White pumpkin-based bioactive compositions for diabetes, dyslipidemia and obesity management

Abstract

Diabetes (DM) is a group of metabolic disorders characterized by chronic hyperglycemia, possibly accompanied by lipemia and obesity. These conditions drive insulin resistance and increase cardiovascular risk. Cucurbita moschata having a chemical name contains a variety of carotenoids, flavonoids, L-arginine, and cucurbitacine, which are biologically active compounds known to humans with a variety of therapeutic effects including anti-inflammatory, anti-oxidation, and health-promoting properties. The present disclosure proposes a white pumpkin powder (WPP)-based composition to improve glucose uptake, accelerate lipid metabolism, and lead to weight loss. In-vitro experiments carried out on human liver HepG2 cells indicate that glucose uptake and insulin sensitivity are remarkably improved, lipid in liver cells is reduced, and the influence on adipogenesis as a weight loss marker is increased. Furthermore, based on the outcome of the disclosure, the proposed WPP composition has the ability to be used at its optimal dose as an effective potential natural therapeutic agent for managing diabetes, dyslipidemia and obesity.

Description

Technical Field

[0001] The present disclosure relates generally to a fruit-based composition for treating diabetes and inducing weight loss, and more particularly to a pumpkin flour-based composition as an effective treatment for diabetes, dyslipidemia, and obesity. Background Art

[0002] Diabetes mellitus (DM) is a long-term metabolic disorder characterized by high blood glucose levels due to the body's inability to produce or properly use insulin, often accompanied by dyslipidemia and obesity (see Alam et al., “Diabetes Mellitus: insights from epidemiology, biochemistry, risk factors, diagnosis, complications and comprehensive management,” Diabetology, 2(2), 36-502021, and Dilworth et al., “Diabetes mellitus and its metabolic complications: the role of adipose tissues,” International Journal of Molecular Sciences, 22, 14, 7644, 2021). The condition can lead to serious complications affecting the heart (see Wong et al., “Cardiovascular risk in diabetes mellitus: epidemiology, assessment and prevention,” Nature Reviews Cardiology, 20(10), 1288-1308, 2019), blood vessels, eyes, kidneys, and nerves. Obesity and dyslipidemia (abnormal lipid levels) further increase insulin resistance, making it more difficult for the body to regulate blood sugar levels. DM is generally divided into two main types: type 1, in which the body's immune system destroys insulin-producing cells; and type 2, in which insulin resistance is often influenced by lifestyle and genetic factors.

[0003] Therefore, effective diabetes management needs to pay attention to both glucose and lipid metabolism, because solving these aspects is essential for reducing metabolic complications associated with the disease. Natural compounds have shown promising potential in preventing or treating the degenerative conditions associated with diabetes. Research supports the therapeutic effects of various plant extracts and isolated compounds, which have bioactive properties that can enhance insulin sensitivity and regulate lipid levels (see Colville et al., " Antioxidant status, peroxidase activity, and PR protein transcript levels in ascorbate-deficient Arabidopsis thaliana vtc mutants [antioxidant status, peroxidase activity and PR protein transcription levels of ascorbate-deficient Arabidopsis thaliana vtc mutants], " Journal of Experimental Botany [Experimental Botany] 59 (14), 3857-3868, 2008). For example, studies on the Cucurbitaceae (a family of plants generally considered to consist of melons, gourds, and pumpkins) indicate that pumpkins are rich in bioactive components (specifically, polysaccharides, proteins and peptides, p-aminobenzoic acid, and sterols) (see Adams et al., "The hypoglycemic effect of pumpkins as anti-diabetic and functional medicines," Food Research International, 44, 862-867, 2011). These findings suggest that natural compounds may provide a complementary approach in diabetes care, particularly in managing metabolic disorders associated with glucose and lipid imbalances.

