GC-MS Analysis of Phytoactive Compounds, Antioxidant and Antibacterial Activity of Citrullus lanatus Seeds

The current study investigated the therapeutic potential of Citrullus lanatus seeds which are commonly discarded after eating the fruit. In this day and age, plant secondary metabolites are preferred therapeutic agents to manage a variety of diseases and disorders. The present study aimed to investigate the bioactive secondary metabolite profile of Citrullus lanatus seeds by investigating total phenolic and flavonoid content, antioxidant potential, and Gas Chromatography-Mass Spectrometry (GC-MS) analysis of bioactive compounds and anti-bacterial properties of four different crude extracts. Alkaloids, flavonoids, phenols, steroids, tannins, saponins, phytosterols, terpenoids, and glycosides were revealed in the seeds after qualitative phytochemical examination utilizing several solvents of varying polarity and established techniques of analysis. DPPH radical scavenging assay was used to assess the antioxidant potential and the total flavonoid and phenolic contents in seed extracts were determined using the spectrophotometric method. Methanolic extract revealed higher extractive yield, antioxidant potential, a higher total phenolic content (132.68 ± 0.861 mg of GAg), and higher total flavonoid content (48.13 ± 0.451 mg of Qg) as compared to other extracts. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of all four seed extracts revealed the presence of 27 high and low molecular weight chemical entities in toto with varying amounts. These bioactive chemical substances have been revealed to be physiologically significant and essential from a pharmaceutical standpoint. This research demonstrates that the Citrullus lanatus seeds are high in bioactive secondary metabolites that are beneficial to human health, have a high antioxidant capacity, and antibacterial action against certain bacterial strains, indicating that these seeds have a lot of therapeutic value.

The major objective of introducing plant-based bioactive compounds into healthcare is to create a healthy interaction with the body's chemistry while avoiding the unwanted and offtarget side effects that medications are known for 1 . Pharmacognostical researchers are being forced to seek out novel plant-based bioactive compounds for use in the treatment of a wide range of diseases and disorders due to an increase in the human population, insufficient drug supply, unmanageable treatment costs, and an increase in antimicrobial resistance to currently used drugs. Plants provide the majority of foods, pharmaceuticals, and nutritional supplements 1, 2 . Primary and secondary metabolites are the chemical compounds found in plants and are termed phytochemicals. Because they are involved in activities like cell division, proliferation, reproduction, metabolism, storage, and development, primary metabolites are vital for plant life. Secondary metabolites in plants aren't merely waste byproducts of primary metabolism; they also have a big influence on plant defenses, ecology, and evolution 2 . Flavonoids are predominant and paramount secondary metabolites found in plants. Flavonoid is a generic name used to describe a family of over 6000 compounds that have a 15-carbon skeleton with two phenyl rings A and B and a heterocyclic ring C. The oxygen with heterocyclic ring C mediates the joining of rings A and B. C6-C3-C6 is the chemical structure used to denote such a compound. Because of their structure, they are essential variable phenolic compounds with strong antioxidant properties. Plants resist oxidative damage because of their inherent secondary metabolites, and as a result, when ingested, they constitute a dietary supply of anti-oxidants 3 . They might minimize the amount of ROS in stressed systems by acting as effective singlet oxygen quenchers. Secondary metabolites have received a lot of attention in pharmacognosy research in the last two decades because they've been shown to have anti-inflammatory, anti-carcinogenic, anti-tumor, anti-oxidative, anti-hypertensive, anti-viral, antiaging, cardioprotective, and immunomodulatory properties, as well as the ability to modulate enzymatic functions, inhibit cell proliferation, induce apoptosis, and inhibit bacterial and fungal growth among others. Current research is also highlighting their role as potent along with their potential to act as substrates for biochemical reactions, cofactors for enzymatic reactions, ligands that antagonize or agonize cellular receptors, act as prebiotics, act as immunomodulators, etc. Bioinformatics and molecular docking information are being applied to forecast the possible use of secondary metabolites in human health and illness 4 .
Plant secondary metabolites (PSM) are naturally occurring physiologically active substances utilized in the traditional type of medicine and diversification of industrial uses. In the case of chronic illnesses, prevention is a better method than therapy. Plant-based foods, such as fruits, have high levels of bioactive phytochemicals, which may have health advantages beyond basic nutrition, such as lowering the risk of chronic illnesses 5 . Watermelon (Citrullus lanatus), a Cucurbitaceae family fruit with over 1,000 cultivars, is a widely farmed fruit worldwide. The large edible fruit, which is a berry with a tough rind and no internal sections and is botanically referred to as a pepo, is grown at optimum conditions all over the world, from subtropical to temperate regions. Although seedless cultivars exist, the luscious, juicy flesh is generally deep crimson to pink, with abundant black seeds. The rind is edible after cooking, and the fruit can be eaten fresh or pickled. It can also be drunk as a juice or as part of a mixed drink 6 . This fruit is a good choice for a healthier diet since it contains phytochemicals such as terpenoids, glycosides, alkaloids, flavonoids, flavonoids, coumarins, quinones, carotenoids, lycopene, anthocyanins, and phenols, as well as vitamins and minerals 7 . Regular consumption of this fruit, which is high in health-promoting substances, may reduce the risk of a variety of deadly diseases, including diabetes, cardiovascular disease, liver problems, obesity, cancer, neurological disorders, and aging-related conditions 8 . Considering the beneficial effects of plant bioactive compounds on human health, this study was organized to determine the presence of secondary metabolites (phenolic and polyphenolic compounds), free radical scavenging activity, and antibacterial activity in Citrullus lanatus seeds that are typically discarded after fruit consumption. These seeds are high in bioactive phenolic and polyphenolic components such as flavonoids, phenols, saponins, terpenoids, glycosides, steroids, alkaloids, coumarins, and quinones, according to the findings of this study. All of the extracts of the seeds were shown to have substantial antioxidant properties as well as antibacterial activity against some of the microbial strains.

