Karthikeyan, R (2018) Evaluation and characterization of Herbal Extract of Halodule Uninervis and Its Anti Diabetic Activity. Doctoral thesis, The Tamilnadu Dr. M.G.R. Medical University, Chennai.
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Abstract
Marine flowering plants Seagrasses are capable of completing their life cycle when they are submerged in sea water. Ecosystem of Seagrass is one of the most widespread coastal vegetation types as compared to the ecosystems of coral and mangrove. Phytochemical study on sea grasses has clearly shown that seagrasses have pharmaceutically active secondary metabolites. Different studies on these seagrass metabolites proven that the extracted active components from saegrasses have alkaloids, glycosides, galactomannan gum, polysaccharides, peptidoglycan, hypoglycans, guanidine, steroids, carbohydrates, glycopeptides, terpenoids, amino acids and inorganic ions which directly or indirectly have a effective action aganst infection, pharmaceutically active hypolipemic agents, reduce blood pressure and blood cholesterol levels. Phenolic compounds are effective secondary metabolites of seagresses which play a main role in various bio relevant activities in human body maintenance. The presence of phytoconstituents, such as flavonoids, phenols and tannin, in seaweeds and seagrasses indicates a possibility that extracts may have antioxidant activity and anti diabetic activity. Shade-dried plant material grounded to coarse powder and the quality of plant powder determined by ash content, acid insoluble ash, acid soluble ash and fluorescence analysis. Positive acceptable results were observed for the total ash values, which show that no inorganic adulteration is present. The Plant powder was treated with various chemical reagents and examined under long UV (254 nm), short UV (366 nm) and visible light. The active constituents were extracted using solvents-Petroleum ether, Chloroform, Ethyl acetate and Methanol in an increasing order of polarity. Preliminary phytochemical Screening (TLC and chemical tests) for the extract of Halodule uninervis was performed to study presence of chemical constituents qualitatively. TLC analysis of methanolic extract of Halodule uninervis shown that there was many phytoconstituents with different composition of mobile phase. Investigation of Petroleum ether, chloroform, ethyl acetate and Methanol extracts have identified the presence of various phytochemical constituents like alkaloid, carbohydrate, glycosides, terpenoids, proteins, amino acids, steroids, tannin, saponin, terpenoid, flavonoid, anthraquinones and phenol. As methanolic fraction responded for the many testes and shown good response against pharmacological activity, This extract tested for the presence of various constituents and nature of the compounds. Upon the investigation of column fraction there are two major components were identified which are Compound 1-ME-HU-C-1 and Compound 2- ME-HU-C-2. Further characterizations of the isolated components were preceded by IR, NMR and mass spectroscopy to identify the isolated components and identified the presence of flavonoid moiety. The methanolic extract was subjected to Column Chromatography on silica (100-200) mesh. Two chemical components were isolated ME-HU-C-1 and ME-HU-C-2. TLC performed for the isolated components and the Rf were identified 0.68 and 0.79 for compound-1 and compound-2 respectively. These compounds were eluted with solvents of increasing polarity like hexane, ethyl acetate and methanol. In the course of isolation procedure, the above named 2 compounds were further processed and characterized. The compound ME-HU-C-1 is Pale yellow residue gave colorless crystalline substance after crystallization with methanol (About 41.25 mg). In TLC chromatogram developed with hexane: Ethyl acetate (8.5:1.5) was homogenous with Rf 0.68. The presence of flavonoid active group is identified by chemical test and identification by IR, 1H NMR, C13 NMR and MASS spectral data. The purity was quantified by reversed phase HPLC and the quantified purity is 99.5%. Based on the spectral data the compound-I was identified as ME-HU-C-1 : 4’, 5-Dihydroxy-6, 7-dimethoxyflavone. The compound ME-HU-C-2 is light brown colour residue which after crystallization converted to Pale yellow white crystals (46 mg). In TLC chromatogram was developed with hexane: Ethyl acetate (8.5:1.5) and it was homogenized with Rf 0.79. Melting range was 95.6-98.2°C. The Chemical test and identification by IR, 1H NMR, C13 NMR and MASS Spectral data were shown the characteristic feature of flavonoid component. The purity was quantified by reversed phase HPLC and the quantified purity is 99.5%. Based on the spectral data the compound-II was identified as ME-HU-C-2 : 4′, 5, 7-trihydroxyflavone Heavy metal analysis performed in selected plant measurements were made for cadmium, lead, arsenic, and mercury and shows absence of heavymetal contents in the extract. The methanolic