Sequence characteristics, genetic diversity and phylogenetic analysis of the Cucurbita ficifolia (Cucurbitaceae) chloroplasts genome.

BACKGROUND: Curcubita ficifolia Bouché (Cucurbitaceae) has high value as a food crop and medicinal plant, and also has horticultural value as rootstock for other melon species. China is home to many different cultivars, but the genetic diversity of these resources and the evolutionary relationships among them, as well as the differences between C. ficifolia and other Cucurbita species, remain unclear. RESULTS: We investigated the chloroplast (cp) genomes of 160 C. ficifolia individuals from 31 populations in Yunnan, a major C. ficifolia production area in China. We found that the cp genome of C. ficifolia is ~151 kb and contains 128 genes, of which 86 are protein coding genes, 34 encode tRNA, and eight encode rRNAs. We also identified 64 SSRs, mainly AT repeats. The cp genome was found to contain a total of 204 SNP and 57 indels, and a total of 21 haplotypes were found in the 160 study individuals. The reverse repeat (IR) region of C. ficifolia contained a few differences compared with this region in the six other Cucurbita species. Sequence difference analysis demonstrated that most of the variable regions were concentrated in the single copy (SC) region. Moreover, the sequences of the coding regions were found to be more similar among species than those of the non-coding regions. The phylogenies reconstructed from the cp genomes of 61 representative species of Cucurbitaceae reflected the currently accepted classification, in which C. ficifolia is sister to the other Cucurbita species, however, different interspecific relationships were found between Cucurbita species. CONCLUSIONS: These results will be valuable in the classification of C. ficifolia genetic resources and will contribute to our understanding of evolutionary relationships within the genus Cucurbita.

Effect of Cucurbita ficifolia Bouché on glutathione level and glycosylated hemoglobin percentage in a Mexican rural population with type 2 diabetes.

ETHNOPHARMACOLOGICAL IMPORTANCE: Cucurbita ficifolia Bouché fruit is widely used in Mexican traditional medicine to treat type 2 diabetes (T2D) because it has been attributed with antioxidant and hypoglycemic properties in different experimental models and T2D patients. An imbalance in physiological glutathione (GSH) concentrations increases the susceptibility to developing complications associated with oxidative stress in T2D patients. AIM OF THE STUDY: To investigate the effect of C. ficifolia on the antioxidant properties of GSH, general health measurements, and biochemical parameters in a Mexican rural population, and to evaluate the changes in socio-affective scores of patients due to improvement in T2D. MATERIALS AND METHODS: Twenty-seven women diagnosed with T2D with poor glycemic control volunteered and were divided into two groups: C. ficifolia (0.5 g/kg of fresh pulp weight) with hypoglycemic pharmacotherapy, and another group with only hypoglycemic pharmacotherapy, for 12 weeks. We evaluated the effect of the fresh pulp of C. ficifolia on body mass index, blood pressure, glucose, glycosylated hemoglobin, cholesterol, triglycerides, and GSH. Expanding the study, we evaluated the quality of life, anxiety, and depression scores before and after the intervention. RESULTS: Treatment with the fresh pulp of C. ficifolia for 12 weeks reduced glycosylated hemoglobin, similar to the hypoglycemic pharmacotherapy group, and significantly increased GSH concentrations. The patients' moods did not change despite increased GSH concentrations and improved T2D control. CONCLUSIONS: The increased GSH concentrations due to the consumption of fresh pulp of C. ficifolia could help to protect against oxidative stress and extend therapeutic benefits in addition to the usual hypoglycemic drugs in patients with T2D.

4-Hydroxybenzoic Acid and ß-Sitosterol from Cucurbita ficifolia Act as Insulin Secretagogues, Peroxisome Proliferator-Activated Receptor-Gamma Agonists, and Liver Glycogen Storage Promoters: In Vivo, In Vitro, and In Silico Studies.

