Pharmacognostic Standardisation and Phytochemical Evaluation on the Seeds of Two Vitis Vinefera L . Varieties Grown in Kashmir Valley , India

Introduction: Grape (Vitis vinifera L.) is one of the largest fruit crop widely grown throughout the world and their seeds are considered a relevant source of polyphenol compounds. Objective: To carry out phytochemical and pharmacognostic evaluation of the seeds of Vitis vinefera L. Var sahibi and hussaini, family vitiaceae as per WHO guidelines. Materials and Methods: In present investigation, the detailed pharmacognostic studies were carried out in terms of organoleptic properties, macroscopical, micro­ scopical characters, physicochemical parameters, preliminary phytochemical screening for major groups of compounds and other WHO recommended parameters for standardization of the seeds of Vitis vinefera L. Var. sahibi and hussaini. Results: Macro and microscopic characters of Vitis vinefera L. seeds Var. sahibi and hussaini were analyzed. Physicochemical parameters such as foreign matter, extractive values, ash content, pH and fluorescent behaviour of seed powder were done by using WHO recommended parameters. A preliminary phytochemical screening was done and revealed the presence of different phytoconstituents like carbohydrates, flavonoids, glycosides, tannins, sterols and fixed oil. Conclusion: This is the first report of its kind on the pharmacognostic studies of Vitis vinefera L. seeds Var. sahibi and hussaini and will serve as valuable source of information towards establishing pharmacognostic standards on identification, purity, quality and classification.


INTRODUCTION
Grapes (Vitis vinifera L.), are one of the world's largest fruit crop, with a total production of approximately 75.1 million tons in 2013.Although, a high part of grapes is made into wine, another part is dried into raisins, a considerable part is consumed as table grapes.Vitis vinifera L. contains large amounts of phytochemicals such as phenolic compounds which offer health benefits via their vital antioxidant activity to neutralize free radicals in biological systems.Grape seeds and skins which are a low-value by-products of wine making or juice making operations are considered to be economical and a good source of these high-quality phenolic compounds. 1The polyphenol compounds are as much as 60-70% in grape seeds compared only 10% in the fruit and 28-35% in the peels; 2 these levels are influenced by the grape variety and ecological factors. 1 Most of the polyphenol compounds found in grape seeds are monomerics catechin, epicatechin and gallic acid, and the polymeric and oligomeric procyanidins. 3These compounds are of interest in pharmaceutical and food industries for therapeutic treatments and health supplements. 2The medicinal and nutritional importance of Vitis vinifera L. has been heralded for thousands of years.Egyptians consumed this fruit at least 6000 years ago, and a number of ancient Greek philosophers praised the healing power of grapes-usually in the form of wine.European folk healers developed an ointment from the sap of grapevines to cure skin and eye diseases.Unripe grapes were used to treat sore throats and dried grapes (raisins) were used to cure constipation and thirst.The ripe, sweet grapes were used to treat a range of health problems including cancer, cholera, smallpox, nausea, eye infections, and skin, kidney, and liver diseases.Moreover, grapes are helpful in bilious dyspepsia, dysuria, haemorrhage, in chronic bronchitis, gout and heart diseases. 4,5he World Health Organisation (WHO) has described guiding principles for the standardisation of medicinal plants with regard to their macroscopic and microscopic description.Several studies have recommended that the pharmacognostic particulars of a plant may provide useful criteria in identification and authentication of plant drugs. 6In view of the importance of Vitis vinefera L. seeds in conventional and contemporary system of medicine, it was thought worthwhile to develop quality standard for the same.As far as botany and chemistry of this plant is concerned, large number of scientific data is accessible but a systematic standardization study is still missing.Hence, in the present investigation an attempt has been made to standardize the Vitis vinefera L. seeds (Var.sahibi & hussaini) 7 using microscopic characters, physicochemical values and heavy metal analysis.

Macroscopic and microscopic analysis
Macroscopic characters of the seeds were studied according to the method of Evans.It refers to evaluation by means of organs of sense and helps to judge various parameters viz.appearance, odour, taste and colour. 8n the other hand microscopic examination of the seeds are not only essential to identify the adulterants but also indispensable in the correct identification of the plant. 9For microscopic studies of the grape seeds, the procedure recommended by Johansen was followed. 10harmacognosy Journal, Vol 8, Issue 5, Sep-Oct, 2016

Physicochemical analysis
The grape seeds were shade dried and powdered using mechanical grinder for determination of physicochemical parameters such as total ash value, water soluble ash value, acid insoluble ash value, foreign organic matter, loss on drying, foaming index, swelling factor, 11 determination of resin and fat content, 12 water soluble extractives and alcohol soluble extractives, 11,13 pH determination. 14Fluorescence analysis study of powdered drug material with different reagents was carried out to observe the color reactions.The drug was treated with various chemical reagents and exposed to visible and ultraviolet light to study their fluorescence behaviour. 15The presence of heavy metals analysis was carried out quantitatively by Atomic absorption spectrophotometer. 16Qualitative phytochemical screening was carried out on the grape seed extracts (petroleum ether, chloroform, ethyl acetate, methanol, aqueous) to determine the presence of carbohydrates, flavonoids, phenolic compounds, glycosides, alkaloids, proteins and amino acids, saponins, sterols, mucilage, resins, lipids/fats etc. by following standard procedures. 17,18

RESULT
The standardization of a crude drug is an integral part of establishing its correct identity.Before any crude drug can be included in a herbal    pharmacopoeia, pharmacognostic parameters and standards must be established.

