Antiradical activity, total phenolic, and total flavonoids extract and fractions of pumpkin (Cucurbita moshata Duch) leaves

Pumpkin (Cucurbita moshata Duch) is one of the Cucurbitaceae plants, which traditionally used to treat skin diseases, measles, jaundice, insomnia, cancer and enhances endurance. Therefore, it was necessary to explore the potential of pumpkin leaves as antiradical. This research aim was to examine the antiradical activity and total phenolic and total flavonoids of pumpkin leaves extract and its fractions using the DPPH method and determined the phenolic and flavonoid contents. Pumpkin leave powder was extracted with methanol. Furthermore, water was added into methanol extract, and be partitioned using n-hexane and ethyl acetate to obtain n-hexane, ethyl acetate, and water fractions. The antiradical activities of pumpkin leave extract and fractions were determined using DPPH (2,2-diphenyl-1-picryhidrazyl) method. Ethyl acetate fraction obtained higher antiradical activities (IC50 6.737±0.196 μg/mL). Correlation of total phenolic and flavonoid contents to inhibit DPPH radical showed that phenolic and flavonoid contents on pumpkin leaves could be inhibited DPPH radical R = 0.8994 and R = 0.9061, respectively. Extracts and fraction pumpkin leaves show strong antiradical activity with DPPH methods, so their potential as antiradical can developed and can be used as a functional food.


Introduction
Since ancient times, plants and their metabolites have been used as medicine. Natural source of antioxidant from Traditional medicine is widely used, which these antioxidant substances may help to develop new potential drug (Bag and Devi, 2015). Antioxidants are defined as a compound that delays or prevents oxidation of biomolecules such as lipid, protein, and DNA, by counteracting free radicals from these biomolecules which prevent cell damage (Proestos et al., 2013;Labiad et al., 2017). In the last several decades, many researchers have traced the antioxidant activity of plants. Where, it is believed that the use of natural antioxidants will reduce the chance of degenerative diseases, such as cardiovascular disease and cancer (Elkhamlichi et al., 2017).
Pumpkin (Cucurbita moschata) is a plant of the family Cucurbitaceae which is widely distributed in tropical, subtropical, temperate, and hot regions (Al-Qaisy and Rathi, 2020). Traditionally, pumpkin (C. moshata) is used to treat skin diseases, measles, jaundice, insomnia, cancer, and can help to enhance endurance. While pumpkin seed oil can be used in hypertension, arthritis, hypercholesterolemia, bladder disorders, and urethral pressure treatment (Suresh and Sisodia, 2018). Phytochemical screening shows flesh and pumpkin seeds containing flavonoids, terpenoids, saponins, and tannins (Marbun et al., 2018).
The objective of the research was to determine the potential antiradical and phenolic and flavonoids total of pumpkin extracts and fractions leaves

Extraction
Pumpkin leaves were dried in the sun and covered with black cloth. Then, the dried leaves were powdered. Pumpkin leaf powder (30 g) was macerated with eISSN: 2550-2166 © 2021 The Authors. Published by Rynnye Lyan Resources FULL PAPER methanol for 3×24 hrs, where every 24 hrs the mixture was filtered, and the solvent was replaced. The extract was evaporated to dryness using Rotary Evaporator. Water (1 L) was added into methanol extract (40 g), then the extract was fractionated with n-hexane and ethyl acetate to obtain the n-hexane, ethyl acetate, and water fraction, as shown in Figure 1.

Yield extract
The yield extract that has been obtained is calculating using Equation 1 (Nurmala and Novianti, 2020)

Yield fraction
The yield fraction that has been obtained is calculating using Equation 2

Phytochemical screening
Phytochemical screening was conducted to determine the profile of secondary metabolites in langsat seeds extracts and fractions. Phytochemical screening methods based on (Yamin et al., 2020).

Alkaloids
The extract and fractions of Lansium domesticum Coor seeds were inserted separately into 1 mL test tubes and added with three drops of Dragendorff's reagent. The formation of brown precipitate indicated the presence of alkaloid.