[0004] In the Cucurbitaceae family, white squash, scientifically known as Cucurbita pepo L., is rich in various carotenoids (lycopene, phytoene, and lutein), flavonoids (apigenin, myricetin, and kaempferol), L-arginine, and cucurbitacins, which are bioactive compounds known for their various therapeutic effects, including anti-inflammatory, antioxidant, and health-promoting properties (see Huerta-Reyes et al., “Selected species of the Cucurbitaceae family used in Mexico for the treatment of diabetes mellitus,” Molecules, 27(11), 3440, 2022 and Sánchez-Velázquez et al., “Nutritional, bioactive components and health properties of the milpatriad system seeds,” Frontiers inNutrition[Frontiers in Nutrition], 10, 2023). Although some studies have investigated the cellular mechanisms of white pumpkin, its overall therapeutic benefits remain uncharted territory.

[0005] In vitro testing is considered a fundamental step in understanding the cellular and molecular effects of compounds to be used for therapeutic purposes. It is essential to evaluate the potential efficacy and safety of potential drugs in a controlled environment before entering animal and / or clinical studies. Among the various in vitro models reported in the prior art, the liver HepG2 cell model is commonly used in in vitro experiments (see Dehn et al., “Characterization of the human hepatocellular carcinoma (hepG2) cell line as an intro model for cadmium toxicity studies,” In Vitro Cellular & Developmental Biology–Animal, 40(5), 172-182, 2004, and Shao et al. “Construction and application of liver cancer models in vitro,” Engineered Regeneration, 3(3), 310-322, 2022) to study the functional sites of white pumpkin to study glucose and lipid metabolism. Similarly, adipogenesis (differentiation of preadipocytes into fat-storing adipocytes) plays a key role in obesity (see Gupta, “Adipocytes,” Current Biology, 24(20), 988-993, 2014 and Sekar et al., “Autophagy: A molecular switch toregulate adipogenesis and lipolysis,” Molecular and Cellular Biochemistry, 477(3), 727-742, 2022).

[0006] Based on a thorough prior art analysis, it has been determined that there is a huge research gap regarding any potential molecular mechanisms by which white pumpkin alters cellular glucose, lipid metabolism, and differentiation pathways. Although some in vivo and in vitro studies have pointed out the hypoglycemic and hypolipidemic properties of white pumpkin (see Sedigheh et al., “Hypoglycemic and hypolipidemic effects of pumpkin on alloxan-induced diabetic rats,” African Journal of Pharmacyand Pharmacology, 5(23), 2620-2626, 2011 and Rahayu et al., “Hypoglycemic and antioxidant activity of yellow pumpkin in diabetic rats,” Indian Journal of Public Health Research & Development, 11(1), 1300-1304, 2020), to date, no studies have directly addressed the effects of white pumpkin extracts on hepatocyte or adipocyte biology. The antioxidant capacity of white pumpkin, which may protect pancreatic cells from oxidative stress and enhance insulin activity, has also not been studied.

[0007] In view of the above shortcomings observed in the prior art, the present disclosure proposes the use of white pumpkin powder, which has been found to be very effective on glucose uptake, lipid metabolism and adipocyte differentiation using HepG2 cells. In vitro experiments have been conducted to demonstrate that white pumpkin powder (WPP) is an effective protective mechanism for the following: glucose regulation in the liver, preventing lipid storage in hepatocytes and lipogenic activity to manage diabetes, dyslipidemia and obesity. Summary of the invention

[0008] A simplified summary of the features disclosed herein is presented below to provide a basic understanding of some exemplary embodiments of the present disclosure. This summary is neither an exclusive overview of all different embodiments of the present disclosure nor an attempt to identify the key elements of the present disclosure. The sole purpose of this summary is to present some concepts disclosed herein in a simplified form as a prelude to a more comprehensive description.

[0009] The object of the present disclosure is to provide a composition and a method for preparing the composition to combat chronic hyperglycemia, dyslipidemia and obesity using carotenoids, flavonoids, L-arginine and cucurbitacin from natural sources.

[0010] According to one embodiment of the present disclosure, the disclosed composition may include a fruit-based natural ingredient, which may belong to, for example, the Cucurbitaceae plant family.

[0011] According to another embodiment of the present disclosure, the disclosed composition may include zucchini in powdered form, hereinafter referred to as white squash powder (WPP).

[0012] According to yet another embodiment, the present disclosure provides a means of producing the disclosed composition, which may include sampling, decontaminating, freeze-drying, and grinding white pumpkin into WPP.

[0013] According to another embodiment of the present disclosure, the disclosed composition at an optimal concentration can serve as an effective agent for managing diabetes, regulating lipid metabolism, and reducing obesity.

