Oxidative stress is an important contributor to the development of degenerative diseases, including cardiovascular diseases, neurodegenerative disorders, diabetes, and cancer (Leyane et al., 2022). It occurs because of an imbalance between free radicals and antioxidants, leading to cellular damage (Barnham et al., 2004). Free radicals, especially reactive oxygen species and reactive nitrogen species, are generated endogenously and exogenously. During aging, the production of endogenous free radicals significantly increases (Barja, 2004). Consequently, elderly individuals are particularly susceptible to oxidative stress and its associated degenerative diseases (Maldonado et al., 2023).

Foods that are rich in antioxidants play an important role in combating oxidative stress in the elderly (Tan and Norhaizan, 2021). According to previous epidemiological studies, antioxidant intake is inversely correlated with the incidence of degenerative diseases (Aune et al., 2018; Mirmiran et al., 2022). For example, foods that are rich in antioxidants, particularly those from fruits, vegetables, and whole grains, contain various vitamins, minerals, and bioactive compounds (e.g., flavonoids, carotenoids, and polyphenols) that neutralize free radicals and protect cells from damage (Lobo et al., 2010).

Pumpkin is a nutrient-dense food and an excellent source of antioxidants (Chuwa and Dhiman, 2023). It is also an essential source of carotenoids, especially β-carotene, which exerts potent antioxidant activities (Ninčević Grassino et al., 2023). Previous studies have demonstrated that pumpkin exhibits significant antioxidant activity (Abbas et al., 2020; Li et al., 2021; Yu et al., 2021). Aside from carotenoids, pumpkin also contains other antioxidant compounds, including lutein, zeaxanthin, and tocopherols (Ninčević Grassino et al., 2023). Besides its antioxidant properties, pumpkin is rich in potassium, magnesium, zinc, vitamin C, linoleic acid, and dietary fiber, making it a promising ingredient for the development of food products (Kulczyński and Gramza-Michałowska, 2019).

A previous study developed an instant pumpkin-based soup that was well accepted by elderly individuals and contains β-carotene, which could meet a significant portion of daily nutritional recommendations (46.9%) (Irwan, 2020). This soup also has an alpha-glucosidase inhibition activity of 94% at a concentration of 5 µg and contains 73.2 µg/100 g of chromium (Irwan et al., 2023). However, this soup has relatively low levels of proteins (Irwan, 2020). Protein is an important contributor to the health of elderly individuals (Shang et al., 2018). Specifically, protein plays an important role in enhancing antioxidant capacity by producing antioxidant enzymes (Ahmad, 1995), preventing muscle damage (Wahjuni and Hartono, 2021; Prodić et al., 2023), and exerting bioactive properties (Prodić et al., 2023). Furthermore, high protein consumption together with high total antioxidant capacity (TAC) has been associated with a low prevalence of frailty in elderly individuals (Kobayashi et al., 2017). In a previous study, the addition of tempeh to soup increased the content of genistein (370.86 mg/100 g) and daidzin (185.61 mg/100 g), superoxide dismutase (SOD) activity, and estradiol hormone levels in ovariectomized rats (Setiawan et al., 2022). However, the addition of tempeh may decrease the acceptability because of its unfavorable color and flavor. Therefore, the present study aimed to evaluate the effects of a newly modified instant pumpkin soup supplemented with chicken breast (IPB) on the antioxidant capacity and oxidative stress levels in elderly individuals.