Flavonoids
The extract and fractions of Lansium domesticum Coor seeds were inserted separately into test 1 mL tubes and added with 0.2 g of magnesium powder and 2 mL of concentrated HCl. The formation of red, orange and green solutions indicated the flavonoid presence.

Tannin
The extract and fractions of Lansium domesticum Coor seeds were inserted separately into 1 mL test tubes and added with 1 mL of 1% Fe (III) chloride solution. The formation of blue to black solution indicated the presence of tannin.

Terpenoid
The extract and fractions of Lansium domesticum Coor seeds were inserted separately into 1 mL test tubes and added with 0.5 mL of acetic acid anhydride and 2 mL of concentrated sulfuric acid. The formation of green, bluish, and brown solutions indicated the terpenoid presence.

Saponin
The extract and fractions of Lansium domesticum Coor seeds were inserted separately into 1 mL test tubes and added with 2 mL of hot water, then cooled and shaken for 10 s. It was declared positive for saponin if the fume generated stabilized in less than 10 mins.

Determination of antiradical activity with DPPH method
The determination of radical activity was done by following the Garcia method (Garcia et al., 2012) which modified the number of samples to use. Methanol p.a (3 mL) and 1 mL of DPPH radical (2,2-diphenyl-1picryhidrazyl) was added to the sample (1 mL). The mixture was shaken until homogeneous, then incubated in a dark room for 30 mins. Then the absorbance of the sample was measured using a UV-Vis spectrophotometer at a wavelength of 513 nm. The antioxidant activity of extract and its fractions to inhibit DPPH radicals can be calculated using the following equation: Where % inhibition = percentage of DPPH radical inhibition, Ac = Absorbance of control and As = Concentration of sample The value of antioxidant strength (IC 50 ) was calculated based on the linear regression equation between % inhibition with the concentration of sample or fractions, where the x-axis was the concentration while the y-axis was % inhibition. The regression equation y = bx + a. Then, the y value was replaced by 50. Where IC 50 was defined as the concentration of the sample needed to FULL PAPER inhibit 50% of DPPH radicals (Rohman et al., 2017).

Determination of total phenolic content
Determination of total phenolic content of pumpkin leaves extracts and fractions using the modifying Folin-Ciocalteau method according to John et al. (2014). Each concentration series of the sample (1 mL, respectively) were taken and added 0.4 mL of the Folin-Ciocalteau reagent, then shaken. After 8 mins later, added 4 mL of 7% Na 2 CO 3 and shaken until homogeneous then sufficient with up to 10 mL volume, allowed to stand for 30 mins and then absorbed by using UV-Vis spectrophotometry at a wavelength of 750 nm. The measurement of each sample concentration series was carried out three times for replication. Total phenolic levels are expressed as Gallic Acid Equivalents (GAE).

Determination of total flavonoid content
Measurement of total flavonoid levels was carried out using the colorimeter method (John et al., 2014;Vyas et al., 2015) by modifying, 10 mg of sample dissolved with methanol p.a. 10 mL. Then, 1 mL was taken with 3 mL of methanol p.a., then 0.2 mL of 10% aluminum chloride and 0.2 mL of potassium acetate 1 M were added and the volume of 10 mL was sufficient with distilled water. The mixture was incubated for 30 mins. Then the absorbance was measured using a UV-Vis spectrophotometer at 435 nm wavelength. Total flavonoid levels are expressed as Quercetin Equivalent (QE).

