INTRODUCTION:

The process of inflammation is a complex response produced by the vascular tissue to protect the tissues against the harmful pathogens, endogenous substances and irritants causing redness, swelling and pain¹. However prolonged and untreated inflammation may cause chronic inflammatory condition as seen in several auto immune disorders such as arthritis and asthma². The term arthritis refers to the inflammation of joints, irrespective of its etiology like infection, genetic defect or some other cause leading to disability due to the destruction of bone and cartilage.

Gouty arthritis is a metabolic disorder, due to obesity, purine rich diet, high blood pressure, and alcohol consumption leading to increased xanthine oxidase expression and hyperuricemia^3,4. The increased concentration of uric acid in blood promotes the formation of monosodium urate crystals (MSU) which gets deposited in the articular cavity and initiate the acute inflammatory reactions leading to the degenerative condition called gouty arthritis. The acute inflammatory reactions are multifaceted which include swelling, pain neutrophils infiltration followed by the release of cytokines (IL-1𝛽, TNF-𝛼), chemokines, prostaglandins, free radicals and matrix metalloproteinase which ultimately destroy the tissue and bones by triggering the inflammatory response^5,6,7.

Gout is most predominantly seen in men than women preceding to menopause since uricosuric effects of oestrogen, however after menopause the occurrence of gout increases significantly in women. Co-morbidities which associated with gout are cardiovascular disease, diabetes mellitus, obesity and chronic kidney disease. Co-morbidities are more common in patients with gout. Increased prevalence of comorbidities as well as causative medications is likely leads to the increased incidence of gout. Gout has able to cured with well endured urate-reducing treatments, which lowering the serum uric acid by inhibiting xanthine oxidase (allopurinol) or by inhibiting the protein denaturation as well as inhibiting proteinase inhibition^8,9.

Treatment includes Uricosuric drugs which increase the urinary excretion of uric acid, or Xanthine oxidase enzyme inhibitors which inhibit uric acid biosynthesis, and can lower the plasma uric acid concentration as well non steroidal anti inflammatory drugs (NSAIDS) to reduce the pain are used for the treatment of gout¹⁰. However long-term usage of these drugs will cause gastrointestinal and liver toxicity. At present alternative medicine in terms of phyto pharmacology is gaining importance all over the world for treating several diseases. In India several medicinal herbs have a claim of treating arthritis. The plant Cadaba fruticosa called as “Vizhuthi” in Tamil classified under the family Capparacae is cultivated in tropical and subtropical regions mainly in Karnataka Tamilnadu and Gujarat in India¹¹. The leaves of Cadaba fruticosa possesses antirheumatic, emmenagogue, antihelminitic and antibacterial properties. The treatment for gouty arthritis requires a potential drugs which can able to reduce the production of uric acid as well as the inhibition of acute immune responses due to MSU deposition. With this aim in mind the present study is executed to evaluate the anti-gout activity of aqueous and ethanolic extract of Cadaba fruticosa leaves through in vitro and in silico analysis^12,13.

MATERIALS AND METHODS:

Chemicals:

In the present study the proteins like Bovine serum albumin, Xanthine oxidase and trypsin were obtained from Hi media chemicals. The ethanol, acetyl salicylic acid and other chemicals were purchased from SD Fine Chemicals Ltd.

Collection of plant material:

The leaves of Cadabafruticosa were collected from Vellore district of Tamil Nadu and the leaves were authenticated by Professor of Pharmacognosy department of K.K. College of pharmacy, Chennai, India. In the institution a voucher specimen is available in the herbarium.

Preparation of Plant extract:

The shade dried leaves(1kg) of Cadaba fruticosa coarse powder was soaked in 90% (v/v) ethanol for three days at room temperature. The filtered and extracted ethanol was subjected to vacuum evaporation to remove the excess solvent. The resultant dry extract yield was calculated. The ethanolic extract of Cadaba fruticosa (EECF) yield was found to be 0.64% (w/v) In the same manner aqueous extract of Cadaba fruticosa (AECF) was prepared and by means of freeze drying it was converted in to powder form and the yield of 0.23% (w/v) was calculated.

