IN VITRO SCREENING OF ANTIOXIDANT AND ANTIAGING POTENTIAL OF CUCUMIS SATIVUS FRUIT EXTRACT

Objective: The present study was designed to screen the anti-aging and anti-wrinkle potential of Cucumis sativus fruit through in vitro estimation of antioxidant, anti-hyaluronidase, anti-elastase, anti-collagenase/anti-matrix metalloproteinase (MMP)-1, and anti-tyrosinase activity. 
Methods: Raw juice of cucumber was taken, filtered and fractionated with ethyl acetate and n-butanol. The obtained extracts were then evaluated for their antioxidant potential through 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging assay taking ascorbic acid as positive control and other enzymatic activities in reference to hyaluronidase inhibition, MMP-1/collagenase inhibition, and elastase inhibition taking catechin as reference standard whereas for tyrosinase inhibition the standard used was quercetin. 
Results: All the evaluations were performed in triplicates and results were noted down. It was observed that aqueous extract of C. sativus fruits showed a maximum DPPH radical scavenging activity (p<0.0001), half-maximal inhibitory concentration (IC50) at a concentration of 122.67 μg/ml. The ethyl acetate fraction of C. sativus fruits exhibited maximum hyaluronidase (p<0.0001), MMP-1/collagenase (p<0.04), and tyrosinase (p<0.04) inhibitory activity, IC50 at a concentration of 59.54, 45.79, and 24.46 μg/ml, respectively. The elastase (p<0.0001) inhibitory activity by n-butanol fraction of C. sativus fruits extract was maximum, IC50 at a concentration of 52.76 μg/ml. 
Conclusion: A potent anti-aging and anti-wrinkle properties were well demonstrated by C. sativus, as depicted from the results obtained.


INTRODUCTION
Aging is a progressive process influenced by both intrinsic and chronological factors and extrinsic or environmental factors which include ultraviolet (UV) rays, air pollution, stress, or smoking [1]. Excessive exposure to UV rays results in the formation of reactive oxygen species (ROS) which causes the adverse effects on the dermal and epidermal connective tissues leading to the damage to cell and cell membranes [2]. Skin physiological changes occurring due to the acceleration of the free radicals, cell contents, and lipid peroxidation stimulated by high exposure to UV radiation directly cause skin aging [3]. Skin shows rugged appearance, hyperpigmentation, dark spots, deep wrinkles, etc. [4]. Hyaluronic acid, elastin, and collagen are important for maintaining skin structure, moisture content, elasticity, and strength of connective tissue of the skin which is decreased in the aging skin [5][6][7]. The increase in the activity of hyaluronidase, elastase, and collagenase/matrix metalloproteinase (MMP)-1 is responsible for decrease in the strength of connective tissue, water holding capacity of the skin, and elasticity of skin. Excessive exposure to UV radiations also results in the development of dark spots which may be due to the overproduction of melanin [8]. Tyrosinase is the main enzyme that catalysis melanin synthesis in melanocytes [9]. Inhibition of these enzymes plays an important role in skin aging. Since traditional medicines and plants provide an extensively large unexplored source for the development of new, potent, and safe cosmetic and skincare products, researchers have paid attention on the exploration of plants and plant extracts for combating skin aging [2]. In the present study, Cucumis sativus commonly known as cucumber was studied for its antiaging and anti-wrinkle potential. It is a commonly used vegetable crop which belongs to the Cucurbitaceae family [10]. Conventionally, it has been used widely for various skin problems and applied topically for swollen eyes, burns, dermatitis, antioxidant, skin whitening and antiwrinkle effect, analgesic, anticancer, and hypoglycemic activity [11,12].

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in juicer. The juice of fruits was fractionated with ethyl acetate and n-butanol (150 ml each) and separated the resultant portions using separating funnel and aqueous portion left in the funnel. The obtained fractions were evaporated and concentrated to get the respective extracts. The extractive values of all the fractions are depicted in Table 1.

