SCREENING OF PHYTOCHEMICAL CONSTITUENTS AND QUANTITATIVE ESTIMATION OF TOTAL FLAVONOIDS AND PHENOLIC COMPOUNDS OF LEAFEXTRACTS OF MITRACARPUS HIRTUS (RUBIACEAE)

Plants have the ability to synthesize mixtures of structurally diverse bio-active compound, with multiple and mutually potential therapeutic effects. The objective of the study is to cover the preliminary phytochemical screening of traditional medicinal plant Mitracarpus hirtus belonging to the family Rubiaceae. The preliminary screening revealed the presence of alkaloids, flavonoids, steroids, tannins, phenolics, glycosides, carbohydrates, proteins and amino acids. In quantitative estimation, among all the extracts, acetone extract exhibited the maximum amount of phenolics (40.26 mg GAE/g extract), and it depicted the maximum quantity of flavonoids (84.43 mg RE /g extract) which explains that the plant must have valuable medicinal properties and so it can be explored.


INTRODUCTION
Origin of medicinal herbs. They have been using them for curative purposes successfully. The records are available in ancient texts. In India itself, there are more than 20000 medicinal plants grown all over the wild forests. Of these, some 60 genus are used immensely in medicinal preparation. Despite their demands today, they are not grown in controlled manner. Rather tribes use them as their livelihood in some belts where they are grown in the wild. Unlike India, in china, the spurts in demand for traditional medicine have made government to allow growth of these plants for further research and development. About 100 units have nearly 600 plant type, grown for their medicinal value. Herbal medicines are used in Ayurveda, Naturopathy and Homeopathy, tradition and Native American medicine (Rangari, 2002). The World Health Organization (WHO) estimates that about 80% of the population living in the developing countries relies almost exclusively on traditional medicine for their primary health care needs. In almost all the traditional medicine, the medicinal plant plays a major role and constitutes the backbone of traditional medicine (WHO, 1978). A medicinal plant is any plant which, in one or more of its organ, contains substance that can be used for therapeutic purpose or which is a precursor for synthesis of useful drugs. The plants that possess therapeutic properties or exert beneficial pharmacological effects on the animal body are generally designated as "Medicinal Plants". Although there are no apparent morphological characteristics in the medicinal plants growing with them, yet they possess some special qualities or virtues that make them medicinally important. It has now been established that the plants, which naturally synthesize and accumulate some secondary metabolites like alkaloids, glycosides, tannins, volatile oils, contain minerals and vitamins, possess medicinal properties (Adnan, 2002).
Mitracarpus hirtus is a generally erect, annual plant that can be simple or sometimes much branched. The relatively stout stems can be 30 -60cm tall. The plant is sometimes gathered from Page 48-52 the wild for local medicinal use. The dried leaves are said to heal old ulcers rapidly. The plant is an antidote for arrow poison (Ken Fern, 2021). Considering the medicinal activity of M. hirtus based on the aforesaid traditional information, the present study was focused on the phytochemical screening and quantitative estimation of secondary metabolites of the selected plant sample to add scientific conclusion to the traditional claims.

Study area
The selected plant Mitracarpus hirtus was collected from Kozhikode District in the Northern part of Kerala. Keezhariyur is a small village situated in Kozhikode district, Kerala state, India. It comes under Keezhariyur Panchayath.
It is located 35 Km from District headquarters Kozhikode. The average annual rainfall of the state ranges from 101.6 to 362cm. The highest temperature recorded was 39.4°C in March and the lowest was 14•C on December. The District has a generally humid climate with a very hot season extending from March to May. The rainy season is during the South West Monsoon, which sets in the first week of June and extend up to September.

Collection of Plant Material
The plant leaf of M.hirtus were collected during the month of December, 2021, Keezhariyur, Kozhikode district, Kerala. The authenticity of the selected plant materials were duly identified and confirmed by Botanical Survey of India, Coimbatore. Fresh and healthy plant leaf of M. hirtus was harvested shade dried and coarsely powdered for extraction.

