Evaluation of nutritive value, phytochemical screening, total phenolic content and in-vitro antioxidant activity of the seed of Prunus domestica L

Rishi Kumar Shukla1, Kishan1*, Abha Shukla2 & Rahul Singh3 1Department of Chemistry, Gurukula Kangri (Deemed to be University), Haridwar 249 404, Uttarakhand, India 2Department of Chemistry, Kanya Gurukula Campus, Gurukula Kangri (Deemed to be University), Haridwar 249404, Uttarakhand, India 3Department of Pharmaceutical Sciences, Gurukula Kangri (Deemed to be University), Haridwar 249 404, Uttarakhand, India *Email: prajapati75kishan@gmail.com


Introduction
Various fruits constitute an important part of the human diet. It is accepted worldwide that a diet enriched with fruits and vegetables lowers the hazard of persistent sicknesses like cardiovascular diseases, cancer and leading a long and healthy life (1). These food products have been regarded as nutraceuticals or functional foods. The foods which are a good source of antioxidants (like phenolic acids, flavonoids etc.) have high demand in today's market as a nutraceutical. Fruits are the source of low fat and low calories while dried fruits have high protein contents, high mineral contents (2).
Prunus domestica L. of the family Rosaceae is generally regarded as plum (2,3). The family Rosaceae is the 19 th largest family of plants (4) and includes about 3000 species whereas the genus Prunus includes about 400-430 species; but only 89 are enlisted in the Genetic Resources Information System. In India, about 36 species of Prunus have been reported of which 18 species can be used for the cultivation of different purposes (5)(6)(7)(8)(9).
It is used in combination with other drugs to treat leucorrhoea, irregular menstrual and miscarriage followed by debility (10). Plums and prunes show laxative stomachic effect. The bark of P. domestica is used as febrifuge while roots are utilized as astringent (11). P. domestica is the source of calcium, magnesium, vitamin A, potassium and fibre (12). This species possesses a discrete place in the Indian medicinal system due to its numerous health benefits (2). It is abundant in different bioactive phytochemicals such as anthocyanins, pectins and carotenoids, lignans, abscisic acid, glucoside, flavonoids, flavonoids glycosides, bipyrrole, dihydroflavonols and carbohydrates (12)(13)(14)(15)(16)(17)(18)(19). These bioactive compounds are found to be varied in concentration depending upon various pre-harvesting factors (20). Plums are an excellent source of nutrients with a significant contribution to human nutrition.
Due to many important phytochemicals, this fruit has an effective antioxidant activity (21). In addition to this, different extracts of P. domestica show many important activities namely antibacterial, anticancer, antihyperlipidemic, blood pressure-lowering activity, anxiolytic activity and antidiabetic activity (2,22,23).
The fruit of the P. domestica is juicy and fleshy which is eaten while the seed is usually discarded. Therefore, the current study aims to evaluate the nutritive value, qualitative phytochemical screening and antioxidant activity of the seed (i.e., kernel) of P. domestica.

Sample collection
For the collection of the seeds, plums were purchased from the local garden situated in Haridwar, Uttarakhand. Seeds were dried under the shade. The plant was recognized and confirmed by the Botanical Survey of India (BSI), Dehradun under accession number 374. For the authentication of the plant material two herbarium specimens were prepared, one of which is deposited in the Botanical Survey of India.

Physical Parameters and Proximate Analysis
Evaluation of the physical parameters and proximate analysis for the calculation of the nutritive value was done according to the Indian pharmacopeia and earlier study on Bombax ceiba (24, 25).

Preparation of Extracts
Soxhlet extractor was employed for the extraction. Briefly, 200 gms of the powdered plant material was put in the thimble of the soxhlet extractor. Extraction was done using the solvents petroleum ether, diethyl ether, ethyl acetate, methanol and double-distilled water in the increasing order of the polarity. Approximately 72 cycles of siphoning were conducted for each solvent or the extraction was continued till the siphoning tube appears colourless. After the extraction, the extracts of different solvents were concentrated under reduced pressure using the rotary evaporator and stored in a refrigerator for further examination.

Phytochemical Screening
Phytochemical screening of the different extracts for the presence of the various phytoconstituents was carried out by standard qualitative methods (26,27). Each concentrate was screened for the nearness of natural dynamic mixes like alkaloids, sugars, glycosides, amino acids, proteins, triterpenoids and steroids, flavonoids, phenolics, gums. Furthermore mucilages, naphthoquinones and so forth.

