Indigenous vegetables of family Cucurbitaceae of Azad Kashmir: A key emphasis on their pharmacological potential.

The antioxidant capacity of extracts of different parts of Cucurbitaceae vegetables was evaluated by DPPH (2, 2-diphenyl-1-picrylhydrazyl) and ABTS (2, 2'-azino bis (ethyl benzothiazoline 6)-sulphonic acid) methods. Total phenolic content (TPC) and total flavonoid content (TFC) were also determined. The correlation of TPC, TFC, DPPH, and ABTS in different extracts of Cucurbitaceae vegetables was analyzed. The peel extracts of studied vegetables had the highest TPC, (C. grandis 3.00±0.86, T. cucumerina 3.24±0.70 and C. moschata 3.12±0.06 mg gallic acid equivalent (GAE) g-1 DW) and TFC (C. grandis 18.96±1.5, T. cucumerina 13.92±1.41 and C. moschata 15.31±0.97 mg rutin equivalent (RE) g-1 DW). The maximum antioxidant potential was obtained by the ABTS method in peel extracts of C. grandis (78.7%) and C. moschata (63.5%) while in pulp extract of T. cucumerina (50.1%) at 10 μg/mL. The percent radical scavenging activity (% RSA) by the DPPH method found maximum for peel and pulp of C. grandis (45.15 and 45.15%, respectively) and peel of T. cucumerina (45.15%) and C. moschata (34.15%). The EC50 obtained in the ABTS method was 0.54 and 7.15 μg/mL for C. grandis and C. moschata, respectively while 0.81 μg/mL for the pulp of T. cucumerina compared to standard ascorbic acid (1.05 μg/mL). The EC50 calculated in the DPPH method was 11.78 μg/mL, 13.34 μg/mL, and 21.00 μg/mL for C. grandis, T. cucumerina, and C. moschata peel respectively compared to the standard Butylated hydroxytoluene (BHT). Among each variable, the correlation between ABTS and TPC provided the highest positive correlation (r = 0.998, p< 0.05) in peel extracts.

1. Introduction

Intake of natural phenolic antioxidants in the diet has a positive correlation with reduced cancer mortality and heart diseases, and longer life expectancy [1-6]. These antioxidants are responsible to nurture immune function, inhibiting malignant transformation, reduce DNA damage and lipid peroxidation [7]. Recently, growing attention has been given to screening out the natural sources of antioxidants like fruits and vegetables compared with synthetic materials [3,6,8-10]. Synthetic antioxidants like butylated hydroxytoluene (BHT) are reported to be carcinogenic and have various side effects [11].

Diseased people need to intake proper foods with functional activities for the fulfillment of their energy and nutritional needs [12]. Vegetables are the cheapest and most easily available source of nutrition which contain bioactive components including minerals, dietary fiber, and vitamins (A, C & E) as well as non-nutritive phytochemicals (carotenoids, alkaloids, terpenoids, bioactive peptides, phenolic compounds, and flavonoids) which reduce the risk of cardiovascular diseases, cancer, obesity, and diabetes [3]. Phenols, flavonoids, and vitamins were reported to contain antioxidant properties [13]. Various researchers have reported the antioxidant properties of vegetables due to the presence of phenolic content in them. However, the extent of antioxidant properties may be affected by many factors including the degree of ripeness, climatic, storage, and geographical conditions [14].

Cucurbitaceae is a large family of plants, with 130 genera and 800 species distributed in the tropical and sub-tropical regions of the world. This family is cultivated all around the world in a variety of environmental conditions having global pharmacological and dietary importance. The main producers of Cucurbitaceae are Turkey, China, India, and the USA [15]. The majority of the plants of Cucurbitaceae has medicinal importance and are used as a medicine for the remedy for ages, urinary ailments, intestinal worms, high blood pressure, kidney stone, headaches, abdominal tumors, fever, diarrhea, and skin allergies [16].

