Metabolic fingerprinting of root , stem and leaf extracts of Phyllanthus amarus

India is one of the richest biodiversity centers in the world with different types of plant species. This huge diversity is due to different agro-climatic conditions throughout. Every higher plant species has got its own metabolic fingerprint with secondary metabolites of pharmaceutical value, which is used in traditional and modern medicines. Herbal products are safe to human and environment in contrast to the synthetic drugs (Panda et al., 2012). In India, plant drug contribution is as much as 80% compared to developed countries (Babu et al., 2011). Traditional systems of medicine are widely practiced due to high population, high treatment cost, side effects of synthetic drugs, insufficient drug supply, and development of resistance to synthetic drugs. In the recent past, the herbal system of medicines has reached a very vital phase. Green plants are used as raw materials as they possess a variety of compounds, which can be extracted and can be used for a variety of scientific investigations. Secondary metabolites present in plant are commercially important in pharmaceutical industries (Joy et al., 2001).


INTRODUCTION
India is one of the richest biodiversity centers in the world with different types of plant species.This huge diversity is due to different agro-climatic conditions throughout.Every higher plant species has got its own metabolic fingerprint with secondary metabolites of pharmaceutical value, which is used in traditional and modern medicines.Herbal products are safe to human and environment in contrast to the synthetic drugs (Panda et al., 2012).In India, plant drug contribution is as much as 80% compared to developed countries (Babu et al., 2011).Traditional systems of medicine are widely practiced due to high population, high treatment cost, side effects of synthetic drugs, insufficient drug supply, and development of resistance to synthetic drugs.In the recent past, the herbal system of medicines has reached a very vital phase.Green plants are used as raw materials as they possess a variety of compounds, which can be extracted and can be used for a variety of scientific investigations.Secondary metabolites present in plant are commercially important in pharmaceutical industries (Joy et al., 2001).
Phyllanthus amarus, commonly called as Bhui Amla, belonging to family Euphorbiaceae, is a small herb in southern India with great medicinal value (Nair and Abraham, 2008), growing to a height of 50-70 cm, bearing herbaceous branches with smooth and light green bark.It has numerous pale green flowers and the fruits are tiny, smooth capsules containing seeds.P. amarus root and leaf extract showed significant inhibition of hepatitis C virus (Ravikumar et al., 2011).Leaves, roots, stem, bark and berries of this genus contain lignans (e.g.phyllanthin and hypophyllanthin) and a variety of other phytochemicals such as alkaloids, flavonoids (e.g.quercetin), glycosides, ellagitannins, and phenylpropanoids (Bagalkotkar et al., 2006).Common lipids, sterols, and flavonols also occur in the plant which makes it an unusual choice for innovating phytopharmaceuticals.The objective of this study is to analyze and quantify phenols and flavonoids in different parts of P. amarus (root, stem and leaf) using different solvents.

Sample Preparation
Fresh plant material was washed under running tap water and separated into three different parts, i.e., stem, root and leaves.They were dried under shade to remove moisture and homogenized to fine powder and stored in air tight bottles.The solvents used for the extraction were ethyl acetate, dimethylformamide, dichloromethane, chloroform, and n-Hexane.About 1 g powder of all plant parts (root, stem, leaves) was weighed separately and ground well.Dried powder was extracted in different solvents in a conical flask and kept on a rotary shaker at 190-220 rpm for 24 h.The filtrates were concentrated under reduced pressure using a Rota Vapor (IKA, Germany).

Phytochemical Screening
The preliminary phytochemical tests on different extracts were performed by specific reagents using standard phytochemical methods.

Estimation of Total Phenolic Content
Total phenolic content was estimated by the procedure of Singleton and Rossi (1965) and McDonald et al. (2001).

Preparation of Standard Solution
Tannic acid was used as standard for estimating the total phenolic content in P. amarus (0.1 mg of tannic acid was taken in 1 ml of methanol to get 0.1 mg/ml standard solution).

Preparation of Calibration Curve
Folin-Ciocalteu reagent method was used for the determination of phenolic content in plant extracts using spectrophotometric analysis (Singleton and Rossi, 1965;McDonald et al. 2001).Different aliquots of tannic acid (50, 100, 150, 200, and 250 µl) were taken from the stock solution (0.1 mg/ml) in test tubes.To that 5 ml of Folinciocalteu reagent was added and mixed well.Later 4 ml of sodium carbonate was added and all the test tubes were vortexed for 50 s.All the test tubes were kept for 30 min in water bath at 40°C.Total phenolic content in the plant extract was estimated using different aliquots of tannic acid as standard.The color developed was measured at 680 nm using UV-VIS spectrophotometer (Elico, India).Blank was prepared with Folin-Ciocalteu reagent and sodium carbonate without plant extract or tannic acid.A graph was plotted by taking the optical density values on Y-axis and concentration of tannic acid on X-axis (Figure 1).