Insulin secretion and GLUT4 expression are two critical events in glucose regulation. The receptors G-protein-coupled receptor 40 (GPR40) and peroxisome proliferator-activated receptor-gamma (PPARγ) modulate these processes, and they represent potential therapeutic targets for new antidiabetic agent's design. Cucurbita ficifolia fruit is used in traditional medicine for diabetes control. Previous studies demonstrated several effects: a hypoglycemic effect mediated by an insulin secretagogue action, antihyperglycemic effect, and promoting liver glycogen storage. Anti-inflammatory and antioxidant effects were also reported. Moreover, some of its phytochemicals have been described, including d-chiro-inositol. However, to understand these effects integrally, other active principles should be investigated. The aim was to perform a chemical fractionation guided by bioassay to isolate and identify other compounds from C. ficifolia fruit that explain its hypoglycemic action as insulin secretagogue, its antihyperglycemic effect by PPARγ activation, and on liver glycogen storage. Three different preparations of C. ficifolia were tested in vivo. Ethyl acetate fraction derived from aqueous extract showed antihyperglycemic effect in an oral glucose tolerance test and was further fractioned. The insulin secretagogue action was tested in RINm5F cells. For the PPARγ activation, C2C12 myocytes were treated with the fractions, and GLUT4 mRNA expression was measured. Chemical fractionation resulted in the isolation and identification of ß-sitosterol and 4-hydroxybenzoic acid (4-HBA), which increased insulin secretion, GLUT4, PPARγ, and adiponectin mRNA expression, in addition to an increase in glycogen storage. 4-HBA exhibited an antihyperglycemic effect, while ß-sitosterol showed hypoglycemic effect, confirming the wide antidiabetic related results we found in our in vitro models. An in silico study revealed that 4-HBA and ß-sitosterol have potential as dual agonists on PPARγ and GPR40 receptors. Both compounds should be considered in the development of new antidiabetic drug development.

Hypoglycemic effect and bioactive compounds associated with the ripening stages of the Cucurbita ficifolia Bouché fruit.

BACKGROUND: The fruit of Cucurbita ficifolia Bouché is known in Mexico as 'chilacayote'. The scientific interest that C. ficifolia Bouché has acquired is due to its important hypoglycemic effect. The present research aimed (i) to discover whether this hypoglycemic property is present at different stages of development of this fruit, and (ii) to characterize some bioactive compounds with antioxidant or anti-inflammatory properties. Ethylene production, respiration rate, and maturity indices were determined during fruit development. The chemical characterization of the aqueous extracts of each stage of maturity studied was determined and their hypoglycemic effects were bioassayed using groups of normal mice with diabetes induced by streptozotocin at a dose of 500 mg-1 kg-1 body weight. RESULTS: Respiration rate and ethylene production showed a typical pattern for non-climacteric fruit and the quality parameters did not show significant changes. Phenolic compounds such as gallic acid and chlorogenic acid were found to have the highest concentration at 15 days of development. Extracts at 15 days showed a hypoglycemic effect that was 11% greater than that of glibenclamide in diabetized mice. CONCLUSION: All stages of development of C. ficifolia fruit had a hypoglycemic effect; however, the aqueous extract from the fruit at 15 days of development showed a better effect than glibenclamide. This finding highlights the potential of this maturity stage, and shows that it is appropriate for inclusion in treatments of type 2 diabetes mellitus. The results also indicate that phenolic compounds are mainly responsible for this effect and not d-chiro-inositol as previously thought. © 2020 Society of Chemical Industry.

CHEMICAL CHARACTERIZATION OF A HYPOGLYCEMIC EXTRACT FROM CUCURBITA FICIFOLIA BOUCHE THAT INDUCES LIVER GLYCOGEN ACCUMULATION IN DIABETIC MICE.

BACKGROUND: The aqueous extract of Cucurbita ficifolia (C. ficifolia) fruit has demonstrated hypoglycemic effect, which may be attributed to some components in the extract. However, the major secondary metabolites in this fruit have not yet been identified and little is known about its extra-pancreatic action, in particular, on liver carbohydrate metabolism. Therefore, in addition to the isolation and structural elucidation of the principal components in the aqueous extract of C. ficifolia, the aim of this study was to determine whether or not the hypoglycemic effect of the aqueous extract of Cucurbita ficifolia (C. ficifolia) fruit is due to accumulation of liver glycogen in diabetic mice. MATERIALS AND METHODS: The aqueous extract from fruit of C. ficifolia was fractionated and its main secondary metabolites were purified and chemically characterized (NMR and GC-MS). Alloxan-induced diabetic mice received daily by gavage the aqueous extract (30 days). The liver glycogen content was quantified by spectroscopic method and by PAS stain; ALT and AST by spectrometric method; glycogen synthase, glycogen phosphorylase and GLUT2 by Western blot; the mRNA expression of GLUT2 and glucagon-receptor by RT-PCR; while serum insulin was quantified by ELISA method. A liver histological analysis was also performed by H&E stain. RESULTS: Chemical fingerprint showed five majoritarian compounds in the aqueous extract of C. ficifolia: p-coumaric acid, p-hydroxybenzoic acid, salicin, stigmast-7,2,2-dien-3-ol and stigmast-7-en-3-ol. The histological analysis showed accumulation of liver glycogen. Also, increased glycogen synthase and decreased glycogen phosphorylase were observed. Interestingly, the histological architecture evidenced a liver-protective effect due the extract. CONCLUSION: Five compounds were identified in C. ficifolia aqueous extract. The hypoglycemic effect of this extract may be partially explained by liver glycogen accumulation. The bioactive compound responsible for the hypoglycemic effect of this extract will be elucidated in subsequent studies.