Collection of citrullus lanatus seeds
In May, Citrullus lanatus fruits were obtained at a local market in Dharmapuri Mandi, Dehradun, and their seeds were harvested. Only healthy-looking fruits were selected. Before analysis, seeds were shade dried and kept in a conducive environment.

Preparation of plant extract
The removal of phytochemicals requires extraction. The solvents utilized and the chemical properties of samples are the two most critical elements that determine the extraction yield under the same time and temperature circumstances. Several studies have shown that extractive yield varies depending on the solvent used 9 . Seeds were collected and cleaned thoroughly with tap water before being sterilized with distilled water. After that, the seeds were shade dried at room temperature for 7-8 days, then homogenized into a fine coarse powder with an electric blender, and lastly kept in airtight containers until needed. Methanol [M], Chloroform [C], Acetone [A], and water [AQ] were the various solvent systems of varying polarity used to extract the fine seed powder using the hot maceration technique. In the conical flasks, 20 grams of dry powder were poured in 100 mL of each solvent, plugged with cotton wool, and shaken at 120 rpm for 38 hours on a rotary shaker. After 38 hours, the extract was filtered with sterilized Whatman Filter Paper Grade No 1. While the solvent was being evaporated, the supernatant was being collected. The resulting blackish gummy exudates residues were weighed using a balance to calculate the extractive yield. The crude extract was maintained at 4°C in sealed Eppendorf tubes before being subjected to qualitative phytochemical analysis, total phenol and flavonoid content, and antioxidant and antibacterial properties testing 10 .

Phytochemical preliminary screening
Traditional procedures established by Trease and Evans in 2002 11 were used to test the extracts for phytochemicals (secondary). The qualitative assessments were carried out to confirm the presence or absence of Flavonoids, phenols, alkaloids, steroids, saponins, glycosides, phytosterols, terpenoids, triterpenoids, anthraquinones, and tannins. The results of all the above tests are summed up in results section.

