Vijayalakshmi, A (2013) Development and Evaluation of Some Bioflavonoids from Indigenous Plants. Doctoral thesis, The Tamil Nadu Dr. M.G.R. Medical University, Chennai.
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Abstract
The thesis entitled development and evaluation of some bioflavonoids from indigenous plants deals with pharmacognostical, phytochemical and pharmacological investigation of medicinal plants Givotia rottleriformis and Cassia tora, traditionally used for the treatment of chronic inflammatory skin disease psoriasis. A perusal of the literature reveled that only fragmentary information was available on Givotia rottleriformis regarding pharmacognosy, phytochemistry and pharmacological activity by any other researchers. This study was designed first time for the isolation of flavonoids from the selected plants, screening of pharmacological activities of the isolated flavonoids in order to establish its folklore claims and the bioactive flavonoids were promoted to formulation and evaluation for their product performance. Plants are becoming potential source for phytoconstituents with varied pharmacological activities. Identification of such plants of potential use in medicine is of significance and as a prelude to this; it becomes necessary to examine the various pharmacognostical characters of the plant before further investigation. In pharmacognostical studies, the macroscopy, microscopy, histochemical studies of the bark of Givotia rottleriformis, physico-chemical constants, fluorescence analysis and inorganic mineral analysis of G. rottleriformis bark and C. tora leaves were carried out. Pharmacognostical standards obtained during the observation are valuable tools for the identification of the plant material. Morphological study had provided a characteristic identity of bark which was smooth brown colour, bitter taste yielding blood red sap from the bruised bark and leaf which was green in colour, bitter taste with characteristic odour. The various distinguishing features of G. rottleriformis bark observed through anatomical studies were 1. The transverse section of the bark exhibits outer periderm and inner secondary phloem. Secondary phloem is the major part of the bark. It is differentiated into outer wider collapsed phloem and inner narrow non collapsed phloem or intact phloem. 2. The tangential longitudinal sections of the bark exhibits uniseriate, narrow and hetero cellular phloem rays with terminal upright cells and middle procumbent cells and long and thick walled straight sieve tube and nodulated sieve plate. Phloem parenchyma cells are vertically rectangular and they occur in vertical strands. Prismatic Calcium oxalate crystals are seen in sclerenchyma elements of the collapsed phloem and druses located in the phloem rays. 3. The micrsocopical examination of the powder showed brachy sclereids, short, narrow thick walled libri form fibres in thick bundles or in small broken pieces and rectangular thin walled parenchyma cells. Histochemistry is mainly used to localize the chemical compounds within the cells and tissues using some chemical reagents have been done and it showed the presence of alkaloids, and flavonoids. Various physico-chemical parameters such as ash values, extractive values, loss on drying and crude fibre content were found to substantiate its standard values. Any significant deviation in the percentage of any parameters reported in this work may indicate adulteration or substitution in the drug. Presence of calcium and iron added up its nutritional value, fluorescence analysis is also a part of diagnostic tool for the presence of chromophore in the particular species. The pharmacognostical details evolved from the present study would help to fix up the standards for Givotia rottleriformis Griff. Ex Wight in relation to its identification, authentication and differentiation from other related species and adulterants. This is the first report on the pharmacognostical standardisation on the bark of Givotia rottleriformis Griff. Ex Wight. From the above mentioned studies it can be concluded that the pharmacognostical standards generated will be useful for the proper identification of the plant G. rottleriformis bark. In Phytochemical evaluation, the ethanolic extract of Givotia rottleriformis and Cassia tora were prepared and studied for qualitative