extract of plant powder extract of Halodule uninervis shows presence of maximum chemical constituents which are responsible for the required invivo and in-vitro pharmacological activity. Effects of Halodule uninervis extract in the single dose study on blood glucose levels was estimated after Streptozotocin administration on the 3rd day. There is no reduction in glucose level due to Halodule uninervis extract (50 mg/kg). At dose levels of 150 and 250 mg/kg, glucose levels were decreased by 24.8% and 29.9% at the 6th hour, respectively. Antidiabetic effect of the extract was slightly decreased at the 8th hour, but remained statistically significant. In parallel experiments, Halodule uninervis extract was administered to diabetic rats for 18 days as per the protocol. The glucose level reduction up to 26% on 9th day of Halodule uninervis (50 mg/kg) treatment started to lower serum glucose on the 9th day. An overall reduction of 26% was observed on the 18th day by 50mg/kg administration 52.5% reduction of glucose level in the serum absorved at 18th day administration at dose level of 150 mg/kg. 250mg / kg extract administration is more effective from 6th day onwards with a reduction rate of 18.9% and maximum reduction of serum glucose level by 61.9% on the 18th day. Between the 12th and 18th days, antidiabetic effect of Halodule uninervis extract was in a concentration dependent manner. The effects of Halodule uninervis extract on hepatic and renal function in Streptozotocin-diabetic rats. As seen, treatment with 50 mg/kg did affect neither the significantly high levels of ALP, GPT, BUN and creatinine, nor the overall oxidative status. Conversely, rats treated with higher doses of the extract (150 and 250 mg/kg) showed significant improvements in hepatic and renal function. Additionally, these two dose levels recovered the weight loss and low white blood cell count observed in Streptozotocin-diabetic rats while decreasing liver glycogen. Halodule uninervis. extract (150 and 250 mg/kg) also showed a protective effect on liver oxidative status. Antioxidants namely GSH, GPx, SOD and catalase were increased by Halodule uninervis extract administration. When compared to Streptozotocin-diabetic rats, MDA formation, as an indirect measure of lipid peroxidation, was found to be significantly low in high dose Halodule uninervis extract-treated rats. Methanolic extract of Halodule uninervis showed up to 62% inhibition of DPPH free radical at 517 nm at high concentration of 1mg/ml. At the concentrations of 0.3mg/ml, 0.5mg/ml and 1mg/ml, quercetin shows similar activity of about 77.6% inhibition of DPPH free radicals. The percentage inhibition of DPPH free radical by methanol extract in the presence of standard quercetin at 517 nm. The in-vitro xanthine oxidase inhibition of methanolic extract revealed 67% of inhibitory activity at 30μg/ml and it was compared with the standard drug quercetin which showed 72% inhibition at 30μg/ml. The Halodule uninervis methanolic crude extract 100, 200, 300 and 400 μg/ml .was capable of scavenging H2O2 in an amount dependent manner. H2O2 scavenging activity was closer to that of standard Ascorbic acid (100μg/ml) at doses 400 μg/ml. Anti microbial activity for crude extract and isolated compounds from the Halodule uninervis methanolic extract were studied. The anti bacterial activity of the crude Methanolic extract and isolated compound ME-HU-C-1 and ME-HU-C-2 at 2000 ppm concentration and the activity against 3 pathogenic bacterial strains, Staphylococcus aureus Gram (+), Escherichia coli Gram (-), Salmonella Gram (-). Antibacterial potential of extracts were assessed in terms of zone of inhibition of bacterial growth. The bacterial strains Staphylococcus aureus Gram (+), Escherichia coli Gram (-) shows the good zone of inhibition in both crude Methanolic extract (15mm) and isolated compound ME-HU-C-2 (12mm) in the dilution of 2000 ppm in replicate manner. Both Compounds ME-HU-C-1 and ME-HU-C-2 was shown predominant microbial growth up to 100ppm. The agar diffusion assay method, there was no growth of E.coli in 1000 ppm for compound ME-HU-C-1 and slight growth observed in compound ME-HU-C-2. In Both dilution assay, there was no growth of E.coli in 2000 ppm for Compounds ME-HU-C-1 and ME-HU-C-2. The antifungal activity of the crude Methanolic extract and isolated compound ME-HU-C-1 and ME-HU-C-2 were mixed with potato dextrose medium and the activity against 2 fungal strains Aspergillus sp and Candida albicans. Antifungal potential of extracts were assessed in terms of percentage of inhibition of fungal growth. The crude Methanol extracts shown 29% inhibition against Candida albicans. No inhibition was observed against Aspergillus sp and Candida albicans due to MEHU-C-1. ME-HU-C-2 had 6% inhibition against Candida albicans and no inhibition was observed against Aspergillus sp. The results were showed that the crude Methanolic extract and isolated compound of Halodule uninervis were found to