DPPH radical scavenging activity
The antioxidant potential of the extract was based on the inhibitory potential of the stable DPPH free radical and the procedure followed was as explained by Braca et al. [14] with little changes. The extracts of C. sativus were prepared in a dilution series (100, 200, and 300 µg/ml) in DMSO. The reaction mixture consisted of 0.1mL test sample with 0.2mL DPPH solution (0.15 mM in 80% methanol solution). The final reaction mixture was shaken dynamically and kept at room temperature for 30 min in the dark. Ascorbic acid was used as a standard. The evaluations were done spectrophotometrically (using ELISA reader) at 517 nm and the % scavenging potential was calculated by the given formula: Inhibitory activity (%) = (Abs control-Abs sample\Abs control)×100.
Where Abs is the absorbance The antioxidant potential of the extracts was demonstrated as halfmaximal inhibitory concentration (IC 50 ) (a concentration [µg/ml] which 50% reduction in the formation of DPPH free radicals are known as IC 50 value of any drug or plant extract). All the observations were taken in the triplicates, graph was plotted by average.

Hyaluronidase inhibitory activity
Fifty microliter bovine hyaluronidase (prepared by diffusing 7900 units/ml of enzyme in 0.1 M acetate buffer; pH 3.5) was added to 50 µl of different dilutions of test extract prepared using 5% DMSO and incubated at 37°C for 20 min. In the control group, 50 µl of DMSO was added instead of plant extract. Hyaluronidase was activated by the addition of 50µl of calcium chloride (12.5 mM) in reaction mixture and the whole mixture was incubated for 20 min at 37°C. The obtained Ca+2 activated hyaluronidase was then treated with 250 µl of sodium hyaluronate (0.1 M acetate buffer; pH 3.5, was used to diffuse 1.2 mg/ml) and kept for 3 min on a water bath at 100°C. The reaction mixture was then cooled to room temperature and added 1.5 ml of PDMAB ([4 g] dissolved in glacial acetic acid [350 ml] and 10 N HCl [50 ml]). The reaction mixture was then kept for 20 min at room temperature [15]. The absorbance was measured at 585nm. Inhibitory effect was calculated as:

MMP-1/Collagenase inhibitory activity
MMP-1/collagenase inhibition assay was carried out on the basis of the method discussed by Kim et al. [16] It involves the use of collagenase (0.8 units/ml) from C. histolyticum and 2 mM of synthetic substrate, FALGPA. The final reaction mixture contained 25 µl of 50 mM tricine buffer, 25 µl of the test sample, and 25 µl of collagenase enzyme (0.1 units). After the addition of 50 µl of 2 mM FALGPA, absorbance was immediately measured at 340 nm by ELISA reader. Catechin was used as a standard. The % enzyme inhibition was calculated by the given formula:

Elastase inhibitory activity
Elastase inhibition activity was analyzed by the procedure discussed by Pientaweeratch et al. [17] Mix 0.1 ml of a 0.2 M Tris-HCl buffer having 1% albumin, 0.025 ml of 10 mM MAAPVN and 0.05 ml of the sample of different concentrations (20 µg/ml, 40 µg/ml, 60 µg/ml, 80 µg/ml, and 100 µg/ml). Then add 0.025 ml of elastase (3 units/ml) to each test sample. Incubate the resultant reaction mixture at 25°C for 20 min and measure the absorbance by ELISA reader at 410 nm. Percentage inhibition was calculated using following formula: Where, A and B represent the absorbance without a test sample after and before incubation, respectively, C and D represent the absorbance with a test sample after and before incubation, respectively.

Tyrosinase inhibitory activity
The determination of tyrosinase was performed using L-DOPA as substrate [18][19][20][21]. First, dissolve 0.8 ml of L-DOPA (2.5 mM) with 2.4 ml of phosphate buffer solution (0.067 M) of pH 6.6. Then incubate the reaction mixture for 10 min at 37sC. After this, 0.8 ml of 2 mg/ml extracts and 0.8 ml tyrosinase solution were mixed. The solution was instantaneously examined for the generation of dopachrome by estimating the linear rise in optical density for 5 min at 475 nm. The observations were taken in triplicates. The tyrosinase inhibitory activity was estimated by the formula: Where, A and B are the absorbance of a sample with L-DOPA and without L-DOPA, respectively, C is the absorbance without sample and with substrate, and D is the absorbance without both sample and substrate.