In vitro anti-arthritic activity of Gouty arthritis:

The use of well-established standard protocols was followed for analyzing the anti-gouty arthritic activity of these two plant extracts. The in vitro Xanthine oxidase inhibitory activity which is responsible for the hyperuricemia was analyzed. Due to hyperuricemia the triggering of protease enzyme, protein denaturation and destabilization of membrane takes place. So the inhibitory activity of protease enzyme, protein denaturation and stabilization of RBC membrane were also assessed. The xanthine oxidase inhibitory activity was performed by the method of Owens and John’s 1999¹⁴. The method of Mizushima and Kobayashi, 1968 ¹⁵was followed for estimating the inhibitory activity protein denaturation by the plant extracts. The protection of RBC in terms of membrane stabilization was carried out according to Sakat et.al., 2010 method¹⁶. The plant extracts inhibitory potential of proteinase enzyme was examined by the method of Oyedapo et, al,.1995¹⁷.

In silico anti arthritic activity:

The anti gouty arthritic activity of EECF was performed in terms of in-silico method also. Molecular docking was performed in patch dock server to identify a suitable ligand from the plant compound to inhibit the cyclooxygenase –I and matrix metalloproteinase-IV which are also involved in pain and degenerative arthritic changes. The three-dimensional structures of the above-mentioned enzymes were retrieved from Protein data bank(http://www.rcsb.org/pdb/). In the present study since ethanolic extract of Cadaba fruticosa (EECF) exhibited better in vitro inhibitory activity, the phytoconstituents present in the ethanolic extract were used for the docking analysis. The researchers Amudha and Rani ¹⁸ as well as Telrandhe and Uplanchiwar¹⁹ have already performed the gas chromatography mass spectrometry (GC-MS) analysis and reported many bioactive molecules in the ethanolic leaf extract of Cadaba fruticosa. From their list 15active compounds two dimensional structure were obtained from PUB chem Site, and their three dimensional structures were drawn using Corina 3D converter. Using the three-dimensional structures of the enzyme and the ligand interaction were analyzed and their docking score was calculated in the patch dock server. Depending on these docking scores the best ligand from Cadaba fruticosa phyto constituent was obtained and further subjected to LIGPLOT analysis to visualize the inhibitory potential in terms of the ligand –amino acid interaction taking place in the active site. The standard drug Indomethacin was used for comparison.

Statistical Analysis:

The values reported are Mean±SE. The statistical analysis was carried out using analysis of variance (ANOVA) followed by Dunnet’s‘t’ test. The < 0.05 p values were considered as significant.

RESULTS:

Xanthine oxidase inhibitory assay:

The Figure-1 shows the inhibitory activity of xanthine oxidase by EECF and AECF. In the present study the ethanolic extract of Cadaba fruticosa showed the higher inhibitory percentage when comparable to aqueous extract. At the concentration of 800 and 1000μg/ml the inhibitory activity was quite similar to the positive drug Allopurinol.

Figure – 1 Effect of EECF and AECF on Inhibition of Xanthine Oxidase

Values are expressed in mean±SD (n=3), statistical significant test for comparison was done by ANOVA followed by Dunnet’s ‘t’test. Comparison between a –Allopurinol vs EECF b – Allopurinol vs AECF *p<0.05, **p<0.1 and ns – Non-Significant

Inhibition of Protein denaturation:

The inhibitory potential of protein denaturation was depicted in Table -1. Here also EECF exhibited better inhibition than AECF and at higher concentration of 800 and 1000μg/ml, the inhibitory percentage was almost 68.5% and 81.5% respectively against the positive control acetyl salicylic acid which showed 71.2% and 83.6% of inhibition for the same concentration.

