Pharmacognostical and phytochemical screening of an Ayurvedic Medicinal Plant ‘ Karunthakali ’ ( Solanum rubrum Mill)

The presence of phytochemicals and the medicinal value of the Ayurvedic medicinal plant Karunthakali or Karimthakali ( Solanum rubrum Mill) is investigated in detail for the first time, both quantitatively and qualitatively. Phytochemical compounds are identified from the samples extracted from the leaf, root and seed of the plant, using standard methods. The ash values of the plant leaf are obtained. Alkaloids, flavanoids, anthronol glycosides, terpenes, carbohydrates, saponins and proteins are found present in this plant parts. Tanins, Free amino acids, Free Anthroquinone and Cartenoids are absent in this plant. Presence of high mineral content is the unique identification observed in this plant. The preliminary investigation of phytochemical study of this plant confirms qualitatively its antimicrobial, antiviral, antidiarrhoel, anthelmintic and anticancer activity.


Introduction
The ancient healing systems of India, Ayurveda and Sidha, are practiced even today worldwide. Ayurveda, which is based on the classical texts, Charaka Samhita and Sushruta Samhita were written around 1000BC. Ayurvedic drugs are used in crude forms like expressed juice, decoction, emulsion, apozems, liniments, electroactive and powdered. Medicinal herbs are the major ingredient in the formulation of Ayurvedic drugs prepared and used by ancient healers and which had remarkable advancement in leading a healthy life and even curing diseases.
Due to the rich complement of phytochemicals and secondary metabolites, plants have been used as sources of medicament against various ailments since very early days. In rural areas where access to modern health facilities is limited by the level of development, plants remain the mainstay of the health care system.
The availability of modern scientific technologies made the medicinal plants having application in pharmaceutical, cosmetic, agricultural and food industry(1,2) and are considered to be chemical factory as they contain multitude of chemical compounds like alkaloids, glycosides, saponins, resins, sesquiterpene lactones and oils. Belewu Besides the roles played in human and animal nutrition, knowledge of proximate, phytochemical and micronutrient composition are fundamental to the understanding of modes of action of medicinal plants in general.
We therefore in this preliminary study, qualitatively and quantitatively analysed the phytochemical constituents of an Ayurvedic medicinal plant Solanum rubrum Mill, which is not yet studied, even though it has potential application in traditional practice of medicine.
The plant Solanum rubrum Mill, was identified in literature as a synonym for Solanum nigrum. In fact, Ayurvedic system pronounces these two plants differently, such as Solanum nigrum as 'Manathakkali' and Solanum rubrum as ' Karunthakali'or Karimthakali. Its phytochemical analysis yielded different results than other plants in the same family 'Solanaceae'. A photograph of the plant is given in Fig.1., for identification.

Fig.1: Solanum rubrum Mill
It is a shrub, and which may be classified as a poisonous plant on the basis of traditional practice and use. It is available in Western Ghats of Peninsular India which has much ecological significance and grows upto one feet to one meter height with dark green coloured leaves and the fruits are red or yellow in colour. Commonly, this plant is not in use nowadays for any purpose. In old palm leaves and manuscripts of Ayurvedic system it is mentioned that this plant can be used for skin diseases, and paralysis disease. With the structure and taste of the leaf, similar to ancient traditional identification of medicinal plants, we suspect that this plant has some more valuable medicinal characters. Hence for the first time, the phytochemical constituents of this plant are presented in this paper. The antioxidant activity of this plant parts were reported recently(6).

Aim and Objective
Ayurvedic drug forms such as Kashaya, Gulika, Ghrita, Arista, Churna, etc. are having their own bioactive nature depending upon the presence of active compounds in the respective plant parts used. In pharmaceutical industries the pharmaceutical aids like binding agent, flavouring agent, sweetening agents, and colouring agents are commonly used in Ayurvedic drug formulation. The scientific understanding of the influence of medicinal plants in curing ailment can be possible through the identification of presence of chemical compounds and hence the phytochemical analysis of medicinal plants became important.