[0014] According to another embodiment of the present disclosure, the disclosed composition at an optimal dose can improve cell viability with very low cytotoxicity, indicating the possibility for therapeutic application.

[0015] According to another embodiment, an optimal dose of the disclosed composition can reduce lipid accumulation, which plays an important role in the treatment of lipid metabolism disorders.

[0016] According to the embodiments, the disclosed compositions can enhance glucose uptake, wherein an optimal dose restores effective levels of glucose to levels comparable to untreated cells.

[0017] According to a final embodiment, the disclosed composition may also increase insulin sensitivity due to the antioxidant properties of WPP. According to the same embodiment, catalase activity may also be increased.

[0018] The above paragraphs are provided by way of general introduction and are not intended to limit the scope of the following claims.The described embodiments, together with further advantages, will be best understood by reference to the following detailed description taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The above and other features, aspects and advantages of the present invention are described in detail below with reference to the accompanying drawings of various embodiments, which are intended to illustrate rather than limit the present invention. The accompanying drawings include the following figures, in which:

[0020] Figure 1 Cell viability of WPP-treated HepG2 cells is shown—values ​​are expressed as standard error of the mean, where p>0.05 was considered significant;

[0021] Figure 2Crystal violet staining of cell viability indicated by WPP-treated HepG2 cells is shown—values ​​are expressed as standard error of the mean;

[0022] Figure 3 Shown are the percentage dead cell counts of Trypan blue staining of WPP-treated HepG2 cells—values ​​are expressed as standard error of the mean;

[0023] Figure 4 The percentage reduction of lipid accumulation in WPP-treated HepG2 cells is shown - values ​​are expressed as standard error of the mean, where p>0.05 was considered significant and '*' shows the significance level;

[0024] Figure 5 Shown are the measured values ​​of triglycerides (expressed in μg / mg protein) in WPP-treated HepG2 cells - values ​​are expressed as standard error of the mean, where p>0.05 was considered significant and '*' shows the significance level;

[0025] Figure 6 The results of glucose uptake in WPP-treated HepG2 cells (expressed as percentage increase) are shown - values ​​are expressed as standard error of the mean, where p>0.05 was considered significant and '*' shows the significance level;

[0026] Figure 7 Shown is the insulin sensitivity observed in WPP-treated HepG2 cells - values ​​are expressed as standard error of the mean, where p>0.05 was considered significant and '*' shows the significance level;

[0027] Figure 8 shows the catalase levels observed in WPP-treated HepG2 cells - values ​​are expressed as standard error of the mean, where p>0.05 was considered significant and '*' shows the significance level; and

[0028] Fig. 9 Shown are the percentage increases in SOD levels in WPP-treated HepG2 cells - values ​​are expressed as standard error of the mean, where p>0.05 was considered significant and '*' shows the significance level. DETAILED DESCRIPTION

[0029] Different embodiments of the present disclosure will now be described in detail by reference to the various components that the disclosed compositions may contain. The term "excipient" may sometimes be used to describe all or some components other than one or more active ingredients, but it should be remembered that some excipients may be active and some active ingredients may have excipient characteristics. In addition, unless otherwise expressly stated, the ingredients, components, excipients, etc. of the disclosed compositions are suitable for one or more of the intended purposes discussed elsewhere herein, such as cosmetically acceptable, environmentally acceptable, pharmaceutically acceptable, acceptable as a food additive, etc.

[0030] The disclosed invention proposes a pharmaceutical composition having white pumpkin powder (WPP) as an active ingredient. The disclosed composition has been analyzed by detailed in vitro studies to investigate the effects of different concentrations of the disclosed composition on glucose uptake and insulin insensitivity, lipid metabolism and weight loss effects as observed by HepG2 cells.

[0031] Experimental methods and setup preparation

[0032] 1. Preparation of WPP

[0033] WPP is prepared by freeze drying Cucurbita oleracea fruit to preserve its bioactive components and then producing a fine powder with a mesh size of about 80. WPP is then dissolved in a suitable amount of dimethyl sulfoxide (DMSO) to prepare a solution of the desired concentration. It is first sterilized and then the solution is used to treat (in vitro) cultured cells.