Design and subjects

In this study, a quasi-experimental design was used to evaluate the effects of two types of instant pumpkin soups on elderly individuals. The research subjects were healthy elderly nursing home residents aged over 65 years. The inclusion criteria were as follows: generally healthy individuals who have the ability to perform daily activities independently; who have no severe chronic diseases, lactose intolerance, severe cognitive impairment, and severe disability; who were non-smokers; and who were not using medications other than aspirin, nonsteroidal anti-inflammatory drugs, vitamin supplements, antihypertensive drugs, or cholesterol-lowering medications (Fuchs et al., 2013). Subjects were excluded if they declined to participate or did not complete the study. The subjects were randomly assigned to one of two groups: the first group received IPB, whereas the second group received instant pumpkin soup prepared with the original recipe (IPO). Each group comprised nine participants. The sample was calculated using Lemeshow’s formula for an experimental study with a level of error (α) of 0.05 and a power of test (1-β) of 95% (Lemeshow et al., 1990). Moreover, having subjects living in a nursing home offers a homogenous environment (Lee et al., 2019), particularly in terms of food consumption, physical activity, daily routine, and medical care and monitoring, which strengthens the study’s reliability.

Production of instant pumpkin soup

The production of instant pumpkin soup was based on Patent No. IDP000082075. Two types of instant pumpkin soups were developed: IPB and IPO. The main ingredients used in the preparation of instant pumpkin soup included pumpkin, carrots, onions, leeks, celery, chicken broth, unsalted butter, cooking cream, salt, pepper, and starch as a filler ingredient. The production process began with the preparation of the chicken broth. Then, pumpkin, carrots, onion, leek, and celery were added to the broth, and the mixture was simmered with rice flour, cooking cream, and a pinch of salt and pepper until a puree was formed. For the preparation of IPB, chicken breast (6.25% of the total pumpkin) was introduced during the simmering phase, and the amount of cooking cream used was reduced. Subsequently, the puree was dried using a drum dryer (Model E-N0 5/5, Changzhou Yibu Drying Equipment Co., Ltd.) to produce sheets. These sheets were then processed and sifted through a 60-mesh screen to form the final instant pumpkin soup powder. Subsequently, the powder was packaged for distribution.

Analysis of nutrient content

The proximate composition of both instant pumpkin soups was analyzed in accordance with standard AOAC methods (AOAC International, 2006). The moisture content was determined using a drying oven at 105°C until a constant weight was achieved, and the weight loss was considered as the water content (AOAC 934.01). The protein content was analyzed using the micro-Kjeldahl method (AOAC 981.10), wherein the sample was digested with sulfuric acid, neutralized with sodium hydroxide, distilled into boric acid, and titrated with hydrochloric acid. The protein content was calculated by multiplying the nitrogen content by 6.25. The fat content was measured using the Soxhlet extraction method (AOAC 920.39) with hexane as the solvent. The ash content was determined by incinerating the sample in a furnace at 550°C until all organic materials were burned off, leaving only inorganic residues (AOAC 923.03). Dietary fiber was measured using the enzymatic-gravimetric method (AOAC 985.29), which involved sequential enzymatic digestion with α-amylase, protease, and amyloglucosidase followed by the precipitation of fiber using ethanol and acetone. Moreover, the β-carotene and vitamin A contents were analyzed using high-performance liquid chromatography (HPLC; Shimadzu Prominence-i LC-2030C, Shimadzu) (Karataş et al., 2019). The sample was treated with ethyl alcohol and centrifuged at 2,400 g for 3 min. The solution was then filtered and extracted using n-hexane, which was subsequently dried using an evaporator. Subsequently, the residue was dissolved in methyl alcohol and prepared for HPLC analysis using a Supelcosil LC-18 column with a mobile phase of acetonitrile, methanol, and water (63:33:4 v/v) at a flow rate of 1.0 mL/min. The detection wavelengths were set at 326 and 454 nm for vitamin A and β-carotene, respectively.

Intervention procedures

Subjects who met the inclusion criteria were provided with the instant pumpkin soup. The daily serving was 15 g (dry weight), rehydrated with 100 mL of water, and consumed once per day for 6 days a week for 4 weeks. The serving was based on the serving size of commercially available soup products. The soup was distributed weekly and administered collectively by caregivers from the nursing home to ensure compliance. The caregiver was responsible for ensuring that each subject consumed a full serving of the soup. All procedures in this study were approved by the Ethics Committee of the University of Muhammadiyah Semarang (No. 174/KE/03/2024).