Assessment of total phenol content (TPC)
The method of assessing the quantity of phenolic content in samples is known as TPC activity. The redox characteristics of phenolic chemicals found in plants allow them to serve as antioxidants 6,10,11 . Total phenolic content was estimated via Folin-Ciocalteau's reagent assay as reported by McDonald et al 12 . 0.2 gram of extracts was dissolved in 1 ml of their respective solvents. 1 ml of this solution and 0.1 ml (0.5 N) Folin-Ciocalteau's reagent was combined and the reaction mixture was incubated at room temperature for about 15-20 min. After that 3 ml, saturated sodium carbonate solution was poured and again incubated for about 30 min. at room temperature. Finally, the absorbance was taken at 760 nm. Gallic acid was employed as a positive control for which a standard curve was developed beforehand.

Assessment of total flavonoid content (TFC)
The flavonoid content was measured using the colorimetric technique with aluminium chloride 13 . The reaction mixture was incubated at room temperature for 30 minutes, with 1.0 ml of sample (1 mg/ml), 1.0 ml methanol, 0.5 ml of (1.2 percent) aluminium chloride, and 0.5 ml of (120 mM) potassium acetate in a final volume of 3 ml. At 415 nm, the absorbance of all the samples was measured. As a positive control, quercetin was utilized.

Antioxidant assay DPPH free radical scavenging activity
The free radical scavenging activity of Citrullus lanatus seed extracts was determined by using the 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method 10,14 . DPPH in oxidized form gives a deep violet color in methanol. An antioxidant compound donates the electron to DPPH thus causing its reduction and in reduced from its color changes from deep violet to yellow 15 . All extracts were measured for hydrogen donating or radical scavenging ability. Extracts were diluted to obtain concentrations of 0.1, 0.3, 0.6, 0.8 and 1.0 mg/ml. Diluted extract solutions (1 ml each) were assorted with an ethanolic solution of DPPH (0.004%). After 30 min of incubation at room temperature, the reduction of the DPPH free radical was spanned by reading the absorbance at 517nm using UV-Visible Spectrophotometer.
Initially, absorption of a blank sample containing an equal amount of ethanol and DPPH solution was prepared and measured as a control. Ascorbic acid was used as standard. The experiment was carried out in triplicate. Percentage inhibition was calculated using equation (1). The data were presented as mean values ± standard deviation (n = 3).
The GC-MS Analysis Agilent Technologies GC system with GC-7890A/MS-5975C model (Agilent Technologies) integrated with HP-5MS column (30 m in length, 250 mm in diameter, 0.25 mm in film thickness) was used to analyze bioactive compounds from various Citrullus lanatus seeds extracts. An electron ionization device using high-energy electrons (70 eV) was used for spectroscopic evaluation by GC-MS. The gas phase was pure helium gas (99.995% purity) at a flow rate of 1 mL/min. The starting temperature was maintained at 50-1500 with a 30 C/min increase rate and a 10-minute hold duration. Subsequently, the temperature was raised to 300°C at a rate of 100°C per minute. After syringe filtration in splitless mode, one microliter of the obtained extracts diluted with appropriate solvents was loaded into the system. Based on the obtained peak area in the chromatogram, the relative quantity of bioactive compounds contained in each of the extracts was represented as a percentage. Based on GC retention time on HP-5MS column and spectral comparison with computer software data of standards (Replib and Mainlab data of GC-MS systems), bioactive chemicals extracted from various extracts of Citrullus lanatus seeds extracts were recognized.

Antibacterial assay
The antibacterial potential of Citrullus lanatus seeds extracts in different solvents was tested against Gram-positive bacteria Bacillus cereus 10451 (BC), S. aureus ATCC29213 (SA), and Gram-negative bacteria Escherichia coli GIM1.708 (EC), Escherichia coli DH5-Alpha (ECá), Salmonella enteritidis10982 (SE). Agar well diffusion assay was incorporated to assess the antibacterial potential of all extracts of Citrullus lanatus seeds 16 . One ml of each bacterial culture was pipetted out and aseptically swab cultured on Mular -Hinton agar plates. Antibiotics chloramphenicol and tetracycline were used as a positive control. DMSO at a concentration of 15% was used as a negative control. All the extracts (0.2 ml) were loaded in the wells punched in the Agar. The MHA plates were incubated for 24 hours at 37 0 C and thereof looked for the zones of inhibition.