[0034] 2. Cultivation of Cell Lines

[0035] Human hepatocyte cell line (HepG2) was grown in Dulbecco's modified Eagle's medium-high glucose (DMEM-HG) supplemented with 100 U / ml penicillin and 10% fetal bovine serum (FBS) until reaching the subculture stage. Subsequent experiments were performed after the medium containing FBS was replaced with serum-free DMEM medium as determined in the prior art.

[0036] 3. Preparation and Dilution of WPP Stock Solution

[0037] Stock WPP was prepared by dissolving 10 mg WPP in 1 ml of DMSO (10 mg / ml) to produce a 10 mg / ml stock solution. Working solutions of 10 μg / ml, 50 μg / ml and 100 μg / ml medium were prepared by diluting the solution in normal (DMEM) medium and filtering through a 0.22 mm sterile syringe filter after mixing.

[0038] 4. Treatment of cell lines with WPP

[0039] The administration of WPP to cultured cells was achieved by inoculating HepG2 cells on 96-well and 24-well plates, respectively. Cells were treated with WPP dilutions at doses of (10 μg / ml, 50 μg / ml and 100 μg / ml) for 24 hours. Cell lysates were prepared for protein evaluation, and cells were analyzed for cell viability as previously described. In all subsequent experiments, the administered cells were treated with an optimal dose. The most suitable dose of white pumpkin would be the level that makes viability close to normal cell viability.

[0040] 5. Cell Viability Assay

[0041] MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed on cells cultured on 96-well plates. Different concentrations of WPP solution were applied to HepG2 cells and after 24 hours of treatment, the cells were subjected to MTT assay to determine cell viability (Senthilraja and Kathiresan 2015). MTT solution (25 μl) was added for 2-3 hours post-treatment and then the purple crystals were dissolved with sodium dodecyl sulfate (SDS) (10%). The absorbance was measured at 570 nm after 3 hours of incubation.

[0042] 6. Trypan blue assay

[0043] Cell viability is evaluated by trypan blue, which is a separation agent for live cells and dead cells. After the cells from each experimental group were washed three times with PBS, these cells were then processed with trypan blue (Invitrogen Inc., USA) for 15 min. The cells were subsequently washed three times with PBS and visualized under a microscope. Dead cells are identified as those stained with trypan blue, and cell viability percentage (dead cells) is determined by dividing the cell number (live cells) excluding trypan blue by the total number of cells multiplied by 100.

[0044] 7. Crystal violet

[0045] Crystal violet staining was also used to assess cell viability. The method was based on 96-well plates. The culture medium of each well of the experimental group was discarded and washed once with PBS. After washing, 0.1% crystal violet dye in 2% ethanol was applied to the wells so that the surface was completely covered. It was allowed to stand at room temperature for 15 minutes. Any dye was carefully removed and the wells were washed thoroughly. Thereafter, 600 μl of 1% SDS was added to each well to dissolve the colorant (for 5–10 minutes) and the absorbance at 540 nm was read on the microtiter plate after incubation.

[0046] 8. Induction of Lipid Accumulation

[0047] The induction of the accumulation and retention of fatty acid-induced lipids in cultured HepG2 cells was performed by plating the cells in 24-well plates and treating them with a medium rich in fatty acids (0.25 mM oleic acid) for 24 hours. After this procedure, the cells were incubated with different concentrations (10 μg / mL, 50 μg / mL, 100 μg / mL) of WPP solutions in the culture medium for 24 hours.

[0048] 9. Oil Red O Staining

[0049] The cells were washed three times with cold PBS and fixed in 4% paraformaldehyde for 30 minutes. The cells were fixed, washed 3 times and incubated at room temperature for 15 min with Oil Red O solution (working solution: 0.5 g of Oil Red O powder dissolved in 60% ethanol). Then, the cells were washed with PBS to remove excessive unbound coloring agents. All samples were mixed with DMSO for the quantification of Oil Red O content. After incubation for 5 min while stirring at room temperature, the optical density (OD) of the samples was measured at a wavelength of 510 nm.

