Antioxidant , antimicrobial activity and phytochemical analysis of the seeds extract of Cucumis sativus Linn

The prehistoric interaction of plants and human is being strengthened by the remarkable use of plants as remedy against diseases. Cucumis sativus L. is extensively cultivated globally including Pakistan. The present study was carried out to find the antioxidant, antimicrobial and phytochemical analysis of Cucumis sativus seed extract. Phytochemical analysis of ethanolic extract revealed the presence of flavonoids, terpenoids, tannins, cardiac glycoside, phenols and carbohydrates. 2,2-diphenyl-1-picrylhydrazyl (DPPH) method was used for antioxidant activity. Crude ethanolic extract showed maximum DPPH scavenging activity of 46.05±1.23 at 500μg/ml, while ascorbic acid showed 92.5%. For Antimicrobial activity against selected bacteria and fungi by agar well diffusion method was used. In the antibacterial activity, crude ethanolic extract was most active against Staphylococcus aureus (21.5mm) and less against Shigella flexneri (17.0mm). The n-haxane fraction was highly active against Salmonella typhi (26mm), DCM against E. coli (16.25mm) and Salmonella typhi showed 16.0mm inhibition with ethyl acetate. Crude extract of ethanol was tested against Alternaria, Acremonium, Verticellium, Pythium and Tricoderma sps. Alternaria showed low zone of inhibition (08mm) while the rest of fungi were highly susceptible with zone of inhibition 15mm, 14mm, 17mm and 15mm respectively. The Pythium spp. was highly susceptible to nhaxane fraction (20.00mm), Acremonium to DCM fraction (20.00mm) and to ethyl acetate (16.00 mm). The results showed significant antimicrobial and antioxidant activity which might be because of flavonoids, terpenoids, tannins and phenols in there, suggesting that C. sativus should be used as an active nutrition to control microbial infections leading to chronic diseases.


Introduction
Plants are basic therapy springs for diseases that can be traced back to time immemorial.Having little or no harm effects of plant materials, therefore herbs based medicines flourished now a days.Plants possess specific biochemical components which exerts curative effects on living organism to treat various diseases.[1].The concrete significance of the medicinal plants is because of antioxidants and antimicrobials properties [2, 3].Microbes become resistant to the available sources of drugs, novel plant sources are needed to handle these resistant strains.It is needed to comprehend the genetics of the resistant microbes for developing new drugs better than the existing ones to reduce threat of infectious diseases. .Plant sources possess chemicals like alkaloids, glycosides, flavonoids, terpenoids etc. in plenty which are potential sources of biological porperties [4].There is an inborn antioxidative mechanism in the human body and many natural activities such as the anticarcinogenic and anti-aging responses, initiate due to antioxidants.Antioxidants soothes or disable free radicals, earlier their attack to the target cells.Disease occurred due to production of free radicals in living system which damage it causing serious and long lasting diseases like diabetes, ageing, low immune system neuro-degeneration etc.The diseased body needed antioxidants in the form of proper nutritional and medicinal supplements to overcome the threat.Plant based research showed that antioxidants are there in the form of chemical components like phenols, flavonoids, proanthocynidine etc. Plants based antioxidants enhances the defense against diseases and slows down the aging process.Natural antioxidants reduces the risks of infectious and chronic diseases .Due to marvelous prospective of the components against prolonged diseases, various researches have proved the energetic character of fruits and vegetables as antioxidants to preserve physique utility in regular manner [25].The purpose of the present project was to explore the underlying curative potentials of the plant in the form of antioxidant, antibacterial, antifungal and preliminarily phytochemical screening of seeds of Cucumis sativus.

Plant Collection
Fresh seeds of Cucumis sativus were collected from ripened dried fruit of the plant.These were properly dried and seed coats were removed from all seeds carefully.Dried seeds were grounded with the help of pistil and mortar to get powdered material.

Preparation of crude extract and its fractions
Extraction is the process in which desired plant tissues will be soaked in solvent for a certain time period, from which medicinally active principles are dissolved in the solvent leaving undissolved materials.In this study 70% ethanol were used for extraction.The grounded material was soaked in 70% ethanol and kept at room temperature for 3-7 days.Filtrate was collected and was concentrated with the help of rotary evaporator.The concentrated crude extract was like a gum.This was the crude ethanolic extract and further fractionation was carried out, subjecting the crude sample to extraction with n-hexane, Dichloromethane and ethyl acetate respectively on polarity bases (Table 1).