chemical analysis, TLC and HPTLC finger print analysis. The total phenol and flavonoid content present in the plants were estimated by Folin-Ciocalteu and AlCl3 method respectively. The total phenolic content and flavonoid content in Givotia rottleriformis bark was found to be 13.80% w/w and 5.7% w/w respectively. The total phenolic content and flavonoid content in Cassia tora leaves was found to be 18.60% w/w and 9.5% w/w respectively. The qualitative preliminary phytochemical analysis was performed to detect the nature of the phyto-constituent present in the plants. The ethanolic extract of Givotia rottleriformis showed the presence of alkaloids, glycosides, proteins, phenols, tannins, flavonoids, steroids and terpenoids. The ethanolic extract of Cassia tora showed the presence of anthroquinone glycosides, proteins, phenols, tannins, flavonoids, saponins, steroids and terpenoids. Qualitative chromatographic analysis (TLC) was performed for the identification of different components in the extracts qualitatively. The HPTLC finger print of ethanolic extract of the plants was also studied. HPTLC was scanned at 280 nm with the best solvent to detect the maximum number of components and peak abundance qualitatively. HPTLC fingerprint is one of the versatile tools for qualitative and quantitative analysis of active constituents. It is also a diagnostic method to find out the adulterants and to check the purity. The defatted ethanol extract of Givotia rottleriformis bark and Cassia tora leaves was subjected to column chromatography separately and eluted with various solvents in the order of increasing polarity. The isolated compounds were characterized by spectral analysis. From Givotia rottleriformis bark, 3 flavonoid glycosides, viz., Rutin, Luteolin-7-O-β-D-Glucuronide, Kaempferol 3-O-[2-O-(6-O-feruloyl)-β-D-glucopyranosyl]-β-D-galactopyranoside, and from Cassia tora leaves, 3 flavonoid glycosides viz., Luteolin-7-O-β-glucopyranoside, Quercetin-3-O- β-d-glucuronide, Formononetin-7-O-Glucoside were isolated. The flavonoids present in both the plants were detected and quantified using HPLC. In Givotia rottleriformis bark, 4 flavonoids were quantified, viz., rutin (0.215 mg/gm), luteolin (8.64 mg/gm), quercetin (1.36 mg/gm), kaempferol (6.36 mg/gm) using peak area by comparison to a calibration curve derived from the standard. Similarly in Cassia tora leaves, 4 flavonoids were quantified, viz., quercetin (10.33 mg/gm), kaempferol (7.43 mg/gm), formononetin (0.46 mg/gm) and luteolin (8.64 mg/gm) at 254 nm using peak area by comparison to a calibration curve derived from the standard. Further the presence of flavonoids was detected and quantified using HPTLC. In Givotia rottleriformis bark, the flavonoids Rutin, Kaempferol and Luteolin were detected and quantified using HPTLC silica gel 60 F254 pre-coated plates with the mobile phase made of Benzene: Methanol: Ammonia (90:10:1). The detection of Rutin, Luteolin and Kaempferol was observed to be linear over a concentration range of 100-500 ng/mL and the concentration of Rutin, Luteolin and Kaempferol was found to be Rutin 285 ng/mg, Kaempferol 360 ng/mg and Luteolin 380 ng/mg. Similarly in Cassia tora leaves, the flavonoids Luteolin, Quercetin and Formononetin were detected and quantified using HPTLC silica gel 60 F254 pre-coated plates with the mobile phase made of Benzene: Methanol: Ammonia (90:10:1). The detection of Luteolin, Quercetin and Formononetin was observed to be linear over a concentration range of 100-500 ng/mL and the concentration of Luteolin, Quercetin and Formononetin was found to be Luteolin 220 ng/mg, Quercetin 160 ng/mg, and Formononetin 210 ng/mg. In vitro antioxidant studies for ethanol extract of Givotia rottleriformis bark, Cassia tora leaves and isolated flavonoids were done by Hydroxyl, DPPH and Nitric oxide radical scavenging method. The tested extracts and isolated flavonoids showed good dose-dependent free radical scavenging property in all the models. The cytotoxic effect of the ethanol extract of Givotia rottleriformis bark, Cassia tora leaves and isolated flavonoids I-VI were evaluated using in- vitro model - HaCaT human keratinocyte cell inhibition, a rapidly multiplying human keratinocyte cell line, as a model of epidermal hyperproliferation in psoriasis. Among the tested flavonoids, II, III, V and VI showed appreciable