INTRODUCTION:

Plants have traditionally played an indispensable role in treatment of various pathophysiologic conditions throughout medical history and is being used in several underdeveloped and developing countries in various forms till date. India being home to some of the world's most biodiverse regions and home to four biodiversity hotspots of world: The Himalayas, The Western Ghats, The Indo-Burma region and The Sundaland (Includes Nicobar group of Islands) is enriched with numerous endemic species¹. Tribal communities residing in this biodiverse region have traditionally used and relied on herbs for treating various ailments².

Exponential increase in population and lack of adequate health care facilities to the rural masses necessitates the need to validate the herbal medicines which can be done by doing a meticulous scientific study on medicinal plants which are under explored.

Hyperhomocysteinemia is looming as an independent risk factor for development of cerebrovascular, peripheral arterial occlusive and coronary disease. 20% to 30% of patients with coronary and peripheral vascular disease have increased levels of homocysteine³. A mere increase of 12% over the normal level of homocysteine has been associated with a threefold increase in risk for myocardial infarction⁴. Neoteric studies on cardiovascular disease confirms a positive association between plasma homocysteine concentration and risk for cardiovascular disorders. Methionine being the only known dietary source of homocysteine is a potent agent that rattles the endothelial integrity. Imbalance in the Methionine-Homocysteine cycle which keeps the levels of homocysteine in check will lead to the development of atherosclerosis and other procoagulant diseases by increasing homocysteine levels⁵.

Rhizophora mucronata Poir belongs to family Rhizophoraceae and commonly known as the looproot mangrove, red mangrove and Asiatic mangrove⁶. Mangroves commonly found in the coastal intertidal zones of Indo-pacific and East Africa. Mangroves have been consistently used in folk medicine for its antiviral, anti-bacterial, anti-diarrheal, antifungal, and antioxidant properties⁷. Extraction of R. Mucronata Poir demonstrated potent anthelmintic activity tested against Indian earthworm Pheretima posthuma⁸. Its bark has been proved to be an effective analgesic and anti-Inflammatory⁹. R. mucronata poir showed some antibacterial activity against S. aureus and E. coli. R. mucronata poir extract of leaves exhibited strong inhibitory action against Bacillus subtilis, Staphylococcus aureus, Candida albicans, Escherichia coli, Aspergillus fumigatus and Aspergillus niger and moderate inhibitory action against Pseudomonas aeruginosa and Proteus vulgaris¹⁰. Its leaf is a natural source for both tannins and flavonoid, but their chemical, biological and pharmacological properties have not yet been determined. Recently scientists are veering in search of effective remedies from mangroves for diseases such as diabetes, asthma, cancer, ulcer, wounds and AIDS¹¹. Ethanol extract of R. mucronata poir showed high antioxidant activity equal to that of Ascorbic Acid which was assessed by means of its ability to convert Fe₃+ to Fe₂+¹². It also exhibited potent ABTS radical cation scavenging activity in a concentration-dependent manner. Ethanol extract showed the highest antioxidant capacity (146.18%) followed by ethyl acetate (123.92%). The IC50 values for the DPPH (1,1‑diphenyl‑2‑picrylhydrazyl), Hydrogen Peroxide, Nitric Oxide, FRAP (Ferric reducing antioxidant powers), LPO (lipid peroxidation) and SOD (superoxide dismutase) showed the potential of free radical scavenging activity of Rhizophora mucronatapoir¹³.

However, no antihyperhomocysteinemic activity has been carried out in Rhizophora mucronata poir owing to its potent antioxidant potential. Therefore, it was thought worthwhile to determine the antihyperhomocysteinemic activity of Rhizophora mucronata poir. Hence, the current study was a pilot study designed to determine whether the ethanol extract of dried leaves of Rhizophora mucronata poir could exert any protective action against methionine-induced hyperhomocysteinemia as judged by biochemical markers and blood parameters.

MATERIALS AND METHODS:

Chemicals:

Methionine and Ascorbic acid were procured from Sai Mirra Innopharm Pvt. Ltd. (Chennai, India). Other chemicals used were of analytical grade. Double-distilled water was used for all biochemical assays.

Plant Materials:

The leaves of Rhizophora mucronata poir were collected from Pichavaram mangrove, Cuddalore district, Tamilnadu. Taxonomic identification of the plant was carried out by Professor Dr. R. Chelladurai, Botanist, Survey of Medicinal plants. Fresh leaves were air dried, powdered and stored in an air tight container at room temperature. The sample analysis was carried out in Sai Mirra innopharm pvt ltd, Chennai.

