INTRODUCTION:

Herbal medicinal plants are widely used all over the world for a number of health conditions. Herbal medicines are prescribed by practitioners who practice based on knowledge and experience gained through prescribing ancient medicine over centuries¹. In India, around 2500 plant species are currently being used as herbal medicaments. For more than 3000 years, herbal medicines are being used either directly as such or as a pharmaceutical preparation². According to WHO, world’s 80% population may be using herbal medicine for their primary health care need³.

Cadaba indica Lam is a traditional medicinal plant commonly known as “Indian Cadaba” or “vizhuthi (in Tamil) and belongs to the family Capparidaceae. It is widely distributed worldwide in arid areas. C.indica is an unarmed, branched shrub up to 3m height. Leaves are simple, elliptic-oblong, varying in size as 12-15 by 8-12mm, dull green in colour. The flower is greenish white in few flowered terminal corymbs, fruit berry and cylindrical torulose. It is commonly distributed in scrub jungles and rocky areas of tropical and subtropical regions. It’s flowering and fruiting season ranges from November to April⁴.

The plant is traditionally used for the treatment of inflammation, syphilis, sores and for worm infestation. Pulipani sidhar has mentioned the medicinal value of this plant in his text, “Jala Thirattu”. Leaf juice is indicated for dysentery, constipation, fever, cough, and lung conditions^5,6. The Cadaba indica leaves contain steroids, lactones, flavonoids, phenols, reducing sugar and tannins^6-9. Its methanolic leaf extract contains a sizable amount of flavonoid and phenolic compounds which are potent antioxidant agents⁹.

Alcoholic extract of Cadaba indica plant has good anti-inflammatory, analgesic and antimicrobial properties¹⁰. The present study is conducted to evaluate its in vitro anti-arthritic activity by estimating its ability to inhibit protein denaturation and proteinase enzyme activity through chemical methods.

MATERIALS AND METHODS:

Collection of plant material:

The plant Cadaba indica was collected during the flowering season in February 2017 from, Melur, Madurai district, Tamil Nadu, India. The plant was authenticated by Dr. V. Chelladurai, Formerly Research Officer of Botany, Central Council for Ayurveda and Siddha, Government of India. A voucher specimen is preserved in our laboratory for future reference.

Preparation of extract:

a) Hot percolation method:

The Cadaba indica extract was prepared by the hot percolation method in Soxhlet apparatus using 100g of coarse leaf powder of the plant. For primary extraction petroleum ether was used. The defatted material obtained through primary extraction was further subjected to secondary extraction with methanol. (Cadaba indica hot maceration methanolic extract-CIHME).

b) Cold maceration method:

In a cold maceration method, 100g of Cadaba indica leaf powder was soaked in petroleum ether in a closed glass jar for a period of 72 hours. Such defatted material was subjected to methanol extraction (Cadaba indica cold maceration methanolic extract- CICME).

Both extracts (CIHME & CICME) were then filtered and dried under reduced pressure in Eyele Rotary evaporator (Japan) at room temperature to a viscous mass, weighed and stored at 4⁰C for further analysis. The percentage yield of hot percolation and cold maceration methanol extracts were 15.8% and 6.2% respectively.

Chemicals and instruments:

All chemicals used in this study were of analytical grade. Methanol, tris HCl, Casein, Perchloric acid, Bovine serum albumin, Trypsin and other chemicals were purchased from “Loba Chemie” private limited, Mumbai. Shimadzu UV-1650 PC UV-Visible spectrophotometer was used to read the absorbance of the assay mixture.

In vitro – anti arthritic activity:

a) Inhibition of protein denaturation assay:

A study on inhibition of protein denaturation was carried out by the method of Mizushima et al¹¹ and Sakat et al¹² with minor modifications. Doses of CIHME & CICME used for this study were 100, 200, 400, 800 & 1000mg. The volume of the test solution was 0.5ml (0.05ml of CIHME and CICME in Sodium Lauryl Sulphate (SLS) with 0.45ml of 5% bovine serum albumin (w/v aqueous solution). 0.5ml of control solution was prepared with 0.45ml bovine serum albumin and 0.05ml distilled water. 0.5ml of the standard solution was prepared with. 45ml of bovine serum albumin and 0.05ml of the standard drug dexamethasone (100,200,400,800 and 1000µg/ml). 1N HCl was added to adjust the pH to 6.3. The samples were incubated at 37⁰C for 20 minutes and then heated at 57⁰C for 3 minutes. After cooling, 2.5ml of phosphate buffer was added to all the above solutions. The wavelength absorbance was measured spectrophotometrically at 660nm. The experiment was performed in triplicate. Percentage inhibition of protein denaturation was calculated as follows:

