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Full Research Article

Preparing Protein Hydrolysates from Pumpkin Seeds and Studying Their Chemical and Antioxidant Properties

A
Ayat Adnan Abbas1
G
Ghydaa H. Aljeboury1
A
Abdulameer Jawad Zayier1
S
Salah M. Alchalab1
H
Hayder Haqi Ismail1
A
Ahmed Flayyih Hasan1,2,*
1Biotechnology Research Center, Al-Nahrain University, Baghdad, Iraq.
2Department of Medical Laboratory Techniques, College of Health and Medical Technology,Al-Farabi University, Baghdad, Iraq.

ABSTRACT

Background: Recent advances in analytical approaches presented new potentials for plant-based drug manufacture within the realms of peptides and proteins.

Methods: Enzymatic hydrolysis of plant-derived parent proteins yields bioactive peptides, which have been investigated for potential applications across diverse experimental contexts. Differences in pH and ionic strength induced significant effects on the solubility of extracted pumpkin seed proteins, as a direct relationship was observed between the solubility of pumpkin proteins and alkaline pH.

Result: Applying the kind of solvent for seed defatting significantly influences protein solubility. Specifically, applying the chloroform-methanol (CM) mixture approach for lipid extraction, as opposed to pentane (P), clearly weakened protein solubility. The objective of the current study is to explore how varied environmental parameters, namely pH and ionic strength, can influence the functional properties of pumpkin seed protein isolate (PSPI), including solubility, interfacial properties and emulsifying activity, compared to pumpkin seed protein hydrolysate (PSPH).

INTRODUCTION

For food and cosmetic purposes, plant-based proteins are a viable substitute for animal proteins since they are renewable and come from a wide range of sources, particularly legumes, grains and oilseeds (Moure et al., 2006). Although oilseeds are cultivated mostly for their oil, they represent a valuable source of proteins as well. Likewise, the protein content may reach 60% in the oilseed, the by-product, hence, it may represents a valuable addition to the protein extraction process (Khayri et al., 2026). The cultivation of pumpkin (Cucurbita moschata Duch., Cucurbitaceae family) is globally widespread across tropical, subtropical and warm regions, where it is employed in a variety of food industries, including juice production, mash preparation, fermented beverages and jam manufacture (Mitić et al., 2018; Al-Taeea et al., 2026). Despite being rich source of a very important nutrients, pumpkin seeds are widely considered as an industrial by-products and are often used in manufacturing of healthy nutritunal vegetable oils due to their high content of oils (Amin et al., 2019; Aktaş et al., 2018).
       
Due to the nutritional importance of the extracted pumpkin seed proteins, they are often added to various food products significantly supporting and improving human health, such as anti-microbial and anti-carcinogenic properties, blood coagulation inhibition and the reduction of the negative consequences of protein malnutrition (Yadav et al., 2010; Tomar et al., 2014). Plant proteins are therefore being used more and more as unusual protein sources to fulfill functional roles in food compositions (Chabanon et al., 2007; Ghiath et al., 2025). However, (Yuliana et al., 2014) reported that plant proteins are known for their poor capacity to dissolve in acidic media which is the reason they are not preferred not to be added to highly or moderately acidic foods (such as dressings, foaming and emulsifying), particularly when solubility is necessary for the desired functional qualities (Tang et al., 2024). Proteins from pumpkin seeds were found to be very poorly soluble (less than 20%) at pH values below 5 (Rezig et al., 2013; Al-Saeedi et al., 2026). Enzymatic hydrolysis of oilseed proteins constitutes a highly effective approach for enhancing their functional properties and increasing solubility, especially at pH close to the isoelectric point (pI) (Tokmakov et al., 2021), better emulsifying capabilities and enriched biological activities are among the functional characteristics of particular relevance (Moure et al., 2006; Luo et al., 2014).
       
To improve functional properties and increase solubility, the described approach of enzymatic hydrolysis of oilseed proteins is applied, although studies on pumpkin seed protein hydrolysates are limited. In a study by (Vaštag et al. 2011), in addition, these proteins hydrolysates exhibit an antioxidants potential as reported by researchers (Xie et al., 2025). On the other hand, a study by (Peričin et al., 2009) demonstrated that highly soluble pumpkin seed proteins can be efficiently obtained through enzymatic hydrolysis approach.
       
The current study aimed to extract and isolate protein from pumpkin seeds (PSPI) in addition to preparing pumpkin seed protein hydrolysate (PSPH) and addressing the functional properties namely solubility, interfacial and emulsifying capacity of both (PSPI) and (PSPH).

MATERIALS AND METHODS

Pumpkin seed powder defatting
 
Pumpkin seeds were thoroughly washed and cleaned to remove any impurities. Seeds were fully dried by placing them in an oven at 45°C for 48 hours. Afterwards, dry seeds kernels were ground into a fine powder using a lab size grinder. Following the method mentioned by (Devi et al., 2019), n-hexane (1:5 w/v) were applied and defatting was carried out. The defatted samples were dried at 40°C and then were stored at 4°C to be later used.
 
Pumpkin seed protein isolation (PSPI)
 
The pumpkin seed powder was suspended in an alkali solution with a pH of 10.00, which was prepared by adding 1 mol L-1 of sodium hydroxide (NaOH) to enable protein suspension. Suspension filtration was carried out after 30 minutes with gentle stirring. In order to assist proteins to be precipitated through increase their dissolving ability, pH needed to be lowered to 5.00 aby adding (1 mol dm-3 HCl) to the suspension. Centrifugation was performed at 4°C and at speed of 10,000 rpm for 20 min. to separate the precipitate from the liquid phase, followed by drying for 48 hours at 30°C (Bučko et al., 2016).
 
