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

Abstract

Diabetes mellitus (DM) is a group of metabolic disorders characterized by chronic hyperglycemia, which may also be followed by lipidemia and obesity. These conditions drive insulin resistance and heig

Description

FIELD OF INVENTION

[001] The present disclosure generally pertains to a fruit-based composition for treating diabetes and inducing weight loss, more particularly, to a composition based on white pumpkin powder as an effective treatment for diabetes, lipidemia and obesity. BACKGROUND

[002] Diabetes mellitus (DM) is a long-term metabolic disorder marked by high blood sugar levels due to the body’s inability to produce or properly use insulin, often followed by lipidemia and obesity (see Alam et al., “Diabetes Mellitus: insights from epidemiology, biochemistry, risk factors, diagnosis, complications and comprehensive management,” Diabetology, 2(2), 36-50 2021, and Dilworth et al., “Diabetes mellitus and its metabolic complications: the role of adipose tissues,” International Journal of Molecular Sciences, 22, 14, 7644, 2021). This condition can cause severe 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 characterized into two primary types: Type 1 where the body’s immune system destroys insulin-producing cells, and Type 2, where the insulin resistance is often influenced by lifestyle and genetic factors.

[003] Hence, effective diabetes management requires a dual focus on both glucose and lipid metabolism, as addressing these aspects is vital for reducing metabolic complications associated with the disease. Natural compounds have shown promising potential in preventing or treating degenerative conditions related to diabetes. Research supports the therapeutic effects of various plant extracts and isolated compounds, which have bioactive properties that may 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,” Journal of Experimental Botany 59 (14), 3857-3868, 2008). For instance, studies on Cucurbitaceae, a plant family generally considered to consist of melons, cucurbits and pumpkins, indicate that pumpkins are rich in biologically active components (specifically, polysaccharides, proteins and peptides, para-aminobenzoic acids 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 could offer a complementary approach in diabetes care, particularly in managing metabolic disorders associated with glucose and lipid imbalances.

[004] Amongst the Cucurbitaceae, white pumpkins, scientifically referred to as Cucurbita pepo L., are abundant with a variety of carotenoids (phytoene, phytofluene, and lutein), flavonoids (apigenin, myricetin, and kaempferol), L-arginine, and cucurbitacin, which are bioactive compounds known for diverse 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 Sanchez-Velazquez et al., “Nutritional, bioactive components and health properties of the milpa triad system seeds”, Frontiers in Nutrition, 10, 2023). Although a few studies have investigated the cellular mechanisms of white pumpkin, its general therapeutic efficacy still remains an unchartered territory.

[005] In vitro testing is considered an essential step towards understanding the cellular and molecular effects of compounds to be utilized for therapeutic purposes. This is considered crucial for evaluating the potential potency and safety of a possible drug within a controlled environment before advancing to animal and / or clinical studies. Amongst the multiple in vitro models reported in the prior-art, the liver HepG2 cells model is commonly used in vitro experiment (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 function site of white pumpkin to study the glucose and lipid metabolism. Likewise, adipogenesis, the differentiation of preadipocytes into fat-storing adipocytes plays an essential role in obesity (see Gupta, “Adipocytes,” Current Biology, 24(20), 988-993, 2014 and Sekaret al., “Autophagy: A molecular switch to regulate adipogenesis and lipolysis,” Molecular and Cellular Biochemistry, 477(3), 727-742, 2022).

[006] Based on the thorough prior-art analysis, large research gaps have been identified to exist concerning any underlying 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 its hypoglycemic and hypolipidemic properties (see Sedigheh et al., “Hypoglycemic and hypolipidemic effects of pumpkin on alloxan-induced diabetic rats,” African Journal of Pharmacy and 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 study has directly addressed the influence of white pumpkin extracts on liver cell or adipocyte biology. Its antioxidative capacity that can shield pancreatic cells against oxidative stress and enhance insulin activity has also not been investigated.