Characteristics of subjects

Data on age, gender, and malnutrition risk were obtained through interviews. The malnutrition risk was assessed using the Mini Nutritional Assessment-Short Form (Kaiser et al., 2009). The nutritional status was evaluated based on the body mass index (BMI), which was determined by measuring the height and weight according to standard procedures (Sánchez-García et al., 2007). A calibrated stature (Seca 213, Seca) and digital body scale (Omron BF 511, Omron) were used to measure the height and weight, respectively.

Blood collection

Blood samples were collected at the beginning and end of the intervention for biochemical analysis. About 5 mL of blood samples was aseptically drawn through venipuncture. The samples were then centrifuged at 1,500 g for 20 min to separate the plasma from the cellular components (Dimopoulos et al., 2008). Subsequently, the plasma was transferred into polypropylene tubes and stored at −20°C until further analysis.

Analysis of antioxidant capacity and oxidative stress

Several plasma antioxidant capacity parameters, including TAC, SOD, glutathione peroxidase (GPx), and catalase (Cat), were evaluated. The oxidative stress parameters that were assessed included malondialdehyde (MDA), oxidized low-density lipoprotein (ox-LDL), 8-isoprostane, tumor necrosis factor-α (TNF-α), and 8-hydroxy-2’-deoxyguanosine (8-OHdG). Enzyme-linked immunosorbent assay (ELISA) kits were used in the analysis following the manufacturer’s instructions. A microplate ELISA reader (Bio-Rad 580, Bio-Rad) was used for the measurements. The kits were produced by Medikbio, Indonesia.

Data analysis

Data were analyzed parametrically to assess differences between groups and between pre- and postintervention. Group differences were evaluated using an independent t-test, whereas pre- and postintervention differences were analyzed using a dependent t-test. Statistical significance was considered at P<0.05.

Nutrient content of instant pumpkin soups

The IPB had a higher protein content (13.40% vs. 2.20%) than the IPO. Moreover, IPB exhibited higher levels of dietary fiber (13.9% vs. 9.21%), β-carotene (6.28 mg/ 100 g vs. 3.38 mg/100 g), vitamin A (96.63 µg/100 g vs. 81.82 µg/100 g), and ash content compared with IPO (5.68% vs. 3.00%). Meanwhile, IPB had lower fat, carbohydrate, and energy contents than IPO (Table 1).

Table 1 . Energy and nutrient contents of instant pumpkin soups per 100 g.

Energy and nutrient	IPB	IPO
Energy (kcal)	405.63	491.69
Ash (g)	5.68	3.00
Fat (g)	10.25	16.50
Water (g)	5.73	3.87
Carbohydrate (g)	69.17	78.30
Protein (g)	13.40	2.20
Dietary fiber (g)	13.90	9.21
β-carotene (mg)	6.28	3.38
Vitamin A (µg)	96.63	81.82

IPB, instant pumpkin soup supplemented with chicken breast; IPO, instant pumpkin soup prepared with the original recipe..

Characteristics of subjects

The present study included subjects with a mean age of over 70 years, with no significant age differences between the two groups (75.0±6.2 years vs. 71.8±6.7 years). The sex distribution in both groups also showed no significant difference. In terms of BMI and malnutrition screening, no significant differences were observed in the nutritional status between groups. Both groups had participants classified as having a normal BMI but at risk of malnutrition (Table 2).

Table 2 . Characteristics of subjects.

Characteristics	IPB (n=9)	IPO (n=9)	P-value
Age (years)	75.0±6.2	71.8±6.7	0.309
Sex			0.052
Male (%)	55.6	11.1
Female (%)	44.4	88.9
MNA-SF score	11.3±1.0	12.0±1.2	0.224
BMI (kg/m2)	22.4±3.5	22.4±2.6	0.998

Values are presented as mean±SD..