Materials and Methods Collection of plant and character identification:
Fresh plants are collected from Kanyakumari district, the southernmost part of India (8.29302Latitude & 77.21828 Longitude) during the month of August ie., the fruiting season of the plant. The collected plants are washed well in fresh water and shadow dried. The leaves and root are separated and made into fine powder using mortar and pestle. The fruits were collected from live plants and the seeds are separated and shadow dried. The shadow dried leaf powder was kept in a closed conical flask with solvents(1:10),(7,8), acetone, benzene, chloroform, ethanol, water, butanol, and ethyl acetate, separately and shook well for 10 min and kept at room temperature for 3 days. After 3 days the extract is filtered, and the filtrate was then dried under room temperature. The same procedure was followed for root and seeds. The ethnomedical information about the plant is searched for and we couldn't trace any literature.
The physical character and the colour analysis of the plant parts are tabulated in Table 1&2. The powdered leaf samples are added with different reagents for behavioral analysis and the possible different chemicals present are identified ( Table 3). The extraction values of solvents such as Water, ethanol, chloroform, ether, acetone, ethyl acetate, butanol and benzene are tabulated in Table  4.    Powder analysis Total ash value: 5 gm powder was ignited in an electric furnace at 500-550 o C in silica crucible until the sample reaches a constant weight.

Water soluble ash value:
The water insoluble matter was collected in an ashless filter paper and ignited in an electric furnace at 450 º C in silica crucible until it reached a constant value. The weight of water soluble ash was calculated by subtracting the weight of insoluble matter from the weight of the total ash.

Acid insoluble ash value:
Total ash obtained was heated with addition of 25 ml of dilute HCl for 10min. It was filtered in an ash less filter paper (Whatman No.41) and the residue was ignited in the furnace to get a constant weight.

Preliminary phytochemical screening of S. rubrum Mill.
Phytochemical screening were carried out for the extracts namely Water, Ethanol, Chloroform, Ether, Acetone, Ethyl acetate, Butanol and Benzene and the powder as such, as per the standard methods(9-15). benzene. The benzene layer was separated and treated with ammonia solution. Formation of rose-pink colour in the ammonical layer indicated the presence of anthranol glycosides.

Detection of alkaloids
B. Cardiac glycosides: 5 ml of extract was treated with 2 ml of glacial acetic acid containing one drop of ferric chloride solution. This was underplayed with 1 ml of concentrated sulphuric acid. A brown ring at the interface indicated the deoxysuger characteristics of cardenolides. A violet ring appeared below the ring while in the acetic acid layer, a greenish ring was formed.

Detection of phlobatannins
Deposition of a red precipitate when an aqueous extract of the plant sample was boiled with 1 % aqueous hydrochloric acid was taken as evidence for the phlobatannins.

Detection of combined anthraquinones
1 gm of powdered sample of the specimen was boiled with 2 ml of 10% hydrochloric acid for 5 mins. The mixture was filtered while hot and filtrate was allowed to cool. The cooled filtrate was partitioned against equal volume of chloroform and the chloroform layer was transferred into a clean dry test tube using a clean pipette. Equal volume of 10% ammonia solution was added into the chloroform layer, shaken and allowed to separate. The separated aqueous layer was observed for any colour change. Delicate rose pink colour showed the presence of an anthraquinone.

Detection of free anthraquinones
5 ml of chloroform was added to 0.5 gm of the powder. The resulting mixture was shaken for 5 minutes after which it was filtered. The filtrate was then shaken with equal volume of 10% ammonia solution. The appearance of a bright pink colour in the aqueous layer indicated the presence of free anthraquinones.

Detection of carotenoids
1 g of each specimen sample was extracted with 10 ml of chloroform in a test tube with vigorous shaking. The resulting mixture was filtered and 85% sulphuric acid was added. A blue colour observed at the interface showed the presence of carotenoids.

Detection of fixed oils and fats
A. Spot test: A small quantity of extract was pressed between two filter papers. The observed oil stain on the paper shows the presence of fixed oil.

Detection of gums and mucilages
Extract was mixed with 10 ml distilled water and 25 ml of alcohol with constant stirring. A White precipitate formed indicates the presence of gums and mucilages.