Cucurbita moschata is a species of the Cucurbitaceae family cultivated all around the world. It is commonly called squash or pumpkin. The pulp of Cucurbita moschata provides a cheaper and good source of food, having health benefits. The seeds of squash contain approximately 15.9 mg/100 g of total tocopherols, and a sufficient quantity of linoleic acid and L-tryptophan, which are largely used for the treatment of depression [17]. Trichosanthes cucumerina is another species widely distributed in the tropical and subtropical regions. It is commonly called snake guard, while the Coccinia grandis is species that grows only in the tropical region and is called ivy gourd. These two species are eaten as vegetables containing a large amount of antioxidants, anti-hypoglycemic agents, and immune systems modulators. Traditionally, these vegetables were largely used to treat leprosy, fever, asthma, bronchitis, scabies, and jaundice [18]. The main chemical constituents present in the Cucurbitaceae family are phytochemicals. The other commonly occurring compounds are carbohydrates, triterpenes, sterols, alkaloids, α-carotene, β-carotene, and lutein zeaxanthin [19].

Various methods are commonly used to measure the antioxidant activities of vegetables, including 2, 2-diphenyl-1-picrylhydrazyl radicals (DPPH) method, ferric reducing antioxidant power (FRAP) method, 2, 2´-azino-bis (3-ethylbenzthio-zoline-6)-sulphonic acid (ABTS) method, and oxygen radical absorbance capacity assay (ORAC) method [19]. Of all these methods, the most convenient and independent of expensive equipment are the DPPH and ABTS methods, thus commonly used.

The present work aimed to investigate the phenolic, flavonoid content, and antioxidant potential of various parts (peel, pulp, seeds) of some Cucurbitaceae vegetables that are commonly consumed and locally grown in Muzaffarabad, Azad Jammu & Kashmir Pakistan for their phytochemical and antioxidant behavior. Phytochemicals present in these vegetables play a protective role from oxidative stress and various other related diseases. The present study will provide information about the medicinal importance and use of studied vegetables highlighting their antioxidant properties.

2. Material and methods

2.1 Chemicals and reagents

Folinciocalteu’s reagent, quercetin, gallic acid, phosphate buffer, 5, 6-Diphenyl-3-(2-pyridyl)-1, 2, 4-triazine-4′, 4″-disulfonic acid sodium salt, 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2, 2-diphenyl-1-picrylhydrazyl (DPPH), methanol, butylated hydroxytoluene (BHT), and potassium persulfate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ascorbic acid was obtained from Fluka (Switzerland) while sodium carbonate, sodium nitrite, aluminum chloride, and sodium hydroxide were purchased from Merck (Darmstadt, Germany). Ultrapure water was prepared using a Millipore System (Millipore, Bedford, MA, USA) and used throughout the experiments. All the used standards were stored in dark at -20°C.

2.2 Sample collection

Three locally grown Cucurbitaceae vegetables including Cucurbita moschata (Voucher No. UR-01) Trichosanthes cucumerina (Voucher No. UR-02) and Coccinia grandis (Voucher No. UR-03) were collected from the capital city Muzaffarabad of Azad Jammu and Kashmir state, Pakistan. Voucher specimens were identified by Dr. Tariq Habib, Assistant Professor, Department of Botany, University of Azad Jammu, and Kashmir (UAJ&K), Muzaffarabad, and were deposited in the herbarium of the Department of Botany, UAJ & K, Muzaffarabad.

2.3 Sample preparation

The collected fresh vegetables were washed with distilled water and the required parts including peel, pulp, and seeds were separated and ground into fine particles by using a mechanical grinder. To extract the antioxidant materials from the samples of vegetables, a reported method was applied with slight modification [3]. Briefly, a specific amount of sample (10.0 g) was treated with 100 mL distilled water in a shaking water bath for 30 minutes at 37°C and 100 rpm. The blended mixture was centrifuged at 4, 200 rpm for 30 minutes. The supernatant was collected, and the solvent was evaporated at room temperature to dryness. The obtained residue was weighed to calculate the extractive yield and stored in an airtight jar at -20°C for further use. The solid aqueous extracts were dissolved in distilled deionized water at a concentration of 10 mg/mL for experimental purposes.

2.4 Total phenolic content determination

TPC present in different parts of vegetables was determined by using the Folin-Ciocalteu method [20]. Briefly, 500 μL of the sample extract was mixed with Folin-Ciocalteu reagent (200 μL) and then mixed with 20% solution of sodium carbonate solution (1mL) after 3 minutes. The solution was incubated for one hour in the dark which turned into deep blue coloration. The absorbance of this mixture solution was monitored at 765 nm by using a blank solution prepared under the same protocol except for the extract solution. TPC was calculated from the trend line of standard gallic acid (0.05–0.25 mg/mL) and expressed in mg of GAE/g dry weight of the extract.