[0050] 10. Intracellular Triglyceride Measurement

[0051] A commercial triglyceride assay kit was used to detect intracellular triglyceride levels in HepG2 cells. After treatment, cells were washed with cold PBS and lysed with a lysis buffer solution containing 1% Triton X-100 in PBS. Cell lysates were harvested by centrifugation and the supernatant was carefully separated for analysis. The lysate was then transferred to a 96-well plate and the triglyceride reagent was added according to the manufacturer's instructions. After incubating the plate according to the manufacturer's instructions, the absorbance at 570nm was measured using a microplate reader. This model allows the evaluation of the lipid-lowering effect of this treatment.

[0052] 11. Glucose Uptake Assay

[0053] HepG2 cells were seeded into 96-well plates and grown to approximately 70%-80% confluence. Subsequently, the cells were starved in serum-free medium (SFM) for 4–6 hours to induce sensitivity to glucose uptake. After starvation, the cells were treated with or without WPP, and insulin (100 nM) was added to the cells for 15-30 minutes to promote glucose uptake. The cells were then incubated with a solution of the glucose analog 2-NBDG for 30 minutes at 37°C (as per the kit instructions) and then washed with cold PBS to eliminate excess glucose plates that were not incorporated. Fluorescence was quantified at a given excitation / emission wavelength (485 / 535 nm) using a microplate reader. Glucose uptake was compared using the relative difference in fluorescence intensity between the treated and control groups.

[0054] 12. Insulin sensitivity measurement

[0055] HepG2 cells were plated in 6-well plates and cultured until 70% to 80% confluency was reached. Subsequently, the cells were starved in serum for 4–6 h before insulin stimulation as a means of increasing their sensitivity to the hormone. After starvation, the cells were incubated with WPP and then stimulated with 100 nM insulin for 15-30 min. After stimulation, the cells were lysed with the lysis buffer included in the assay kit and collected as lysate. For Akt (p-Akt), a phosphorylation-specific ELISA was performed according to the instructions of the kit. The absorbance at 450 nm was measured in a microplate reader, and the changes in insulin sensitivity represented by p-Akt levels in treated and control cells were compared.

[0056] 13. Superoxide dismutase (SOD) assay

[0057] The antioxidant activity of HepG2 cells treated with white pumpkin powder was assessed using superoxide dismutase (SOD) assay. SOD activity was determined by the performance of the enzyme measured using a commercially available test kit in neutralizing superoxide free radicals. After collecting the cell lysate after WPP treatment, SOD was assayed according to the kit instructions. Reaction monitoring was performed by a colorimetric method that measured the inhibition of the reaction induced by superoxide. This demonstrates the influence of white pumpkin powder in regulating the antioxidant defense mechanism of cells.

[0058] 14. Catalase Assay

[0059] The antioxidant activity of WPP-treated HepG2 cells was screened by catalase assay based on the reduction of hydrogen using a commercially available kit. The cells were lysed to prepare the samples and after treatment with white pumpkin powder, the catalase assay was performed according to the protocol provided by the kit. The H 2 O 2 Catalase activity is measured as a decrease in absorbance, which reflects the effect of the compound on maintaining intracellular redox balance.

[0060] Results and Analysis

[0061] The data of the experimental groups are expressed as mean ± SEM of three experimental replicates. For statistical analysis of the data, the mean values ​​of each group were compared by one-way ANOVA, followed by Bonferroni test to determine the differences between the groups. Graph Pad software was used to perform statistical evaluation of the quantitative data from the experimental groups by using two-way ANOVA. Statistical significance was determined by a p value of less than 0.05.

[0062] 1. Cell Viability Assessment

[0063] MTT assay was performed to observe the viability of HepG2 cells after treatment with different concentrations of WPP, and the results are shown in Figure 1 For the untreated control group, cell viability was measured to be 100% ± 8.03. After treatment with 10 μg / ml WPP, viability increased to 109% ± 5.45, while exposure to 50 μg / ml WPP caused it to drop to -91.6% ± 5.16. More interestingly, at 100 μg / ml of WPP, viability went back up to 106% ± 8.24. The data suggest that at limited doses of WPP, cell viability varied slightly, indicating a nonlinear response relative to the μg / ml concentration of the WPP solution administered.