Preparation of successive extract of Rhizophora mucronate:

The dried and coarsely powdered drug (100g) was packed in a Soxhlet apparatus and subjected to hot percolation with solvents of increasing polarity using n-hexane, chloroform, ethyl acetate and ethanol. Each filtrate was evaporated under vacuum, and hexane (green), chloroform (dark green), ethyl acetate (green) and ethanol (dark brown) residue obtained was stored at 4^◦ C for further use^16-18. The average yield of the leaves of Rhizophora mucronata poir extract was approximately 1.2% w/w for hexane, 2.3 %w/w for chloroform, 6.8 for ethyl acetate %w/w and 8.8 %w/w for ethanol. For experimental studies, the weighed amount of ethanolic extract residue of Rhizophora mucronata poir (200 and 400 mg/kg) was dissolved in 1% Tween 80 in normal saline and administered to adult male Wistar albino rats by the oral route.

Standardization of Extract:

Preliminary phytochemical screening of succesive extracts with n-hexane, chloroform, ethyl acetate and ethanol of dried leaves was carried out for the detection of phytoconstituents, using standard chemical tests. Alkaloids, flavanoids, Phenols, saponins and carbohydrates were detected in the extract^19-23. High Performance liquid Chromatography (HPLC) was used for the estimation of Vitamin A, E, and C for the crude extracts using (Waters 2495/2996,2487 –Empower³software.

Table.1 Quantitative estimation of phytoconstituents of Rhizophora mucronata poir

S. No	Extract	Total Flavanoid Content	Total Phenolic Content	Total Alkaloid Content
1.	n-Hexane	85.23 mg/ 100 g	78.87 mg/ 100 g	28.26 mg/100 g
2.	Chloroform	61.81 mg/ 100 g	66.36 mg/ 100 g	27.80 mg/100 g
3.	Ethyl acetate	40.01 mg/ 100 g	73.36 mg/ 100 g	51.66 mg/100 g
4.	Ethanol	51.07 mg/ 100 g	1882.0 mg/ 100 g	143.6mg/100 g

High Performance Liquid Chromatography (HPLC) Analysis of Vitamin A, E and C^[24-29]:

All chromatographic solvent are HPLC grade. The crude Ethanol leaf extract of R.mucronata poir was dried at 70°C in water bath. The dry sample filtrated through membrane filter with an aperture size of 0.5 µm. Mobile phase used for the analysis of Vitamin A and E was Acetonitrile and methanol in the ratio 75:25. In the case of vitamin C, Phosphate buffer and acetonitrile in the ratio of 25:75 was used as mobile phase. The liquid chromatography was equipped with 280-nm detector and 4.6-mm × 15-cm column that contains 5-μm packing C18 . The flow rate was about 1.5 ml per minute. Retinyl palmitate (7.5μg/ml), USP alpha -tocopherol (2 mg/ml) and ascorbic acid (1 mg/ml) were used as standards for Vitamin A, E and C respectively. Equal volumes (about 40 μL) of the standard preparation and the sample preparation were separately injected into the chromatograph. The chromatograms were recorded and the estimation of the amount of antioxidant vitamins were calculated as shown in the table no.2

Table.2: Composition of antioxidant vitamins in the leaves of Rhizophora mucronata poir

S. No	Extract	Vitamin A mg/100g	Vitamin E mg/100g	Vitamin C mg/100g
1.	Hexane	1.93	1069.24	35.64
2	Chloroform	0.7696	143.62	64.51
3	Ethyl acetate	0.684	64.51	1069.24
4.	Ethanol	0.4035	28.26	2941.8

Rich source of the antioxidant Vitamins A, C and E in leaves of R. mucronata Poir makes this plant an additional dietary means for prevention of oxidative stress disorders.

Animal:

Healthy, male, adult, albino Wistar rats (150–200g) procured from the Institutional animal house facility, Madras Medical College, Chennai and acclimatized under standard laboratory conditions at 25±2^◦C, relative humidity (50±15%), and normal photoperiod (12-h light-dark cycle) for 7 days were used for the experiment. Commercial rat pellet diet and water were provided ad libitum. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Madras Medical College, Chennai, which is registered with Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India (registration no. 1917/ReBi/S/16/CPCSEA/25.10.2016)

Experimental procedures^[30-33]

After acclimatization, all the animals were randomly divided into five groups of 6 animals each and treated as follows:

Group I, normal healthy control: Rats received only 1% Tween 80 in normal saline.

Group II, Disease control: Rats received only methionine (1g/kg, p.o.) for 30 days.