Percentage of inhibition = 100- [(Absorbance of control- Absorbance of sample) x 100 / Absorbance of control]

b) Inhibition of proteinase enzyme activity:

Proteinase enzyme inhibitory assay was carried out by the method of Oyedepo et al¹³ and Sakat et al¹². 2.0ml of test solution consisted of 0.06 mg trypsin, 1.0ml 25mM Tris-HCl buffer at pH 7.4 and 1.0ml of each dose of (100,200,400,800 and 1000μg/ml) CIHME and CICME in SLS. 0.5ml of Dexamethasone in the doses of 100, 200, 400, 800 and 1000μg/ml was used as standard. These mixtures were incubated at 37°C for a period of 5min. 1.0ml of 0.8% (w/v) casein was added next. Then these mixtures were incubated at 37°C for another 20min. To terminate the reaction, 2.0 ml of 70% perchloric acid was added. The resultant cloudy suspension was centrifuged. The wavelength absorbance of the supernatant was found to be 280nm against buffer and was termed as blank. The percentage inhibition of proteinase enzyme activity was calculated as below:

Percentage of inhibition = 100- [(Absorbance of control- Absorbance of sample) x 100 / Absorbance of control]

Statistical analysis:

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

RESULTS:

Inhibition of protein denaturation:

The inhibition of protein denaturation activity of both CICME and CIHME was studied and compared statistically with that of the standard drug dexamethasone. Table -1 shows the percentage inhibition of protein denaturation of both extracts and dexamethasone with respect to their different dose ranges (100, 200, 400, 800 and 1000μg/ml). The percentage inhibition of CICME was found to be higher than CIHME at all dose ranges. However, the percentage inhibition of both extracts was lower than that of the standard drug dexamethasone.

Table 1: Percentage inhibition of protein denaturation of CICME & CIHME

Dose (µg/ml)	Dexamethasone (%Inhibition)	CICME (% Inhibition)	CIHME (%Inhibition)
100	39.68±1.3	17.29±1.8**	7.46±2.2**
200	61.82±0.9	35.32±3.9**	30.97±1.9**
400	73.88±0.7	56.34±3.6*	51.99±2.5**
800	84.20±0.9	69.53±0.9	68.41±2.8*
1000	91.54±0.9	77.99±0.9	74.75±1.3*

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 Dexamethasone versus CICME and Dexamethasone versus CIHME,*p< 0.05, **p<0.01

Figure 1: Inhibition of protein denaturation

Inhibition of proteinase enzyme activity:

The percentage inhibition of proteinase enzyme activity of CICME & CIHME was statistically compared to that of dexamethasone. Table -2 shows the percentage of proteinase enzyme inhibition activity of CICME, CIHME and standard drug dexamethasone in different dose ranges. Although the percentage inhibition of CICME was statistically not significant compared to dexamethasone, they were higher than that of CIHME.

Table 2: Percentage inhibition of Proteinase enzyme activity of CICME & CIHME

Dose (µg/ml)	Dexame-thasone (% Inhibition)	CICME (% Inhibition)	CIHME (% Inhibition)
100	31.96±0.6	23.55±1.7	8.12±0.5*
200	50.65±1.3	38.51±0.7	18.99±0.6**
400	63.41±1.7	62.25±1.2	38.59±1.6*
800	87.10±0.9	70.69±0.6	69.35±1.2
1000	89.75±0.4	77.21±0.3	74.53±0.4

Figure 2: Inhibition of proteinase enzyme

DISCUSSION:

The extract of the traditional herbal plant Cadaba indica was prepared by both cold maceration and hot percolation methods in our study. Both these extraction methods are most commonly used as a standard procedure to obtain a high yield of active ingredients. The quantity of bioactive compounds varies according to each method of extraction and the type of solvent used. Pharmacological activities of herbal plants depend upon the number of active principles present in it. Here in our study, we compared the two different types of methanolic extract of Cadaba indica leaf for its inhibitory action on protein denaturation and proteinase enzyme activity. Mohan et al. reported that total amount of phenol and flavonoids were found to be higher in methanolic extract of Cadaba indica than other solvent extracts. They also concluded that the antioxidant activity of Cadaba indica was greater with the methanolic extract⁹. The amount of rutin, gallic acid and quercetin in various types of extracts of Cadaba indica leaf was quantified by Thirumalai et al, who have reported that quercetin and rutin were found to be in higher quantities in cold macerated methanolic leaf extract than in hot percolation method. In addition, they also revealed the presence of gallic acid in the cold macerated methanolic leaf extract of Cadaba indic¹⁴. Rutin, quercetin and gallic acid have anti inflammatory, antiarthritic, anti allergic, anticancer and antioxidant properties^15-17.