2.5 Determination of total flavonoid content

The TFC in vegetable samples was quantified according to the reported method [21]. The sample solution consisting of extract (1 mL), distilled water (2 mL), and 5% sodium nitrite (0.15 mL) was incubated at room temperature for 6 minutes. Aluminum chloride (10%, 0.15 mL) and sodium hydroxide (4%, 2 mL) were added to the sample solution followed by incubating again for 6 minutes at room temperature. The final volume of the sample solution was made up to 10 mL by addition of ultrapure water, mixed thoroughly, and kept for 15 minutes at room temperature. Absorbance was recorded at 510 nm using UV-spectrophotometer (Shimadzu UV 1800) against the blank solution prepared under the same protocol except for the extract solution.

The total flavonoid content present in the peel, pulp, and seeds of Cucurbitaceae vegetables was calculated from the linear equation of quercetin (0.066–0.0166 mg/mL) taken as a standard and the results were represented as mg QE/g dry weight of the extract.

2.6 Antioxidant activities by ABTS method

The ABTS●+ free radical scavenging assay was used to determine the antioxidant potential of extracts [22]. The ABTS●+ free radicals in the ABTS stock solution were generated by mixing potassium persulphate (2.5 mM) and ABTS (7 mM) solution (1:1, v/v) and let to stand at room temperature for 24 hours. The solution was diluted until the absorbance (Ao) of 0.90±0.04 was obtained at 734 nm. Extracts in a range of concentrations (2.0–10.0 μg/mL) were added in ABTS●+ free radical solution and absorbance of sample solution (Ai) was measured. The percentage radical scavenging activity (% RSA) was calculated by using Eq 1, and half-maximal effective concentration (EC50 μg/mL) was calculated by trend line equation taking ascorbic acid as a standard.

Where Ao is the absorbance of the blank solution, Ai is the absorbance of the sample solution.

2.7 Analysis of antioxidant activities by DPPH method

DPPH assay [23] was performed where different concentrations of the extracts (2–10 μg/mL) were added to 1 mL of a 0.008% methanol solution of DPPH. This solution was incubated for 30 minutes at room temperature, and UV absorbance was taken at 517 nm against a blank solution kept in the reference compartment. The control was prepared using the same protocol as described above without any extract. The BHT was used as a positive control. The percentage of DPPH free radical scavenging potential of extracts was calculated using the following equation.

Where Ab is the absorbance of control having all reagents except the extract and As is the absorbance of the test.

2.8 Statistical analysis

All experiments were performed in triplicates and the data were expressed as the mean ± standard error (SD) of three independent results. One-way analysis of variance (ANOVA) with a statistical significance level set at p < 0.05 correlations between the total phenolic, flavonoid content, and antioxidant capacities were made using the Pearson procedure (p < 0.01).

3. Results and discussion

3.1 Extraction yield

The percentage yield of aqueous extracts from the peel, pulp, and seeds of studied Cucurbitaceae vegetables (photographic images are included in Fig 1) were shown in Table 1. The extractive yield varied among all vegetable parts where the peel extracts provided the maximum percentage yield i.e., C. grandis (36.80%), T. cucumerina (43.87%), and C. moschata (29.31%). Reported literature suggests that extractive yield may vary from plant to plant and depends upon the nature of secondary metabolites [24-27].

Fig 1

The whole fruit of a) C. grandis, b) T. cucumerina and c) C. moschata from the Cucurbitaceae family.

Table 1

The percentage yield of extracts from the peel, pulp, and seeds of studied Cucurbitaceae vegetables.

English name of vegetable	Scientific name of vegetable	Part used	% Yield of extracts
Ivy Gourd	Coccinia grandis	Peel	36.80
		Pulp	13.70
		Seed	6.22
Snake Gourd	Trichosanthes cucumerina	Peel	43.87
		Pulp	22.43
		Seed	25.60
Butternut squash	Cucurbita moschata	Peel	29.31
		Pulp	25.56
		Seed	12.42

