SCREENING OF Andrographis Paniculata EXTRACT FOR ANTIOXIDANT AND GENOTOXIC ACTIVITIES

Andrographis paniculata is an important medicinal plant which has been used to treat various ailments. The present study was undertaken to analyze the phytochemical compounds and evaluated the antioxidant and genotoxic potential of Andrographis paniculata leaves and whole plant extracts. Phytochemical compounds analyzed by qualitative and quantitative analysis of methanol extracts of the leaves and whole plant extracts which showed the presence of Alkaloids, Carbohydrate, Resins, Saponins, Flavonoid, Steroids, Glycosides and Tannin. Quantitative analysis were also conducted to determine the amount of Alkaloids, Flavonoids, Saponins and Tannin by HPTLC Finger printing methods in A. paniculata ( A.P.) leaves and whole plant .The Antioxidant activity of A. paniculata Methanolic extract was evaluated by Fenton methods which showed the dose dependent Inhibition of TBARS formation. The Genotoxicity was evaluated by Micronucleus Assay. The dose dependent prevention of bone marrow micronucleus formation by Andrographis paniculata leaves and whole plant extracts was observed. Therefore, Andrographis paniculata leaves and whole plant extract caused antioxidant and genotoxic potential.


Introduction
Plants have been an important source of medicine to living organism for thousands of years.Plants are contemplated not only as dietary supplement to living organism but also traditionally used for treatment of many health problems 1 .Andrographis is an important genus of the Acanthaceae family that is medicinally important taxa containing 26 species.Andrographis paniculata is also called as Kalmegh or "King of Bitters."This plant is an extremely bitter in taste that is used to treat liver disorders, bowel complaints of children, colic pain, common cold and upper respiratory tract infection 2,3,4 .It is also used for the treatment of snake bite, bug bite, diabetes, dysentery, fever, and malaria 5 .Whole plant leaves and roots are also used as a folklore remedy for different diseases in Asia and Europe.AP has been reported to have a broad range of pharmacological effects including anticancer, antihepatitis, antihyperglycemic, hepatoprotective, antiinflammatory, antimicrobial, antidiarrheal, cardiovascular, cytotoxic, anti-HIV, immunostimulatory, and sexual dysfunctions 6 .Andrographis paniculata is used in Asia from centuries in traditional medicine to treat gastrointestinal (GI) tract and respiratory infectious diseases.It has been reported that Andrographis has a broad range of pharmacological effects 7 .It has been suggested to safe in controlled clinical trials report for treating upper respiratory tract infections.It also showed significant cardio protection by inducing antioxidant activity in myocardium 8 .Cytotoxic activity against cancer cell lines has been reported by Compounds of Andrographis paniculata 9 .Antimicrobial activity against eleven bacterial strains by ethanol extract of Andrographis paniculata have been reported 10 .Andrographolide have been reported to hypoglasmic activity in rats 11 .Antiulcer activity was reported in duodenal ulcer model in rats 12 .Hepatoprotective effect was reported on acetaminophen induced hepatotoxicity in albino rats 13 .An andrographolide was also reported to induce apoptosis in TD-47 human breast cancer cell line in a time and concentration-dependent manner by increase expression of p53, bax, immuno histochemical parameters such as caspase-3 and decrease expression of bcl-2 8 .Andrographis paniculata 14.Dry leaf powder was reported to cause spermatogenesis, cessation of degenerative changes in the seminiferous tubules.The extract also produced significant muscarinic activity, which accounts for its antivenom effects 15 .Many of the conditions commonly treated with Andrographis paniculata in traditional medical systems are important, which requires further investigations for benefit in cancer treatment.

Chemicals
All the Materials and Reagents used for the study were purchased from CDH, Renchem and Hi-Media Ltd., India.

Animals
The experimental study was conducted on random bred, 6-7 weeks old and 24-28 gm body weight bearing, male Swiss albino mice.Animals were maintained under controlled conditions of temperature (24 ± 3 0 C) and light (Light: dark, 10 hrs: 14 hrs.).The animals were provided with standard mice feed and tap water ad libitum.

Preparation of Andrographis paniculata Leaves and Whole Plant Extract
Plant material of Andrographis paniculata was collected and the specimen was authenticated by the botanist of Deendayal Research Institute, Chitrakoot, Satna, Madhya Pradesh(India).The noninfected leaves and whole Plant was washed, air dried, powdered and extracted separately using 50 % methanol in a separating funnel.Extract thus obtained were vacuum evaporated into powder.These extract was again dissolved in DDW immediately prior topical application.