Preparation of Plant Extracts
The powdered plant samples (40 g/200 ml) were extracted successively with hexane, acetone and ethanol using Soxhlet apparatus at 55-86℃ for 9-10 hr in order to extract the polar and nonpolar compounds. The powder was air dried and packed then used for each solvent extraction. The solvent of respective extracts were reduced under room temperature and stored at 5℃ for further use.

Qualitative screening of Phytochemical Compounds: 3.4.1. Extractive yield
The air dried leaves were exhaustively extracted with successive solvent extraction using soxhlet apparatus viz., petroleum ether, benzene, chloroform, ethyl acetate, ethanol and hot water was which indicates the presence of earthy materials in the sample. The water soluble ash is used to estimate the amount of inorganic  Table 2. Physico-chemical analysis of Kedrostis foetidissima leaves performed as per Indian Pharmacopoeia (Peach and Tracey, 1955). The extracts were filtered and concentrated to dryness under reduced pressure using rotary vacuum evaporator (RE 300; Yamato, Japan), Lyophilized (4KBTXL -75; Vir Tis Benchtopk, New York, USA) to remove traces of water molecules and their extractive yield percentage wascalculated.

Qualitative phytochemical evaluation
Phytochemical screening for crude solvent extracts were carried out and their bioactive compounds were determined using standard methods Trease and Evans, 1983;Harborne, 1984).

Quantitative phytochemical analysis 3.5.1. Determination of total flavonoids
The total flavonoid content of samples was determined by following the modified colorimetric method of Zhishen et al. (1999). 0.5 ml extract was mixed with 2 mL of distilled water and subsequently with 0.15 mL of 5% NaNO2 solution. After 6 min, 0.15 mL of 10% AlCl3 solution was added and allowed to stand for 6 min, then 2 mL of 4% NaOH solution was added to the mixture. Immediately distilled water was added to bring the final volume to 5 mL, and then the mixture was thoroughly mixed and allowed to stand for another 15 min. Absorbance of the mixture was recorded at 510 nm versus prepared water blank. Rutin was used as a standard compound for the quantification of total flavonoid. All the values were expressed as milligram of rutin equivalent (RE) per gram of extract.

Determination of total phenolics
The total phenolic content was determined according to the method described by Siddhuraju and Becker (2003). Aliquots of each extract were taken in test tubes and made up to the volume of 1 mL with distilled water. Then 0.5 mL of folinciocalteu phenol reagent (1:1 with water) and 2.5 mL of sodium carbonate solution (20%) were added sequentially in each tube. Soon after vortexing the reaction mixture, the test tubes were placed in dark for 40 min and the absorbance was recorded at 725nm against the reagent blank. The analysis was performed in triplicate and the results were expressed as gallic acid equivalents (GAE).

Extractive Yield Percentage
The yield of sequential extracts (%) is shown in Figure 2.

Qualitative Phytochemical analysis
Phytochemical screening of the sequential extract of M. hirtus revealed the presence of various bioactive components of which phenolics, saponins, alkaloids, tannin, Glycosides, Proteins, Carbohydrates, and Amino acids are the most prominent components and the result of phytochemical test given in the Table 1.

Quantitative Phytochemical analysis
Total flavonoids and phenolic content of leaves of Mitracarpus hirtus in rutin and gallic acid equivalents are presented in Table 2. The highest phenolic and flavonoid content was noted in the acetone leaf extract, 84.43 mgRE/g extract, and 40.26 mgGAE/g extract flavonoids and phenols were present in leaves respectively. The flavonoids are hydroxylated phenolic substances known to be synthesized by plants in response to microbial infection and they have been found to be antimicrobial substances against wide array of microorganisms in vitro. Their activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell wall (Marjorie, 1996.). They are also effective antioxidants and show strong anticancer activities  Values are expressed as mean±SD (n=6).)

CONCLUSION
The results revealed the presence of medicinally important constituents such as flavonoids and phenol in the study plant. Further the plant could be considered for antioxidant, anticancer, immunomodulatory activities.