Macroscopical characters
The two varieties of Vitis vinefera L.: sahibi and hussaini differ only in their size.In Sahibi variety, the shape of the seed was pear shaped, the colour of seed was dark brown, the surface is smooth with ridge on back surface, apex is discoidal, size is 4-8 mm long, taste is bitter.In hussaini variety, the shape of the seed was also pear shaped, the colour of seed was dark brown, the surface is smooth with ridge on back surface, apex is discoidal, size is 4-6 mm long, taste is bitter.

Microscopic analysis
The transverse section of the seeds of both the varieties has been found to be almost same and revealed five zones (Figure 1 & 2): cuticle and epidermis; outer integument or soft seed coat, composed of large parenchymatous cells; medium integument or hard seed coat, composed of two layers of cells; inner integument; (v) endosperm and embryo.At the first observation, the cuticle was found thin and, the epidermis was constituted of rectangular cells.The outer integument was constituted of parenchymatous cells.The epidermis and the outer integument were made of a soft layer of cells, which covered the seed.The raphe was  The inner integument is between the medium integument and the endosperm.The centre of the seed was occupied by parenchymatous tissue, surrounded by integuments and containing the embryo sac.

Physicochemical analysis
Various physicochemical parameters of Vitis vinefera L. seeds (Var.sahibi and hussaini) like loss on drying, total ash value, water soluble ash, acid insoluble ash, swelling index, resin content, fat content, foreign content, loss on drying, foaming index and pH values were determined and are shown in Table 1.

Extractive values
Extracts were prepared with various solvents.Percentages of the extractive values were calculated with reference to air dried drug and are given in Table 2.

Phytochemical analysis
The results of qualitative phytochemical analysis of the different seed extracts of the Vitis vinefera L. (Var.sahibi and hussaini) are shown in Table 3.Preliminary phytochemical analysis revealed the presence of medicinally important secondary metabolites like flavonoids, glycosides, tannins and sterols, whereas alkaloids and saponins were absent.

Fluorescence analysis of extracts
Fluorescence study of Vitis vinefera L. seeds (Var.sahibi and hussaini) were performed as per standard procedure.Powdered seeds were treated with various chemical reagents and exposed to visible and ultraviolet light (254 and 365 nm wavelengths) to study their fluorescence behaviour.The characteristic fluorescence colours of the powdered seeds with various chemical reagents are stated in Table 4.

TLC analysis
The TLC of Ethyl acetate extract of the two varieties of Vitis vinefera L. seeds on Silica gel 60 F254 pre-coated sheets using different solvent systems showed highest number of spots in Chloroform : Ethyl acetate: Methanol : Formic acid (6:2:1:0.5).Methanolic extract of these two varieties showed highest number of spots in Chloroform : Ethyl acetate: Formic acid (10:8:2) using Iodine vapour as viewing medium (Table 5).

Microbial load and Heavy metal analysis
At the stock dilutions of 1:1,1:10 and 1:100, the number of microbial colonies which were visible to the naked eye were 131, 81 and 51 for sahibi variety and 24, 15 an 6 for hussaini variety respectively.All these microbial colonies were round in shape and white in colour in both sahibi and hussaini varities (Figure 3).Heavy metal analysis results of both these varieties showed that the Cadmium, Lead and Cobalt were not detected which indicates that it is safe for human consumption.

DISCUSSION
The source and quality of raw materials play a pivotal role in guaranteeing the quality and stability of herbal preparations.Many factors can affect the quality and consequently the therapeutic value of herbal medicines, such as light exposure, temperature, water availability, nutrients, period and time of collection, method of collecting, drying, packing, storage and transportation of raw material, age and part of the plant collected. 19For this reason it is of utmost importance to have raw materials with a consistent and reproducible quality standard.Pharmacognostic standardization including physico-chemical parameters are the primordial steps meant for identification, authentication, and detection of adulteration and also compilation of quality control of crude drugs.In this regard, the microscopic and macroscopic features of Vitis vinefera L. seeds (Var.sahibi and hussaini) have been studied.The extractive values give an idea about the chemical constitution of the drug and from the study, the extractive value of methanol was found to be highest followed by chloroform in case of sahibi variety and aqueous followed by methanol in case of hussaini variety.The ash value determines the earthy matter or inorganic composition and other impurities present along with the drug.The analysis of ash values indicates that foreign inorganic matter in considerably normal amounts in both the varieties.Heavy metal analysis and microbial load for the drug was in normal values as set down by WHO.When physical and chemical methods are inadequate, the plant material may be identified from their adulterants on the basis of fluorescence characteristics.The fluorescent analysis under visible light and UV light by treatment of different chemical reagents showed different colour.This is attributed to the ultra violet light which produces fluorescence in many natural products that do not visibly fluoresce in daylight.Thus fluorescence is used for qualitative assessment of crude drug. 20Behaviours of the powdered drug with different chemical reagents and preliminary phytochemical analysis are helpful for detection of various phytoconstituents.

CONCLUSION
In conclusion, these pharmacognostic standards for the seeds of Vitis vinefera L. (Var.sahibi and hussaini) are laid down for the first time in this study.These would not only help in setting indices for identification of raw material and preparation of plant monograph but also will serve as a diagnostic tool in development of pharmacopoeial standards for the future studies.
dorsal face of this tissue.Two layers of rectangular cells with thin walls constituted the medium tissue or medium integument.