Identification of Secondary Metabolites, Antimicrobial and Antioxidant Activity of Grape Fruit (Vitis vinifera) Skin Extract

Plant collection

Edible V. vinifera or black grapes variety is largely cultivated in household gardening. The black grapes are widely grown in the State of Tamil Nadu, India. The grapes were collected from a Kamaraj market in Thanjavur, Tamil Nadu, India.

Extraction

Fresh and healthy plant fruits of V. venifera were washed thoroughly with running tap water and grape skin were peeled and allowed to natural drying under shade for several days. Finally the dried material was pulverized to powdering form. The 20g of pulverized sample was dissolved in 200ml of distilled water, heated at 80°C for 30min and filtered using Whattman No.1 filter paper to obtain the extract. The filtrates were then lyophilized by freeze drying technique using Christ Alpha 1-2 LD Freeze Dryer.

Phytochemical screening

The preliminary phytochemical assays were carried out for the detection of secondary metabolites such as polyphenols, flavonoids; alkaloids, terpenoids, steroids, tannins, saponins, and cardiac glycosides were profiled using standard phytochemical methods [9, 10].

Antibacterial activity

The Aqueous extracts of grape skin were screened for the antibacterial activity against gram-negative bacteria such as Aeromonas hydrophilic, Escherichia coli, Salmonella typhi O, Salmonella typhi H, Klebsiella pneumonia and gram-positive bacteria viz., Bacillus subtillis, Staphylococcus aureus using agar-well diffusion method [11]. The antibacterial activity was presented by minimum inhibitory concentration through zone of inhibition (mm).

GC MS analysis of grape skin

The Gas chromatography mass spectroscopy analysis of the grape skin aqueous extract was performed by Shimadzu GCMS - QP2010. The inert gas helium (99.9995%) is used as carrier gas. The ionization and flow through the column was fixed as 1.0ml/min. The sample split ration was set as 10:1; the injector volume is set as 1.0 µL. The column used is fused capillary silica (30m × 0.25mm × 0.25 µm) column. The temperature was programmed as injector: 260°C, detector: v 300°C, column: 70°C, 10°C min-1, 260°C (10min). The total GC run time is up to 2 hours. The Mass range was m/z 40-1000 at scan interval of 0.5s, speed of 2000 amu s-1 and detector voltage of 1.0kV. The post run was analyzed in the Nits library database provided in the GCMS system [12].

Fourier transform infrared (FTIR) analysis

FTIR spectrophotometer was used to identify the characteristic functional groups in the grape skin extracts. The aqueous extract (5.0mg), were added with potassium bromide (KBr) in a mortar and pressed at pressure of 6 bars within 2min in order to prepare a thin translucent sample discs. The FTIR spectrum was obtained using Perkin Elmer 2000 spectrophotometer system with a scan range from 400 to 4000 cm−1 [13, 14].

In vitro antioxidant activity

The Stable 1, 1-diphenyl-2-picryl hydrazine (DPPH) free radical scavenging activity of the AE was measured according to the methods of Blois, (2001) but with minor modifications. One mL of 0.2 mM DPPH solution in methanol was mixed with the 1mL extracts of 50, 100, 250, 500, and 1000 _µ_g/ml The mixture was incubated in dark for 20min at 27°C and the absorbance was measured at 517nm. The free radical scavenging activity was determined by comparing its absorbance with that of a blank solution. Ascorbic acid was used as a standard. The ability to scavenge the DPPH radical was calculated using the following equation [15, 16, 17, 18, 19], A=13, A=20.

Antimicrobial activity and photochemistry of grape fruit skin aqueous extract

The antibacterial efficacy of Grape Skin aqueous Extract was presented in (Table 1). The aqueous extract of grape skin was found to inhibit all the screened broad spectrum bacteria with notable inhibitory activity. Among bacteria, significant inhibition was observed in S. aureus, K. Pneumonia and Salmonella typhi O & H whereas E. coli was inhibited to least extent. The positive control Ciproflacin showed higher antibacterial activity than the grapes skin extract. The extracts showed good bactericidal activity against both the gram-negative and gram-positive bacteria. The preliminary photochemical studies of AE of grape skin extract was profiled and presented in the (Table 2). The phytochemical profile showed the presence of alkaloids, carbohydrates, flavonoids, phenols, proteins, terpenoids, tannins, and sterols. Saponins and cardiac glycosides were found to be present in the aqueous extract of grape skin.

(+) denotes presence of the secondary metabolites, (-) absence of the secondary Metabolites.

GC MS analysis

Click to enlarge

Figure 1 a&b: Identification of compounds from Grape skin aqueous extract using Gas Chromatography Mass Spectroscopy The GCMS analysis of the grape skin aqueous extract was displayed in the (Figure 1a&b). The peaks obtained in the GCMS spectrum showed the presence of 17 vital bioactivity rich compounds. The compounds were found as α-Tocopherol, β-D-mannoside, Lupenone, glycerin, cymene, ethyl Oleate, L-glucose, norolean-12-ene, Palmitic acid, Cis oleic acid, norolean-12-ene , ϒ-sitosterol, D- Allose ,(+-)-p-menth-1-en-4-ol, Cis-Dimethyl morph line, flavon-3-ol, Procyanidin B1, Lano sterol, stigma sterol, Procyanidin B2 and Procyanidin C1. Previous reports revealed that, the skin and seeds of grapes are known to be rich sources of phenolic compounds, both flavonoids and non-flavonoids [20, 21].

Fourier transform infrared fingerprint analysis

The FTIR spectroscopic studies revealed the presence of various functional groups by peak observed at 665.4, 1030, 1408, 1659, 2060, 2523, 2954 and 3385 presented in the (Figure 2) (Table 3). The respective peaks expressed the chemical group such as alkyl, ketone, aldehyde, carboxylic acids, esters, and amide in aqueous extracts of V. vinifera fruit skin [22].

In vitro antioxidant activity

The free radical scavenging activity of grape fruit skin extracts were determined by the DPPH method. Antioxidant molecules can quench DPPH free radicals and convert them to a colorless product, resulting in a decrease in absorbance at 517nm. In our study, the grape skin extracts exhibited significant scavenging activity when compared with standard ascorbic acid (Table 4). The IC50 values of grape skin extract was observed to be

Grapes are one of the palatable, sweet in taste and exceedingly nutritious rich in flavonoids, polyphenols and vitamin, consumers from all parts of the world devour this organic product to a great extent. Yet, the assessment of the medical advantages was less. Therefore the present study showed that grape skin extracts contained substantial bioactive compounds, which possessed antimicrobial, antioxidant and anti-obesity properties.