Membrane stabilization study:

The protective effect of the plant extract on RBC membrane against the heat and hypotonic saline induced damage was shown in the Table-2.There was a significant RBC membrane stability observed in the EECF treated group comparable (**p<0.1 ) to the AECF treated group.This protection can be comparable to the acetyl salicylic acid treated group, since from concentration 400μg/mlthe EECF and Acetyl salicylic acid groups exhibited almost similar type of protection in the present study.

Table – 1 Effect of EECF and AECF on Inhibition of Protein Denaturation

Conc of Extract (in µg )	% stabilization of EECF	% stabilization of AECF	% stabilization of Acetyl Salicylic Acid
100	52.5±1.65 a **	29.1 ± 2 .06 b**	72.9±3.3
200	62.6±2.08 a **	41.4 ± 2.10 b**	74.2±0.95
400	72.9±2.25 a *	52.6±2.31 b**	76.23 ±2.0
800	81.33±1.92 a ^ns	56.3±2.45b **	82.8±1.62
1000	82.6±1.10 a ^ns	67.8±2.4 b **	83.8 ±1.5

Values are expressed in mean±SD (n=3), statistical significant test for comparison was done by ANOVA followed by Dunnet’s ‘t’ test. Comparison between a –Allopurinol vs EECF b – Allopurinol vs AECF *p<0.05, **p<0.1 and ns – Non-Significant

Table – 2 Effects of EECF and AECF on Membrane Stabilization

Conc of Extract (in µg )	% Inhibition of EECF	% Inhibition of AECF	% Inhibition of Acetyl salicylic acid
100	29.3±2.43 a **	22.26± 2.9 b**	50.3±1.7
200	36.6±1.57 a**	28.3±1.69 b**	62.9±1.2
400	50.5±3.05 a**	36.2±2.15 b**	68.2±0.76
800	68.5±1.60 a ^ns	42.5± 2.46 b **	71.2 ±1.15
1000	81.53±1.35 a ^ns	46.9 ±2.20 b**	83.76 ±1.49

Values are expressed in mean±SD (n=3), statistical significant test for comparison was done by ANOVA followed by Dunnet’s ‘t’ test. Comparison between a –Allopurinol vs EECF b – Allopurinol vs AECF *p<0.05, **p<0.1 and ns – Non-Significant

Proteinase Inhibition study:

The proteinase enzyme inhibitory potential of EECF and AECF extracts were shown in the Figure-2.Both the extracts have the potential to inhibit the protease enzyme. But the EECF has the higher protease inhibitor activity than the AECF. From the lower concentration EECF inhibited the protease enzyme.

Figure – 2 Effect of EECF and AECF on Inhibition of protease enzyme

Values are expressed in mean±SD (n=3), statistical significant test for comparison was done by ANOVA followed by Dunnet’s ‘t’ test. Comparison between a –Acetyl salicylic acid vs EECF b – Acetyl salicylic acid vs AECF *p<0.05, **p<0.1 and ns – Non-Significant

In silico studies:

To study the In silico anti gout activity, the cyclooxygenase -I and matrix metalloproteinase enzyme inhibitory activity of the ligands Cadabicine, Quercetin and Cadabicinetriacetate, which were selected based on the docking score among 15 ligands were evaluated by Ligplot analysis Table-3. The Table-4, Figure -3 and Figure -4 explain the nature of enzyme amino acid interaction with their number of hydrogen and hydrophobic bonding with the above-mentioned ligands. The positive drug Indomethacin was used as a positive drug in the docking analysis. There is similar type of hydrophobic interaction exists between the lysine, Valine, alanine, and arginine amino acid residues of enzyme cyclooxygenase -I with that of the ligand Quercetin and Cadabicine Diacetate as well as the standard drug Indomethacin. There are amino acid interactions seen between the three ligands and the enzyme matrix metalloproteinase but all interactions are diversified and no common amino acid is involved either in the ligands or in the standard drug Indomethacin.