[0064] Figure 1 The results presented in reveal that the WPP solution exhibited an abnormal increase in cell viability at lower (10 μg / ml) and higher (100 μg / ml) concentrations, and furthermore, a decrease in viability at a concentration of 50 μg / ml indicated the complexity of the interaction between WPP and HepG2 cells. This behavior may be due to a dose-dependent biphasic effect on cell viability, and may be a result of differences in the activity of antioxidants or bioactive compounds at different concentrations as reported in the prior art. The decrease at 50 μg / ml may also be due to mild cytotoxicity, while the improvement at 100 μg / ml may indicate a toxicohormesis effect, in which low-level stress activates a transcellular protective mechanism.

[0065] A crystal violet assay was performed to assess the viability of HepG2 cells exposed to graded concentrations of WPP. Figure 2 As shown in the graph of , cells not incubated with any sugar solution presented an absorbance equal to 1 ± 0.0884 (control). In contrast, a slight decrease in the measured absorbance was observed with 10 μg / ml WPP solution (0.912 ± 0.0952), further decreased to a lower level (0.884 ± 0.0446) at 50 μg / ml, and decreased to 0.901 ± 0.0545 at 100 μg / ml WPP concentration.

[0066] As from Figure 2 The lower HepG2 cell viability observed in, although significant only at the highest WPP concentration, seems to indicate that the cytotoxicity of WPP is very low at doses corresponding to the doses used. The observed reduction in cell viability is comparable to previous work showing that certain plant-derived compounds have limited effects on cell proliferation without substantial cytotoxicity to hepatocytes. These results suggest that WPP may be safe to use at up to 100 μg / ml and may even be sensitive in therapeutic and research applications without causing severe cytotoxic effects.

[0067] Trypan blue exclusion assay was performed to determine the viability of HepG2 cells after treatment with different doses of WPP. Figure 3 As shown, in the untreated control group, the proportion of dead cells was 11.4% ± 2. After treatment with 10 μg / ml WPP, there was a small increase in dead cells (11.7% ± 1.79), and the percentage of dead cells at 50 μg / ml WPP (11.4% ± 2.5) did not rise. In contrast, treatment with 100 μg / ml of WPP resulted in an increased cell death recorded as 10.1% ± 2.64.

[0068] Figure 3 The results show that whether or not the cells are treated with WPP does not seem to affect the rate at which HepG2 cells are killed at any tested concentration. However, the higher percentage of dead cells observed at a concentration of 100 μg / ml WPP indicates potential cytotoxicity, but since very little cell death was found overall, the overall effect can be considered non-cytotoxic. This is related to previous studies that have shown that some plant extracts exhibit low cytotoxicity and support cell health and vitality at lower doses, but show toxicity and deleterious effects on cells at higher concentrations. The percentage of dead cells remained stable at lower concentrations (10 and 50 μg / ml), which is consistent with studies indicating that low doses of bioactive compounds are most likely to protect liver cells, emphasizing the prospects of WPP as a safe therapeutic agent, especially at low concentrations.

[0069] 2. Lipid Accumulation

[0070] To determine lipid accumulation, Oil Red O staining assay was performed on HepG2 cells treated with different concentrations of WPP, and the results are shown in Figure 4 The lipid accumulation level of the group not exposed to the treatment was measured to be 7.93% ± 1.71. At higher concentrations, WPP significantly reduced lipid accumulation at 10 μg / ml (58.5% ± 3.01) and 50 μg / ml (29.9% ± 2.32), with a reduction of 29.0% and an average range of 63.7%. At the highest WPP concentration of 100 μg / ml, lipid accumulation was reduced by 75.6% (20.1% ± 3.06).

[0071] Figure 4The results show that WPP dose-dependently reduces lipid accumulation in HepG2 cells. WPP appears to have anti-adipogenic properties, as indicated by a significant reduction in lipid levels (especially at higher concentrations). The significant reduction in lipid accumulation at 100 μg / ml is consistent with the observation that specific bioactive plant compounds can trigger adipogenic regulatory pathways, resulting in better hepatic lipid profiles, suggesting a potential role for WPP in treating lipid metabolism disorders.