(PSPI) moisture, ash and protein content determination
 
Moisture and ash content of (PSPI) was determined according to the approach described by (Helrich, 1990). Kjeldahl method was adopted to quantify crude protein content in (PSPI), through applying a conversion factor of 6.25 (N × 6.25) according to the assumption that proteins contain about 16% of nitrogen (Goyal et al., 2022; Ahmed et al., 2026). 
 
Enzymatic hydrolysis
 
To prepare (10 g dm-3) (PSPI) suspension, a pH 3 solution was prepared by adding (1 mol dm-3 HCl) followed by adding certain amount of the obtained (PSPI) to the pH 3 solution at 37°C with enzyme to substrate ratio (0.02:1) g. The enzymatic hydrolysis proceeded for 90 minutes. The resulting hydrolyzed suspension was subjected to vacuum filtration followed by drying process in an oven at an inlet temperature of 85°C and an outlet temperature of 50°C following the method described by (Bučko et al., 2016; Al-Ameri et al., 2026) with minor modifications.
 
Degree of hydrolysis (DH)
 
Formaldehyde titration method mentioned by (Li et al., 2013) was implemented for the determination of DH as follows: a dilution was achieved to 5 ml of hydrolysates by adding 60 ml of distilled water while being placed on magnetic stirrer. 0.05M of NaOH (standard titration solution) was used to titrate the hydrolysates to a pH of 8.2. After adding 10 milliliters of 14% formaldehyde to the beaker, 0.05 M NaOH was added to proceed the titration until a pH of 9.2 was attained. The volume of consumed NaOH was recorded. Value of DH was calculated using the following equation:

 
Where,
C = Concentration of the standard (0.05M) NaOH titration solution.
V1 = Amount of (0.05 M) NaOH used in titration solution reaching pH of 9.2.
V2 = Amount of (0.05 M) NaOH used in titration solution reaching pH of 8.2.
V = Collagen hydrolysate’s total volume.
m = Raw material’s mass.
 
Protein determination
 
Bovine serum albumin was used as the reference for the determination of the crude extract’s protein concentration as (mg/ml), according to the biuret method (Gornall et al., 1949).
 
Determination of amino acid constituents
 
The content of amino acids present in the freeze-dried collagen samples was quantified employing  High Performance Liquid Chromatography (HPLC) (Shimadzu, Japan) approach. 0.5M acetic acid at a concentration of 5 µg/mL was used to dissolve amples. According to (Ersser and Davey, 1991), O-phthalaldehyde (OPA) produces a luminous derivative for primary amino acids at least 10 times more sensitively than ninhydrin was applied. At room temperature, the reaction proceeds rapidly when mobile phases A and B (Methanol (Me-OH), Tetrahydrofuran (THF) and 0.02 M Sodium Acetate) buffer solution adjusted to pH 5.9 with a ratio of (80:2.5:17.5) and 0.02 M Tetrahydrofuran (THF) and Methanol (Me-OH) with a ratio of (20: 2.5: 77.5), respectively are used. Proline and hydroxyproline exhibited no observable reactivity, whereas cysteine displays minor reactivity. The concentration of amino acids and the residues are determined by the following formulas:




DPPH radical scavenging activity assay
 
Following the procedure described by (Salman et al., 2019) with minor modifications, the scavenging potential of α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical was determined. 1.5 milliliters of extract were mixed to 1.5 milliliters of DPPH. The mixture was stirred and subsequently allowed to stand at ambient temperature for 30 minutes in a dark place. Spectrophotometric assay was carried out at wavelength of 517 nm and the absorbance of the resulting solution was achieved. (DPPH) radical scavenging activity was estimated according to following equation:

 
Fourier transform infrared spectroscopy (FTIR) assay
 
The FTIR analysis was achieved by employing a (Shimadzu, Japan) FTIR spectrometer spanning 400-4000 cm-1. Samples were prepared via the potassium bromide disc KBr pallets method, incorporating 2 mg of Moringa (Muyonga et al., 2004).
 
Water absorption capacity (WAC) and oil absorption capacity (OAC)
 
For the determination of (WAC) and (OAC), the approach described by Habib et al. (2025) was employed with minor modifications. The procedure involved mixing 1 g of the sample with 10 ml of distilled water and/or 10 ml of oil for 30 seconds and were allowed to stand at room temperature for 30 minutes. Subsequently, samples were exposed to centrifuging for 10 minutes at speed of 5000 rpm. The supernatant was recovered for the estimation of (WAC) and (OAC) /gram of samples.
 
Denaturation temperature (Td)
 
Chen et al. (2021) defined the Denaturation Temperature (Td) as the precise temperature where a substance, usually a protein solution, exhibits a changes in its structure through denaturation. A research study performed by (Pati et al., 2010) reported the use of Ostwald viscometer, which is a tool utilized for assessing variations in viscosity due to temperature changes, which enabled the measurement of collagen degradation temperature. For the preparation of (0.1% w/v) collagen with a concentration at (0.997 g/ml), (0.1 M) acetic acid was used followed by incubation of samples at 10°C for 30 min.  
• Solubility = Total protein in the sample + Protein content of the supernatant.
• Current solubility NaCl ×100 = Relative solubility percentage.
• Maximum solubility in the range sample.
• The ratio of the protein content in the upper liquid to the overall protein content in the sample was used as a foundation to calculate solubility.
       
To measure viscosity, the temperatures were incrementally raised to 20, 25, 30, 35, 40, 45, 50 and 55°C, with an incubation for 30 min. for each temperature. The below equation was used to quantify viscosity:

 
Where,
t1 = Water flow duration.
P1 = Water density.
t2 = Sample flow duration.
P2 = Sample density (0.997 g/ml).
       
