JOURNAL OF CRITICAL REVIEWS
ISSN- 2394-5125 VOL 7, ISSUE 07, 2020
DETERMINATION OFPHYTOCHEMICAL CONSTITUENTS BYQUANTITATIVE ANALYSIS FROM ANISOMELESMALABARICAEXTRACT.
Dr.S.Selvakumar 1, S. Vimalanban2,Merina Paul Das 3
Professor 1 , UG Scholar 2, Assistant Professor 3 Department of Industrial Biotechnology 1,2,3 Email id: [email protected] BIST, BIHER, Bharath Institute of Higher Education & Research, Selaiyur, Chennai -73.1,2,3
Abstract Background: Plantderived secondary metabolites make up a significant segment of natural productbased pharmaceuticals especially from the herbal medicinal plants. Hence, it is of interest to investigate the quantification of Phytochemicals such as total content of alkaloids, flavanoids, tannins and phenolic compounds are of our interest.Methods:Quantitative analysis of phytocontituents of chloroform extract of A.malabarica were used to find out the quantification of Phytochemicals. Results:The results of the present study envisages that the presence of alkaloids,flavanoids,tannins and poly phenol compounds. Conclusion:The chloroform extract of Anisomelesmalabarica contains pharmacologically active phytochemicalconstituents. Key words: Phytochemical quantification, Anisomelesmalabarica, Tannins, Phenols, Flavanoids.
1. Introduction Traditional system of Indian medicine is the most ancient healthcare system describes thousands of medicinal plants with their medicinal properties. In recent times, developed countries are turning to the use of traditional medicinal systems because the phytochemicals are potent in different therapeutic applications as they show defensive mechanism of action against a number of chronic diseases including cancer, cardiovascular disease, diabetes, neurodegenerative disease. Plant based components are also involved in anti viral as well as antimicrobial activity and also show efficacy in radioprotection. But still there are some difficulties in proper therapeutic administration of phytochemicals due to their low water solubility, low absorptivity and bioavailability. So a strategy of engineered phytochemicals has been developed to enhance solubility, cellular permeability, proteolytic stability and self life of plant based components. Still further research is required to ensure high yield as well as viability and bio availability of the plant derived components in different therapeutic applications [1]. Anisomelesmalabarica found in Western Ghats of southern India. This plant has traditional medicinal value with phytochemical components which have antioxidant potential [2].Anisomelesmalabarica (L.) R. Br. is perhaps the most useful traditional medicinal plant. It is a highly aromatic plant belonging to the family Lamiaceae (Labiatae). Anisomelesmalabarica is a species of herbaceous plant native to tropical and subtropical regions. Mosquitoes act as a vector for most of the life threatening diseases like malaria, yellow fever, dengue fever, filariasis, encephalitis [3]
2. Materials and methods 2.1. Collection of samples The medicinal plants used for the experiment were whole plant of Anisomelesmalabarica which were collected from the local medicinal farms and identified, authenticated by botanist. 2.2. Preparation of extracts 1000 grams of plant material was packed in three separate round bottom flask for sample extraction using solvents namely Aqueous, Chloroform and Methanol. The extraction was conducted by 250 ml of the each solvent mixture for a period of 24 hours. At the end of the extraction the respective solvents were concentrated under reduced pressure and keep it in water bath (at 50°c). Now the extracted experimental solutions were stored in refrigerator. 2.3. Chemicals and Reagents All chemicals were used for this project were purchased from M/s. Sigma Chemicals, USA.
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2.4. Quantification of Phytochemicals Determination of total phenolic content, Determination of total tannin Content, Determination of total Alkaloid content and Determination of total flavanoid content were analysed [4-16]. 2.5. Determination of Total content of Alkaloids The plant extract (1mg) was dissolved in dimethylsulphoxide (DMSO)and added 1ml of 2N hydrochloric acid and filtered. This solution was transferred to a separating funnel, 5 ml of bromocresol green solution and 5 ml of phosphate buffer were added. The mixture was shaken with 1, 2, 3 and 4 ml chloroform by vigorous shaking and collected in a 10-ml volumetric flask and diluted to the volume with chloroform [5-6]. A set of reference standard solutions of atropine (20, 40, 60, 80 and 100 μg/ml) were prepared in the same manner as described earlier [7],[9]. The absorbance for test and standard solutions were determined against the reagent blank at 470 nm with an UV/Visible spectrophotometer. The total alkaloid content was expressed as mg of AE/g of extract [8]. 2.6. Determination of Total content of flavanoids Total content of flavanoid was measured by the aluminium chloride colorimetric assay. The reaction mixture consists of 1 ml of extract and 4 ml of distilled water was taken in a 10 ml volumetric flask [10-12]. To the flask, 0.30 ml of 5 % sodium nitrite was treated and after 5 minutes, 0.3 ml of 10 % aluminium chloride was mixed [39-40]. After 5 minutes, 2 ml of 1M Sodium hydroxide was treated and diluted to 10 ml with distilled water. A set of reference standard solutions of quercetin (20, 40, 60, 80 and 100 μg/ml) were prepared in the same manner as described earlier [13,14]. The absorbance for test and standard solutions were determined against the reagent blank at 510 nm with an UV/Visible spectrophotometer. The total flavanoid content was expressed as mg of QE/g of extract [15-16] . 2.7. Determination of Total content of Tannins The tannins were determined by Folin-Ciocalteu method. About 0.1 ml of the sample extract was added to a volumetric flask (10 ml) containing 7.5 ml of distilled water and 0.5 ml of Folin-Ciocalteu phenol reagent, 1 ml of 35 % Na2CO3 solution and dilute to 10 ml with distilled water [19-20]. The mixture was shaken well and kept at room temperature for 30 min. A set of reference standard solutions of gallic acid (20, 40, 60, 80 and 100 μg/ml) were prepared in the same manner as described earlier. Absorbance for test and standard solutions were measured against the blank at 725 nm with an UV/Visible spectrophotometer. The tannin content was expressed in terms of mg of GAE /g of extract [21-23]. 2.8. Determination of total content of phenolic compounds The concentration of phenolics in plant extracts was determined using spectrophotometric method. Folin- Ciocalteu assay method was used for the determination of the total phenol content [24-26]. The reaction mixture consists of 1 ml of extract and 9 ml of distilled water was taken in a volumetric flask (25 ml). One millilitre of Folin-Ciocalteu phenol reagent was treated to the mixture and shaken well. After 5 minutes, 10 ml of 7 % Sodium carbonate (Na2CO3) solution was treated to the mixture [27], [29]. The volume was made up to 25 ml. A set of standard solutions of gallic acid (20, 40, 40, 60, 80 and 100 μg/ml) were prepared in the same manner as described earlier [28], [30]. Incubated for 90 min at room temperature and the absorbance for test and standard solutions were determined against the reagent blank at 550 nm with an Ultraviolet (UV) /Visible spectrophotometer. Total phenol content was expressed as mg of GAE/gm of extract [31-33].