Rheumatoid arthritis (RA) is an autoimmune condition and a chronic systemic inflammatory disease that affects many tissues and organs^18,19. Denatured tissue protein can lead to inflammation. Denatured proteins lose their biological functions. External stress or compounds such as strong acid or base, concentrated organic salt, an organic solvent or heat alters the tertiary and secondary structure of proteins. In vivo denaturation of proteins can generate autoantigens in arthritis²⁰. In our present study, both CICME & CHIME extracts inhibited the protein denaturation in a dose dependent manner. CICME resulted in the maximum inhibitory percentage of 77.99% at 1000μg/ml while, for the same dose, CIHME caused only 74.75% inhibition. The maximum inhibitory percentage of the standard drug dexamethasone at 1000μg/ml was 91.54%. Although both extracts resulted in statistically significant action, compared to dexamethasone, CICME extract was found to be more effective and more potent than CIHME extract in inhibiting protein denaturation.

Tissue damage in arthritis is mainly due to inflammation caused by leukocyte proteinase. Hence, proteinase inhibitors provide significant relief in arthritis through their anti-inflammatory action²¹. In fact, an agent possessing proteinase inhibitory activity can be therapeutically exploited in arthritis^23,24. Neutrophils contain serine proteinase enzymes in their lysosomal storage sites. In patients with arthritis, elastases and proteases released from leukocytes degrade the proteoglycan present in the superficial layer of cartilage ^22,23. The depletion of proteoglycan precipitates the formation of immune complexes resulting in the release of matrix metalloproteinases. MMPs such as stromelysin and collagenases are the main mediators that degrade connective tissue matrix and cause joint damage in Rheumatoid arthritis.

In our study, the percentage inhibition of CICME was higher than the CIHME at all concentrations. Standard drug dexamethasone shows high efficacy in the proteinase inhibitory activity.

CONCLUSION:

Inhibition of protein denaturation and proteinase enzyme activity were the most accepted parameters for anti-inflammatory and anti-arthritic activities. In the present study, results indicate that the methanolic leaf extracts of Cadaba indica significantly inhibited the protein denaturation and proteinase enzyme activity. Based on the results, the cold macerated methanolic extract (CICME) of Cadaba indica showed better activity than hot percolated methanolic extract (CIHME). The higher amount of bioactive compounds such as quercetin, gallic acid, and rutin in the cold macerated methanolic extract may be the reason for this. Further studies are needed to establish its safety, efficacy and mechanism of action.

CONFLICT OF INTEREST:

The authors declare they have no conflict of interest.

REFERENCE:

1. Vispute S, Khopade A. Glycyrrhizaglabra Linn.- “Klitaka”: A review. Int J Pharm Bio Sci. 2011; 2: 42–51.

2. Thirumal M, Vadivelan R, Kishore G, Brahmaji VS. Aristolochia bracteolata: An overview on pharmacognostical, phytochemical and pharmacological properties. Earth J. 2012; 1: 66–78.

3. Sudha K, Mathanghi SK. Traditional underutilized green leafy vegetables and its curative properties. Int J Pharm. 2012; 2: 786–793.

4. Nadkarni AK. Indian Materia Medica- 1, 3rd ed., Popular Prakashan, Bombay; 2002: 225-226.

5. Rastogi, RP and Mehrotra, BN., In; Compendium Indian Medicinal Plants Vol-V, Publications and Information Directorate, NewDelhi,1991, 116.

6. Selvamani M, Latha D (2005). Antimicrobial activity of Cadaba indica Lam. Indian Journal Pharmaceutical Sciences. 2005: 637-638.

7. Mythreyi R, Sasikala E, Geeta A and Madhavan V. Antipyretic activity of leaves of Cadaba fruticosa. Pharmacology online. 2008; 3:136-142.