Group III: Rats received ethanolic extract of Rhizophora mucronata poir (200mg/kg, p.o.) coadministered with methionine (1g/kg, p.o.) for 30 days

Group IV: Rats received ethanolic extract of Rhizophora mucronata poir (400mg/kg, p.o.) coadministered with methionine (1g/kg, p.o.) for 30 days.

Group V: Rats received Ascorbic acid (100mg/kg, p.o.) coadministered with methionine (1g/kg, p.o.) for 30 days.

At the end of the experiment, blood samples were withdrawn from the retroorbital plexus using microcapillary technique from all the groups of overnight-fasted rats, and serum was separated for biochemical estimation of homocysteine, lactate dehydrogenase and blood clotting parameters.

Table.3 Effect of ethanolic Rhizophora mucronata Poir extract on Homocysteine and Lactate Dehydrogenase levels in blood.

Treatment	Homocysteine (µg/mL)	LDH(IU/L)
Normal Healthy control	9.630 ± 0.342^b	29.457 ± 0.752^b
Methionine control	23.763 ±0.452^a	59.755 ±0.920^a
Ethanol extract (200mg/kg)	16.692±0.071^b	39.035 ±0.563^b
Ethanol extract (400 mg/kg)	16.086 ±0.055^b	36.412 ±0.298^b
Ascorbic acid (100 mg/kg)	16.761 ±0.046 ^b	34.533 ±0.112^b

^a p < 0.01 as compared with group I, ^bp < 0.01, compared with group II.

Statistical analysis:

All data were expressed as mean±SEM. All the groups of data were analyzed by one-way analysis of variance followed by Dunnett t -test using GraphPad Prism 3.0 (Graph-Pad Software; San Diego, CA, USA). p<0.01 values were considered as statistically significant.

RESULTS:

Methionine administration in the disease control group resulted in significant (p<0.01) elevation of homocysteine, lactate dehydrogenase (LDH) and significant (p<0.01) decrease in thrombin, prothrombin and aptt time in blood compared with normal healthy control group.

Ethanolic Rhizpora mucronata extract and ascorbic acid acid treatment in hyperhomo-cysteinemic rat significantly (p<0.01) decreased the homocysteine, LDH, pro-coagulant factors and increased the in thrombin, prothrombin and aptt time in the blood compared with methionine control group

Table. 4 Effect of Ethanolic extracts of Rhizophora mucronata poir on blood clotting parameters.

TREATMENT	APTT	PT	TT
Normal Healthy control	38.8±1.11^b	10.6±0.33^b	16.55±0.88^b
Methionine control	29.3±0.71^a	9.8±0.30^a	115±0.57^a
Ethanol extract (200mg/kg)	36.1±0.60^b	11.6±0.33^b	195±0.25^b
Ethanol extract (400 mg/kg)	33.1±0.48^b	10.3±0.21^b	165±0.51^b
Ascorbic acid (100 mg/kg)	32.17±0.48^b	10±0.25^b	155±0.2^b

^aP< 0.01 as compared with group I, ^bP < 0.01, compared with group II.

APTT –Activated plasma thromboplastin time TT- Thrombin Time, PT- prothrombin time.

DISCUSSION:

The current study examined the antihyperhomocysteine and procoagulant factors lowering potential of ethanol Rhizophora mucronata poir extract (200 and 400 mg/kg, p.o.) in methionine-induced hyperhomocysteinemia in rats. The mechanisms associated with homocysteine-induced endothelial dysfunction are mediated by increased oxidative stress leading to increased levels of oxidized LD. Hyperhomocysteinemia may promote the gen-eration of reactive oxygen species (ROS) such as H₂O₂ and hydroxyl radicals via the autooxidation of sulfhydryl (-SH) grou by decreasing the intracellular levels of Glutathione peroxide, which is involved in the elimination of free radicals³⁴.

Homocysteine, a thiol containing amino acid derived from demethylation of dietary methionine, may generate partially reduced ROS that are able to stimulate the lipid peroxidation involved in atherosclerotic process. Thus, an imbalance in dietary methionine may contribute to the de-velopment of atherosclerosis by increasing homocysteine levels.

The data in our current study showed that methionine (1g/kg, p.o.) treatment in disease control group rats significantly (p<0.01) elevated the levels of homocysteine and lactate dehydrogenase Free radicals generated by hyperhomocysteinemia initiate lipid peroxidation of the membrane-bound polyunsaturated fatty acids, leading to impairment of the membrane structural and functional integrity³⁵. Furthermore, the results of test drug were comparable with Ascorbic acid, a standard positive control.