Table -3 -Docking score values of compounds with Cyclooxygenase –I and Matrix Metalloprotease IV

S. No	Compound Name	Cyclooxy-genase-I	Matrix Metalloprotease IV
1	Cadabicine	-0.08	-0.02
2	Quercetin	-0.09	-0.12
3	Cadabicine triacetate	-0.11	-0.12
4	Isoquercetin	-0.03	-0.13
5	Thiazolidine	-2.92	2.67
6	1,2 Benzene dicarboxylic acid mono 2 ethyl ester	-2.07	-1.01
7	9.12 octadecatrienoic acid	-1.90	-0.90
8	Benzene 1 methyltrdecayl	-0.39	-1.01
9	n-Hexadectrinoic acid	-0.33	-1.29
10	Hexadecanoic acid ethyl ester	-0.24	-0.09
11	Cadabacine methyl ester	-1.23	-0.03
12	Strchidine	-0.96	-0.51
13	Vanillic acid	-0.13	-0.76
14	Oleic acid	-0.59	0.26
15	phytol	-0.36	-0.68

Figure: 3 Amino acid Interaction of cyclooxygenase -I enzyme with specific ligands

Table 4- Interaction with the cyclooxyganase-I and Matrix Metalloprotease IV enzymes with phyto chemicals of C.Fruticosa

S. No	Compound	Cyclooxygenase –I enzyme amino acid bindingsite		Matrix Metalloprotease IV amino acid binding site
S. No	Compound	H bonding sites	Hydrophobic contact sites	H bonding Sites	Hydrophobic contact sites
1	Cadabicine	1	47	0	72
1	Cadabicine	ASN 266 (A)	PHE 234(A), ARG236(A), ASP237(A), GLN238(A), ASN266(A) and LEU269(A)	-	THR283(A), PHE284(A), ASP285(A), GLY327(A)LEU328(A), GLU329(A), ASP378(A), LYS425(A), ASP427(A), HIS440(A) and GLY441
2	Quercetin	1	30	5	85
2	Quercetin	GLU33(A)	LYS26(A), GLU33(A), ARG38(A), VAL43(A)and ALA44(A)	THR230(A)	HIS238(A), SER229(A), THR230(A)ASP231(A), ASP251(A), GLN273(A) THR274(A)LYS276(A)
3	Cadabicine Diacetate	3	111	2	166
3	Cadabicine Diacetate	GLU30(A) ARG38(A) ASP 57(A)	LYS 26(A), VAL27(A), GLU30(A)GLU33(A), ARG38(A)VAL43(A) ALA44(A) andASP57(A)	GLN35(A), LYS36(A)	ASP32(A), GLN35(A)LYS36(A), GLU39(A)LYS40(A), ASN46(A)ASP47(A)ARG54(A)
4	Indomethacin	2	102	1	118
4	Indomethacin	LYS26(A) ARG38(A)	LYS26(A), LYS29(A), ARG38(A)VAL43(A), ALA44(A), GLY45(A), ILE5(A), LEU55(A)ASP57(A)and ASP58(A)	GLN35(A)	LYS85(A), ILE290(A)ARG291(A)GLU293(A), ARG304(A), VAL319(A), PHE320(A)and GLY353

Figure:4 Aminoacid Interaction of Matrix metalloproteinase IVenzyme with specific ligands

DISCUSSION:

In the gouty arthritis the deposition of MSU crystals in the articular tissues will trigger the immune response in the neutrophils and initiate a variety of molecular events leading to the pain and inflammation. It is mandatory to follow two treatment strategies for gouty arthritis. Initially the drug should be capable of reducing the hyperuricemia by inhibiting the enzyme xanthine oxidase and secondly it should reduce the immune response to reduce inflammation, pain and protect the bones and tissues from auto destruction. In the present investigation comparable to aqueous extract the ethanolic extract of Cadaba fruticosa exhibited a dose dependent inhibitory activity of xanthine oxidase enzyme. The researchers Arokiyaraj et al.,¹¹ have reported the presence of terpenoids, phenols, gums, flavanoids, saponins, sugars, alkaloids and steroids in the ethanolic extract could be responsible for the xanthine oxidase enzyme inhibitory activity observed in the present study. In the present investigation we evaluated the ability of our extracts in terms of the inhibition of protein denaturation, membrane stabilization and proteinase inhibition since these are the common cause of inflammation, synovial proliferation and joint tissue destruction which occurs in gouty arthritis also²⁰.