[007] In light of the aforementioned shortcomings observed in the prior-art, the present disclosure proposes the usage of white pumpkin powder which has been researched to be highly effective against glucose uptake, lipid metabolism, and fat cell differentiation using HepG2 cells. In vitro experiments have been carried out to prove that the white pumpkin powder (WPP) is an effective protective mechanism for: glucose regulation in the liver, prevention of lipid storage in the hepatocytes, and adipogenic activity to manage diabetes, dyslipidemia, and obesity. SUMMARY OF THE PRESENT DISCLOSURE

[008] The following presents a simplified summary of features disclosed herein to provide a basic understanding of some exemplary embodiments of the present disclosure. This summary is neither an exclusive overview of all the different embodiments of the present disclosure, nor intended to identify the critical elements of this disclosure. Its sole purpose is to present some concepts disclosed herein in a simplified form as a precursor to a more comprehensive description.

[009] It is an object of the present disclosure to provide a composition and method of making the same, utilizing naturally occurring sources of carotenoids, flavonoids, L-arginine and cucurbitacin to fight chronic hyperglycemia, lipidemia and obesity.

[010] 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.

[011] According to another embodiment of the present disclosure, the said disclosed composition may include powdered form of Cucurbita pepo L. hereinafter referred to as the white pumpkin powder (WPP).

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

[013] According to another embodiment of the present disclosure, the optimum concentration of the disclosed composition may act as an effective agent for managing diabetes, regulating lipid metabolism, and decreasing obesity.

[014] According to another embodiment of the present disclosure, the disclosed composition in its optimal dosage may improve cell viability and very low cell cytotoxicity suggesting the possibility for use in therapeutic application.

[015] According to another embodiment, the optimum dosage of the disclosed composition may reduce lipid accumulation playing a major role in the treatment of lipid metabolism disorders.

[016] According to an embodiment, the disclosed composition may enhance glucose uptake with the optimum dosage restoring glucose validity levels like those in untreated cells.

[017] According to one last embodiment, the disclosed composition may also increase insulin sensitivity due to the antioxidant properties of the WPP. According to the same embodiment, the catalase activity may also increase.

[018] The foregoing paragraphs have been 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

[019] The foregoing and other features, aspects, and advantages of the present invention are described in detail below with reference to the drawings of various embodiments, which are intended to illustrate and not to limit the invention. The drawings comprise the following figures in which:

[020] FIG. 1 shows the cellular viability of WPP-treated HepG2 cells - values expressed as standard error of mean, where p >0.05 is considered as significant;

[021] FIG. 2 shows the crystal violet staining for cell viability expressed by WPP-treated HepG2 cells - values expressed as standard error of mean;

[022] FIG. 3 shows the trypan blue staining for WPP-treated HepG2 cells for the percentage count of dead cells - values expressed as standard error of mean;

[023] FIG. 4 shows the percentage reduction in lipid accumulation in WPP-treated HepG2 cells - values expressed as standard error of mean, where p >0.05 is considered as significant and shows the level of significance;

[024] FIG. 5 shows the measurement of triglycerides in the WPP-treated HepG2 cells in terms of pg / mg of proteins - values expressed as standard error of mean, where p >0.05 is considered as significant and shows the level of significance;

[025] FIG. 6 shows the glucose uptake results in WPP-treated HepG2 cells in terms of percentage increase - values expressed as standard error of mean, where p >0.05 is considered as significant and shows the level of significance;

[026] FIG. 7 shows the insulin sensitivity observed in WPP-treated HepG2 cells - values expressed as standard error of mean, where p >0.05 is considered as significant and shows the level of significance;

[027] FIG. 8 shows catalase levels as observed in WPP-treated HepG2 cells - values expressed as standard error of mean, where p >0.05 is considered as significant and shows the level of significance; and

[028] FIG. 9 shows percentage increase in SOD levels in WPP-treated HepG2 cells - values expressed as standard error of mean, where p >0.05 is considered as significant and shows the level of significance. DETAILED DESCRIPTION

[029] The different embodiments of the disclosure will now be described in detail by reference to the various components which the disclosed composition may comprise. The term “excipient” may be used occasionally to describe all or some of the components other than the active principle(s) bearing in mind that some excipients can be active and that some active principles can have excipient character. Additionally, if not explicitly otherwise stated, ingredients, components, excipients etc. of the disclosed composition are suitable for one or more of the intended purposes discussed elsewhere herein e.g. are cosmetically acceptable, environmentally acceptable, pharmaceutically acceptable, acceptable as food additives etc.