3.2 Evaluation of total phenolic content

The phenolic content present in plants is responsible to reduce the reactive oxygen [O] via an H atom donated by the phenolic OH group and electrons transfer from phenoxide anions [28]. The polyol enzymes that are necessary for the nutrition and health of humans are also affected by polyphenols [29]. The TPC present in different species of the Cucurbitaceae family was determined by using a standard gallic acid calibration curve and results (expressed in mg GAE/g of dry extract weight) were presented in Table 2. Khatana et al reported that the TPC in the peel of C. moschata extract was 6.4±0.1 mg GAE/g DW [30], which was higher than our results (3.12±0.06 mg GAE/g DW) while in the same study the total phenolic content in pulp extract was less (2.5±0.3 mg GAE/g DW) than our results (2.95±0.04 mg GAE/g DW). Kondhare and Lade reported the total phenolic composition of the aqueous extracts of C. grandis fruit (8.2±0.2 mg GAE/g DW) [31], which were found to be in close agreement with the sum of the phenolic content of peel, pulp, and seed extracts (8.56±2.57 mg GAE/g DW) found in the current study. Higher values of phenolic content (8.18±1.56 mg GAE/g DW) were found in T. cucumerina peel, pulp, and seed extracts collectively when compared to T. cucumerina fruit aqueous extract (4.64±0.3mg GAE/g DW) [32]. The differences in the phenolic composition of the same species reported in different studies could be due to the variation in growing conditions, indicating that phenolic content may vary with variation in geographical region and climatic conditions.

Table 2

TPC and TFC in the aqueous extracts of various parts of studied Cucurbitaceae vegetables.

VegetableScientific name	TPC (mg GAE/g dry extract)			TFC (mg RE/g dry extract)
	Peel	Pulp	Seed	Peel	Pulp	Seed
Coccinia grandis	3.00±0.86	2.87±0.91	2.69±0.83	18.96±1.5	9.50±0.90	7.76±0.20
Trichosanthes cucumerina	3.24±0.70	2.51±0.41	2.43±0.45	13.92±1.41	9.14±0.46	8.73±0.05
Cucurbita moschata	3.12±0.06	2.95±0.04	2.88±1.6	15.31±0.97	14.38±0.73	9.71±0.16

3.3 Evaluation of total flavonoid content

Flavonoids are among the polyphenols, which gained significant interest because of their various biological effects as anti-inflammatory, antimicrobial, anticarcinogenic, antioxidant, and vasorelaxant [33]. Flavonoids have a broad spectrum of antioxidant properties particularly due to radical scavenging activity.

The highest flavonoid content was found in the peel extracts of all studied Cucurbitaceae vegetables (Table 2) where C. grandis, T. cucumerina, and C. moschata exhibited 18.96±1.5, 13.92±1.4, and 15.31±0.97 mg RE/g DW flavonoid content respectively. The lowest flavonoid content was obtained from the seed extracts., C. grandis (7.76±0.20 mg RE/g DW), T. cucumerina (8.73±0.05 mg RE/g DW), and C. moschata (9.71±0.16 mg RE/g DW). No reported literature was available for the total flavonoid content of any species of the Cucurbitaceae family.

3.4 Antioxidant activities by ABTS method

The ABTS method has been broadly used for the assessment of the antioxidant capabilities of natural products. In this method, the dark blue color of 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS•+), is reduced by an antioxidant (which transfer an electron) into colorless ABTS solution. The change in concentration of ABTS•+ after the reduction process is measured spectrophotometrically. In the current study, the antioxidation potential of the peel, pulp, and seed extracts from three Cucurbitaceae vegetables was evaluated and presented in Table 3. The EC50 (concentration of extract scavenging 50% of free radicals) was calculated from the % RSA values by using the linear equation of ascorbic acid taken as a standard (Table 5).

Table 3

Antioxidant activity of aqueous extracts of various parts of studied Cucurbitaceae vegetables evaluated by ABTS●+ radical scavenging method.

Conc e- ntrat ion μg/mL	% ABTS Radical Scavenging Activity
	Ascor bicacid	Coccinia grandis			Trichosanthes cucumerina			Cucurbita moschata
		Peel	Pulp	Seed	Peel	Pulp	Seed	Peel	Pulp	Seed
2.0	53.0±1.05	53.2±0.92	51.4±0.99	25.5±0.32	27.4±0.21	27.6±0.43	14.7±0.09	27.1±0.89	26.9±0.85	4.7±0.03
4.0	68.0±1.08	60.9±0.75	60.8±0.82	27.5±0.40	31.0±0.25	35.1±0.50	15.2±0.02	37.9±0.98	29.7±0.92	10.8±0.32
6.0	80.0±1.20	69.9±0.93	64.9±0.87	28.3±0.69	34.2±0.45	41.0±0.67	18.1±0.53	46.7±1.06	32.6±0.97	17.8±0.50
8.0	92.0±1.21	72.1±1.15	69.9±0.92	30.2±0.72	38.7±0.70	47.4±0.58	19.3±0.45	50.3±1.21	38.8±1.5	20.9±0.65
10.0	98.0±1.30	78.7±1.05	74.4±1.20	32.1±0.77	40.9±0.68	50.1±0.98	20.9±0.91	63.5±1.34	42.2±1.30	25.9±0.89

Table 5

Means EC50 values for ABTS●+ and DPPH radical scavenging potential of the aqueous extracts of various parts of studied Cucurbitaceae vegetables.