8. Tenpe CR, Upaganlawar AB, Yeole PG (2006) Anti diabetic activity of Cadaba indica in Alloxan induced diabetic rats. Ind J Nat. Prod 22:14–17

9. Mohan VR, Lincy MP, Devi GS. Evaluation of phenolic and flavonoid contents and antioxidant activity of various solvent extracts of Cadaba indica lam. Int J Adv Pharm Sci. 2015; 6(3): 2849-2853.

10. Gupta JK, Pal TK, Ghosh K, Sen DJ, Sureshbabu P, Latha S, Selvamani P. Analgesic and anti-inflammatory activities of leaf extract of Heliotropium zeylanicum and Cadaba indica, Drug lines. 2006; 8(2): 6-9.

11. Mizushima Y and Kobayashi M. Interaction of anti‐inflammatory drugs with serum preoteins, especially with some biologically active proteins. J of Pharma Pharmacol 1968; 20:169‐ 173.

12. Sakat S, Juvekar AR, Gambhire MN. In vitro antioxidant and anti-inflammatory activity of methanol extract of Oxalis corniculata Linn. International Journal of Pharma and Pharmacological Sciences 2010; 2(1):146-155.

13. Oyedepo OO and Femurewa AJ. Anti‐protease and membrane stabilizing activities of extracts of Fagrazanthoxiloides, Olaxsubscorpioides and Tetrapleuratetraptera. Int J of Pharmacong 1995; 33: 65‐69.

14. Thirumalai V, Nirmala P, Venkatanarayanan R, Detection and Estimation of Phenolic Acid and Flavonoids in Leaves of Cadaba indica Lam by High Performance Thin Layer Chromatography. Sch. Acad. J. Pharm., Sept, 2018; 7(9): 417-424.

15. Middleton E, Kandaswami C, Theoharides TC. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacological reviews. 2000; 52(4): 673-751.

16. Guardia T, Rotelli AE, Juarez AO, Pelzer LE. Anti inflammatory properties of plant flavonoids. Effects of rutin, quercetin and hesperidin on adjuvant arthritis in rat. Il farmaco. 2001; 56(9): 683-687.

17. Jackson JK, Higo T, Hunter WL, Burt HM. The antioxidants curcumin and quercetin inhibit inflammatory processes associated with arthritis Inflammation Research. 2006; 55(4):168-175.

18. Malaviya. A.N, Kapoor. S.K, Singh. R.R, Kumar.A, Pande.I, Prevalence of rheumatoid arthritis in the adult Indian population, Rheumatol Int 1993; 13:131-134

19. Subramaniam Mohana Devi, Vellingiri Balachandar, Keshavarao Sasikala, Pappusamy, Manikantan, Meyyalagan Arun, Shafi ahammed khan Mustaq ahamed, Balasubramanian Balamurali Krishnan, Shanmugam Sureshkumar, Sellappa Sudha, Elevated rheumatoid factor (RF) from peripheral blood of patients with rheumatoid arthritis (RA) has altered chromosomes in Coimbatore population, South India, International Journal of Arthritis and Rheumatism 2010; 2 (10): 167-174

20. G. Leelaprakash, S.Mohan Dass. In vitro anti-inflammatory activity of methanol extract of Enicostemma axillare Int. J. Drug Dev. & Res., 2011, 3 (3): 189-196.

21. Das SN and Chatterjee S. Long term toxicity study of ART-400. Indian. J. Indg Med. 1995;16(2): 117-123

22. Patel SS and Zaveri MN: Trypsin and Protein Denaturation Inhibitory Activity of Different Fractionation and Isolated Compound of Leaf and Root of Justicia gendarussa. Int J Pharm Sci Res 2014; 5(12): 5564-5571.

23. Narayani P. C, Anbu J, Vasuki R, Rajeswari Hari. Invitro and In vivo Anti–Arthritic Activity of Combined Ethanolic Extracts of Calotropis gigantea and Cardiospermum halicacabum in Wistar Rats. Journal of Natural Remedies Jan 2015 :14(1): 58-66

24. Asin HE, Taurog JD. Mechanisms of disruption of the articular cartilage surface in inflammation: neutrophil elastage increases availability of collagen tye II epitopes for binding with antibody on the surface of articular cartilage. J Clin Invest. 1991; 87(5):1531–1536.

Received on 17.07.2019 Modified on 10.09.2019

Research J. Pharm. and Tech 2020; 13(3): 1219-1223.

DOI: 10.5958/0974-360X.2020.00224.3