[030] The disclosed invention presents a pharmaceutical composition with white pumpkin powder (WPP) as an active ingredient. The disclose composition has been analyzed through detailed in vitro studies to investigate the effect of different concentrations of the disclosed composition on glucose uptake and insulin insensitivity, lipid metabolism, and weight loss effects as observed through HepG2 cells. [031 ] Experiment Methodology and Setup Preparation

[032] 1. Preparation of the WPP

[033] WPP is prepared by freeze-drying white pumpkin fruit to preserve its bioactive components and subsequently followed by producing a fine powder with an approximate 80 mesh size. The WPP is then dissolved in the appropriate amount of dimethyl sulfoxide (DMSO) to prepare a solution of the desired concentration. This is first sterilized, and then this solution is used to treat (in vitro) the cultured cells.

[034] 2. Culturing of the Cell Line

[035] The human liver cell line (HepG2) was grown in Dulbecco's Modified Eagle Medium-High Glucose (DMEM-HG) that is supplemented with 100 ll / rnl penicillin and 10% fetal bovine serum (FBS) until reaching the sub culturing phase. The ensuing experiments were carried out after switching the FBS containing medium to serum-free DMEM medium as identified in the prior-art.

[036] 3. Preparation of Stock Solution and Dilutions for WPP

[037] The preparation of stock WPP was done by dissolving 10mg WPP in 1ml of DMSO (10mg / ml) producing a 10mg / ml stock solution. Working solutions of 10pg / ml, 50 pg / ml, and 100 pg / ml medium were prepared by diluting the solutions in plain (DMEM) medium and filtered through a 0.22 mm sterile syringe filter after mixing.

[038] 4. Treatment of Cell Line with WPP

[039] Administration of WPP on the cultured cells was achieved by seeding the HepG2 cells onto 96- well and 24-well plates respectively. Cells were treated with (10pg / ml 50 pg / ml and 100 pg / ml) doses of WPP dilutions for 24 hours. Cellular lysates were prepared for protein evaluation, and the cell viability assay was analyzed on cells as described previously. Administered cells were treated with one optimum dose in all subsequent experiments. The most suitable dosage of white pumpkin would be at the level at which the viability is close to that of normal cells.

[040] 5. Cell Viability Assay

[041] 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 administered to HepG2 cells and after 24 hours of treatment, these cells were subjected to MTT assay for cell viability (Senthilraja and Kathiresan 2015). An MTT solution (25ul) was added for 2-3 hours post-treatment, and the purple crystals were then solubilized with sodium dodecyl sulfate (SDS) (10%). Absorbance was measured at 570nm after incubating for 3 hours.

[042] 6. Trypan Blue Assay

[043] Cell viability was evaluated by trypan blue, which was a separator agent of live and dead cells. The cells, after being washed three times with PBS from various experimental groups, were then treated in trypan blue (Invitrogen Inc., USA) for 15 min. Cells were subsequently washed three times with PBS and visualized under a microscope. Dead cells were identified as stained with trypan blue and the percentage cell viability (dead cells) was determined by dividing the number of cells which excluded trypan blue (live cells) with total number of cells multiplied by 100.

[044] 7. Crystal Violet

[045] The Crystal Violet Staining Method was also used to assess cell viability. Method was performed based on a 96-well plate. The media was discarded from each well of the experimental groups and washed once with PBS. After washing, 0.1 % crystal violet dye in 2% ethanol was applied to the wells so that the surface is completely covered. This was allowed to stand at room temperature for 15 mins. Any dye was removed carefully, and wells were washed thoroughly. Following this, 600pl 1% SDS was added in each well to solubilize the stain (for 5-10 min) and the absorbance at 540nm was read on a microtiter plate after incubation.

[046] 8. Induction of Lipid Accumulation

[047] Induction of accumulation and retention of fatty acid-induced lipids in cultured HepG2 cells was done by plating cells in a 24-well plate and treated with fatty acid (0.25 mM oleic acid) enriched medium for 24 hours. Following this procedure, cells were incubated with WPP solution in the medium at different concentrations (10 pg / mL, 50 pg / mL, 100 pg / mL) for 24 hours.