be more effective against all the microbes tested. CONCLUSION: Marine organisms offer a wide source to discover useful therapeutics with cost effective. Recently, numerous marine metabolites with potent pharmacological properties have been identified. • Among them seagrass is one of the most widespread coastal vegetation types as compared to the ecosystems of coral and mangrove. Phytochemical study on sea grasses has clearly shown that seagrasses have pharmaceutically active secondary metabolites. • Different studies on these seagrass metabolites proven that the extracted active components from saegrasses have alkaloids, glycosides, galactomannan gum, polysaccharides, peptidoglycan, hypoglycans, guanidine, steroids, carbohydrates, glycopeptides, terpenoids, amino acids and inorganic ions which directly or indirectly have a effective action aganst infection, pharmaceutically active hypolipemic agents, reduce blood pressure, and blood cholesterol levels. Phenolic compounds are effective secondary metabolites of seagresses which play a main role in various bio relevant activities in human body maintenance. The presence of phytoconstituents, such as flavonoids, phenols, and tannin, in seaweeds and seagrasses indicates a possibility that extracts may have antioxidant activity and anti diabetic activity. • Diabetes mellitus. a major disorder in the world leading to massive economic losses. Diabetes is a chronic disorder of carbohydrate, fat and protein metabolism characterized by increased fasting and fed blood sugar levels in the human body. World health organization predicted that the developing countries will face major burden due to Diabetes. Studies conducted in India, highlighted that the prevalence of diabetes is high and also the rapid increase in the urban population. • Prolonged medications required for the treatment of diabetes mellitus, considering the limitations and side effect in the synthetic drugs, natural products derived from medicinal plants and marine products are being looked for the treatment of diabetes. However components of medicinal plants and marine products are the base of many conventional drugs. Among 1200 plants and marine products which used for the treatment of diabetes mellitus there 200 pure compounds have showed lowering blood glucose activities. considering low toxic, ready availability and cost effective, usage of natural products for the treatment of diabetes mellitus has recommended and encouraged by WHO (World Health Organization) worldwide. • The seagrass Halodule uninervis which is easily available from the coastal area of south Tamilnadu and identified that contain rich pharmaceutically effective secondary metabolites and is the evident that the presence of many phytochemical constituents in the marine plant extract. Multiple analyses shown the presence of various active phytoconstituents like alkaloid, carbohydrate, tannin, Saponin, terpenoid, flavonoid, Coumarin and phenols are present. • The isolated phytochemical components were characterized and the Methanol extract obtained from the successive extraction of Halodule uninervis was shown a well response for most of the qualitative chemical tests which confirms that the effective extraction of maximum constituents from the plant material and shows effective in-vivo and in-vitro pharmacological activity. As the better response derived from the methanolic extract this extract was chosen for the further investigation and shown significant anti diabetic activity respect to the protocol studies. • A well response observed against the induced diabetic. Based on the multi concentration against response, higher concentration shown well response against high concentration of 250mg/kg of body weight. Also the extract was well responded for anti-oxidant and antimicrobial activities. • Atomic absorption spectrophotometric analysis of methanolic extract of Halodule uninervis conformed the absence of Cd, Pb, As and Hg. Further investigation of the on the isolation and identification of component of the plant may lead to chemical entities with potential for clinical use with cost effective manner. • Based on the above study it was concluded that the investigation of sea grass Halodule uninervis optained from marine source have active phyto-constituents which are pharmaceutically effective and less toxic. Considering low toxic, ready availability and cost effective, usage of Halodule uninervis for the treatment of diabetes mellitus is mostly recommended.
| Item Type: | Thesis (Doctoral) |
|---|---|
| Additional Information: | Reg.No.141440009 |
| Uncontrolled Keywords: | Evaluation, characterization, herbal extract, Halodule uninervis, anti diabetic activity. |
| Subjects: | PHARMACY > Pharmaceutical Chemistry |
| Depositing User: | Subramani R |
| Date Deposited: | 12 Nov 2019 05:24 |
| Last Modified: | 24 Oct 2022 05:18 |
| URI: | http://repository-tnmgrmu.ac.in/id/eprint/11318 |
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