Total Phenolic Content
The folin-ciocalteau method was adopted for the determination of the total phenolic content of each extract with some modifications. Briefly, 100 µl extract dilution at a concentration (1000 µg/ml) or gallic acid (10-400 µg/ml) put in a test tube, containing 7.9 ml of the distilled water. Distilled water also serves as blank. Now, added 500 µl of the folin's reagent to the volumetric flask and shaken properly and incubated for 8 min at room temperature. Now, 1.5 ml of 20% Na2CO3 was added to the above mixture to make the final volume 10 ml and incubated for 2 hrs at room temperature. Absorbance was measure at 765 nm with UV-Vis double beam spectrophotometer (Systronics 2205). Calculations were carried out using the calibration curve of gallic acid. The total phenolic content of each extract was expressed as mg of gallic acid equivalents (GAE) per gms dried weight (mg GAE/g dw) (28).

DPPH Free Radical Scavenging Assay
All the obtained extracts of the seed of the P. domestica were evaluated for their antioxidant power by the DPPH method according to standard methodology with some modification (29). Briefly, 1 ml of the extract (100 -5000 µg/ml) was mixed with 0.004% DPPH solution and leave at room temperature for 1 hr. After 1 hr absorbance was measured at 517 nm using the Systronics 2205 double beam UV-Visible spectrophotometer. The change in the color from pink to yellow is directly proportional to the scavenging of the DPPH radical. Ascorbic acid was used as standard. 1 ml of the solvent and 3 ml of the DPPH solution serve as blank.

Ferric Reducing Antioxidant Power (FRAP Assay)
FRAP assay was carried out according to standard methodology with some modification (30). Stock solution include 300 mM acetate buffer (pH 3.6), 40 mM HCl solution, 20 mM FeCl3.6H2O (Ferric chloride hexahydrate) solution, 10 mM TPTZ (2,4,6-tri-(2pyridyl)-1,3,5-triazine) solution. Working FRAP reagent was prepared by mixing Acetate buffer, ferric chloride hexahydrate solution and TPTZ solution in the 10:1:1 ratio. Antioxidant potential was evaluated by reacting 1 ml of extract (500 µg/ml) and 10 ml of working FRAP reagent. The absorbance of the coloured product was taken at 593 nm after incubating at 37 °C after 30 min. 1 ml methanol and 10 ml working FRAP reagent act as the control. Ascorbic acid was used as standard. carbohydrates, fat as well as fibre, which constitutes the major essential for the living being. On the basis of the above values, the nutritive value of the seed was calculated and its nutritive value is found to be 341.991±2.106 Kcal/100 gm. High nutritive value is suggestive that plum seed can be the source of the feed and fodder. Table 3, displays the yield of the extract of different solvents based on the total material. Petroleum ether, diethyl ether, ethyl acetate, methanol and water yield 13.752%, 10.045%, 7.251%, 11.934% and 14.625% respectively concerning the total material. Fig. 1, represents the yield of the different obtained extracts based on total extract. Table 4, representing the results of the qualitative phytochemical screening. Phytochemical screening has shown the presence of carbohydrates, glycosides, protein, steroids and terpenoids, fixed oil and fats, flavonoids and phenolic compounds. Bioactivity and the therapeutic value of the plant extract are attributed to the phytoconstituents of the plant extract.  Table 6, depicts the results of the DPPH free radical scavenging assay. The IC50 (concentration of the plant extract required to scavenge the 50% of DPPH concentration) values of diethyl ether, ethyl acetate, methanol and aqueous fractions are 3730.567±0.914 µg/ml, 1837.399±0.377 µg/ml, 2520.596±0.398 µg/ml, 3490.380±0.777 µg/ml respectively whereas the IC50 value of the petroleum ether fraction was beyond our dilutions prepared and IC50 value of ascorbic acid was determined in the same way as that of the sample and it is found to be 13.296±0.075 µg/ml. Fig.  2, shows the variation of the percentage inhibition against the concentration of the plant extract and standard.

Conclusion
Various experiments conducted on the seed of this plant revealed it to be an excellent source of energy because of its excellent nutritional value. Phytochemical screening shows the presence of many   Aqueous secondary metabolites. The total phenolic content and the antioxidant study revealed that the seed of the P. domestica has antioxidant potential. Total phenolic content in plant extract is responsible for its various biological activities including antioxidant activity. We have found a strong correlation between the total phenolic content and the antioxidant activity by the DPPH method. The antioxidant potential is attributed to phenolic content, plays an important role in drug development and supplements, which could be very important in preventing or decreasing the rate of oxidative stress.  Each experiment was performed in triplicate. All the values are represented as the mean ± SD. Each experiment was performed in triplicate. All the values are represented as the mean ± SD.