[0072] 3. Triglyceride Measurement

[0073] HepG2 cells were monitored to quantify triglyceride levels after treatment with different concentrations of WPP, as Figure 5 As shown. The triglyceride of the untreated control was 31.4 ± 3.15 μg / mg protein. Inducing lipid accumulation resulted in a significant increase in triglyceride content (80.5 ± 5.03 μg / mg protein). When treated with 10 μg / ml, the triglyceride content was 73.4 ± 2.89 μg / mg protein, and when treated at WPP equal to or higher than 50 μg / ml, it had a significantly lower level (57.9 ± 2.27 μg / mg protein). Importantly, a significant protective effect of 100 μg / ml WPP against lipid accumulation was observed, as evidenced by the reduced triglyceride levels reaching 34.4 ± 4.04 μg / mg protein (P < 0.001).

[0074] Figure 5 The results showed that WPP reduced triglyceride levels in HepG2 cells in a dose-dependent manner. The sharp decrease noted at a concentration of 100 μg / ml is consistent with previous studies that demonstrated the alleviation of lipid accumulation due to enhanced fatty acid oxidation and improved hepatic lipid metabolism by plant extracts. In addition, the significant reduction in triglycerides at low concentrations means that the bioactive compounds in WPP may have lipid-lowering activity and is consistent with previous evidence showing that natural products may play an important role in metabolic disorders.

[0075] 4. Glucose Uptake Assay

[0076] Glucose uptake in HepG2 cells after treatment with different concentrations of WPP was evaluated via 2-NBDG glucose uptake assay, and the results are presented in Figure 6Regarding glucose uptake, the glucose uptake level of untreated control cells was 68.6% ± 3.15. In the absence of serum (CFM), glucose uptake was significantly reduced by 3-5 times (19.5% ± 5.03). In the presence of 10 μg / ml WPP, glucose uptake reached 26.6% ± 2.89, while the use of a further concentration of 50 μg / ml raised this level to 42.1% ± 2.27, which further increased to 65.6% ± 4.04 after treatment with 100 μg / ml WPP.

[0077] The results indicate that WPP enhanced glucose uptake in HepG2 cells in a dose-dependent manner, with the highest concentration of 100 μg / ml completely restoring glucose effective levels to levels similar to those observed in untreated cells. This indicates the possibility of an insulin mimetic or insulin sensitizing effect of WPP. The significant rise in glucose uptake at elevated WPP concentrations correlates with other studies indicating that plant-derived antioxidants and polyphenols can improve insulin sensitivity and glucose metabolism.

[0078] 5. Insulin Sensitivity Assay

[0079] p-AKT ELISA assay was used to measure the assessment of insulin sensitivity in HepG2 cells treated with different levels of WPP, as Figure 7 As shown. It was observed that the untreated control group was 1.39 ± 0.271 (p-AKT level per unit of insulin), which corresponds to baseline insulin sensitivity. In SFM, p-AKT levels were significantly reduced to 0.151 ± 0.0419 (p < 0.001), indicating reduced insulin signaling. At 10 μg / ml WPP, WPP treatment resulted in 0.571 ± 0.084 p-AKT, which increased to 0.722 ± 0.082 at 50 μg / ml WPP, and finally reached 1.36 ± 0.228 at 100 μg / ml WPP concentration.

[0080] The substantial increase in p-AKT at 100 μg / ml suggests that WPP may improve insulin signaling, preferably by activating the Akt pathway. Various findings in the prior art demonstrate that plant extracts with antioxidant properties can activate the PI3K / Akt pathway, a major pathway for insulin action. This is consistent with other plant-based therapies that increase p-AKT expression and appear to improve glucose metabolism, which may be beneficial in restoring insulin sensitivity. These findings suggest that WPP may be effective in improving insulin sensitivity and glucose homeostasis.

[0081] 6. Catalase Assay

[0082] The antioxidant capacity of catalase in HepG2 cells was determined after treatment with different concentrations of WPP. The level of catalase activity in untreated cells was taken as 100% (i.e., 98.3 ± 6.05%). After treatment with 10 μg / ml WPP, catalase activity decreased to 29.3% ± 7.54, with a marginal improvement of 39.8% ± 4.33 observed. When treated with 50 μg / ml WPP, the measured levels recovered significantly (76.5% ± 3.77), eventually reaching above baseline (103% ± 4.73) at a concentration of 100 μg / ml WPP, as shown in Figure 2. Figure 8 shown.