Sample	Vegetable Part	EC50 for ABTS●+radical scavenging potential (μg/mL)	EC50 for DPPH radical scavenging potential (μg/mL)
Ascorbic acid	-	1.05	-
BHT	-	-	7.35
Coccinia grandis	Peel	0.54	11.78
	Pulp	0.81	12.10
	Seed	32.76	13.51
Trichosanthes cucumerina	Peel	14.96	13.34
	Pulp	9.40	14.62
	Seed	45.22	17.80
Cucurbita moschata	Peel	7.15	21.0
	Pulp	14.04	22.41
	Seed	18.09	23.41

The highest antioxidant property was presented by the peel extracts of three Cucurbitaceae vegetables. C. grandis and C. moschata exhibited the scavenging activity in the range of 53.2±0.92–78.7±1.05% and 27.1±0.89–63.5±1.34% respectively in the concentration range of 2–10 μg/mL. Whereas T. Cucumber in a pulp extract provided the radical scavenging activity in the range of 27.6±0.43–50.1±0.98% with the same concentrations. A moderate level of % scavenging radical activities was noted in the pulp extracts of C. grandis (51.4±0.99–74.4±1.20%), C. moschata (26.9±0.85–42.2±1.30%), and peel extracts of T. cucumerina (27.4±0.21–40.9±0.68%). The lowest scavenging potential was exhibited by the seed extracts of three studied samples. A similar trend was reported by Xu et al. [34] and Zhang et al. [35] where peel extract provided the maximum antioxidant potential than pulp and seeds. Variation in antioxidant potential among different fruit tissues of the same vegetable could be due to the presence of the maximum amount of phenolic and flavonoid content in the peel.

A group of researchers reported the % ABTS radical scavenging potential of C. grandis whole fruit extracts prepared in different solvents including petroleum ether (43.2±0.2), dichloromethane (61.0±0.5), acetone (57.4±0.4), methanol (95.8±1.0), and water (88.4±0.6) at a concentration of 100 μg/mL [31]. In the present study, almost the same results were observed by 10 μg/mL of peel and pulp extracts indicating that the C. grandis species in the current work has far greater antioxidant potential. This can be due to the various number of antioxidants present in the same species grown in different areas under different conditions. No study was found in the reported literature regarding the % RSA of T. cucumerina and C. moschata measured by the ABTS method.

3.5 Antioxidant activities by DPPH method

The antioxidant ability of any material can be evaluated by various methods, in which the DPPH essay is very simple and rapid. This method is frequently used to assess the ability of a material to scavenge the free radical and provides valuable information about hydrogen donors compounds [36]. Free electron generated by DPPH reagent show absorption peak at 517 nm [37]. However, when the odd electron of the DPPH reagent is reduced by the H atom of any hydrogen donor substance, the purple color of DPPH fades to yellow. The color change is the indication of the generation of reduced DPPH [38]. The extent of decolorization of DPPH indicates the radical-scavenging potential of the antioxidant. The resulting decolorization is stoichiometric with respect to the number of electrons captured. Results of percent scavenging activity and the obtained EC50 values were presented in Tables 4 and 5 respectively.

Table 4

The antioxidant activity of the aqueous extracts of various parts of studied Cucurbitaceae vegetables evaluated by the DPPH radical scavenging assay.