To conclude, the In-Vivo animal studies showed that elevated levels of Homocysteine, Lactate dehydrogenase (LDH) were reduced significantly (p<0.01) along with decrease in prothrombotic factors by treatment with ethanolic extract of Rhizophora mucronata Poir, suggesting its defence mechanism against hyperhomocysteine induced Cardiovascular diseases (CVD) and the abundance of antioxidant vitamins exemplifies its high nutritional value.

REFERENCES:

1. Keerti Shrivastava, Skand K. Mishra. Ethnomedicine for Jaundice used by the Tribals of Achanakmar-Amarkantak Biosphere Reserve, India. Res. J. Pharmacology and Pharmacodynamics.2017; 9(1): 27-30.

2. Krishna Mohan G.V., K. Kameswara Rao, P.V.V. Prasada Rao. The Assessment of Sediment Ages Determination of Bhavanapadu Mangrove Ecosystem, North Coastal Andhra Pradesh, East Coast of India. Asian J. Research Chem. 4(7): July, 2011; Page 1137-1140.

3. Rahul Rawat, Yogesh Josh. Effect of Antihypertensive Drugs on Homocysteine level among Hypertensive Patients. Asian J. Res. Pharm. Sci. 2018; 8(4): 219-222.

4. Hyperhomocysteinemia: An independent risk factor for vascular disease; N Engl J Med 1991;324: 1149– 1155.

5. Toborek M, Hening B: Is methionine an atherogenic amino acid? J Ophthalmol Nutr 1994; 3: 80–83.

6. Sunita C. Mesta, R. Onkarappa, Meghana S.H, Manu H.M, Kavana S, Mohamed Talib. Phosphate solubilizing endophytic actinomycetes from mangrove plants Rhizophora mucronata and Sonneratia caseolaris and its effect on Seedling vigour.Research J. Pharm. and Tech. 2018; 11(3): 1172-1178.

7. Pitamber J, Hitender P, Pradeep S, S.R Sathish Kumar, Veena S, K.V. Bhaskara Rao. Production of L-Asparaginase by Marine Actinobacteria Isolated from the Rhizosphere of Mangrove Plants. Research J. Pharm. and Tech. 7(5): May, 2014; Page 526-530.

8. Y. Narasimha Rao et al., In-vitro anthelmintic activity of Rhizophora mucronata leaf extract. Scholar Research Library Der Pharmacia Lettre, 2016, 8 (6):33-36.

9. Ravi Parimi, K E Pravallika. Studies on Phytochemical Screening, Total Phenolic Content and In vitro Antioxidant activity of Different Extracts of Euphorbia thymifolia Roots. Research J. Pharm. and Tech. 2017; 10(2): 551-556.

10. Manickavelu Manimaran, Krishnan Kannabiran. Alpha amylase activity of Streptomyces sp. VITMK1 isolated from marine soil sample of Pichavaram, Tamil Nadu, India. Research J. Pharm. and Tech 2018; 11(6): 2180-2182.

11. Mariappan premananthan et al., In vito anti human immune deficiency virus activity of polysaccharide from Rhizophora mucronata. Biosci Botech Biochem, 63(7), 1187-1191,1999.

12. Vipul Singh, Arvind Kumar Singh, Rohit Tripathi, Preeti Mishra, Roopes Kumar Maurya. 1, 2, 4- Triazole Derivatives and its pharmacological activities. Asian J. Research Chem. 6(5): May 2013; Page 429-437.

13. Suganthy Nand Pandima Devi K. In vitro antioxidant and anti-cholinesterase activities of Rhizophora mucronata. Pharmaceutical Biology 2015, 54:1, 118-129.

14. G. V. Krishna Mohan, K. Kameswara Rao, P. V. V. Prasada Rao. Environmental Impact Assessment of Sediment Quality Analysis of Bhavanapadu Mangrove Ecosystem, East Coast of India. Asian J. Research Chem. 4(7): July, 2011; Page 1067-1072.

15. Shrada. B. Kumar, Dhanraj. M. Role of Vitamin C in Body Health. Research J. Pharm. and Tech 2018; 11(4): 1378-1380.

16. Josphine A., Senthilkumar G., Madhanraj P, Panneerselvam A. Diversity of Fungi from Drift Wood of Muthupet Mangroves. Asian J. Pharm. Ana. 1(3): July-Sept. 2011; Page 53-55.

17. Dima Al Diab, Nour Al Asaad. Comparative Analysis of Ascorbic Acid Content and Antioxidant Activity of Some Fruit Juices in Syria. Research J. Pharm. and Tech 2018; 11(2): 515-520.