Statistical analysis
Data are expressed as IC 50 . Linear regression was used to determine the IC 50 values.

Determination of extractive value
In the present study, the fruits of C. sativus were collected and juice was prepared followed by fractionation using ethyl acetate and n-butanol. The extracts were dried; stored and extractive value was evaluated. The results are summarized in Table 1.

In vitro DPPH radical scavenging activity
The absorbance of all extracts was measured at 517 nm for different concentrations. The percentage antioxidant activity of ethyl acetate, N-butanol, and aqueous extract of C. sativus at 300 µg/ml exhibited the maximum antioxidant potential of 47.13, 49.64, and 72.40 µg/ml, respectively, while ascorbic acid at 300 µg/ml concentration exhibited the antioxidant potential of 91.11. C. sativus extracts (ethyl acetate, N-butanol, and aqueous) have dose-dependent significant (p<0.0001) DPPH radical inhibitory activity with IC 50 value, at a concentration of 358.04, 294.64, and 122.67 µg/ml, respectively, in comparison to ascorbic acid (IC 50 ) at 24.57µg/ml. Fig. 1 shows the DPPH radical inhibitory potential of C. sativus extracts as compared to ascorbic acid.

Hyaluronidase inhibitory activity
The absorbance of all extracts was read at 585 nm for different concentrations and the percentage of the total anti-hyaluronidase potential of ethyl acetate, n-butanol, and aqueous extracts of C. sativus was calculated. The ethyl acetate, n-butanol, and aqueous extracts of C. sativus at 100 µg/ml exhibited a maximum total hyaluronidase inhibition (Fig. 2)

Elastase inhibitory activity
The absorbance of all extracts was read at 410 nm for different concentrations and the percentage of total anti-elastase potential of ethyl acetate, n-butanol, and aqueous extracts of C. sativus was calculated. The ethyl acetate, n-butanol, and aqueous extracts of C. sativus at 100 µg/ml exhibited a maximum total elastase inhibition ( Fig. 3)

MMP-1/Collagenase inhibitory activity
The absorbance of all extracts was read at 340 nm for different concentrations and the percentage of the total anti-collagenase potential of ethyl acetate, n-butanol, and aqueous extracts of C. sativus was calculated. The ethyl acetate, n-butanol, and aqueous extracts of C. sativus at 100 µg/ml exhibited a maximum total Collagenase inhibition ( Fig. 4) Table 2.

DISCUSSION
Photoaging is mainly stimulated by the excessive exposure of skin to UV radiations, which causes the release of ROS, thereby rendering

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severe damage to proteins, lipids, and DNA. C. sativus flesh mainly consists of water, ascorbic acid (Vitamin C), gallic acid, caffeic acid, flavonoids, phenolic compounds, etc., and is known to be used in many skincare products to reduce the signs of skin aging and relieve skin irritations [22]. In the present study, the anti-aging and anti-wrinkle potential of C. sativus fruit was studied through in vitro estimation of antioxidant, anti-hyaluronidase, anti-elastase, anti-collagenase/anti-MMP, and anti-tyrosinase activity. The aqueous extract of C. sativus was fractionated into ethyl acetate and n-butanol fraction to correlate the possible constituents responsible for the observed activities. The maximum antioxidant activity was exhibited by the aqueous fraction as it is rich in ascorbic acid, cucurbitacin a, b, c, and 96% water. The ethyl acetate fraction contains organic compound such as citric acid, malic acid, gallic acid, caffeic acid, and α-linoleic acid and may be responsible for the anti-hyaluronidase, anti-collagenase/anti-MMP, and anti-tyrosinase activity. The elastase inhibition was maximum shown by the n-butanol fraction. This may be attributed to the presence of phenolic compound such as p-hydroxybenzoic acid, hydroxycinnamic acid, flavones, β-carotene, flavanols, squalene, and β-sitosterol. The presence of carotenoids, phenolic flavonoids, tannins, polyphenols, and lycopene was confirmed by the various phytochemical screening studies carried out by different researchers [23][24][25]. The main acids found are citric and malic acids. Phenolic compounds present are p-hydroxybenzoic acid, hydroxycinnamic acid, flavones, and flavonols. Cucumber is rich in Vitamin C and Vitamin B [26].