[0083] Figure 8 The results suggest that WPP restores the increase in catalase activity in HepG2 cells in a dose-dependent manner, thereby counteracting the oxidative stress caused by SAMP (serum deprivation and / or partial hepatectomy of hepatocellular carcinoma cell lines). The significant increase in catalase activity, especially at a dose of 100 μg / ml, indicates the antioxidant potential of WPP, which is a key factor in oxidative defense. In summary, these results emphasize WPP as a promising candidate molecule for antioxidant therapeutics for diseases associated with oxidative stress.

[0084] 7. SOD assay

[0085] HepG2 cells were treated with different concentrations of WPP to determine the antioxidant activity of SOD, where the results are shown in Fig. 9 In. The SOD percentage is the relative level of SOD in treated cells compared to SOD in untreated cells, wherein 100% is defined as 57.3% ± 1.76 activity measured for untreated (i.e., only culture medium is added). In serum-free culture medium (SFM), SOD activity is significantly reduced to 23.5% ± 3.51, meaning a high level of oxidative stress. SOD levels are improved (29.6% ± 4.49 SOD) after applying 10 μg / ml WPP solution. A further increase of 43.1% ± 3.26 was observed at 50 μg / ml WPP concentration, wherein the highest SOD activity (63.7% ± 3.98) was obtained when cells were incubated with the maximum concentration of WPP (100 μg / ml).

[0086] Fig. 9 The results showed that WPP increased the SOD activity level in HepG2 cells at the highest dose with statistical significance. The results showed that SOD levels increased significantly, especially at 100 μg / ml, indicating that WPP has the ability to effectively enhance the cellular antioxidant defense system.

[0087] As will be apparent from the description, numerous modifications and variations of the disclosed embodiments may be made.It should be noted that the various embodiments of the present disclosure disclosed herein may be implemented in a manner different from the specific description provided herein, as long as the embodiments fall within the scope defined by the following claims.

Claims

1. A composition based on natural fruit powder for improving human health, comprising: bioactive compounds that enhance cell viability; biologically active compounds with reduced cytotoxic effects; bioactive compounds that reduce lipid accumulation; biologically active compounds with increased fatty acid oxidation; biologically active compounds that exhibit increased insulin sensitizing effects; as well as Bioactive compounds that have the ability to increase the cellular antioxidant defense system.

2. The composition based on natural fruit powder as claimed in claim 1, wherein the natural fruit powder further comprises: White pumpkin powder; and Organic solvents.

3. The white pumpkin powder as claimed in claim 2, wherein: The white pumpkin is freeze-dried; and Converted to a powder with a mesh size ranging between 75 and 84.

4. The composition of claim 2, wherein the organic solvent is dimethyl sulfoxide (DMSO).

5. The natural fruit powder-based composition as claimed in claim 1, wherein the optimal dosage concentration is between 10 μg / ml and 110 μg / ml.

6. The natural fruit powder-based composition according to claim 1, wherein the composition: Possesses the ability to act as an antidiabetic agent; It is lipid-lowering in nature; Has the ability to resist oxidative stress; and Has the ability to induce weight loss.

7. The natural fruit powder based composition as claimed in claim 1, wherein when administered at an optimal dose of the composition, lipid accumulation in vitro is reduced by up to 76%.

8. The natural fruit powder based composition as claimed in claim 1, wherein when administered at an optimal dose of the composition, the in vitro triglyceride level is reduced to 34.4±4.04 μg / mg protein.

9. The natural fruit powder based composition as claimed in claim 1, wherein in vitro glucose uptake is reduced up to 65.6% ± 4.04 when administered at an optimal dose of the composition.

10. The natural fruit powder based composition of claim 1, wherein when administered at an optimal dose of the composition, in vitro insulin sensitivity increases to 1.36±0.228 p-AKT.

11. The natural fruit powder based composition as claimed in claim 1, wherein in vitro catalase level increases up to 103% ± 4.73 when administered at an optimal dose of the composition.

12. The natural fruit powder based composition as claimed in claim 1, wherein when administered at an optimal dose of the composition, in vitro SOD level increases up to 63.7% ± 3.98.

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