Cucurbita ficifolia (Cucurbitaceae) modulates inflammatory cytokines and IFN-γ in obese mice.

This study investigated the effect of aqueous extract of Cucurbita ficifolia Bouché on systemic chronic inflammation in an obesity model induced by monosodium glutamate (MSG) via modulating the expression of adipokines (TNF-α, IL-6, resistin, and adiponectin) and immune-regulatory cytokines (IFN-γ and IL-10). Cucurbita ficifolia extract was administered daily by gavage to lean and MSG-obese mice for 30 days. At the end of treatment, cytokine mRNA expression in adipose tissue was determined by real-time polymerase chain reaction (PCR), and the protein levels of these cytokines were also quantified by enzyme-linked immunosorbent assay (ELISA). Cucurbita ficifolia extract decreased body mass and inflammation in MSG-obese mice by reducing the expression of TNF-α and IL-6; these decreases were parallel to significant reductions in protein levels. The extract also increased protein levels of IL-10 in lean mice and IFN-γ in both lean and MSG-obese mice. In conclusion, C. ficifolia extract modulates systemic chronic inflammation in MSG-obese mice and could have a beneficial effect on the adaptive immune system in obesity.

Cucurbita ficifolia Bouché increases insulin secretion in RINm5F cells through an influx of Ca(2+) from the endoplasmic reticulum.

ETHNOPHARMACOLOGICAL IMPORTANCE: Cucurbita ficifolia Bouché(C. ficifolia) is a plant used in Mexican traditional medicine to control type 2 diabetes (T2D). The hypoglycemic effect of the fruit of C. ficifolia has been demonstrated in different experimental models and in T2D patients. It has been proposed that D-chiro-inositol (DCI) is the active compound of the fruit. Additionally, it has been reported that C. ficifolia increases the mRNA expression of insulin and Kir 6.2 (a component of the ATP-sensitive potassium (K(+)ATP) channel, which is activated by sulphonylurea) in RINm5F cells. However, it remains unclear whether C. ficifolia and DCI causes the secretion of insulin by increasing the concentration of intracellular calcium ([Ca(2+)]i) through K(+)ATP channel blockage or from the reservoir in the endoplasmic reticulum (ER). MATERIAL AND METHODS: The aqueous extract of C. ficifolia was obtained and standardized with regard to its DCI content. RINm5F pancreatic ß-cells were incubated with different concentrations (50, 100, 200 and 400µM) of DCI alone or C. ficifolia (9, 18, 36 and 72µg of extract/mL), and the [Ca(2+)]i of the cells was quantified. The cells were preloaded with the Ca(2+) fluorescent dye fluo4-acetoxymethyl ester (AM) and visualized by confocal microscopy. Insulin secretion was measured by an ELISA method. Subsequently, the effect of C. ficifolia on the K(+)ATP channel was evaluated. In this case, the blocker activator diazoxide was used to inhibit the C. ficifolia-induced calcium influx. In addition, the inositol 1,4,5-trisphosphate (IP3)-receptor-selective inhibitor 2-amino-thoxydiphenylborate (2-APB) was used to inhibit the influx of calcium from the ER that was induced by C. ficifolia. RESULTS: It was found that DCI alone did not increase [Ca(2+)]i or insulin secretion. In contrast, treatment with C. ficifolia increased [Ca(2+)]i 10-fold compared with the control group. Insulin secretion increased by 46.9%. In the presence of diazoxide, C. ficifolia decreased [Ca(2+)]i by 50%, while insulin secretion increased by 36.4%. In contrast, in the presence of 2-APB, C. ficifolia increased [Ca(2+)]i 18-fold, while insulin secretion remained constant, indicating an additive effect. Therefore, C. ficifolia was not found to block the K(+)ATP channel. However, it did exert an effect by increasing [Ca(2+)]i from the ER, which may partly explain the insulin secretion observed following treatment with C. ficifolia. CONCLUSIONS: The hypoglycemic properties of C. ficifolia can be explained in part by its effect as a secretagogue for insulin through an increase in [Ca(2+)]i from the calcium reservoir in the ER. Therefore, the mechanism of action of C. ficifolia is different to those of the currently used hypoglycemic drugs, such as sulfonylureas. These results support that C. ficifolia may be a potential natural resource for new agents to control T2D.