The following equation is utilized to calculate fractional viscosity:

 
(PSPI) and (PSPH) solubility determination
 
The solubility of (PSPI) and (PSPH) was determined by preparing suspensions at different concentrations (csusp, 10-100 g dm-3) of (PSPI) and (PSPH) and employing buffer solutions with a range of pH ranged between (3-8) and ionic strength of (Ic=0.1 mol dm-3).
       
To achieve complete dissolution of the proteins, suspension solutions were set in a water bath at 50°C for 1 hour with continuous stirring. Sartorius membrane filtration with (0.45 μm) of pore size was used for the isolation of soluble proteins to acquire (PSPI) and (PSPH) suspension solutions. Following Lowry method, the amount of dissolved proteins in the protein solution (csol) was measured expressed in g dm-3 (Lowry et al., 1951).
 
Ethical approval
 
Approval was obtained from the Research Ethics Committee of (Biotechnology Research Center, Al-Nahrain University, Baghdad, Iraq).
 
Statistical analysis
 
Statistical Analysis System (SAS) was adopted to distinguish the influence of different groups on research factors (Cary, 2012). To calculate the smallest significant overall differences, Least Significant Difference (LSD) was applied.

RESULTS AND DISCUSSION

Determination of moisture, ASH and protein
 
According to Table 1, the contents of moisture, ash, fat and carbohydrates were 6.97%, 2.87%, 1.31% and 2.82%, respectively. It was found that (PSPI) has an 86.89% protein content. The findings are consistent with those obtained for other oil seeds, such as cashew nuts (91%), sesame seeds (59%), sunflower seeds (70%) and sesame seeds (20%).

Table 1: Chemical composition of (PSPI).


 
DPPH scavenging (2, 2-Diphenyl-1-Picrylhydrazyl) of pumpkin extract
 
The DPPH radical scavenging of pumpkin hydrolysate (62%) concentration.
 
Amino acid composition
 
FTIR assay
 
Liver function
 
Liver function trial included 4 groups of male albino rats in a total of 20 rats (5 rats\group). The first group represents the control group and was given only water and stander pellet. The second group was given (25 mg/kg) of CCl4. The third group was fed with (25 mg/kg) of CCl4 + (100 mg/kg) aqueous extract of pumpkin. The fourth group was given only (100 mg/kg) aqueous extract of pumpkin. Test period was 35 days. Laboratory animals were anesthetized using 10% ketamine to facilitate the process of drawing blood samples from the heart. Blood samples were centrifuged at 3000 rpm for 10 minutes. The serum was kept in the refrigerator at -20°C until the biochemical analysis was performed, which included Aspartate Aminotransferase (ALT), Alanine Aminotransferase (AST), lipid profile, blood urea nitrogen (BUN), Creatinine, Malondialdehyde (MDA), Catalase (CAT), Superoxide Dismutase (SOD) tests.
       
According to Table 2, A notable elevation in (ALT) activity was observed in the group exposed to (25 mg/kg) CCl4, reaching (39.67±1.20 IU/L), in contrast to the control group exibiting (18.33±1.20 IU/L). Additionally, the group receiving a combination of CCl4 (25 mg/kg) and pumpkin (100 mg/kg) demonstrated an ALT activity of (29.00±2.08 IU/L). On the other hand, a significant increase in (AST) activity in the CCl4 group was noted, recording (47.00±4.04 IU\L) in comparison to the control group, the group treated with CCl4 + (100 mg\kg) aqueous extract of pumpkin and the group treated with (100 mg\kg) pumpkin only recording (47.00 ±4.04 IU\L), (31.00±1.00 U\L) and (19.00 ±1.00 IU\L), respectively.

Table 2: Comparison between difference groups in AST and ALT.


 
Lipid profile
 
Results showed in (Table 3) indicate a substantial elevation in cholesterol levels in the CCl4 group recording (105.00 4.51) compared to the control group and CCl4 group treated with (100 mg\kg) aqueous extract of pumpkin and with group treated with (100 mg\kg) pumpkin only (87.00±2.64), (87.67±1.20) and (85.67±2.96), respectively. Results indicate a notable reduction in triglyceride levels within the CCl4 group + aqueous extract of pumpkin at (100 mg/kg) yielding (87.00±1.73 mg/dL), in contrast to (94.33±2.97 mg/dL) observed in the CCl4 group. A significant raise in High-Density Lipoprotein (HDL) levels was observed in the CCl4 group with the addition of (100 mg/kg) pumpkin aqueous extract, reaching (28.67±0.66 mg/dL), in comparison with the CCl4 group (free of addition), which exhibited (18.67 ± 0.67 mg/dL). The findings indicated a statistically significant elevation of Low-Density Lipoprotein (LDL) levels exclusively in the CCl4)group (67.66±3.84 mg/dL) in comparison to the control group (43.00±2.64 mg/dL) and the CCl4 group with the addition of (100 mg/kg) aqueous pumpkin extract, which amounted to (42.00±1.52 mg/dL). Furthermore, results showed no significant difference in the level of very low-density lipoprotein VLDL in the CCl4 group (18.76±0.62 mg\dl) in comparison to the group treated with (100 mg\kg) aqueous extract of pumpkin (17.40±0.34 mg\dl).

Table 3: Comparison between difference groups in lipid profile.