Preliminary Phyto-Chemical Screening
Alkaloids 1 gram of dried powder was added with 10 ml of 1M-HCl and ultrasonicated for 15 min at 30ºC.The mixture was filtered and 3 ml of filtrate was treated with few drops of either Dragendorff's Resins 10 ml of distilled water were added to 1 g of dried powder, and ultrasonicated for 15 min at 30ºC.The mixture was filtered.Occurrence of turbidity showed the presence of rasins.

Saponins
About 0.5mg of the extract was shaken with five ml of distilled water.Formation of frothing (appearance of creamy miss of small bubbles) shows that the presence of saponins.

Starch
Small quantity of extract was taken and add 2-3 drop of iodine solution on it.Observe the colour of solution.Blue black colour indicates the presence of starch.

Flavonoids
Ferric chloride test: Test solution when treated with few drops of Ferric chloride solution would result in the formation of blackish red color indicating the presence of flavonoids.
Alkaline reagent Test: Test solution when treated with sodium hydroxide solution, shows increase in the intensity of yellow color which would become colorless on addition of few drops of dilute Hydrochloric acid, indicates the presence of flavonoids.

Steroids
2ml of chloroform and concentrated H2SO4 were added with the 5ml aqueous plant crude extract.
In the lower chloroformlayer red color appeared that indicated the presence of steroids.

Glycoside
Borntrager s Test: About five mg of the extract was boiled with 10% HCl for few minutes in a water bath.It was filtered and allowed to cool.Equal volume of CHCl3 was added to the filtrate.Few drops of 10% NH3 were added to the mixture and heated.Formation of pink colour indicates that the presence Glycosides.

Antioxidant Activity
Antioxidant activity of Andrographis paniculata Leaves and While Plant extract (10-100µg/ml) were determined by De-oxyribose Method (Fenton Reaction) of Halliwell et al., (1987).The hydroxyl radical attacked to deoxyribose and initiated a series of reaction that eventually resulted in the formation of Thiobarbeturic Acid Reaction Substances (TBARS).

Micronucleus Asaay
It was done by the method reported by Schmid (1975)  17 , modified by Aron et al 18 and standardised by us 19  (Agrawal etal ,1998).In Micronucleus Assay, the extract of Andrographis paniculata Leaves and Whole plant at the volume of 0.2 ml at different dose level such as 1000.1500, 2000 mg/kg body weight was injected 24 hrs before the treatment of Cyclophoshamide, to three animals.Singlre ip.Injection of 50 mg/kg Cyclophasphamide in 0.9% saline was injected 24 hours before the Andrographis extract treatment.The animals were sacrificed by cervical dislocation and bone marrow cells were harvested.The slides were prepared essentially as described by Schmid (1975)   .
After staining with May-Gruenwald and Giemsa Stain, a total 1000 cells were scored at the magnification of X 1000 (100 x 10x) for each group.The data are expressed as the average number of micro nucleated cells polychromatied erythrocytes cells (PCE) cells / animals.

Results and Discussions
The therapeutic properties of medicinal plants are perhaps due to the presence of various secondary metabolites that are phenols, flavonoids, alkaloids, glycosides, steroids, saponins etc.The Leaves and Whole plant of Andrographis paniculata extract have revealed the presence of Alkaloids, Carbohydrate, Resins, Saponins, Flavonoid, Steroids, Glycosides and Tannin.Protein

Antioxidant Activity
The free radical scavenging capacity of the methanolic extract of Andrographis paniculata Leaves and Whole palnt extract were determined by using TBARS method.A. paniculata showed High Performance Thin Layer Chromatography) fingerprinting analysis bands were observed on the HPTLC plates and the Rf values were calculated, where Rf value is Retention factor value.The observed fingerprint, rather than the presence of the compounds, represents a unique pattern and is given by a set of Rf values and the specific colours observed for these compounds.(Fig 1 & 2) extract; Track B: Ethanol extract; Track C: Benzene extract and Track D: Chloroform extract

[Agrawal et. al., Vol.7 (Iss.6): June 2019] ISSN-2350-0530(O), ISSN-2394-3629(P) DOI: 10.5281/zenodo.3262211
Take 1ml of sample in test tube and take 1ml of distilled water in another tube as control.Add 2ml of anthrone reagent to all the tubes.Mix thoroughly all the content of the tube.Observe for color change in bluish green.That indicates all carbohydrate give test positive result.To 0.5 mg of extract equal volume of 40% NaoH solution and two drops of one percent copper sulphate solution was added.The appearance of violet colour indicates that the presence of protein.

Table 1 :
Preliminary phyto-chemical screening of Andrographis paniculata Leaves & Whole The quantitative analysis that shows the percentage of Alkaloids, Saponins, Flavonoids, and Tannin present in extract.The result of Quantitative Phyto-chemical Analysis of Andrographis paniculata Whole plant & Leaves extract shown in Table No.-2.