[048] 9. Oil Red O Staining

[049] The cells were washed three times with chilled PBS and fixed for 30 minutes in 4% paraformaldehyde. Cells were fixed followed by 3 washes and incubated with Oil Red O solution (working solution: 0.5g of Oil Red O powder dissolved in 60% ethanol) at room temperature for 15min. Cells were then washed with PBS to remove excess, unbound staining. All samples were mixed with DMSO for quantification of Oil Red O content. After incubating at room temperature for 5 min while agitating, the optical density (OD) of the sample was measured on a wavelength 510 nm.

[050] 10. Intracellular Triglycerides Measurement

[051] A commercial triglyceride assay kit was used to detect intracellular triglyceride levels in HepG2 cells. After treatment, the cells were washed using chilled PBS and a lysis buffer solution containing 1% Triton X-100 in PBS was used to lyse the cells. The cell lysate was harvested by centrifugation and the supernatant was separated carefully for analysis. The lysate was then transferred into a 96-well plate and triglyceride reagent was added as per the manufacturer's instructions. Absorbance at 570nm was measured following incubation of the plate as per the instructions of the manufacturer using a microplate reader. This model allowed the assessment of the hypolipidemic effects of this treatment.

[052] 11. Glucose Uptake Assay

[053] HepG2 cells were seeded into a 96-well plate and grown to approximately 70-80% confluency. Subsequently, the cells were starved in serum-free medium (SFM) for 4-6 hours to induce sensitivity for glucose uptake. After starvation, cells were treated with or without the WPP treatment and insulin (100 nM) was added to cells for 15-30 minutes to promote glucose uptake. The cells were then incubated for 30 minutes at 37°C with a solution of the glucose analog 2-NBDG (following kit instructions) and afterwards washed with cold PBS to eliminate unincorporated excess glucose panels. Fluorescence was quantified with a microplate reader at the given excitation / emission wavelengths (485 / 535 nm). The relative difference in fluorescence intensity between the treated and control groups was used to compare glucose uptake.

[054] 12. Insulin Sensitivity Assay

[055] HepG2 cells were placed into a 6-well plate and cultured until reaching the confluency of 70 to 80%. Cells were subsequently starved in serum 4-6 h prior to the insulin stimulation, as a means of increasing their sensitivity to the hormone. After starvation, cells were incubated with WPP and then stimulated with 100 nM insulin for 15-30 min. After stimulation, cells were lysed with the lysis buffer included in the assay kit and collected as lysates. For Akt (p-Akt), a phosphorylation-specific ELISA was performed following the instructions of the kit. The absorbance at 450 nm was measured in microplate reader and the change of insulin sensitivity represented by p-Akt level in treated and control cells were compared.

[056] 13. Superoxide Dismutase (SOD) Assay

[057] The antioxidant activity in white pumpkin powder-treated, HepG2 cells was assessed using a Superoxide Dismutase (SOD) assay. SOD activity was determined by the enzyme performance in neutralizing superoxide radicals, measured using a commercially available kit. The SOD assay was performed as per the kit instructions after collecting the cell lysates posttreatment with WPP. The reaction monitoring was performed by a colorimetric method measuring the inhibition of reactions induced by superoxide. This showcased the impact of white pumpkin powder in regulating cellular antioxidant defense mechanisms.

[058] 14. Catalase Assay

[059] Screening antioxidant activity by catalase assay in WPP-treated HepG2 cells, based on the decrease in hydrogen, was performed with a commercially available kit. Cells were lysed for preparation of the sample and catalase assay was done following protocol supplied with the kit after treating with white pumpkin powder. Catalase activity was measured by monitoring the decrease in H2O2 absorbance at 340 nm, which reflects this compound effect on maintaining redox balance within the cell.

[060] Results and Analysis

[061] The data of experimental groups were expressed as mean ± SEM for three experiment replicates. To analyze the data statistically, group means were compared by one-way ANOVA followed by Bonferroni's test to determine differences between groups. Graph Pad software was used to statistically evaluate quantitative data from the experimental groups by using two ways ANOVA. Statistical significance was determined by a p-value of less than 0.05.