The EECF extract exhibited a better anti-gout arthritic activity when comparable to AECF in the present study. Upon the recognition of the MSU crystals the neutrophils infiltration occurs followed by the release of the lysosomal enzymes in the form of proteases leading to further inflammation and injury to the cell membrane. The lysosomal membrane mimics the RBC membrane and any substances which protect the RBC membrane and inhibit the hemolysis will also protect the lysosomal membrane and prevent the release of inflammatory mediators mainly the protein degrading enzymes. Additionally injury to the lysosomal membrane initiates the secretion of phospholipase A2 (PLA2) which in turn digests phospholipids to release the fatty acid arachidonic acid²¹. Arachidonic acid being the substrate for the enzyme cyclooxygenase produces the prostaglandins which is one of the inflammatory mediator and a pain causing agent in any type of tissue injury²². In the present investigation the ethanolic leaf extract of Cadaba fruticosa exhibited not only membrane stabilizing effect but also inhibit the protease enzymes and protein denaturation. Significant analgesic activity may be expected for the plant extract by the inhibition and the release of phospholipase A2 and thereby the synthesis of prostaglandin is also halted. The bioactive substances Stachydrine, 3-hydroxyl stachydrine, cadabine, terpenoids, flavones present in the EECF extract specifically inhibit these inflammatory mediators has the protective effect as an antigout agent²³.

Complex inflammatory reactions play predominant role in any type of arthritis, which ultimately induce the immune system to produce several mediators, which will cause destruction of cells and severe pain leading to disability. A potential drug should act in multifaceted manner to treat this chronic pain causing and disabling disease. So in the present investigation by Insilico methods. we analyzed whether the phyto constituents present in the leaf extract of Cadaba fruticosa could act as an antagonist ligand to the enzyme the cyclooxygenase –I (Cox-I) and matrix metalloproteinase IV and thereby reduce the pain and tissue destruction. Based on the Ligplot analysis two components namely Quercetin and Cadabicine Diacetate served as a better antagonistic ligand in inhibiting the above enzymes thereby protect the joints from pain and destruction.

CONCLUSION:

The successful treatment for gouty arthritis requires the management of pain and joint destruction as the consequence of the complex immunochemical reactions against the MSU deposition in the joints. The ethanolic extract of Cadaba fruticosa leaves contain several phyto constituents which have the ability to inhibit these immune mediated inflammatory reactions as evidenced in the present study. So this plant extract can be used as a potential drug for the treatment of gouty arthritis.

REFERENCE:

1. Sherwood ER, Toliver-Kinsky T. Mechanisms of the inflammatory response. Best Practice and Research Clinical Anaesthesiology. 2004; 18(3): 385-405.

2. Nathan C, Ding A. Nonresolving Inflammation. Cell. 2010; 140(6): 871-882.

3. Bieber JD, Terkeltaub RA. Gout: on the brink of novel therapeutic options for an ancient disease. Arthritis & Rheumatism.2004; 50(8): 2400–2414.

4. Choi HK, Atkinson K, Karlson EW, Willett W, Curhan G. Purine-rich foods, dairy and protein intake, and the risk of gout in men. New England Journal of Medicine.2004; 350(11): 1093–1103.

5. Akahoshi T, Murakami Y, Kitasato H. Recent advances in crystal-induced acute inflammation. Current Opinion inRheumatology. 2007; 19(2): 146–150.