Cucurbita ficifolia†Bouché (Cucurbitaceae) and D-chiro-inositol modulate the redox state and inflammation in 3T3-L1 adipocytes.

OBJECTIVES: Cucurbita ficifolia (characterised by its D chiro inositol (DCI) content) and of synthetic DCI on the redox state, mRNA expression and secretions of proinflammatory cytokines. Additionally, we evaluated the insulin-mimetic action of both treatments by assessing protein kinase B (PKB) activation in 3T3-L1 adipocytes. METHODS: Adipocytes were treated with C. ficifolia and synthetic DCI. The redox state was determined by spectrophotometry as changes in the reduced glutathione/oxidised glutathione (GSH/GSSG) ratio, glutathione peroxidase and glutathione reductase activities; H2 O2 levels were measured by flow cytometry. The mRNA expression and the protein level of cytokines were determinate by real-time reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The activation of PKB activation was detected by Western blot. KEY FINDINGS: C. ficifolia extract and synthetic DCI reduced oxidative stress by decreased H2 O2 levels, increased glutathione peroxidase activity and changes in the GSH/GSSG ratio. Furthermore, DCI decreased the mRNA expression and secretion of tumour necrosis factor-α, interleukin 6 (IL-6) and resistin, while C. ficifolia reduced protein levels of resistin and increased IL-6 levels. Only DCI demonstrated insulin-mimetic action. CONCLUSIONS: The antioxidant and anti-inflammatory effects of C. ficifolia extract can be explained in part by its DCI content, which modulates the GSH/GSSG ratio and contributes to a reduced proinflammatory state. C. ficifolia and DCI treatments may reduce the disturbances caused by oxidative stress. Additionally, DCI may improve insulin sensitivity through its insulin-mimetic effects.

Influence of two hypoglycemic Cucurbitaceae (Cucurbita ficifolia Bouché and Ibervillea sonorae Greene) on ATP-sensitive potassium channels in rat aortic rings / Influencia de dos Cucurbitaceae (Cucurbita ficifolia Bouché e Ibervillea sonorae Greene) sobre los canales de potasio sensibles a ATP en anillos aórticos de rata

Cucurbita ficifolia Bouché fruit containing D-chiro-inositol and Ibervillea sonorae Greene root containing cucurbitane-type glycosides are used to control diabetes in Mexico. Although the hypoglycemic effect of both plants has been demonstrated and some active compounds proposed, their mechanisms are still unknown. The aim of this study was to determine if the incubation with both aqueous extracts avoids the inhibition of contraction induced by phenylephrine similarly to glibenclamide in rat aortic rings. The hypoglycemic aqueous extracts of C. ficifolia and I. sonorae were characterized for their content of either D-chiro inositol or cucurbitanes respectively, and then we assayed the characterized extracts in vitro on the diazoxide-induced relaxation of rat aortic rings precontracted with phenylephrine, using as positive control glibenclamide. I. sonorae extract blocked the KATP channels in a concentration-dependent manner (p < 0.05), whereas C. ficifolia extract had no effect on these channels. I. sonorae extract produces a hypoglycemic effect through a similar mechanism to sulphonylureas in this experimental model; however, hypoglycemic action of C. ficifolia extract should be explained by an independent KATP channels mechanism.

Los frutos de Cucurbita ficifolia conteniendo D-quiro-inositol y las raíces de Ibervillea sonorae conteniendo glucósidos tipo cucurbitano son empleados en el control de la diabetes en México. Aunque el efecto hipoglucémico de ambas plantas ha sido demostrado y se han propuesto algunos de sus compuestos activos, aún se desconoce su mecanismo de acción. El objetivo de este estudio fue determinar si la incubación con ambos extractos acuosos evita la inhibición de la contracción inducida por fenilefrina de manera similar a la glibenclamida en anillos aórticos de rata. Los extractos acuosos hipoglucémicos de C. ficifolia e I. sonorae fueron caracterizados en su contenido de D-quiro inositol o cucurbitanos, respectivamente y entonces fueron estudiados en un modelo in vitro en la relajación inducida por diazóxido en anillos aórticos previamente contraídos con fenilefrina, usando como control positivo glibenclamida. El extracto de Ibervillea sonorae bloqueó los canales KATP de manera dosis-dependiente (p < 0.05), mientras que Cucurbita ficifolia no tuvo efecto en esos canales. El extracto de I. sonorae produce efecto hipoglucémico a través de un mecanismo similar al de las sulfonilureas en este modelo experimental; por su parte, la acción hipoglucemiante del extracto de C. ficifolia debe ser explicado mediante un mecanismo independiente de los canales KATP.