Cucurbitaceae family has a member named pumpkin which includes several food and medicinal plants that remain largely unvalued and underused. Growing scientific interest in pumpkin is driven by its remarkable nutritional composition and antioxidant potential, making it valuable both as a functional food and a medicinal resource. The seeds of pumpkin are often discarded as waste; despite they are rich in nutritionally important bioactive compounds, possessing anti-carcinogenic, anti-inflammatory, antihypertensive, antidiabetic and antimicrobial properties. The present study aimed to assess the nutritional composition and antioxidant capacity of pumpkin seeds. Antioxidant activity was quantitatively analyzed using DPPH assay in both n-hexane and aqueous extracts, and the nutritional profile was determined using standard analytical methods. The nutritional analysis showed that pumpkin seeds contained moisture (6.40 ± 0.50%), ash (1.50 ± 0.06%), fat (30.60 ± 2.50%), fiber (9.20 ± 1.20%), protein (31.80 ± 2.55%), and carbohydrates (20.50 ± 1.70%). The total polyphenolic content (TPC) of the n-hexane extract was 38.75 ± 2.80 mg GAE/g, while the aqueous extract contained 20.68 ± 1.72 mg GAE/g. At concentrations ranging from 1 to 2.5 mg/ml, the aqueous extract exhibited DPPH radical scavenging activity between 14.16 ± 0.90% and 40.23 ± 2.90%, whereas the n-hexane extract showed higher inhibition values ranging from 29.41 ± 2.10% to 62.75 ± 4.10%. A statistically significant difference between the extracts was observed (P < 0.05). These findings emphasize the potential of pumpkin seeds as valuable sources of bioactive compounds and natural antioxidants rather than being considering pumpkin seeds as useless.

Due to the substantial nutraceutical and therapeutic potential of bioactive components, the demand for seeds and nuts for vegetable fats and oils has increased recently. In promising countries, this demand is expected to rise due to population and income growth, as well as the desire for improved nutrition and fuel [1]. Pumpkin is grown worldwide and it is the member of the cucurbitaceae family (Figure 1). In Pakistan, it is primarily used as a vegetable, but it has also been used often as a functional food. Recently, pumpkin seeds have drawn a lot of interest due to their high nutritional value and potential health benefits. Generally pumpkin seeds seen as a waste product and it are abundant in bioactive compounds with nutraceutical qualities [2]. 

                                                                                                      Figure 1: Pumpkin and Pumpkin seeds

A considerable extent of squander is produced during the processing of pumpkins, the pulp comprises 72–76% of the finished product, peel (2.6–16%) and seed (3.1–4.4%). Trash from homes, farms and the food industry are the primary sources of pumpkin waste. The processing industries employ pumpkin waste for a number of things, such as producing electricity, biogas and wholesome and valuable foods. The food industry has made considerable use of pumpkin peels as a natural colorant and dietary fiber source [3].

Natural antioxidants frequently have phenolic moieties in their chemical structure that contribute to an increase in their antioxidant activity [4]. 

Pumpkin seeds have highly phenolic which are main contributors to plants' antioxidant potential. These include phenolic acid, tannins and flavonoids [5]. According to a study by Singh and Kumar, (2022) [6] using LC-ESI-MS a number of phenolic substances were discovered to have peaks in the positive mode: kapempferol p-coumaric acid, ferulic acid, protocatechuic acid, tyrosol, sinapinic acid, syringic acid, apigenin, and chlorogenic acid and their respective illusion times were 1.52, 9.40, 11.60, 14.24, 15.53, 15.82, 19.29, 20.07 and 25.87 minutes (Figure 2). 

                                                                                       Figure 2: Phenolic compounds in pumpkin seed [6]

The seeds typically have a lofty amount of polyunsaturated and monounsaturated fatty acids and are substantial in size. A significant amount of pumpkin seed oil is made up of linoleic acid, oleic acid, palmitic acid, ECN-44, ECN-46, tocopherols, ß-sitosterol, and delta-7-sterols [7]. For decades, various research investigations have been undertaken on the active rudiments of pumpkin peel, pulp and seeds to provide a thumbnail sketch of their health-related consequences, which have proved their anti-inflammatory [8], antibacterial [9] and anticarcinogenic [10], antidiabetic [11] and antihypertensive [12] (Figure 3). 