Statistical data
All the experiments were performed at least three times. The results were presented as mean ± S.E.M. (standard error of the mean).

Extractive yield result
The yield of crude extracts was determined in percentage using the following formula 9, 17 .
Yield (%) = (Dry weight of extract ÷ Dry weight of plant material) × 100 The extractive yield of samples in four different solvents viz., methanol, chloroform, acetone, and water are given in Table 1.

Qualitative phytochemical screening result
All the seed extracts were undertaken for qualitative phytochemical analysis using different tests previously described. The results of the phytochemical analysis showed the presence of flavonoids, steroids, alkaloids, phenols, terpenoids, quinones, tannins, glycosides, and saponins in high amounts, while anthraquinones were not detected ( Table 2).

Total phenolic content result
As a basis, phenolic content was measured using the Folin-Ciocalteu's reagent in each extract. The results were derived from a calibration curve (y = 0.0079x -0.2866, R 2 = 0.9861) of gallic acid (50-250 µg/mL) (Fig.1) and expressed in gallic acid equivalents (GAE) per gram dry extract weight. The total phenolic contents of all the samples ranged from 54.87 to 132.68 mg/g gallic acid equivalent ( Table 3). The content of phenolic compounds was higher in methanol extract and lower in chloroform extract Total flavonoid content result The concentration of flavonoids in all the four Citrullus lanatus seeds extracts was analysed via the spectrophotometric method with aluminium chloride. The concentration of flavonoids was expressed in terms of Quercetin equivalents (mg of Q/ g of extracted compound) for which the standard  (Fig.3). The percentage inhibition (% inhibition) at various concentrations (0.1-1.0 mg/ml) of all 4 samples as well as standard Ascorbic acid (40 to 200 ìg/ ml ) were calculated and plotted in graphs using Microsoft Office Excel 2018. Our result revealed that % inhibition of methanol extract is higher among all when compared to standard L-ascorbic acid (Fig. 4 & 5).
All the extracts in different solvents showed significant antioxidant potential when compared to the reference antioxidant ascorbic acid in a dosedependent manner. IC50 value, representing the amount of extract which scavenged/reduced 50% of the DPPH radical, was calculated from the percent scavenging versus concentration curve. A higher concentration to reduce 50% of DPPH solution showed lower antioxidant activity. In this assay, the IC50 value of the reference standard ascorbic acid was found to be 114.62µg/ml while the IC50 value of different values varied. (Fig. 4)

Bioactive compounds contained in the extracts
Tables 5-8 list the bioactive chemicals found in methanol, water, chloroform, and acetone extracts of Citrullus lanatus seeds. Their elution sequence in an HP-5MS column was used to identify and characterize them. These bioactive chemicals' elution time, molecular formula, and quantity also are reported. Figures 6-9 show the GC chromatograms of the four extracts, which indicate the retention time in the column as well as the observed peaks that correlate to the bioactive chemicals contained in the extracts. Watermelon seeds when extracted with methanol  were found to be rich in Cis-7-Dodecen-1-yl acetate (17.29%), n-Hexadecanoic Acid (15.78%), beta -Sitosterol (14.16%), and Lupeol (8.48%).

Antibacterial activity result
Antimicrobial activity was assessed by the development and measurement of the inhibition zone around the discs after the incubation time. The interpretation of the results is shown in Table 9.

