracts of flowers of Tridex procumbens

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In-vitro anti-inflammatory activity of different extracts of flowers of Tridex procumbens

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