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TOTAL PHENOLIC AND FLAVONOID CONTENT OF DIFFERENT EXTRACTS OF INDIGOFERA TINCTORIA LINN


About Authors:
Nagasaraswathi.M*, Rafi khan.P, Aleemuddin.MA, Gopi Chand.K, Nagaprashanthi.Ch
Faculty of Pharmacy, PRIST University,
Thanjavur-614901, Tamilnadu (INDIA)
abdulaleemuddin@gmail.com

ABSTRACT
The importance for medicinal herbs is increasing day by day due to its high availability and lower side effects. The present study investigated the total phenolic and flavonoid content of various extractions of Indigofera tinctoria linn. The total phenolic content was determined by folin-ciocatechu reagent using Gallic acid as standard and total flavnoid content by aluminium chloride assay using Quercetin as a standard. The present study showed the ratio of phenolic and flavonoid content in different extracts of Indigofera tinctoria linn. Aqueous extract showed the more flavonoid and phenolic content in comparision with other extracts.


Reference Id: PHARMATUTOR-ART-1591

INTRODUCTION :
Medicinal herbs are known to produce certain bioactive substances and secondary metabolites such as flavonoids, phenolic compounds to maintain healthy system of humans and animals. ( Amandeep Kaur et al., 2011, Jeevan K. Prasain et al.,2004 ). Phenolic compounds have one or more hydroxyl groups attached directly to an aromatic ring. Polyphenols are the compounds that have more than one phenolic hydroxyl group attached to one or more benzene rings(Wilfred vermerries, Ralph Nicholson,2006). Flavonoids are a group of polyphenolic compounds, which are widely distributed throughout the plant Kingdom. Flavonoids are largely planar molecules and their structural variation comes in part from the pattern of substitution: hydroxylation, methoxylation, prenylation, or glycosylation. (Sujatha V  et al.,2005). Flavonoids are responsible for many activities like Hepatoprotective activity, CNS activity, cardio tonic activity, lipid lowering activity, antioxidant activity, anti neoplastic activity and anti microbial activity.( Raj Narayana K et al., 2001). Flavonoids are having the greatest antioxidant activity and the order of antioxidant power of flavonoids are catechins > dihydrochalcones > proanthocyanidins > flavonones > flavonols > flavones > anthocyanidins > flavonols. (david Hoffman,2003).

Reactive oxygen species (ROS) such as singlet oxygen (O-), superoxide anion (O2-) and hydroxyl (.OH) radical and hydrogen peroxide (H2O2) are often released by  biological reactions or from exogenous factor. These ROS play major role in the degenerative diseases such as cancer, hypertension, atherogenesis, Alzheimers disease, and Parkinsons disease. flavonoids and other polyphenolic constituents have been reported to be act as potent antioxidant. (  Nagulendran KR et al., 2007,Maria Kratchanova et al.,2010). Due to chemical diversity and shape flavonoids interact with targets in different locations to influence the changes in biological activity(Charles S. Buer et al.,2010).

The plant Indigofera tinctoria Linn (fabaceae) is popularly known as neeli in tamil and common name is indigo (Amrith Singh, 2006). Indigofera tinctoria was traditionally using to treat various kinds of diseases such like constipation, liver diseases, heart palpitation, and gout (Amrith Singh, 2006) stimulant, deobstruent, purgative, antiseptic, astringent, antibacterial, antioxidant, cytotoxic effect, hepato protective  activity (Renuka devi K.P et al., 2011), Antidyslipidemic activity (Anju Puri et al., 2007), Anti-hyperglycemic activity (Amarnath V Bangar et al., 2011), Anti-proliferative activity (Thiruvamiyoor Ravichandran Kameswaran et al., 2008), Anti-inflammatory activity (Pramod  K Tyagi et al., 2010), Antihelmenthic activity (Gunasekaran Balamurugan et al., 2009). The juice of I.tinctoria leaves and indigo powder mixed with honey is used for enlargement of liver and spleen. Juice is also used in asthma, whooping cough, lung diseases and kidney disorders as in dropsy (Nadkarni et.,al 2002).The current study is to investigate the phenolic and flavonoidal content of different extracts of Indigofera tinctoria Linn.