Moreover, pumpkin seeds have demonstrated anti-ulcerative qualities by preserving the stomach mucosa [13]. Researchers are constantly working to find more efficient and economical ways to repair wounds because medications don't seem to be enough to promote healing [14]. However, the high concentrations of polyunsaturated fatty acids (PUFA), tocopherols and plant sterols in pumpkin seed oil have a positive impact on wound healing [15]. 

Additionally, it has been demonstrated in vivo that the naturally occurring bioactive compounds in pumpkin seeds, such as carotenoids, tocopherols and sterols, have a wide variety of biological effects in avoiding a number of chronic diseases [16-17]. So, this study was done to ascertain the nutritional facts and TPC of the seed that was extracted in water and n-hexane solvents from locally accessible pumpkin and to assess its capacity to scavenge free radicals. 

                                                                                                  Figure 3: Biological activities of pumpkin seed [18]

Preparation of pumpkin seed powder After being bought from a local market Lahore, Pakistan, the pumpkin (Cucurbita maxima) was cleaned, chopped and its seeds removed. To get rid of the yellow fibrous bits that were connected, the seeds were thoroughly cleaned. The seeds were then dried in a tray dryer for eighteen hours at 60°C. For later usage, the dried seeds were separated from its kernel and then were trodden into a powder that stored in container at 8°C. Proximate analysis Proximate analysis was performed for the pumpkin seed powder and procedures [19] were used to analyze the moisture and ash content. The Micro-Kjeldahal technique was used to analyze the protein content of the samples. The digestion unit was used for both digestion and distillation. Total fat content was determined by using the Soxhlet apparatus with n-hexane as a solvent (boiling point: 60°C to 80°C). Samples free of fat and moisture were used to assess the sample's crude fiber content by using an acid (H2SO4) and subsequent alkali (NaOH) digestion by boiling in a fiber tech system. Using the difference method, the amount of carbohydrates was determined. The energy, which was reported as Kcal/100g, was determined [20] by multiplying the % of protein and carbohydrate by 4.0 and fat by 9.0.

Total phenolic content

According to Saeed et al. (2021), the TPC of pumpkin seeds was determined using Folin-Ciocalteu's technique, which involved calculating the gallic acid standard curve between absorbance and concentrations [21-22]. The TPC was represented as mg GAE/g.

Radical scavenging activity by DPPH assay 

The Brand-Williams et al., (1995) method was used to evaluate 2,2-Diphenyl-1-picrylhydrazyl's (DPPH) capacity to scavenge radicals [23]. Briefly 2.9 ml of a 0.1 mM methanolic solution of DPPH was added to test tubes that contained 100 µl of pumpkin seed extract at varying concentration. The tubes were then violently shaken. The tubes were allowed to stand at room temperature for thirty minutes. The control was prepared as previously mentioned, but without any extract. The absorbance changes of the samples were measured at 517 nm. The inhibition % for radical scavenging activity was calculated using the following formula.

DPPH scavenging activity (%) = Absblank – Abssample × 100 

                                           Absblank 

Statistical Analysis

Each experiment in this study included three replications and the results were displayed as ± standard deviation. Graphs were plotted using SPSS software version 21.0. The significance of mean differences between the extracts was examined using the "t" test. A significance criterion of p<0>

Proximate composition One of the healthiest vegetables consumed worldwide is the pumpkin (Cucurbita spp.), which is now recognized as a serviceable food [24]. Pumpkin seeds are rich source of high-quality fat, protein, fiber, antioxidants and other phytochemical compounds. Although these miracle seeds are typically regarded as agro industrial waste, they have intriguing nutraceutical qualities. Table 1 displays the proximate analysis of pumpkin seeds. Pumpkin seed powder was found to have 6.40±0.50% moisture, 1.50±0.06% ash, high protein content (31.80±2.55%), high fat content (30.60±2.50%) and crude fiber content (9.20±1.20%). Its carbohydrate content was 20.50±1.70% while the seed powder's energy value was 484.6± 6.80 Kcal/100g. 