[062] 1. Cellular Viability Assessment

[063] The MTT assay was done to see the viability of HepG2 cells after treatment with different concentrations of WPP with the results shown in FIG. 1. Cell viability was measured to be 100% ± 8.03 for the untreated control group. Viability after treatment with 10 pg / ml WPP increased to 109% ± 5.45, whereas exposure to 50 pg / ml WPP caused depletion of it down to -916% ± 5.16. More interestingly, WPP at 100 pg / ml viability also increased back to 106% ± 8.24. The data suggests a slight variation of cell viability with limited doses of WPP indicating a non-linear response with respect to pg / ml concentrations of the administered WPP solution.

[064] The results presented in FIG. 1 reveal that the WPP solution exhibited a paradoxical increase in cell viability at lower (10 pg / ml) and higher (100 pg / ml) concentrations, moreover a decreased viability at a concentration of 50 pg / ml suggests the complex nature of interactions WPP and HepG2 cells. This behavior may be due to the dose-dependent, biphasic effects on cell viability and might be a result of the differences in antioxidant or bioactive compound activity at differing concentrations as reported in the prior-art. The reduction at 50 pg / ml may also be due to mild cytotoxicity, while the improvement at 100 pg / ml may represent an effect of hormesis, in which low-level stress activates protective mechanisms across cells.

[065] The crystal violet assay was performed to assess the viability of HepG2 exposed to graded concentrations of the WPP. As shown in the chart of FIG. 2, cells that were not incubated with any of the sugar solution presented an absorbance equal to 1 ± 0.0884 (control). In contrast, a slight decline in the measured absorbance (0.912 ± 0.0952) was observed with the 10 pg / ml WPP solution, which further decreased to lower levels (0.884 ± 0.0446) at 50 pg / ml and 0.901 ± 0.0545 at 100 pg / ml WPP concentrations.

[066] As observed from FIG. 2, lower HepG2 cell viability, although significant only at the highest WPP concentrations, appears to indicate a very low cytotoxicity of WPP at doses corresponding to those used. The observed decrease in cell viability is comparable with prior work that has shown the modest impact of certain plant- derived compounds on cell proliferation, without substantial cytotoxicity to hepatocytes. These results imply that WPP is likely safe for use up to 100 pg / ml and may even have sensitivity therapeutic and research applications without causing severe cytotoxic effects.

[067] A trypan blue exclusion assay was performed to determine the viability of HepG2 cells upon treatment with different doses of WPP. As shown in FIG. 3, in the untreated control group, the proportion of dead cells was 11.4% ± 2. There was a small increase (11.7% ±1.79) in dead cells after 10 pg / ml WPP treatment, and the percentage of dead cells (11.4% ±2.5) did not go up at 50 pg / ml WPP. Contrastingly, treatment with 100 pg / ml of WPP resulted in an increased cell death recorded at 10.1% ± 2.64.

[068] The results of FIG. 3 show that whether or not cells were treated with WPP seemed not to affect the rate at which HepG2 cells were killed in any of the concentrations tested. However, the higher percentage of dead cells seen at 100 pg / ml WPP concentration is suggestive of potential cytotoxicity, but since overall, very few cells were found to be dead, the general effect can be considered as non-cytotoxic. This correlates to prior-art studies showing some plant extracts exhibiting low cytotoxicity and supporting cell health and viability at lower doses but displaying toxicity as well harmful effects on cells at higher concentrations. Dead cell percentages stay stable at lower concentrations (10 and 50 pg / ml) which is in accordance with studies indicating that low doses of bioactive compounds are most likely protective of the hepatic cells, underlining the promise of WPP as a safe therapeutic agent, especially at low concentrations.

[069] 2. Lipid Accumulation

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

[071] The results of FIG. 4 show that WPP dose-dependently decreases lipid accumulation in HepG2 cells. The WPP appears to have anti-lipidogenic properties, as indicated by the pronounced decrease in lipid levels (particularly at higher concentrations). The significant decrease in lipid accumulation at 100 pg / ml is consistent with the observation that specific bioactive plant compounds could trigger lipogenic regulatory pathways leading to better hepatic lipid profiles, suggesting a potential role of WPP in the treatment of lipid metabolism disorders.