MATERIALS AND METHODS
Plant material

A fresh aerial part of Indigofera tinctoria Linnwas collected from Thanjavur (India) and it was identified and authenticated by Dr.G.V.S.Murthy, Scientist ‘F’ & Head of Botanical Survey Of India, Coimbatore. Specimen number BSI/SRC/5/23/2011-12/Tech-1051

Instruments
UV/Vis double beam spectrophotometer (Techcomp UV-2310 spectrophotometer) and 1 cm quartz cells were used for all absorbance measurements.

Reagents and solutions
The extracting solvents used were all of analytical grade, including ethanol, ethyl acetate, acetone, chloroform, petroleum ether and other reagents and standards: Gallic acid   , sodium nitrite, aluminium chloride, sodium hydroxide, sodium carbonate, folin-ciocalteu reagent, Quercetin and Gallic acid, were purchased from Merck mumbai.

Preparation of plant extracts :
Aqueous extraction( Decoction method)

150 g of coarse powder of Indigofera tinctoria Linnleaves and mixture of stem and root powder were  boiled with 600ml of distilled water (Sukhdev Swami Handa et al.,2008). Then it was cooled to room temperature and filterate was filtered.

Solvent Extraction:
100gm of powder was subjected to successive soxhlet extraction by various solvent such as petroleum ether, chloroform, acetone, ethylacetate and ethanol. The percentage yield was showed in table I .

Extractive values calculated by following formulae:


Preparation of Standard Solutions:
Gallic acid and Quercetin 10mg were accurately weighed into a 10 mL volumetric flask, dissolved in 10mL methanol and the solution was made up to 10 mL with the same solvent [1mg/mL].

Determination of total phenolics
To analyze the total phenolic content (TPC), the method of   Folin Ciocalteu assay was carried out (Marinova.D et al.,2005). In this method Gallic acid was used as standard and Total phenolic content was expressed as mg gallic acid equivalent (GAE)/100 g fresh weight.. For this purpose, the calibration curve of Gallic acid was drawn (Figure I). 1ml of standard or extract solution (20, 40, 60, 80,100 mg/l) was taken into 25ml volumetric flask, containing 9ml of HPLC grade distill water.1ml of Folin-Ciocalteu’s reagent was added to the mixture and shaken. After 5min, 10ml of 7%Na2Co3 solution was added to the mixture. The solution was diluted upto 25ml with HPLC grade distill water. Incubate the solution at room temperature for 90 min . A reagent blank using HPLC Grade distill water was prepared.  The absorbance was noted at 750nm using UV-Visible spectrophotometer.

Determination of total flavonoids
Colorimetric aluminum chloride method was used for flavonoid determination (Marinova.D et al.,2005). in this method Quercetin was used as standard and flavonoid contents were measured as quercetin equivalent. For this purpose, the calibration curve of quercetin was drawn(Figure II). 1ml of standard or extract solution (20, 40, 60, 80,100 mg/l) was taken into 10ml volumetric flask, containing 4ml of HPLC grade distill water. 0.3ml of 5%NaNo2 added to the flask. After 5min, 0.3ml 10%AlCl3 was added to the mixture. At the 6th min add 2ml of 1M NaOH was added and volume made up to 10ml with HPLC grade distills water.  The absorbance was noted at 750nm using UV-Visible spectrophotometer. A reagent blank using HPLC Grade distill water was prepared.  The absorbance was noted at 415nm using UV-Visible spectrophotometer.

Results and Discussion :

Phytochemical analysis
The results of preliminary phytochemical  studies are as follows for different extracts.

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