 
Kidney function
 
Outcomes from (Table 4) demonstrate significant increase in (BUN) levels in the CCl4 group (45.66±2.18 mg\dL) compared to the CCl4 group treated with aqueous extract of pumpkin (100 mg\kg) (28.67±2.40 mg\dL) and control group (24.67±2.60 mg\dL). As for the creatinine level, a significant increase was observed in the CCl4 group recording (2.21±0.11mg\dL) compared to the CCl4 group treated with aqueous extract of pumpkin (100 mg\kg) and control group amounted to (0.980±0.04) and (0.466±0.08mg\dL), respectively.  

Table 4: Comparison between difference groups in B. urea and creatinine.


  
Anti-oxidant
 
According to Table 5, findings displayed that MDA levels were significantly higher in the CCl4 group (5.40±0.36 IU\L) than in the CCl4 control group treated with (100 mg\kg) aqueous extract of pumpkin, group treated with (100 mg\kg) pumpkin only and control group (3.067±0.07 IU\L), (1.466±0.13 IU\L) and (1.783±0.24 IU\L), respectively. Additionally, a substantial increase in the activity of (CAT) enzyme was recorded in the group treated of CCl4 (7.60±1.23 IU\L) compared with CCl4 group treated with (100 mg\kg) aqueous extract of pumpkin (3.73±0.44 IU\L) and control group (2.89±0.23 IU\L). Moreover, similar increase was observed in the SOD enzyme in the CCl4 group (12.81±1.44 IU\L) compare with CCl4 group treated with aqueous extract of pumpkin100 mg\kg (8.19 ±0.58 IU\L) and control group (5.69±0.28 IU\L).

Table 5: Comparison between difference groups in anti-oxidant.


       
A balanced amino acid content was observed in the (PSPI) as shown in Table 6. The highest quantities of Lysine, Alanine, Tyrosine, Histidine and phenylalanine (10.80, 10.80, 9.80, 9.80 and 9.88) µg, respective were observed compared to other essential amino acids. In contrast, high amounts of the non-essential amino acids, cysteine (4.52 µg) and glycine (3.08 µg) were detected. From a hydrophobic perspective, amino acid sequence, structure and concentration all affect a protein’s antioxidant capacity (Chi et al., 2015; Alyasiri et al., 2025). The high content of hydrophobic amino acids namely (glycine, valine, alanine and leucine) might be the reason behind the increase in lipid solubility of (PSPI). Additionally, a study by (Ferenczy and Kellermayer, 2022; Hasan et al., 2021; Abd El-Rahmana et al., 2024) reported that hydrophobic amino acids the elevated concentration may improve protein stability by compressing the inner core of the protein, preventing structural disturbances and promoting molecular integrity. Histidine, phenylalanine, tyrosine and tryptophan are examples of aromatic amino acids that have been shown to act as antioxidants by converting free radicals into stable molecules by providing them with an electron. The ability of histidine (2.312%) and arginine (3.182%) to donate hydrogen ions justifies their existence, influencing the protein’s buffering capacity and possibly enhancing protein’s functional properties. 

Table 6: (PSPI) Amino acid analysis.


       
FTIR assay was conducted to distinguish between different functional groups present in the (PSPI). Fig 1 illustrates the functional groups associated with the sample’s active components, according to the positions of peaks present in the infrared region. The (FTIR) of the (PSPI) spectra were examined in the 4000-600 cm-1 frequency range. The distinctive peaks observed in the (PSPI) FTIR analysis result resemble amide groups (I, II and III) in addition to β-sheet structure of proteins. The fingerprint zone of protein amide (I and II) bands are known to be in the range of 1633 to 1452 cm-1 (Kaur et al., 2024; Alankooshi et al., 2023). Li et al., (2017) reported that 3276 to 3011 cm-1 wide absorption peak resembles the molecular vibrations observed of N-H and alkyl group. Moreover, (Yang et al., 2021) study results demonstrated that N-H stretching vibrations may resembles peaks within the 1600-1700 cm-1 region, which is equivalent to the proteins secondary structure.

Fig 1: FTIR spectra of (PSPI).


       
It is worth noting that the most significant absorption peak is at 1633 cm-1 is as it is consistence with the β-sheet in proteins. Furthermore, the 1633 cm-1 peak embraces the amide group (amide I, -CO-NH‚ ) signals, as it is noted from stretching vibrations of the C-O and C-N bonds (Peng et al., 2014). In contrast to amide I, the amide II band resembles the peptide bond (-CO-NH) and further represents the secondary amide group with superior complexity, resembling 1525 cm-1 peak. On the other hand, amide III is represented by the peaks suggested to be derived from both C-N stretching and N-H bending in the range between 1300 and 1200 cm-1. Peaks in the range of 1390, 1231 and 1160 cm-1 are suggested to be an indication of the stretching vibrations of C-N groups exhibited both aromatic and aliphatic amines. Lastly, according to a study by Liu (2021); Alyasiri et al. (2018), C-O stretching vibrations exist in the range of 2800 to 3300 cm-1 while the C-H stretching vibrations occurs in the range between 1000 to 1320 cm-1.

CONCLUSION

Results obtained from the current study show that (PSPI) has an abundant nutritional and functional plant based proteins. Its high content of amino acids make it a promising source of alternative protein supplement. High content of amino acids, minerals and essential antioxidants can be incorporated into food products. The results of the study can also be used to develop our understanding of the properties of protein isolated from pumpkin seeds, to maximize nutritional benefits from these seeds in order to enhance food products towards making the most of these seeds to improve the quality of food products’ to promote human health.
 
Funding
 
None.

CONFLICT OF INTEREST

The authors warrant that there is no conflict of interest between them.