Preparation of Test Sample Solution
To 1 ml of the plant extract, 5 ml of FC reagent and 4 ml Na 2 CO 3 were added and incubated for 30 min at 40°C.OD values were taken at 680 nm.Quantification of phenols was done for root, stem and leaf extracts of P. amarus based on the absorbance measured and by comparing the results with a standard curve of tannic acid.Phenol concentration was estimated from the standard curve in root, stem and leaf extracts separately and was expressed in terms of tannic acid (mg of tannic acid/g of extract).

Preparation of Standard Solution
Quercetin was used as standard for estimating the total flavonoid content of the plant P. amarus (1 mg of quercetin was dissolved in 1 ml of methanol and was taken as standard to get 1 mg/ml solution).

Preparation of Calibration Curve
Quercetin method was used for the determination of total flavonoid content in plant extracts using spectrophotometric analysis (Satishkumar et al., 2008;Patel et al., 2010;Patel et al., 2012;Pallab et al., 2013).Different aliquots of quercetin (15, 30, 60, 90, 120, 150, 180, and 210 µl) were taken from the stock solution (1 mg/ml) in test-tubes.To those respective dilutions of methanol was added and made the volume up to 1500 µl and mixed well.Later 0.1 ml of 2% aluminum chloride and 0.1 ml of 1 M potassium acetate was added and mixed well.All the test-tubes were allowed to stand for 30 min at room temperature.Blank was prepared with aluminum chloride and potassium acetate without plant extract or quercetin.Absorbance was measured for the color that was developed at 420 nm using UV-VIS spectrophotometer.A graph was plotted by taking the optical density values on Y-axis and concentration of quercetin on X-axis (Figure 2).

Preparation of Test Sample Solution
To 1 ml of the plant extract, 0.5 ml of AlCl 3 was added to the plant extract and incubated for 1 h at room temperature.To that 0.1 ml of potassium acetate (CH 3 COOK) was added and mixed well.Quantification of flavonoids in root, stem and leaf extracts of P. amarus was done based on the absorbance measured at 420 nm and by comparing the results with standard curve of quercetin.Flavonoid concentration was estimated from the standard curve in root, stem and leaf extracts separately and was expressed in terms of quercetin (mg of quercetin/g of extract).

Calculation
Test concentration = OD of Test/Dilution Factor.
Dilution Factor = OD of Standard/Concentration of Standard.

RESULTS AND DISCUSSION
Phenols and flavonoids in different parts (root, stem and leaf) of P. amarus were evaluated in different solvents, i.e., ethyl acetate, dimethylformamide, chloroform, dichloromethane and n-Hexane (Shah and Yadav, 2015).Leaf was found to possess the highest activity of phenols and flavonoids compared to root and stem.Dimethylformamide was found to be the best solvent for extraction of phenols and flavonoids in root, stem and leaf extracts among the solvents tested.Dimethylformamide leaf extract exhibited highest phenol percentage of 46.77% (Figure 3) and highest flavonoid percentage of 56.18% (Figure 4).
There was report on qualitative and quantitative phytochemical screening of different plant parts of P. amarus with water, methanol, ethyl acetate and petroleum ether (Awasthi et al., 2015).Similar studies have been reported in Phyllanthus fraternus (Kavit et al., 2013) and different species of Phyllanthus leaf extracts for preliminary phytochemical evaluation (Gopinath et al., 2012).
Gas chromatography-mass spectrometry (GC-MS) analysis of P. amarus in dimethylformamide leaf extract revealed the presence of compounds displayed in Table 1 (Figure 5).Carissanol dimethyl ether and fumaric acid were detected in maximum concentration by GC-MS analysis.Fumaric acid has been reported to possess exceptional anti- microbial and antioxidant properties.Hence, it was mostly used in a number of industrial processes (Huan et al., 2002).Our reports are in corroboration with the reports of Veena Gayathri in Tridax procumbens (Krishnaswamy and Christina, 2015) and Dib et al., (2013) in Arbutus unedo.Sylvatesmin detected in this extract was reported to have high radical scavenging activity (Saha et al., 2015) and the compound 1-Heptacosanol was reported to have antimicrobial, anti-oxidant and nematicidal properties in some marine algae (Murugan and Iyer, 2014).Methoxy group containing compounds have been reported to exhibit antimicrobial action by distorting cell surface (Baluja et al., 2015).GC-MS analysis of our present study also revealed the presence of methylenedioxy compound which could be liable for anti-microbial activity.

CONCLUSION
Medicinal and aromatic plants comprise the basis of primary health care for bulk of the population and are an important source of income for rural population.Ancient traditional systems of medicine are practiced in Asia mainly because of historical circumstances and cultural beliefs.Medicinal plants are accessible, affordable and culturally appropriate source of primary health care for more than 80% of the Asian population according to the World Health Organization.There is a need for harmonization among various institutes of the region working on medicinal plants and traditional medicines to develop drugs for health care (Swami et al., 2006).The whole plant of P. amarus was best suitable for herbal medicine because of many active constituents present in it and opens a gate way for novel drug discovery.

Figure 1 :
Figure 1: Standard graph for tannic acid

Table 1 : List of compounds detected by GC-MS analysis in Phyllanthus amarus dimethylformamide leaf extracts Rt (min) Compound name CAS
GC-MS: Gas chromatography-mass spectrometry, RT: Retention time