[072] 3. Triglyceride Measurement

[073] HepG2 cells were monitored to quantify triglyceride levels after treatment with different concentrations of WPP as shown in FIG. 5. Untreated controls had triglycerides of 31.4 ± 3.15 pg / mg protein. Induction of lipid accumulation caused significant elevation of triglyceride content (80.5 ± 5.03 pg / mg protein). The triglyceride contents were 73.4 ± 2.89 pg / mg protein with treatment of 10 pg / ml with dramatically lower levels (57.9 ± 2.27 pg / mg protein) when treated at WPP equal to or above WPP 50pg / ml. Importantly, it was observed that a significant protective effect of 100 pg / ml WPP against lipid accumulation as evidenced by the reduced triglyceride levels reached 34.4 ± 4.04 pg / mg protein (P<0.001).

[074] The results of FIG. 5 suggest that WPP reduces triglyceride levels in HepG2 cells in a dose-dependent manner. The drastic decrease noted at the concentration of 100 pg / ml agrees with previous studies that demonstrated ameliorated lipid accumulation due to enhanced fatty acid oxidation and improved hepatic lipid metabolism by plant extracts. In addition, the significant reduction of triglycerides at small concentrations signifies that bioactive compounds in WPP may have lipid-lowering activities and is consistent with previous evidence showing the important role natural products could play in metabolic disorders.

[075] 4. Glucose Uptake Assay

[076] The glucose uptake of HepG2 cells after WPP treatment at different concentrations were evaluated via the 2-NBDG Glucose Uptake Assay with the results presented in FIG. 6. Untreated control cells had a glucose messenger of 68.6% ±3.15 with respect to glucose uptake. In absence of serum (CFM), glucose uptake was markedly reduced by 3-5 folds (19.5% ± 5.03). The glucose uptake reached 26.6% ± 2.89 in the presence of 10 pg / ml WPP, while this level raised to 42.1% ± 2.27 using further concentration of 50 ug / ml, increasing further to 65.6% ± 4.04 after treatment with 100 pg / ml WPP.

[077] Results indicate that WPP enhances glucose uptake in HepG2 cells in a dosedependent manner, with the highest concentration of 100 pg / ml fully restoring glucose validity levels similar to those seen in untreated cells. This indicates the possibility of the insulinmimetic or insulin-sensitizing effect of WPP. The remarkable rise in glucose uptake at elevated WPP concentrations correlates with other studies indicating that plant-derived antioxidants and polyphenols may ameliorate insulin sensitivity and glucose metabolism.

[078] 5. Insulin Sensitivity Assay

[079] The assessment of insulin sensitivity in HepG2 cells treated with differential levels of WPP using a p-AKT ELISA assay was measured as shown in FIG. 7. The untreated control group was observed to be at 1.39 ± 0.271 (p-AKT level per unit of insulin) corresponding to baseline insulin sensitivity. In SFM, p-AKT levels decreased significantly to 0.151±0.0419 (p<0.001) indicating decreased insulin signaling. WPP treatment led to 0.571 ± 0.084 p-AKT at 10 pg / ml WPP, increasing to 0.722 ± 0.082 at 50 pg / ml WPP, eventually reaching 1.36 ± 0.228 at 100 pg / ml WPP concentration.

[080] The large increase in p-AKT at 100 pg / ml shows that WPP may improve insulin signaling preferably by activating the Akt pathway. Various findings in the prior-art have demonstrated that plant extracts with antioxidant properties may activate the PI3K / Akt pathway, a major pathway of insulin action. This is consistent with other plant-based therapies that raise the expression of p-AKT and appear to ameliorate glucose metabolism, which may all contribute favorably towards restoring insulin sensitivity. These findings suggest that WPP might be effective for the amelioration of insulin sensitivity and glucose homeostasis.

[081] 6. Catalase Assay

[082] The antioxidant capacity of catalase in HepG2 cells was determined after treatment with various concentrations of WPP. The level of catalase activity in untreated cells was taken as 100% (i.e., 98.3 ± 6.05%). Catalase activity was reduced to 29.3% ± 7.54with a marginal improvement of 39.8% ± 4.33 observed after treatment with 10 pg / ml WPP. The levels were measured to be significantly restored (76.5% ± 3.77) when treated with 50 pg / ml WPP, eventually reaching above baseline (103% ± 4.73) at 100 pg / ml WPP concentration as shown in FIG. 8.