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    Full Research Article

    Preparing Protein Hydrolysates from Pumpkin Seeds and Studying Their Chemical and Antioxidant Properties

    A
    Ayat Adnan Abbas1
    G
    Ghydaa H. Aljeboury1
    A
    Abdulameer Jawad Zayier1
    S
    Salah M. Alchalab1
    H
    Hayder Haqi Ismail1
    A
    Ahmed Flayyih Hasan1,2,*
    1Biotechnology Research Center, Al-Nahrain University, Baghdad, Iraq.
    2Department of Medical Laboratory Techniques, College of Health and Medical Technology,Al-Farabi University, Baghdad, Iraq.

    ABSTRACT

    Background: Recent advances in analytical approaches presented new potentials for plant-based drug manufacture within the realms of peptides and proteins.

    Methods: Enzymatic hydrolysis of plant-derived parent proteins yields bioactive peptides, which have been investigated for potential applications across diverse experimental contexts. Differences in pH and ionic strength induced significant effects on the solubility of extracted pumpkin seed proteins, as a direct relationship was observed between the solubility of pumpkin proteins and alkaline pH.

    Result: Applying the kind of solvent for seed defatting significantly influences protein solubility. Specifically, applying the chloroform-methanol (CM) mixture approach for lipid extraction, as opposed to pentane (P), clearly weakened protein solubility. The objective of the current study is to explore how varied environmental parameters, namely pH and ionic strength, can influence the functional properties of pumpkin seed protein isolate (PSPI), including solubility, interfacial properties and emulsifying activity, compared to pumpkin seed protein hydrolysate (PSPH).

    INTRODUCTION

    For food and cosmetic purposes, plant-based proteins are a viable substitute for animal proteins since they are renewable and come from a wide range of sources, particularly legumes, grains and oilseeds (Moure et al., 2006). Although oilseeds are cultivated mostly for their oil, they represent a valuable source of proteins as well. Likewise, the protein content may reach 60% in the oilseed, the by-product, hence, it may represents a valuable addition to the protein extraction process (Khayri et al., 2026). The cultivation of pumpkin (Cucurbita moschata Duch., Cucurbitaceae family) is globally widespread across tropical, subtropical and warm regions, where it is employed in a variety of food industries, including juice production, mash preparation, fermented beverages and jam manufacture (Mitić et al., 2018; Al-Taeea et al., 2026). Despite being rich source of a very important nutrients, pumpkin seeds are widely considered as an industrial by-products and are often used in manufacturing of healthy nutritunal vegetable oils due to their high content of oils (Amin et al., 2019; Aktaş et al., 2018).
           
    Due to the nutritional importance of the extracted pumpkin seed proteins, they are often added to various food products significantly supporting and improving human health, such as anti-microbial and anti-carcinogenic properties, blood coagulation inhibition and the reduction of the negative consequences of protein malnutrition (Yadav et al., 2010; Tomar et al., 2014). Plant proteins are therefore being used more and more as unusual protein sources to fulfill functional roles in food compositions (Chabanon et al., 2007; Ghiath et al., 2025). However, (Yuliana et al., 2014) reported that plant proteins are known for their poor capacity to dissolve in acidic media which is the reason they are not preferred not to be added to highly or moderately acidic foods (such as dressings, foaming and emulsifying), particularly when solubility is necessary for the desired functional qualities (Tang et al., 2024). Proteins from pumpkin seeds were found to be very poorly soluble (less than 20%) at pH values below 5 (Rezig et al., 2013; Al-Saeedi et al., 2026). Enzymatic hydrolysis of oilseed proteins constitutes a highly effective approach for enhancing their functional properties and increasing solubility, especially at pH close to the isoelectric point (pI) (Tokmakov et al., 2021), better emulsifying capabilities and enriched biological activities are among the functional characteristics of particular relevance (Moure et al., 2006; Luo et al., 2014).
           
    To improve functional properties and increase solubility, the described approach of enzymatic hydrolysis of oilseed proteins is applied, although studies on pumpkin seed protein hydrolysates are limited. In a study by (Vaštag et al. 2011), in addition, these proteins hydrolysates exhibit an antioxidants potential as reported by researchers (Xie et al., 2025). On the other hand, a study by (Peričin et al., 2009) demonstrated that highly soluble pumpkin seed proteins can be efficiently obtained through enzymatic hydrolysis approach.
           
    The current study aimed to extract and isolate protein from pumpkin seeds (PSPI) in addition to preparing pumpkin seed protein hydrolysate (PSPH) and addressing the functional properties namely solubility, interfacial and emulsifying capacity of both (PSPI) and (PSPH).

    MATERIALS AND METHODS

    Pumpkin seed powder defatting
     
    Pumpkin seeds were thoroughly washed and cleaned to remove any impurities. Seeds were fully dried by placing them in an oven at 45°C for 48 hours. Afterwards, dry seeds kernels were ground into a fine powder using a lab size grinder. Following the method mentioned by (Devi et al., 2019), n-hexane (1:5 w/v) were applied and defatting was carried out. The defatted samples were dried at 40°C and then were stored at 4°C to be later used.
     
    Pumpkin seed protein isolation (PSPI)
     
    The pumpkin seed powder was suspended in an alkali solution with a pH of 10.00, which was prepared by adding 1 mol L-1 of sodium hydroxide (NaOH) to enable protein suspension. Suspension filtration was carried out after 30 minutes with gentle stirring. In order to assist proteins to be precipitated through increase their dissolving ability, pH needed to be lowered to 5.00 aby adding (1 mol dm-3 HCl) to the suspension. Centrifugation was performed at 4°C and at speed of 10,000 rpm for 20 min. to separate the precipitate from the liquid phase, followed by drying for 48 hours at 30°C (Bučko et al., 2016).
     