Antioxidant assay: Scavenging activity of free radicals
Antioxidant assay of seeds of Cucumis sativus was calculated using 1, 1-diphenyl-2-picryl hydrazyl (DPPH) assay following the method of Ahmad and Patel & Patel [31, 32].0.1 mM concentration of DPPH in methanol was arranged.1ml of this concentration was used in 3ml of crude extract to different concentration (250 & 500µg/ml).This mixture was shaken briskly and stand at room temperature for 30 minutes.Then by using spectrophotometer, absorbance was calculated at 517 nm.Ascorbic acid was used as standard.The percent DPPH was measured by using the following equation: DPPH scavenging effect (%) or Percent inhibition = A0 -A 1 / A0× 100.Where A0 was the Absorbance of control reaction and A1 was the Absorbance in the presence of the test or standard sample [33].

Results and discussion
Antimicrobial activity of the crude ethanolic extract and three fractions n-hexane, dichloromethane and ethyl acetate were studied against five bacterial and five fungal strains.Extracts showed potent activity against all the microorganisms.Antimicrobial potential was assessed by recording the zone of inhibition of the microbial growth in mm.The (Table 2) showed antibacterial activity and (Table 3) shows antifungal activity.It is reported that medicinal plants can be good sources of antibacterial agents [34] The microbic susceptibility to different extract of the seeds of Cucumis sativus, expresses its anti-infection ability.All the solvent extracts of the seeds of C. sativus was found active against the tested organisms, which indicated the presence of broad spectrum antibiotic compounds.These extracts also showed active potentials against tested fungus which revealed the presence of potent antimicrobial constituents in all the solvent extracts.Regular intake of this fruit may decrease the possibilities of infections.Preliminary phytochemical analysis showed (Table 4) the presence of Alkaloids, Flavonoids, Terpenoids, Tannin, Cardiac glycosides, phenols and Carbohydrates.The ethanolic extracts of seeds of C. sativus was screened for different Phytochemicals like anthraquinones, alkaloids, steroids, tannin, terpenoid, flavonoids, saponin and cardiac glycosides.The presence of flavonoids, cardiac glycosides, tannin and saponin as shown in (Table 4), explain its dietary and therapeutic significance.The present study is in accordance with the previous work and the slight change may be because of soil mineral composition, cultivation and climatic differences [18, 43-45].
Antioxidants help to protect living system from being damage.Fruits, vegetables and other wild plants are good sources of these antioxidants [46, 47].In vitro, free radical scavenging effects of C. sativus was evaluated by DPPH method, ascorbic acid was used as standard and a spectrophotometer was uses at 517 nm.The results showed (Table 5) that at 125 µg/ml conc.the effect was 28.1%, 250 µg/ml concentration the effect was 32.0% and at 500µg/ml, it was 46.05%.So, the extract showed having a clear effect on scavenging the free radicals.Free radicals are said to be actively involved in many chronic diseases, such as cardiac and cancer among others [18].The antioxidants, hunt the free radicals which formed in living organisms.The antioxidative ability of natural products has been extensively studied by DPPH test.From the preliminary phytochemical screening of the seeds ethanolic extract of Cucumis sativus showed to consist of glycosides, steroids, carbohydrates, saponin and tannins, it is concluded that, the occurrence of flavonoids & tannins in the extract proposes that these complexes might be responsible for scavenging free radicals and said to be as an effective antioxidant [48, 49].

Table 1 . Amount of different extracts of seeds of Cucumis sativus Linn
Method described bySamie et al. [27]was used for activity.Using the micropipette, 100μl of different fungal cultures were spread over the agar plates with the help of inoculating loop in double distil water.Using a sterile cork borer, hole) were made in each of the culture plates.75μl of crude and different fractions of Cucumis sativus extract was poured added and inoculated plates were incubated at 28 o C for one day or two day showing clear inhibitions.Inhibiting zones were calculated in millimeter, indicating the activity.Each test was triplicated and standard deviation was calculated.

36, 37].
. In the antibacterial activity, the crude extract was the most active against Escherichia coli, Staphylococcus aurous (22.5mm) and less against Shigella flexneri (17.0mm).The n-hexane fraction was the most active against Salmonella typhi (26.0mm),DCM was 16.25mm against Escherichia coli and Salmonella typhi showed 16.0mm zone of inhibition with ethyl acetate fraction.Similarly ethanol and methanol extracts of P. emblica reduces the growth of bacteria [35].The bacterial organism were susceptible to medicinal plant extracts.It is reported that as compared to gram positive, gram negative bacterial strains are very vulnerable to plant extracts [