The differences in age, MNA-SF score, and BMI were analyzed using an independent t-test, whereas gender was analyzed using the Spearman test..

IPB, instant pumpkin soup supplemented with chicken breast; IPO, instant pumpkin soup prepared with the original recipe; MNA-SF, Mini Nutritional Assessment-Short Form; BMI, body mass index..

Effects of instant pumpkin soups on the antioxidant capacity and oxidative stress in the elderly

Both types of instant pumpkin soups demonstrated potential benefits for the antioxidant status and oxidative stress in the elderly. However, IPB showed more potent effects than IPO. Plasma SOD levels significantly increased in the IPB group (432.63±396.03 pg/mL; P=0.011), whereas no significant change was observed in the IPO group (127.30±364.69 pg/mL; P=0.326). Although other parameters, including plasma TAC, GPx, and Cat, showed improvement in both groups, these changes were not statistically significant. Furthermore, plasma MDA, ox-LDL, TNF-α, and 8-OHdG levels were significantly reduced in the IPB group, whereas only plasma ox-LDL, TNF-α, and 8-OHdG levels were significantly reduced in the IPO group (P<0.05). Notably, the reductions in plasma ox-LDL (−555.03±155.86 vs. −83.09±66.45 pg/mL; P<0.001) and TNF-α levels (−58.24±22.85 vs. −30.59±18.08 ng/mL; P=0.012) were significantly more pronounced in the IPB group than in the IPO group (Fig. 1).

Figure 1. Effects of two types of instant pumpkin soups on the antioxidant capacity and oxidative stress in the elderly. Differences in the antioxidant capacity and oxidative stress between baseline and endline were analyzed using a dependent t-test. The changes between the IPB and IPO groups were compared using an independent t-test (*P<0.05, **P<0.01). IPB, instant pumpkin soup supplemented with chicken breast; IPO, instant pumpkin soup prepared with the original recipe; LDL, low-density lipoprotein.

The present study demonstrated an increase in the protein, β-carotene, vitamin A, fiber, and ash contents in instant pumpkin soup when supplemented with chicken breast. Compared with the findings in a previous study, the modified soup (IPB) exhibited higher levels of protein, fiber, and β-carotene (Irwan, 2020). Chicken, particularly breast fillet, is known to enhance the nutritional value of soups, especially by increasing the protein content (Thuy et al., 2020, 2023). Chicken breast fillet contains approximately 30% protein and 10 µg of vitamin A per 100 g (USDA, 2019). Aside from adding chicken breast, the use of cooking cream was reduced in the present study, which decreased the fat and energy content. This modification is particularly beneficial for the elderly, for whom the fat intake should be limited, whereas the intake of protein, vitamins, minerals, and fiber needs to be increased (Yeung et al., 2021). Furthermore, instant cream soup is a potential type of food with a high rate of acceptance by the elderly because of its texture, which aligns with oral motor function (Irwan, 2020).

The IBP and IPO exhibited beneficial effects on the antioxidant status of the elderly participants. Although statistical analysis revealed few significant increases, the mean antioxidant capacity and levels of antioxidant enzymes in both groups showed improvements. Notably, the SOD levels in the IPB group significantly increased. These findings are in line with those of previous studies, suggesting that pumpkin is a valuable source of antioxidants when incorporated into various food products, including bread, noodles, biscuits, and cakes (Akter et al., 2020; Wahyono et al., 2020; Indrianti et al., 2021; Hussain et al., 2022). In a previous study, the methanol extracts of pumpkin peel demonstrated an antioxidant activity equivalent to 151 mg Trolox/100 g (Kulczyński and Gramza-Michałowska, 2019). Moreover, pumpkin contains various bioactive compounds, including 10 phenolic compounds (e.g., kaempferol, salicylic acid, and ferulic acid) and two flavonoid compounds (catechin and kaempferol) (Stryjecka et al., 2023). Pumpkin polysaccharides also exhibit strong scavenging activity against free radicals (ABTS+ and OH•) in MIN6 cells (Gao et al., 2019). Additionally, animal studies have shown that pumpkin-based products may improve oxidative stress by increasing the levels of GPx (Ghahremanloo et al., 2018), Cat (Abou Seif, 2014), and SOD (Chen et al., 2020).