6. Terkeltaub R. Pathogenesis and treatment of crystal-induced inflammation in Arthritis and Allied Conditions. Lippincott Williams and Wilkins, Philadelphia, Pa, USA WJ. Koopmanand LW. Moreland Eds; 20042357–2372.

7. Sabina EP, Rasool M, Mathew L, EzilRani P, Indu H.6-Shogaol inhibits monosodium urate crystal-induced inflammation-an in vivo and in vitro study. Food and Chemical Toxicology. 2010; 48(1): 229–235.

8. Robinson PC. Gout – an update of aetiology, genetics, co-morbidities and management. An international Journal of Midlife Health and Beyond. 2018; 118(1): 67-73.

9. Abhishek A, Roddy E, Doherty M. Gout – a guide for the general and acute physicians. Clinical Medicine 2017; 17(1): 54–59.

10. Terkeltaub R. Gout. Novel therapies for treatment of gout and hyperuricemia. Arthritis Res Therapy. 2009;11(4): 236.

11. Arokiyaraj S, Radha R, Martin S, Perinbam K.Phytochemical analysis and anti-diabetic activity of Cadabafruticosa. Indian Journal of Science and Technology. 2008; 1(6): 1-4.

12. Yoganarasimhan SN. Medicinal Plants of India. Bangalore: Interline Publishing; 1996.p. 8.

13. Chopra RN, Nayar SL, Chopra IC. Glossary of Indian Medicinal Plants (Raw Materials), New Delhi: CSIR Publications; 1979.p.43.

14. Owen P, JohnsT. Xanthine oxidase inhibitory activity of north eastern North American plant remedies used for gout. Journal of Ethnopharmacology. 1999; 64(2): 149-160

15. Mizushima Y, Kobayashi M. Interaction of anti-inflammatory drugs with serum proteins especially with some biologically active proteins. Journal of Pharmacy and Pharmacology. 1968; 20(3): 169-173.

16. Sakat SS, Archana RJ, Manoj NG. Invitro antioxidant and anti-inflammatory activity of methanol extract of oxalis corniculatalinn. international Journal of Pharmacy and Pharmaceutical Sciences. 2010; 2(1); 146-155.

17. Oyedapo OO, Famurewa AJ. Anti-protease and membrane stabilizing activities of extracts of Fagrazanthoxiloides, Olaxsubscorpioides and Tetrapleuratetraptera. International Journal of Pharmacognosy.1995; 33(1): 65-69.

18. Amudha M, Rani S. Assessing the bioactive constituents of Cadabafruticosa (L.) Druce through GC-MS. International Journal of Pharmaceutics and Pharmaceutical Science. 2014;6(2): 383-385.

19. Telrandhe UB, Uplanchiwar V. Phyto-pharmacological perspective of Cadaba farinosa forsk. American Journal of Phytomedical Clinical Therapy. 2013;1(1): 11-22.

20. Umapathy E, Ndebia EJ, Meeme A, Adam B, Menziwa P, Nkeh-Chungag BN, Iputo JE. An experimental evaluation of Albucasetosa aqueous extract on membrane stabilization, protein denaturation and white blood cell migration during acute inflammation, Journal of Medicinal Plants Research. 2010; 4(9): 789-795.

21. Meyer MC, Rastogi P, Beckett CS, Mc Howat J. Phospholipase A2 Inhibitors as Potential Anti-Inflammatory Agents. Current Pharmaceutical Design.2005; 11(10): 1301-1312.

22. Yousif G, Iskander GM and Eisa EB. Alkaloids of cadaba farinose and cadabarotundifolia. Fitoterapia. 1984: 55(1): 117-118.

23. Koeberle A, Werz O. Multi-target approach for natural products in inflammation. Drug Discovery Today. 2014;19(12): 1871-1882

Received on 26.11.2019 Modified on 05.02.2020

Research J. Pharm. and Tech. 2021; 14(5):2587-2592.

DOI: 10.52711/0974-360X.2021.00455