Concentrationμg/mL	% DPPH radical scavenging assay
	Butylated hydroxytoluene (BHT)	Coccinia grandis			Trichosanthes cucumerina			Cucurbita moschata
		Peel	Pulp	Seed	Peel	Pulp	Seed	Peel	Pulp	Seed
2.0	53.0±1.05	22.46±0.21	21.61±0.84	18.69±0.47	28.53±0.34	18.53±0.98	15.3±0.95	22.76±0.51	18.3±0.22	6.23±0.06
4.0	68.0±1.08	29.76±0.61	27.38±0.95	24.69±0.90	30.69±0.55	26.46±0.87	20.84±0.65	25.23±0.59	21.5±0.81	13.07±0.05
6.0	80.0±1.20	33.46±0.70	32.3±1.02	29.76±0.85	32.15±1.01	32.07±1.50	26.15±0.38	28.04±0.98	24.76±0.45	16.3±0.62
8.0	92.0±1.21	39.53±1.09	37.0±1.15	34.46±0.55	38.38±0.65	35.15±1.00	29.76±0.78	31.26±1.02	27.76±0.90	19.01±0.59
10.0	98.0±1.30	45.15±1.10	45.15±1.08	40.84±1.09	45.15±1.18	37.46±1.20	32.15±1.15	34.22±1.05	30.65±1.11	23.05±0.43

DPPH radical scavenging ability increased as the concentration of extracts was increased. The percent DPPH radical scavenging potential (% RSA) of peel extracts of all studied vegetables was found to be maximum which might be attributed to the greater amount of phenols and flavonoids present in peel extracts. The % RSA of the aqueous peel extracts ranged for C. grandis (22.46±0.21–45.15±1.10%), the pulp (21.61±0.84–45.15±1.08%) and seed (18.69±0.47–40.84±1.09%), T. cucumerina peel (28.52±0.34–45.15±1.18%), pulp (18.53±0.98–37.46±1.20%), and seed (15.3±0.95–32.15±1.15%), C. moschata peel (22.76±0.51–34.22±1.05%), pulp (18.3±0.22–30.65±1.11%) and seed (6.23±0.06–23.05±0.43%) at the concentration range of 2.0–10.0 μg/mL. The order for the DPPH radical scavenging activity of the tissues of all studied vegetables was peel > pulp > seed. Liyanage et al reported the DPPH radical scavenging potential of the aqueous extracts of T. Cucumerina fruit, leaves, and flower with the % RSA values of 10.83 ± 0.7, 3.08 ± 0.2, and 4.16 ± 0.1 respectively, at a concentration of 100 μg/mL [32].

3.6 Statistical analysis

To identify the chemical content responsible to give the antioxidant capacity to the vegetables of the Cucurbitaceae family, correlation coefficients among the TPC, TFC, and antioxidant capacity by ABTS and DPPH methods were analyzed. Table 6 compiled the Pearson correlation as indicated with the coefficient of correlation (r) values among IC50. The higher the r-value, the higher the correlation of variables. In the case of peel extract, among each variable, the correlation between ABTS and total phenolic content showed the highest positive correlation (r = 0.998, p<0.05) which was like the study of other researchers [39] for the cucurbit family while in the study of seed the ABTS and TPC had statistically no significant correlation. In the case of the seed sample, a moderate and positive correlation between DPPH radicle scavenging capacity and TPC (r = 0.686, p<0.05) was observed. In another report, the highest and most positive correlation exists between DPPH scavenging capacity and total flavonoid (r = 0.910, p<0.01) for the species of the Cucurbitaceae family [40].

Table 6

Pearson correlation among EC50 of DPPH and ABTS radical scavenging activity and TPCs and TFCs.

Correlation (r) among variable	Sample	TPC	TFC
ABTS	Peel	0.998**(0.030)	-0.954 ns(0.127)
	Pulp	-0.92**(0.02)	-0.915**(0.058)
	Seed	0.260 ns(0.23)	0.984 ns(0.876)
DPPH	Peel	0.234**(0.012)	-0.47 ns(0.93)
	Pulp	0.24**(0.005)	0.87 ns(0.09)
	Seed	0.66**(0.01)	0.959 ns(0.07)

r, correlation coefficient; TPC, total phenolic content; TFC, total flavonoid content; DPPH, DPPH radical scavenging activity; ABTS, 2, 2´-azino-bis (3-ethylbenzthiozoline-6)-sulphonic acid method. The numbers in parentheses are p-values.