3. Results and Discussion Figure 1: Standard calibration curve of total content of Alkaloids. Alkaloids Standard Calibration Curve
140 . 120 100 80 y = 97.63x - 99.90 60 R² = 0.842 40 20 Concentration in in µg/ml Concentration 0 0 0.5 1 1.5 2 2.5 Absorbance at 470 nm.
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Figure 2: The total content of alkaloids in A.malabarica. Total Content of Alkaloids in Anisomeles malabarica 140
. 120
100
80
60 y = 63.45x - 29.75 R² = 0.681 40
Concentration in in µg/ml Concentration 20
0 0 0.5 1 1.5 2 2.5 Absorbance at 470 nm.
Figure 3: Standard calibration curve of total content of Tannins. Tannins standard calibration curve
140 120 100 80 60 40 y = 47.96x - 0.428 R² = 0.037
Concentration µg/ml Concentration 20 0 0 0.5 1 1.5 2
Absorbance at 725nm Figure 4: The total content of Tannins in A.malabarica. Total content of Tannins in Anisomeles malabarica
140 120 100 80 60 y = -92.39x + 236.5 R² = 0.711
40 concentration µg/ml concentration 20 0 0 0.5 1 1.5 2 2.5 3 Absorbance at 725nm
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Figure 5: The standard calibration curve of total content of Flavanoids. Flavanoids standard calibration curve 140 120 100 80 60 40 y = -1.324x + 72.64
Concentration µg/ml Concentration 20 R² = 0.000 0 0 0.5 1 1.5 2 2.5 3 Absorbance at 510nm
Figure 6: The total content of Flavanoids in A.malabarica. Total content of Flavanoids in Anisomeles malabarica 140 120 100 80 y = -97.01x + 311.7 60 R² = 0.976 40
Concentraion µg/ml Concentraion 20 0 0 0.5 1 1.5 2 2.5 3 3.5 Absorbance at 510nm
Figure 7: The standard calibration curve of total content of Phenolic compounds. Phenolic compounds standard calibration curve 140 120 100 80 60 y = -356.2x + 1442. R² = 0.700 40 20
0 Concentration µg/ml Concentration 3.75 3.8 3.85 3.9 3.95 4 4.05 Absorbance at 550nm
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Figure 8: The total content of Phenolic compounds in A.malabarica. Total content of Phenolic compounds in Anisomeles malabarica 140 120 100 80 60 40
y = -38.37x + 220.1 Concentration µg/ml Concentration 20 R² = 0.004 0 3.82 3.84 3.86 3.88 3.9 3.92 3.94 3.96 3.98 4 4.02 Absorbance at 550nm
The present study was performed to evaluate the total content of phenols, tannins, alkaloids and flavanoids in chloroform extract ofAnisomelesmalabarica(figure 1-8). Figure 1 and 2 shows that the total alkaloids content in standard and chloroform of extract ofAnisomelesmalabarica[37-38].Total alkaloids content in the extract expressed in terms of atropine equivalent (mg of AE/g of extract) (y =63.45x-29.75, R2=0.681), (y=97.63x- 99.90, R2=0.842). Figure 3 and 4 shows that the total flavanoids content in standard and chloroform of extract of Anisomelesmalabarica. Total flavanoid content in the extract expressed in terms of quercetin equivalent (mg of Q E/g of extract) (y= 0.001x+ 0.806, R2= 0.890), (y= 0.001x+ 0.847, R2= 0.929) [34-36]. Figure 5 and 6 shows that the total tannins content in standard and chloroform of extract of AnisomelesmalabaricaTotal Tannins content in the extract expressed in terms of gallic acid equivalent (mg of GAE/g of extract. (y =0.000x +1.28, R2=0.901), (y=0.001x+0.93, R2=0.957). Figure 7 and 8 shows that the total phenolic compound content in standard and chloroform of extract ofAnisomelesmalabarica Total phenolic contents in the extract expressed in terms of gallic acid equivalent (mg of GAE/g of extract) (y=0.000x+1.888, R2=0.960), (y= 0.000x+1.779, R2 =0.904). The Pharmacological,Toxicological and Biochemical mechanism of action of chloroformic extract ofAnisomelesmalabarica was determined by the nature of these phytocompounds which are responsible for the desired therapeutic properties and definite physiological effects [17-18].
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