    (PSPI) moisture, ash and protein content determination
     
    Moisture and ash content of (PSPI) was determined according to the approach described by (Helrich, 1990). Kjeldahl method was adopted to quantify crude protein content in (PSPI), through applying a conversion factor of 6.25 (N × 6.25) according to the assumption that proteins contain about 16% of nitrogen (Goyal et al., 2022; Ahmed et al., 2026). 
     
    Enzymatic hydrolysis
     
    To prepare (10 g dm-3) (PSPI) suspension, a pH 3 solution was prepared by adding (1 mol dm-3 HCl) followed by adding certain amount of the obtained (PSPI) to the pH 3 solution at 37°C with enzyme to substrate ratio (0.02:1) g. The enzymatic hydrolysis proceeded for 90 minutes. The resulting hydrolyzed suspension was subjected to vacuum filtration followed by drying process in an oven at an inlet temperature of 85°C and an outlet temperature of 50°C following the method described by (Bučko et al., 2016; Al-Ameri et al., 2026) with minor modifications.
     
    Degree of hydrolysis (DH)
     
    Formaldehyde titration method mentioned by (Li et al., 2013) was implemented for the determination of DH as follows: a dilution was achieved to 5 ml of hydrolysates by adding 60 ml of distilled water while being placed on magnetic stirrer. 0.05M of NaOH (standard titration solution) was used to titrate the hydrolysates to a pH of 8.2. After adding 10 milliliters of 14% formaldehyde to the beaker, 0.05 M NaOH was added to proceed the titration until a pH of 9.2 was attained. The volume of consumed NaOH was recorded. Value of DH was calculated using the following equation:

     
    Where,
    C = Concentration of the standard (0.05M) NaOH titration solution.
    V1 = Amount of (0.05 M) NaOH used in titration solution reaching pH of 9.2.
    V2 = Amount of (0.05 M) NaOH used in titration solution reaching pH of 8.2.
    V = Collagen hydrolysate’s total volume.
    m = Raw material’s mass.
     
    Protein determination
     
    Bovine serum albumin was used as the reference for the determination of the crude extract’s protein concentration as (mg/ml), according to the biuret method (Gornall et al., 1949).
     
    Determination of amino acid constituents
     
    The content of amino acids present in the freeze-dried collagen samples was quantified employing  High Performance Liquid Chromatography (HPLC) (Shimadzu, Japan) approach. 0.5M acetic acid at a concentration of 5 µg/mL was used to dissolve amples. According to (Ersser and Davey, 1991), O-phthalaldehyde (OPA) produces a luminous derivative for primary amino acids at least 10 times more sensitively than ninhydrin was applied. At room temperature, the reaction proceeds rapidly when mobile phases A and B (Methanol (Me-OH), Tetrahydrofuran (THF) and 0.02 M Sodium Acetate) buffer solution adjusted to pH 5.9 with a ratio of (80:2.5:17.5) and 0.02 M Tetrahydrofuran (THF) and Methanol (Me-OH) with a ratio of (20: 2.5: 77.5), respectively are used. Proline and hydroxyproline exhibited no observable reactivity, whereas cysteine displays minor reactivity. The concentration of amino acids and the residues are determined by the following formulas:




    DPPH radical scavenging activity assay
     
    Following the procedure described by (Salman et al., 2019) with minor modifications, the scavenging potential of α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical was determined. 1.5 milliliters of extract were mixed to 1.5 milliliters of DPPH. The mixture was stirred and subsequently allowed to stand at ambient temperature for 30 minutes in a dark place. Spectrophotometric assay was carried out at wavelength of 517 nm and the absorbance of the resulting solution was achieved. (DPPH) radical scavenging activity was estimated according to following equation:

     
    Fourier transform infrared spectroscopy (FTIR) assay
     
    The FTIR analysis was achieved by employing a (Shimadzu, Japan) FTIR spectrometer spanning 400-4000 cm-1. Samples were prepared via the potassium bromide disc KBr pallets method, incorporating 2 mg of Moringa (Muyonga et al., 2004).
     
    Water absorption capacity (WAC) and oil absorption capacity (OAC)
     
    For the determination of (WAC) and (OAC), the approach described by Habib et al. (2025) was employed with minor modifications. The procedure involved mixing 1 g of the sample with 10 ml of distilled water and/or 10 ml of oil for 30 seconds and were allowed to stand at room temperature for 30 minutes. Subsequently, samples were exposed to centrifuging for 10 minutes at speed of 5000 rpm. The supernatant was recovered for the estimation of (WAC) and (OAC) /gram of samples.
     
    Denaturation temperature (Td)
     
    Chen et al. (2021) defined the Denaturation Temperature (Td) as the precise temperature where a substance, usually a protein solution, exhibits a changes in its structure through denaturation. A research study performed by (Pati et al., 2010) reported the use of Ostwald viscometer, which is a tool utilized for assessing variations in viscosity due to temperature changes, which enabled the measurement of collagen degradation temperature. For the preparation of (0.1% w/v) collagen with a concentration at (0.997 g/ml), (0.1 M) acetic acid was used followed by incubation of samples at 10°C for 30 min.  
    • Solubility = Total protein in the sample + Protein content of the supernatant.
    • Current solubility NaCl ×100 = Relative solubility percentage.
    • Maximum solubility in the range sample.
    • The ratio of the protein content in the upper liquid to the overall protein content in the sample was used as a foundation to calculate solubility.
           
    To measure viscosity, the temperatures were incrementally raised to 20, 25, 30, 35, 40, 45, 50 and 55°C, with an incubation for 30 min. for each temperature. The below equation was used to quantify viscosity:

     
    Where,
    t1 = Water flow duration.
    P1 = Water density.
    t2 = Sample flow duration.
    P2 = Sample density (0.997 g/ml).
           