**Correlation is significant at the 0.01/0.05 level (two-tailed), ns = not significant.

For peel, ABTS values were correlated with the phenolic content (r = 0.998). The TFC and ABTS in the case of peel were not significantly correlated as p values were higher than the 0.05 significance level. However, studies have been found to report a high correlation between ABTS values with the total phenolic (TP) and total flavonoid (TF) content of other Cucurbitaceae species [39]. The difference may arise due to the difference in specific solvents used. Other compounds could be involved in the antioxidant capacity apart from phenols, such as vitamin C. For pulp the ABTS have negative correlation with TPC and TFC (r = -0.92 and -0.915 respectively). In the case of seed, ABTS was not significantly correlated with the phenolic content and flavonoid content (p> 0.05). For peel, pulp, and seed, DPPH values were correlated with the phenolic content, while TFC was not significantly correlated with the EC50 value of DPPH (p> 0.05). There is no correlation between DPPH and TFC might be due to different mechanism abilities and the use of different standards. While the contradictory report also found such as the antioxidant activities of T. cucumerina well correlated with the amount of total phenolic and flavonoid contents [32]. This study indicated that phenolic compounds significantly affected all antioxidant activities while flavonoids did not contribute significantly.

4. Conclusion

To evaluate the antioxidant capacity of the sample, various methods must be used in parallel. The aqueous extracts of peel, pulp, and seed had the lowest EC50 values in the ABTS method and were considered strong antioxidants. The correlation between ABTS and TPC exhibited the highest positive correlation in peel extracts. Phenolic compounds were the major contributors to ABTS antioxidant capacity in peel extracts. Not all variables in the peel, pulp, and seed extracts from three species of Cucurbitaceae were linear. Results showed that the vegetables of the family Cucurbitaceae have a high potential for use in pharmacy and phytotherapy. It could be concluded that the studied vegetables are natural sources of phytochemicals and antioxidant substances of high biological importance. Antioxidant activities of the extracts may be attributed to these phytochemicals. The edible part of the vegetable is its pulp, but our results indicated the presence of maximum phenolic and flavonoid content in the peel of all studied vegetables based on which, we strongly recommend the use of the peel of all studied vegetables as an economical and valuable source of phytochemicals and antioxidants rather than discarded as waste material. Moreover, in view of phytochemical screening and antioxidant potential presented by these vegetables of the Cucurbitaceae family, their optimum consumption in the diet has been strongly recommended to reduce the risk of oxidative stress and its related diseases.

14 Feb 2022

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Muhammad Ishtiaq

Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1.Why selected these plants from the whole family?

2. Provide the importance and recommendations of the study.

3. Cross check the reference with main body text.

4. Typographical and English language corrections need improvement.

Reviewer #2: The abstract is very concise but abbreviation used more. So, correct it. Introduction is very brief. it does not cover whole topic. Add relevant data in introduction in sequence. Methods are designed in good form. Results description showed lack of justifications. Please add suitable discussion. Conclusion is not properly indicated key findings. All References must be formatted according to given format of the journal. Some spelling mistakes are observed. So, I think, major revision of the paper is required

Reviewer #3: Indigenous vegetables of family Cucurbitaceae of Azad Kashmir: A key emphasis to their pharmacological potential

I congratulate authors for presenting a nice MS. The MS is well written. However there are some issues to be corrected before publishing.

Grammatical mistake

µgm/ mL;

Genus and species must have gap;

Mistake in ref 15 and 26

Please make reference crosschecking for similar type of work,

What is the difference between this work and previous works (Ref 24, Ref 25, Ref 26, Ref 31)?

What is the reason for positive, negative and no correlation of peel, pulp and seed. Is there any evidence from this research?

What is take home message from this study? Please state this nutshell in conclusion.

What is the global/regional inferences of this study?

All the best!

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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14 Mar 2022

yes. we have accommodate all possible changes

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

11 Apr 2022

Please correct paper as guided now for improvement, see comments section

25 Apr 2022

Thank you for the opportunity to submit a revised version of the manuscript. As requested, we provide here a rebuttal to the comments, as well as a detailed description of how we have met the suggestions for improvement. Please note that we have made almost the proposed changes.

Thanks again for your consideration.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

23 May 2022

Indigenous vegetables of family Cucurbitaceae of Azad Kashmir: A key emphasis on their pharmacological potential

PONE-D-21-38940R2

Dear Dr. Kalsoom Akhter ,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Muhammad Ishtiaq

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The paper is well written and properly updated

Reviewers' comments:

27 May 2022

PONE-D-21-38940R2

Indigenous vegetables of family Cucurbitaceae of Azad Kashmir: A key emphasis on their pharmacological potential

Dear Dr. Akhter:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Muhammad Ishtiaq

Academic Editor

PLOS ONE