Assessment of phytochemicals and quantification of primary and secondary metabolites of Artabotrys hexapetalus (L.f.) Bhandari leaves

The main goal of the research was to explore the existence of phytochemicals, quanti ication of primary and secondary metabolites of leaves extract of Artabotrys hexapetalus (L.f.) Bhandari. The phytochemical activity of leaves of Artabotrys hexapetalus was assessed using different solvent extracts like water, ethanol, acetone, chloroform and petroleum ether. Among the different solvent extracts, aqueous leaves extract revealed thehigh content of phytochemicals. So the aqueous leaves extract was used for further investigations. Aqueous leaves extract of Artabotrys hexapetalus was subjected to quantitative analysis of primary metabolites like carbohydrates, proteins and amino acids. Quantitative analysis of secondary metabolites like lavonoids, tannins and phenols were performed using aqueous leaves extract of Artabotrys hexapetalus. Qualitative screeningof phytochemicals reported the existenceof carbohydrates, amino acids, proteins, lavonoids, alkaloids, saponins, phenols, glycosides, tannins and diterpenes. Quantitative analysis showed the presence of carbohydrates (43.16±1.0 mg/g extract), proteins (60.4±0.88 mg/g extract), amino acids (19.33 ± 1.30 mg/g extract), lavonoids (28.3 ±0.91 mg/g extract), tannins (24.53±1.02 mg/g extract) and phenols (7.63±0.85 mg/g extract). The present study concluded that aqueous leaves extract of Artabotrys hexapetalus as a potential source of phytochemicals, primary and secondary metabolites.

Artabotrys hexapetalus, Primary metabolites, Secondary metabolites, Flavonoids, Phenols, Phytochemicals, Tannins A The main goal of the research was to explore the existence of phytochemicals, quanti ication of primary and secondary metabolites of leaves extract of Artabotrys hexapetalus (L.f.) Bhandari. The phytochemical activity of leaves of Artabotrys hexapetalus was assessed using different solvent extracts like water, ethanol, acetone, chloroform and petroleum ether. Among the different solvent extracts, aqueous leaves extract revealed the high content of phytochemicals. So the aqueous leaves extract was used for further investigations. Aqueous leaves extract of Artabotrys hexapetalus was subjected to quantitative analysis of primary metabolites like carbohydrates, proteins and amino acids. Quantitative analysis of secondary metabolites like lavonoids, tannins and phenols were performed using aqueous leaves extract of Artabotrys hexapetalus. Qualitative screening of phytochemicals reported the existence of carbohydrates, amino acids, proteins, lavonoids, alkaloids, saponins, phenols, glycosides, tannins and diterpenes. Quantitative analysis showed the presence of carbohydrates (43.16±1.0 mg/g extract), proteins (60.4±0.88 mg/g extract), amino acids (19.33 ± 1.30 mg/g extract), lavonoids (28.3 ±0.91 mg/g extract), tannins (24.53±1.02 mg/g extract) and phenols (7.63±0.85 mg/g extract). The present study concluded that aqueous leaves extract of Artabotrys hexapetalus as a potential source of phytochemicals, primary and secondary metabolites.

INTRODUCTION
Plants contain natural bioactive compounds called phytochemicals. Phytochemicals are classi ied into two categories primary metabolites and secondary metabolites (Krishnaiah et al., 2009).
Carbohydrates, amino acids, chlorophyll and proteins are primary metabolites. Secondary metabolites include alkaloids, lavonoids, terpenoids, tannins, phenols, glycosides, saponins (Krishnaiah et al., 2007). The medicinal property of a plant is due to secondary metabolites (Cowan, 1999). Secondary metabolites are free radicals scavengers (Mradu et al., 2012). Analysis of phytochemicals and quanti ication of primary and secondary metabolites will be helpful to identify bioactive compounds. These bioactive compounds have therapeutic value in treating diseases.
Artabotrys hexapetalus (L.f.) Bhandari is a member of the Annonaceae family. It is located in India, South China and Srilanka. Within India, it is indigenous to south India and very commonly  (Li et al., 1997) and scrofula (Li and Yu, 1998).This study aims to analyse the phytoconstituents, quanti ication of primary metabolites (carbohydrates, proteins and amino acids) and secondary metabolites ( lavonoids, tannins and phenols) in aqueous leaves extract of Artabotrys hexapetalus.

Plant sample collection and authentication
The leaves of Artabotrys hexapetalus were gathered from Erode district, Tamilnadu, India. The plant was validated (BSIS/RC/5/23/2016/Tech/2101) by Botanical Survey of India Taxonomist, Southern Regional Centre, Coimbatore, Tamilnadu, India.