    The following equation is utilized to calculate fractional viscosity:

     
    (PSPI) and (PSPH) solubility determination
     
    The solubility of (PSPI) and (PSPH) was determined by preparing suspensions at different concentrations (csusp, 10-100 g dm-3) of (PSPI) and (PSPH) and employing buffer solutions with a range of pH ranged between (3-8) and ionic strength of (Ic=0.1 mol dm-3).
           
    To achieve complete dissolution of the proteins, suspension solutions were set in a water bath at 50°C for 1 hour with continuous stirring. Sartorius membrane filtration with (0.45 μm) of pore size was used for the isolation of soluble proteins to acquire (PSPI) and (PSPH) suspension solutions. Following Lowry method, the amount of dissolved proteins in the protein solution (csol) was measured expressed in g dm-3 (Lowry et al., 1951).
     
    Ethical approval
     
    Approval was obtained from the Research Ethics Committee of (Biotechnology Research Center, Al-Nahrain University, Baghdad, Iraq).
     
    Statistical analysis
     
    Statistical Analysis System (SAS) was adopted to distinguish the influence of different groups on research factors (Cary, 2012). To calculate the smallest significant overall differences, Least Significant Difference (LSD) was applied.

    RESULTS AND DISCUSSION

    Determination of moisture, ASH and protein
     
    According to Table 1, the contents of moisture, ash, fat and carbohydrates were 6.97%, 2.87%, 1.31% and 2.82%, respectively. It was found that (PSPI) has an 86.89% protein content. The findings are consistent with those obtained for other oil seeds, such as cashew nuts (91%), sesame seeds (59%), sunflower seeds (70%) and sesame seeds (20%).

    Table 1: Chemical composition of (PSPI).


     
    DPPH scavenging (2, 2-Diphenyl-1-Picrylhydrazyl) of pumpkin extract
     
    The DPPH radical scavenging of pumpkin hydrolysate (62%) concentration.
     
    Amino acid composition
     
    FTIR assay
     
    Liver function
     
    Liver function trial included 4 groups of male albino rats in a total of 20 rats (5 rats\group). The first group represents the control group and was given only water and stander pellet. The second group was given (25 mg/kg) of CCl4. The third group was fed with (25 mg/kg) of CCl4 + (100 mg/kg) aqueous extract of pumpkin. The fourth group was given only (100 mg/kg) aqueous extract of pumpkin. Test period was 35 days. Laboratory animals were anesthetized using 10% ketamine to facilitate the process of drawing blood samples from the heart. Blood samples were centrifuged at 3000 rpm for 10 minutes. The serum was kept in the refrigerator at -20°C until the biochemical analysis was performed, which included Aspartate Aminotransferase (ALT), Alanine Aminotransferase (AST), lipid profile, blood urea nitrogen (BUN), Creatinine, Malondialdehyde (MDA), Catalase (CAT), Superoxide Dismutase (SOD) tests.
           
    According to Table 2, A notable elevation in (ALT) activity was observed in the group exposed to (25 mg/kg) CCl4, reaching (39.67±1.20 IU/L), in contrast to the control group exibiting (18.33±1.20 IU/L). Additionally, the group receiving a combination of CCl4 (25 mg/kg) and pumpkin (100 mg/kg) demonstrated an ALT activity of (29.00±2.08 IU/L). On the other hand, a significant increase in (AST) activity in the CCl4 group was noted, recording (47.00±4.04 IU\L) in comparison to the control group, the group treated with CCl4 + (100 mg\kg) aqueous extract of pumpkin and the group treated with (100 mg\kg) pumpkin only recording (47.00 ±4.04 IU\L), (31.00±1.00 U\L) and (19.00 ±1.00 IU\L), respectively.

    Table 2: Comparison between difference groups in AST and ALT.


     
    Lipid profile
     
    Results showed in (Table 3) indicate a substantial elevation in cholesterol levels in the CCl4 group recording (105.00 4.51) compared to the control group and CCl4 group treated with (100 mg\kg) aqueous extract of pumpkin and with group treated with (100 mg\kg) pumpkin only (87.00±2.64), (87.67±1.20) and (85.67±2.96), respectively. Results indicate a notable reduction in triglyceride levels within the CCl4 group + aqueous extract of pumpkin at (100 mg/kg) yielding (87.00±1.73 mg/dL), in contrast to (94.33±2.97 mg/dL) observed in the CCl4 group. A significant raise in High-Density Lipoprotein (HDL) levels was observed in the CCl4 group with the addition of (100 mg/kg) pumpkin aqueous extract, reaching (28.67±0.66 mg/dL), in comparison with the CCl4 group (free of addition), which exhibited (18.67 ± 0.67 mg/dL). The findings indicated a statistically significant elevation of Low-Density Lipoprotein (LDL) levels exclusively in the CCl4)group (67.66±3.84 mg/dL) in comparison to the control group (43.00±2.64 mg/dL) and the CCl4 group with the addition of (100 mg/kg) aqueous pumpkin extract, which amounted to (42.00±1.52 mg/dL). Furthermore, results showed no significant difference in the level of very low-density lipoprotein VLDL in the CCl4 group (18.76±0.62 mg\dl) in comparison to the group treated with (100 mg\kg) aqueous extract of pumpkin (17.40±0.34 mg\dl).

    Table 3: Comparison between difference groups in lipid profile.