The high contents of β-carotene, vitamin A, vitamin C, and phenolic compounds in pumpkin play an important role in neutralizing free radicals and reducing oxidative stress, thereby contributing to its strong antioxidant properties. Furthermore, pumpkin may facilitate the upregulation of key antioxidant enzymes. Pumpkin polysaccharides have been shown to enhance the expression of dauer formation-16, superoxide dismutase-1, and skinhead-1, which are key regulators of SOD (Gao et al., 2019), and nuclear factor-erythroid 2-related factor-2 (Nrf2), which is a transcription factor that promotes the expression of GPx and Cat (Chen et al., 2015). Moreover, the activation of Nrf2 has been linked to the suppression of nuclear factor-kappa B, resulting in reduced TNF-α levels, as observed in the present study (Wang et al., 2023). TNF-α is a major proinflammatory cytokine that exacerbates oxidative stress (Pickering and O’Connor, 2007). A significant decrease in TNF-α levels suggests that instant pumpkin soup may alleviate oxidative stress through its anti-inflammatory effects.

The present study also incorporated chicken breast into the soup, increasing the protein content, which may contribute to the soup’s enhanced biological activity. Protein, particularly cysteine, plays an important role in glutathione production and muscle cell support, contributing to a reduction in oxidative stress (Lu, 2009). Adequate protein intake is also essential for modulating the inflammatory response, especially in the regulation of proinflammatory cytokines, such as TNF-α (Hruby and Jacques, 2019). Several amino acids, including glutamine, glutamate, cysteine, and aspartate, can stimulate the activation of Nrf2 in the cell by modulating Kelch-like ECH-Associated Protein 1 (KEAP1), which allows Nrf2 to translocate to the nucleus and activate the enzymes (Egbujor et al., 2024). Therefore, the enhanced effects of IPB on antioxidant capacity and oxidative stress are likely attributed to its high protein, β-carotene, vitamin A, and fiber contents.

The nutritional profile of IPB was significantly improved compared with IPO. IPB had higher protein, dietary fiber, β-carotene, vitamin A, and ash contents and had lower fat, carbohydrate, and energy contents compared with IPO. Additionally, IPB demonstrated more potent effects on oxidative stress and inflammation in the elderly, with significant increases in plasma SOD levels and more pronounced reductions in plasma MDA, ox-LDL, TNF-α, and 8-OHdG levels compared with IPO. These results suggest that IPB offers greater antioxidant and anti-inflammatory benefits, making it a promising functional food for enhancing the lifespan of and reducing oxidative damage in older adults. However, the use of a quasi-experimental design without a control or placebo group limits the ability to establish causal inferences regarding the effects of the soup on the antioxidant capacity of the elderly. Although the subjects lived in a nursing home, several factors (e.g., food consumption, physical activity, and daily habits) were not strictly controlled. Future studies should include a more controlled environment, a larger and more diverse sample of subjects, and a longer intervention period to enhance the generalizability of the findings and evaluate long-term outcomes.

We would like to thank the Jakarta Santa Anna Elderly Home and the Bogor Hanna Elderly Home for providing the participants for this study. We also extend our gratitude to Dr. Al Mukhlas Fikri for his invaluable comments on this manuscript.

None.

The authors declare no conflict of interest.

Concept and design: WSI, BS, AS, H, TM. Analysis and interpretation: WSI, BS, AS, H, TM. Data collection: WSI, BS, AS, H, TM. Writing the article: WSI, BS, AS, H, TM. Critical revision of the article: WSI, BS, AS, H, TM. Final approval of the article: all authors. Statistical analysis: WSI, BS. Obtained funding: None. Overall responsibility: BS.