A study on the proximate composition of pumpkin seed was carried out in 2022 [25] which showed that crude protein (32.47±0.15), crude fat (46.39±0.06), crude fiber (12.29±0.17), ash (7.38±0.05) and carbohydrate (4.18±0.21) of Cucurbita maxima seeds. Our findings are comparable to those of literature [26], who revealed that seeds of Cucurbita pepo contain fat 33.13%. A key factor in the safe storage of food items is moisture content and ash concentration provides insight into the mineral elements it contains. It shows the makeup of inorganic components following the combustion of organic materials (fats, proteins, and carbohydrates) and moisture. In essence, a food sample's mineral content is what is anticipated to enhance growth and development and accelerate metabolic processes [27]. Seeds are useful as a robust economic supply of edible oil due to its grater fat. The value of its protein is in line with the findings of a review [28], which found that pumpkin seeds had a high protein content of 25.4%, which is the foundation for body development. According to researcher [29], the most notable protein found in pumpkin seeds that exhibits the primary anthelmintic activity. Protein digestibility and corrected amino acid scores ranging from 0.48 to 0.67 for lysine, pumpkin seeds are a moneyed cause of the majority of essential amino acids [30].

             Data are represented ± SD

                                                                                                Table 1: Proximate composition of Pumpkin Seed

Total Polyphenolic contents (TPC)

The hexane extract of the pumpkin seed had the highest TPC at 38.75±2.80 mg GAE/g, whereas the water extract had lowest TPC 20.68±1.72 mg GAE/g. These outcomes concur with literature [31], who found that the Nigerian pumpkin seed cultivar had TPC 32.90 mg GAE/g. In another study it was found that pumpkins of the Cucurbita pepo species have high total polyphenol content than those of the Cucurbita maxima species [32]. Because of the reactivity of their moiety, phenolic compounds are naturally occurring antioxidants. They can be classified as tannins, phenolic acids and flavonoids based on the structural components' linkage to benzene rings and the phenolic hydroxyl groups' attachment. As non-enzymatic antioxidants, phenolic and flavonoid chemicals primarily aid in the elimination of reactive oxygen species (ROS), which is advantageous for both human and plant health [33-34].

DPPH Radical Scavenging Activity: 

The DPPH radical test is one way to measure antioxidant activity. The extracts' antioxidants neutralize the violet DPPH radical in this test, which causes the solution to become discolored [35-36]. Pumpkin seed water extract showed the lowest DPPH radical scavenging activity 14.16±0.90–40.23±2.90%, whereas n-hexane extract showed the highest radical scavenging activity i.e., 29.41±2.10–62.75±4.10% at concentration of 1–2.5 mg/ml, (Figure 4). There were notable variations (p < 0>

The polyphenolic chemicals found in pumpkin seeds are responsible for these antioxidant properties. Additionally, the seeds are a good source of, an organic substance squalene which is a key component of the Mediterranean diet and has a chemo preventative effect on cancer prevention by lowering lipid peroxidation. Squalene shields healthy cells from the harmful effects of chemotherapy, but not tumor cells. Anticancer, antidiabetic, antitumor, antimicrobial, anti-mutagenic, antioxidant, anti-inflammatory and antiulcer characteristics have also been identified in pumpkin seeds [41-42].

                                                                    Figure 4: % Inhibition (DPPH) of water and n-hexane extract of pumpkin seed powder

Pumpkin seeds (wastes) have high antioxidants i.e. of polyphenols and protein, fat, dietary fiber and minerals, because they are high in protein, fat and antioxidants, these pumpkin wastes could be used as a food supplement or further utilized for value addition.

Statement Of Ethics

Ethical approval of this study was taken from the Food & Biotechnology Research Centre, PCSIR Laboratories Complex, Lahore Pakistan.

Conflict Of Interest Statement 

The authors declare no conflict of interest.

Contribution 

The authors contributed equally