     
    Kidney function
     
    Outcomes from (Table 4) demonstrate significant increase in (BUN) levels in the CCl4 group (45.66±2.18 mg\dL) compared to the CCl4 group treated with aqueous extract of pumpkin (100 mg\kg) (28.67±2.40 mg\dL) and control group (24.67±2.60 mg\dL). As for the creatinine level, a significant increase was observed in the CCl4 group recording (2.21±0.11mg\dL) compared to the CCl4 group treated with aqueous extract of pumpkin (100 mg\kg) and control group amounted to (0.980±0.04) and (0.466±0.08mg\dL), respectively.  

    Table 4: Comparison between difference groups in B. urea and creatinine.


      
    Anti-oxidant
     
    According to Table 5, findings displayed that MDA levels were significantly higher in the CCl4 group (5.40±0.36 IU\L) than in the CCl4 control group treated with (100 mg\kg) aqueous extract of pumpkin, group treated with (100 mg\kg) pumpkin only and control group (3.067±0.07 IU\L), (1.466±0.13 IU\L) and (1.783±0.24 IU\L), respectively. Additionally, a substantial increase in the activity of (CAT) enzyme was recorded in the group treated of CCl4 (7.60±1.23 IU\L) compared with CCl4 group treated with (100 mg\kg) aqueous extract of pumpkin (3.73±0.44 IU\L) and control group (2.89±0.23 IU\L). Moreover, similar increase was observed in the SOD enzyme in the CCl4 group (12.81±1.44 IU\L) compare with CCl4 group treated with aqueous extract of pumpkin100 mg\kg (8.19 ±0.58 IU\L) and control group (5.69±0.28 IU\L).

    Table 5: Comparison between difference groups in anti-oxidant.


           
    A balanced amino acid content was observed in the (PSPI) as shown in Table 6. The highest quantities of Lysine, Alanine, Tyrosine, Histidine and phenylalanine (10.80, 10.80, 9.80, 9.80 and 9.88) µg, respective were observed compared to other essential amino acids. In contrast, high amounts of the non-essential amino acids, cysteine (4.52 µg) and glycine (3.08 µg) were detected. From a hydrophobic perspective, amino acid sequence, structure and concentration all affect a protein’s antioxidant capacity (Chi et al., 2015; Alyasiri et al., 2025). The high content of hydrophobic amino acids namely (glycine, valine, alanine and leucine) might be the reason behind the increase in lipid solubility of (PSPI). Additionally, a study by (Ferenczy and Kellermayer, 2022; Hasan et al., 2021; Abd El-Rahmana et al., 2024) reported that hydrophobic amino acids the elevated concentration may improve protein stability by compressing the inner core of the protein, preventing structural disturbances and promoting molecular integrity. Histidine, phenylalanine, tyrosine and tryptophan are examples of aromatic amino acids that have been shown to act as antioxidants by converting free radicals into stable molecules by providing them with an electron. The ability of histidine (2.312%) and arginine (3.182%) to donate hydrogen ions justifies their existence, influencing the protein’s buffering capacity and possibly enhancing protein’s functional properties. 

    Table 6: (PSPI) Amino acid analysis.


           
    FTIR assay was conducted to distinguish between different functional groups present in the (PSPI). Fig 1 illustrates the functional groups associated with the sample’s active components, according to the positions of peaks present in the infrared region. The (FTIR) of the (PSPI) spectra were examined in the 4000-600 cm-1 frequency range. The distinctive peaks observed in the (PSPI) FTIR analysis result resemble amide groups (I, II and III) in addition to β-sheet structure of proteins. The fingerprint zone of protein amide (I and II) bands are known to be in the range of 1633 to 1452 cm-1 (Kaur et al., 2024; Alankooshi et al., 2023). Li et al., (2017) reported that 3276 to 3011 cm-1 wide absorption peak resembles the molecular vibrations observed of N-H and alkyl group. Moreover, (Yang et al., 2021) study results demonstrated that N-H stretching vibrations may resembles peaks within the 1600-1700 cm-1 region, which is equivalent to the proteins secondary structure.

    Fig 1: FTIR spectra of (PSPI).


           
    It is worth noting that the most significant absorption peak is at 1633 cm-1 is as it is consistence with the β-sheet in proteins. Furthermore, the 1633 cm-1 peak embraces the amide group (amide I, -CO-NH‚ ) signals, as it is noted from stretching vibrations of the C-O and C-N bonds (Peng et al., 2014). In contrast to amide I, the amide II band resembles the peptide bond (-CO-NH) and further represents the secondary amide group with superior complexity, resembling 1525 cm-1 peak. On the other hand, amide III is represented by the peaks suggested to be derived from both C-N stretching and N-H bending in the range between 1300 and 1200 cm-1. Peaks in the range of 1390, 1231 and 1160 cm-1 are suggested to be an indication of the stretching vibrations of C-N groups exhibited both aromatic and aliphatic amines. Lastly, according to a study by Liu (2021); Alyasiri et al. (2018), C-O stretching vibrations exist in the range of 2800 to 3300 cm-1 while the C-H stretching vibrations occurs in the range between 1000 to 1320 cm-1.

    CONCLUSION

    Results obtained from the current study show that (PSPI) has an abundant nutritional and functional plant based proteins. Its high content of amino acids make it a promising source of alternative protein supplement. High content of amino acids, minerals and essential antioxidants can be incorporated into food products. The results of the study can also be used to develop our understanding of the properties of protein isolated from pumpkin seeds, to maximize nutritional benefits from these seeds in order to enhance food products towards making the most of these seeds to improve the quality of food products’ to promote human health.
     
    Funding
     
    None.

    CONFLICT OF INTEREST

    The authors warrant that there is no conflict of interest between them.

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