antiproliferant activity in HaCaT cell line. The compound II and V exhibited similar antiproliferant activity in HaCaT cell line, were of same flavonoid (luteolin), the yield of compound II was high when compared with compound IV and hence compound II was subjected to further studies along with compound III and VI. The results were validated using asiaticoside as positive control. The ethanol extract of Givotia rottleriformis bark, Cassia tora leaves was found to be safe up to 2000 mg/kg body weight and isolated flavonoids II, III and VI up to 500 mg/kg body weight by acute toxicity model study as per the OECD guidelines 423. In sub acute toxicity study, ethanol extract of G. rottleriformis (200 mg/Kg and 400 mg/Kg) treated via oral route over a period of 28 days have no toxic effect on rats. Toxicity profile of Cassia tora leaves reported by Ambali [et al.], revealed that the twenty eight days of oral administration of methanol extract of Cassia tora did not result in death of the animals and clinical signs of toxicity include diarrhoea. The ethanol extract of Givotia rottleriformis bark, Cassia tora leaves and isolated flavonoids II, III and VI were selected for the screening of in-vivo anti-psoriatic activity using Perry’s mouse tail model. The ethanol extract of both the plants at higher dose (400 mg/Kg) and compound II, III, VI (50 mg/Kg) and combination of compound (30 mg/Kg) also showed significant change in epidermal thickness compared to control while ethanol extract at lower doses did not produce any significant change in epidermal thickness. Bioactive flavonoid combination II, III, VI (1:1:1) were promoted to formulation of combinational tablet dosage form and evaluation for their product performance using various evaluation parameters such as weight variation, hardness, friability, thickness, disintegration & dissolution study. From the studies - appearance, weight variation test, thickness, hardness, friability, disintegration, dissolution and assay were recorded and it was found that formulated tablet results met Pharmacopoeial specifications limit, and the tablets were suitable for oral administration. The standardized formulation (30 mg/Kg) along with ethanol extract of G. rottleriformis and C. tora (400 mg/Kg) was evaluated for anti-psoriatic activity using ultraviolet-B-induced psoriasis in rat model and cytokine inhibition assay against IL-1, IL-6, IL-8, IL-17 and TNF -α and. The developed formulation has shown antipsoriatic activity by good reduction in the thickness of epidermis, significant retention of the stratum granulosum and the absence of movement of neutrophils in UV-B induced psoriasis. The developed formulation containing bioactive flavonoids showed remarkable inhibitory activity against the cytokines TNF-α amd IL-17 at higher concentration (30 μg/ml). In conclusion, we could demonstrate that the formulation containing flavonoids Kaempferol, Luteolin and Formononetin exert strong anti-TNFα and anti-IL-17 effects in ex vivo LPS-stimulated blood. Therefore, additional clinical investigation of these compounds is indicated to evaluate the efficacy and safety of their application as dietary supplements with health benefits to psoriatic patients. With the support of in-vitro assay and in-vivo pharmacological activity the ethanolic extract of both the plants at the dose level of 400 mg/kg and isolated flavonoids II, III, VI (50mg/Kg) and its combination (30 mg/Kg) showed significant anti-psoriatic activity. Further studies were focused on structural activity relationship of phytoconstituents isolated from the ethanolic extract. This scientific study revealed the efficacy of the drug and it would definitely have wide scope in future. So, the study can be concluded that the developed formulation containing bioflavonoids can be effective and safety as dietary supplements with health benefits to psoriatic patients, justifying the use of these plants in traditional medicine.
| Item Type: | Thesis (Doctoral) |
|---|---|
| Uncontrolled Keywords: | Development, evaluation, some bioflavonoids, indigenous plants. |
| Subjects: | PHARMACY > Pharmaceutics |
| Depositing User: | Subramani R |
| Date Deposited: | 18 Nov 2019 17:16 |
| Last Modified: | 22 Sep 2022 09:38 |
| URI: | http://repository-tnmgrmu.ac.in/id/eprint/11745 |
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