Results and discussion
In this study, the extraction method used was maceration, then followed by fractionation using solvents with different polarities. The purpose of using solvents with different polarities was to extract completely compounds whether non-polar, semipolar, and polar. The same compound can be found in different solvents, but the levels are different in each solvent. This depends on the distribution coefficient of the compound (Yamin et al., 2010). The results of pumpkin leave extraction and fractionation are presented in Table 1.
Based on the data in Table 1 shows that the crude extract yield is 25.57%. This result indicates that the pumpkin leaves can be utilized. Even after fractionation, the ethyl acetate fraction shows the highest yield of 40%, which means that the compounds in pumpkin leaves are mostly semi-polar. The compound will be easily attracted to a suitable solvent, according to the principle of "like dissolved like", where compounds will be attracted to solvents with the same polarity (Amaro et al., 2015).
Phytochemistry screening showed the extract and fraction of pumpkin leaves contained secondary metabolite alkaloids, flavonoids, tannin, saponin and terpenoids. The result of phytochemistry screening shown in Table 2.
Utilization radical DPPH (2,2-diphenyl-1picryhidrazyl) in determining the strength of free radical caused by DPPH is a stable radical (Shekhar and Anju, 2014), which receives electrons or hydrogen radical to become a stable diamagnetic molecule (Arina and Rohman, 2013). Besides, DPPH radicals are also considered a standard colorimetric technique to measure the antioxidant activity of extracts and pure compounds from plants (Mishra et al., 2012). The reduction ability of DPPH free radicals is based on a decrease in wavelength at 513 nm after adding antioxidants. In this study, the standard used is vitamin C and solvent for dissolving DPPH using methanol. The use of methanol is based on its sensitivity compared to other solvents (Rohman et al., 2017).
The antiradical activity was determined using vitamin C as a positive control. The result in Table 3, shows the IC 50 values of pumpkin leave extracts and fractions with vitamin C as positive controls. Pumpkin leave extract and fractions showed antiradical activity based on the IC 50 value. The smaller IC 50 value of the sample indicates that the stronger functions as an antiradical. Based on the data in Table 3 ethyl acetate fraction is the most powerful fraction as antiradical. This is in line with different research which the ethyl acetate fraction of the red fruit (Panandus conoideus L.) (Rohman et al., 2010) (Rohman et al., 2017). Figure 2 shows the correlation of ethyl acetate fraction inhibition with the amount of DPPH radical, ethyl acetate fraction can be further separated to obtain an active isolate.
Phenolic and flavonoid compounds are known to have antiradical activity. These compounds can donate hydrogen radicals contained in the hydroxy group (-OH) to attached in radical DPPH, hence the DPPH radical becomes stable (Pietta, 2000;Heim et al., 2002;Agati et al., 2012). Therefore, some antioxidant activity of natural substances associated with phenolic compounds and flavonoids (Rohman et al., 2017) Phenolic and flavonoids are the most abundant compounds in plants that are beneficial in human health (Wijaya et al., 2017). Total phenolic and flavonoid levels have a correlation with antioxidant power in extracts and fractions (Nur et al., 2019). Table 4 and Table 5 shows the total phenolic and flavonoid content of pumpkin leaf extracts and fractions. The ethyl acetate fraction obtained high phenolic and flavonoid levels compared to methanol extract, n-hexane, and water fractions. Phenolic and flavonoid levels of ethyl acetate fraction were 56.43 g GEA/100 g sample and 55.07 g QE/100 g sample, respectively. These results indicated that the value of antioxidant strength correlates with phenolic and flavonoid levels contained in the extract and fraction of pumpkin leaves.
The correlation between antiradical strength (IC 50 ) with phenolic and flavonoid levels of extracts and pumpkin leaf fraction is shown in Figure 3. The correlation coefficient (R 2 ) is used to determine the   Figure 3 shows the correlation between IC 50 values with total phenolic content and flavonoids. Absis (x) is the IC 50 value and ordinate (y) is the phenolic and flavonoid content of the methanol extract and its fractions. The R 2 value obtained is 0.8994 and 0.9061. This indicates that 89.94% of antiradical strength is influenced by phenolic content and 90.61% is influenced by flavonoid content contained in pumpkin leave extract and fraction.

Conclusion
Pumpkin leaves have antiradical abilities, which the ethyl acetate fraction exhibits the strongest antiradical. The correlation between total phenolic, flavonoid content, and radicals inhibition is very high. These show the compounds most responsible as antiradical are phenolic and flavonoid compounds. Thus, pumpkin leaves can be developed as a functional food.

Conflict of interest
The authors declare no conflict of interest.