18. Harborne JB, Phytochemical methods: A Guide to Modern Techniques of Plant Analysis, 2nd ed, London; Chapman and Hall Ltd; 1973: 49-188.

19. Rohini r m and Amit kumar Das. A comparative evaluation of analgesic and anti-inflammatory activities of Rhizophora mucronata bark Extracts. pharmacologyonline 1: 780-791 (2009).

20. Yunita E. Puspitasar, Aniek M. Hartiati, Eddy Suprayitno. The Potency of Rhizophora mucronata Leaf Extract as Antidiarrhea. Journal of Applied Sciences Research, 8(2): 1180-1185, 2012.

21. Vichare Vijaya, Morade Priya, Kokane Jyoti, Tambe Vrushali, Dhole S.N. A Comparative Study of Estimation of Ascorbic Acid by Different Methods in Fresh Fruit Juices and Marketed Preparations. Research J. Pharm. and Tech. 4(11): Nov. 2011; Page 1690-1692.

22. A. Moumita ray, et ala., Pharmacognostic and anti-hyperglycemic evaluation of the leaves of sunderban mangrove, Rhizophora mucronata l. International Journal of Advances in Pharmaceutical Sciences Volume 5|Issue 5| September-October 2014.

23. D G S Andayani1 et al., Isolation, identification of alkaloid from Rhizophora mucronata and the activity of its methanol extract against barnacles. IOP Conf. Series: Earth and Environmental Science 160 (2018) 012005.

24. Harborne JB, Phytochemical methods: A Guide to Modern Techniques of Plant Analysis, 2nd ed, London; Chapman and Hall Ltd; 1973: 49-188.

25. Hima V., Rubesh Kumar S., Duganath N., Devanna N. A Novel Validated Stability Indicating Chromatographic Method for the Simultaneous Estimation of Ascorbic acid and Gallic acid in the Ayurvedic Capsule Dosage form of Amla by UFLC. Asian J. Research Chem. 6(9): September 2013; Page 826-831.

26. N. Suganthy and k. Pandima devi Nutritional evaluation of Asiatic mangrove Rhizophora mucronata – its proximate composition, amino acid profiles and physico-chemical properties: ijpsr (2016), vol. 7, issue 6

27. Batool et al., Asiatic Mangrove (Rhizophora mucronata) – An overview. European Academic Research (2014). vol. II, Issue 3/ June 2014.

28. Patrick B. Durst. Mangrove trees and shrubs. FAO Regional Office for Asia and the Pacific 2007.

29. M. Elayarani, P. Shanmuganathan, P. Muthukumaran. In Vitro Anti-Oxidant Activity of the Various Extracts of Cassia auriculata L. Flower by UV Spectrophotometer. Asian J. Pharm. Tech. 1(3): July-Sept. 2011; Page 70-72.

30. M. Nazam Ansari and U. Bhandari Antihyperhomocysteinemic Activity of an Ethanol Extract from Embeliaribes. in Albino Rats, Pharmaceutical Biology, (2008) 46:4, 283-287.

31. Shaker E, Manaa S, Mubarak A, Hady Ferdous AE Hypo- Homocysteinemia Effect for Some Ethanolic Plant Extracts. Journal of Nutrition and Food Science (2013) 3:200.

32. Mrunali Potbhare, Nikhita Phendarkar, Deepak Khobragade. In vitro Evaluation of Antioxidant Potential of N –Acetyl-D-Glucosamine. Asian J. Res. Pharm. Sci. 2017; 7(2): 120-122.

33. Sandeep Goyal, V.K. Bansal, Dhruba Sankar Goswami, Suresh Kumar. s Vascular Endothelial Dysfunction: Complication of Diabete Mellitus and Hyperhomocysteinemia. Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 657-664.

34. Nappo F1, De Rosa N, Marfella R, et,al. Impairment of endothelial functions by acute hyperhomocysteinemia and reversal by antioxidant vitamins. JAMA1999; Jun 9;281(22):2113-8.

35. Ch. Madhu, J. Swapna, K. Neelima, Monic V. Shah. A Comparative Evaluation of the Antioxidant Activity of Some Medicinal Plants Popularly Used in India. Asian J. Res. Pharm. Sci. 2(3): July-Sept. 2012; Page 98-100.

Received on 06.04.2019 Modified on 10.05.2019

Research J. Pharm. and Tech. 2019; 12(10):4807-4811.

DOI: 10.5958/0974-360X.2019.00831.X