[083] The results of FIG. 8 imply that in a dose-dependent manner, WPP restores the increased catalase activity in HepG2 cells counteracting SAMP (serum deprivation of hepatocellular carcinoma cell line and / or partial hepatectomy) with oxidative stress. The significant elevation of catalase activity especially at 100 pg / ml dose indicates antioxidant potential of WPP which is a key factor in oxidative defense. Collectively, these results underscore WPP as a promising candidate molecule for an antioxidative therapeutic agent in diseases associated with oxidative stress.

[084] 7. SOD Assay

[085] HepG2 cells were treated with different concentrations of WPP to determine the antioxidant activity of SOD with the results shown in FIG. 9. Percentage SOD is the relative levels of SOD in treated cells compared to that in untreated cells, where 100% was defined as activity measured for untreated (i.e. media only added) cells at 57.3% ± 1.76. In serum-free medium (SFM), the SOD activity decreased markedly to 23.5% ± 3.51, implying a high level of oxidative stress. SOD levels improved (29.6% ± 4.49 SOD) upon administration of 10 pg / ml WPP solution. A further increase of 43.1% ± 3.26 was observed at 50 pg / ml WPP concentration with the highest SOD activity (63.7% ± 3.98) obtained when cells were incubated with the maximal concentration of WPP (100 pg / ml).

[086] The results of FIG. 9 show that WPP increases SOD activity levels in HepG2 cells with statistical significance at the highest doses. Results showed a significant increase of SOD levels, especially at 100 pg / ml, indicating that WPP has an effective ability to enhance the cellular antioxidant defense system.

[087] As is evident from the description, numerous adjustments and alterations can be made to the disclosed embodiments. It should be noted that the multiple embodiments of the present disclosure, disclosed herein, may be implemented differently from the specific description provided herein, as long as the said implementation falls within the boundaries defined by the following claims:

Claims

1. A natural fruit powder based composition for improving human health, comprising: bioactive compounds which enhance cell viability;bioactive compounds with reduced cytotoxic effects;bioactive compounds reducing lipid accumulation;bioactive compounds with increased fatty acid oxidation;bioactive compounds exhibiting increased insulin sensitizing effects; and bioactive compounds having the ability to increase cellular antioxidant defense system.

2. The natural fruit powder based composition of Claim 1, wherein the natural fruit powder further comprises of:a white pumpkin powder; and an organic solvent.

3. The white pumpkin powder of Claim 2, wherein: the white pumpkin is freeze dried; and converted into 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 of Claim 1, wherein optimum dosage concentration is between 10 pg / ml and 110 pg / ml.

6. The natural fruit powder based composition of Claim 1, wherein the composition; has the ability to act as an anti-diabetic agent;is hypolipidemic in nature;23 04 26has the ability to fight oxidative stress; and has the ability to induce weight loss.

7. The natural fruit powder based composition of Claim 1, wherein the in vitro lipid accumulation is decreased by up to 76% when administered with the optimum dosage of the said composition.

8. The natural fruit powder based composition of Claim 1, wherein the in vitro triglyceride levels are decreased to 34.4 ± 4.04 pg / mg protein when administered with the optimum dosage of the said composition.

9. The natural fruit powder based composition of Claim 1, wherein the in vitro glucose uptake is reduced to up to 65.6% ± 4.04 when administered with the optimum dosage of the said composition.

10. The natural fruit powder based composition of Claim 1, wherein the in vitro insulin sensitivity is increased to 1.36 ± 0.228 p-AKT when administered with the optimum dosage of the said composition.

11. The natural fruit powder based composition of Claim 1, wherein the in vitro catalase levels are increased up to 103% ± 4.73 when administered with the optimum dosage of the said composition.

12. The natural fruit powder based composition of Claim 1, wherein the in vitro SOD levels are increased up to 63.7% ± 3.98 when administered with the optimum dosage of the said composition.

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