Result
Plant material is composed of primary metabolism such as water, minerals, organic compound, protein lipid and carbohydrates, and secondary metabolism such as phenol, tannins, flavonoids, vitamin C & E. In order to establish the identity, purity, safety and quality of the plant, standardization is an important tool for the herbal drugs. In order to standardize a drug, various macroscopic, physiochemical and phytochemical analysis are to be performed.
As per the analysis performed on the Solanum rubrum, the physical contents such as total ash, acid soluble and water insoluble ash values give a specific identification to this plant since these values are much high compared to many other plants. The total ash of Solanum rubrum at 500-550 o C is 24% which is very high and the acid soluble ash is 40%; the water insoluble ash is 78%, (with Loss on heat: 2%), which are the special characters as far as medicinal plants are concerned.
The phytochemical screening of leaf, root and seed of the plant revealed the presence of protein, carbohydrate, reducing sugar, phenol, flavonoids, saponins and alkaloids (Table 5). The presence of these secondary metabolities suggests that the plant might be medicinal importance (16). It is found that the presence of Alkaloids, Flavanoids and Anthranol glycosides are high in the plant leaf, followed by Diterpene, Triterpene, Carbohydrates, Saponins and Protein (Table 6). (Depending upon the appearance of the darkness of the colour, the concentration of phytochemicals are identified as high, moderate and low). Flavanoids are phenolic compounds act as primary antioxidant or free radical scavenger. Terpinoids act as regulators of metabolism and play a protective role as antioxidant. Saponins are glycosides of both triterpene and steroids having hypotensive and cardiodepresent properties. Tannins, free aminoacid, free anthroquinone and Carotenoids are not identified. Whereas in root, only Alkaloids, Carbohydrates and Anthranol glycosides are present more than saponins and Diterpene (Table 7). The experiment on the seed shows only the presence of Alkaloids, Carbohydrates, Anthranol glycosides and phenolic compounds (Table 8).     Medicine, 2013, 4(4), 328-341 Flavonoids    Table 11, and the phytomedicinal value of the compounds found in this plant are discussed in the next section. Antidiarrhoeal Enhance intestinal absorption of Na + and water.

Discussion
The physio-chemical evaluation of drugs is an important parameter in detecting adulteration or improper handling of drugs. It can serve as a valuable source of information and provide appropriate standards to establish the quality of the plant material in future study or application. Plant extract containing alkaloids, flavanoids, phenolic and /or terpenes showing active trypanocidal activity (26). These compounds are potent bioactive compounds that could be used for therapeutic purpose or which are precursors for the synthesis of useful drugs (27).
Alkaloids are known to play some metabolic roles and control development in living system (28). These have protective role in human and animal and it is used in medicine especially the steroidal alkaloids which constitutes most of the valuable drugs. Though one of their biological property is cytotoxicity (29), alkaloids have analgesic (30,31), antispasmodic and antibacterial (32,33) properties.
Flavanoids are found to be useful in disease resistance (34). They are hydroxylated phenolic 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 (35). They also are effective antioxidant and show strong anticancer activities (36)(37)(38).
The phenolic compounds are one of the largest and most ubiquitous groups of plant metabolites (39). They possess biological properties such as antiapoptosis, antiaging, anticarcinogen, antiinflammation, antiatherosclerosis, cardiovascular protection and improvement of endothelial function, as well as inhibition of angiogenesis and cell proliferation activities (40). The phenols are reported to inhibit alpha-amylace, sucrose, as well as the action of sodium glucose-transporter 1 (SGLUT-1) of the intestinal brush border. The polyphenol compound has antimicrobial and antifungal effect and has been used in disinfections.
Steroids have been reported to have antibacterial properties(41) and are important especially due to their relationship with compounds such as sex hormones (42). Plant sterols could act as a natural preventive dietary product (43). It helps in the lowering plasma cholesterol and LDB cholesterol. Hence, it will assist in reducing drastically the morbidity and mortality caused by cardiovascular disease.
Saponins, which are known to produce inhibitory effect on inflammation (44) and has the property of precipitating and coagulating red blood cells. Crude saponins are having inhibition of glucose transport. Glycosides are known to lower the blood pressure (45).
The major parts of the medicinal plants used for various treatments are leaf, root and seed and to some extent flower and fruits. The pharmaceutical industry first extract the active ingredients before being used in the manufacturing of drugs, hence, discovering the evolution of drugs in the medicinal plants may highly useful for synthesis of new drugs.
The physico-chemical and phytochemical studies of Solanum rubrum yielded a set of qualitative and quantitative pharmaco-botanical parameters or standards that can serve as an important source of information to ascertain the identity and to determine the quality and purity of the plant material in future studies. However, in designing specific drugs, it is essential to isolate the bioactive fractions from these major groups.

Conclusion
For the first time, the phytochemical constituents of Solanum rubrum are identified qualitatively as a preliminary study. The presence of triterpenoids, saponin, steroids, flavanoids, carbohydrates and phenols show the high medicinal value of this plant as antimicrobial, antiviral, antidiarrhoeal, anthelmintic, and aticancer. The high ash value (both acid and water soluble) of this plant is a unique character identified, which suggest the higher availability of minerals in the plant leaves, and also  Medicine, 2013, 4(4), 328-341