Preparation of plant extract
Fresh leaves of Artabotrys hexapetalus were collected from Erode district, Tamilnadu, India. Collected fresh leaves were cleaned with sterile water and dried under shade. Dried leaves were subjected to mechanical grinding to obtain a coarse powder.
The coarse powder of A.hexapetalus leaves (20 grams) was soaked with 200 ml of different solvents like water, ethanol (alcohol), acetone, chloroform and petroleum ether in the ratio of 1:10 for three days. Then the plant sample was extracted with a muslin cloth and used to analyse the phytoconstituents.

Preparation of aqueous leaves extract of Artabotrys hexapetalus
Large scale aqueous leaves extract, was prepared by soaking 40g leaves powder in 400ml distilled water for three days. Then the plant sample was extracted with a muslin cloth. The ilterate obtained was evaporated to dryness in a microwave oven under controlled temperature. Finally, 8 gram of greenishbrown powdered crystals were obtained, and it was kept for later analysis in tightly sealed desiccators.

Analysis of total carbohydrates
Quantitative evaluation of carbohydrates in Artabotrys hexapetalus leaves was performed with anthrone reagent using a standard protocol (Hedge and Hofreiter, 1962).

Analysis of total proteins
The protein content of Artabotrys hexapetalus leaves was evaluated according to the Lowry method (Lowry et al., 1951).

Analysis of total amino acids
Standard procedure was followed to determine the amino acid content of Artabotrys hexapetalus leaves (Moore and Stein, 1954) .

Analysis of lavonoids
Aluminium chloride method was used to assess the lavonoids of Artabotrys hexapetalus leaves (Woisky and Salatino, 1998).

Analysis of tannins by Folin-Denis method
The tannin content of Artabotrys hexapetalus leaves was analysed by Folin-Denis method (Mohan, 2017).

Analysis of total phenols
Assessment of phenols in Artabotrys hexapetalus leaves was done by standard procedure (Malik and Singh, 1980).

Statistical analysis
Quantitative estimation of primary and secondary metabolites were done in triplicates with standards. The results of the quantitative analysis were given as mean ± standard deviation.

RESULTS AND DISCUSSION
The result of phytochemical analysis of different solvent extracts of A.hexapetalus leaves has been listed in Table 2.
Among the various solvent extract of Artabotrys hexapetalus leaves, aqueous extract revealed more phytoconstituents. Carbohydrates, amino acids, proteins, alkaloids, lavonoids, tannins, diterpenes, phenols, saponins, and glycosides were reported in the aqueous leaves extract of Artabotrys hexapetalus. When compared to aqueous extract, other extracts showed fewer phytoconstituents. So the aqueous leaves extract of Artabotrys hexapetalus was taken for further studies.

Analysis of total carbohydrates
The amount of carbohydrate in aqueous leaves extract of A.hexapetalus was found to be 43.16± 1.0 mg/g extract (Figure 1).

Analysis of total proteins
The total protein content of aqueous leaves extract of A.hexapetalus was found to be 60.4 ± 0.88 mg/g extract.

Analysis of total amino acids
The amount of amino acid in aqueous leaves extract of A.hexapetalus was found to be 19.33± 1.30 mg/g extract.

Analysis of lavonoids
Flavonoid content of aqueous leaves extract of A.hexapetalus was found to be 28.3 ± 0.91 mg/g extract. Flavonoids are a potent radical scavenger and possess the metal chelating ability (Michalak, 2006;Winkel-Shirley, 2002;Rivero et al., 2001). Flavonoids possess anticancer activity, antiin lammatory activity and lower the risk of heart disease (Okwu and Okwu, 2004).

Analysis of tannins
The tannin content of aqueous extract of A.hexapetalus leaves was found to be 24.53 ± 1.02 mg/g extract. Tannin plays a protective role against oxidative stress (Ness and Powles, 1997). Tannins are very effective against microorganisms and parasites. It is used in the treatment of in lammation in mouth and diarrhoea (Ofokansi et al., 2005).

Analysis of total phenols
Phenol content of aqueous extract of A.hexapetalus leaves was found to be 7.63 ± 0.85 mg/g extract. Plant phenols possess radical scavenging ability and protection against UV radiation (Bennett and Wallsgrove, 1994).

CONCLUSIONS
Phytochemical analysis of A.hexapetalus leaves was performed using different solvent extracts like aqueous, ethanol, acetone, chloroform and petroleum ether. Among the different solvent extracts, an aqueous extract of A.hexapetalus leaves revealed the presence of phytoconstituents like carbohydrates, amino acids, proteins, alkaloids, lavonoids, tannins, diterpenes, phenols, saponins and glycosides. Quantitative analysis of carbohydrates, proteins, amino acids, lavonoids and phenols were done using aqueous extract of A.hexapetalus leaves. This study concludes that aqueous extract of A.hexapetalus leaves is a potent source of phytochemicals, primary metabolites and secondary metabolites. Secondary metabolites act as antioxidants and scavenge free radicals. A.hexapetalus leaves can act as a natural antioxidant.