DISCUSSION
In May 2021, Citrullus lanatus seeds were gathered and analyzed. The presence or absence of secondary metabolites, antioxidant, and antibacterial properties of Citrullus lanatus seeds were investigated in this study. Phytochemicals exhibit biological features such as antioxidant activity, antibacterial activity, detoxification enzyme modulation, immune system modulation, and general hormonal activity regulation 3,9,13 . The extraction of phytochemical content is affected by the kind of solvent used and the procedure used to prepare the extract. Methanolic extract resulted in the highest extraction yield and a more complex phenolic content 17,18 . Methanolic and water extracts showed the greatest extraction yield, high secondary metabolite extraction, high flavonoid content, high antioxidant potential, and effective antibacterial activity, according to the findings of this study. Terpenoids, glycosides, steroids, alkaloids, flavonoids, coumarins, and quinones were found in high concentrations in this research, however, phytosterols and anthraquinones were not. This is consistent with Ali et al., 2012 19 , who found that alkaloids and terpenes are extensively dispersed throughout the Citrullus genus. For methanol extract of Citrullus lanatus seeds, These seeds contain significant free radical scavenging activity and consequently antioxidant activity, according to our research. The amount and kind of bioactive compound produced by medicinal plants determine its anti-ailment action and different physiological impacts on the human body system. The range of this work, however, did not include an exploration into the precise functions of the extracted phytochemicals from watermelon seed; studies have revealed that these secondary metabolites are subject to a range of pharmacological effects in fruits and vegetables 22,23 .
Because of the companionship of alkaloids, watermelon seeds can be handed-down as basic therapeutic agents for analgesic, antispasmodic,  24 . According to research, alkaloids affect the central nervous system and can act as pain relievers in some cases like morphine.
The presence of alkaloids has been discovered in phytochemical screens of most plants traditionally used to treat malaria 14,25 . Saponins are found in plants  and indicate that they have the ability to precipitate and coagulate red blood cells 11,20 . Phytosterols are one of several nutrients that are said to be beneficial to the heart. Low-density lipoprotein (LDL) cholesterol can be reduced by roughly 10% by taking 2-3 grams of phytosterols each day for 3-4 weeks 26 According to human studies, people who consumed the most phytosterols had a lower incidence of stomach, lung, breast, and ovarian cancer 27 . Phenolic compounds have been shown to act as antioxidants with a wide range of therapeutic effects, including anticancer, anti-inflammatory, and diabetic activities. Some molecular targets of pro-inflammatory mediators in inflammatory reactions are known to be inhibited by phenolic substances such as gallotannins, condensed tannins, and flavonoids. The phytochemicals also function as antioxidants, scavenging free radicals and therefore reducing inflammation 28 .
The GC-MS results showed that the seeds contain many phytochemicals with Lupeol, 9,12octadecadienoyl chloride, and Bis(2-ethylhexyl) phthalate having the least retention times and 11-Dodecenol, Methyl tetradecanoate, and Oxazole having the highest retention times. Phytochemical analysis of the seeds showed many compounds, apparently with diverse pharmacological and biological significance. However, Phenol, 2,2-methylenebis [6-(1,1-dimethylethyl)-4-ethyl], n-Hexadecanoic Acid, Cyclopropanecarboxylic Because the risk of infection by antibiotic-resistant microorganisms is increasing dramatically, the identification and search for chemicals with antimicrobial action has become more important in recent years. Citrullus lanatus seeds show antibacterial action against many strains, according to our research, which might be attributable to

CONCLUSION
In this study, these seeds, which are typically thought of as a waste product of the fruit, were found to be an excellent source of physiologically important phytochemicals. Alkaloids, flavonoids, phenols, steroids, tannins, saponins, phytosterols, terpenoids, and glycosides were found in a qualitative phytochemical examination of these seeds utilizing several solvents of varying polarity and established techniques of analysis. Bioactive compounds revealed quantitively in GC-MS analysis have been revealed to be physiologically significant and essential from a pharmaceutical standpoint by an ample amount of research. Seed extracts showed substantial antioxidant and anti-bacterial action due to these phenolic and polyphenolic components. As a result, it is recommended that these compounds be analyzed as potential therapeutic agents in the management of oxidative stress-related disorders, infectious diseases, and other ailments that have taken a toll on human health.

ACkNOwLEDGEMENT
We acknowledge the support of the Department of Life Sciences of our institution Graphic Era (Deemed to be) University for providing requisites and all the help in conducting and submitting this research article.

Conflict of Interest
There were no commercial or financial links that may be deemed a potential conflict of interest during the research.

Funding Sources
The author(s) received no financial support for the research.