Animal models and natural products to investigate in vivo and in vitro antidiabetic activity

Diabetes mellitus is a chronic disease which has high prevalence. The deficiency in insulin production or impaired insulin function is the underlying cause of this disease. Utilization of plant sources as a cure of diabetes has rich evidence in the history. Recently, the traditional medicinal plants have been investigated scientifically to understand the underlying mechanism behind antidiabetic potential. In this regard, a substantial number of in vivo and in vitro models have been introduced for investigating the bottom-line mechanism of the antidiabetic effect. A good number of methods have been reported to be used successfully to determine antidiabetic effects of plant extracts or isolated compounds. This review encompasses all the possible methods with a list of medicinal plants which may contribute to discovering a novel drug to treat diabetes more efficaciously with the minimum or no side effects

In vitro adipogenic potential and glucose uptake stimulatory effect of the terpenoids isolated from Tetracera indica Merr.

In vitro adipogenic potential and glucose uptake stimulatory effect of the terpenoids isolated from Tetracera indica Merr

An in vitro adipogenic potential and glucose uptake stimulatory effect of betulinic acid and stigmasterol isolated from Tetracera indica in 3T3-L1 cell line

Aerial parts of Tetracera indica Merr. (Dilleniaceae) are rich in betulinic acid and stigmasterol and traditionally used to treat diabetes. This study was aimed to evaluate an in vitro antidiabetic potential of betulinic acid and stigmasterol to ascertain whether they may contribute antidiabetic effect to T. indica. Initially, betulinic acid and stigmasterol were isolated from the most effective subfraction (ethyl acetate) and subjected to an in vitro antidiabetic investigation through adipogenesis and fluorescence glucose (2-NBDG) uptake assays using 3T3-L1 fibroblast. MTT viability assay was performed at 0.78 to 100 μg/mL for 48 h to determine the safe concentration. Both compounds were subjected to 2-NBDG uptake test on the differentiated adipocytes. The cells were treated in safe concentrations (25-100 μg/mL) as well as in different adipogenic cocktails, which were modified by the addition of compounds to be investigated and in the presence or absence of insulin (10 μM). Rosiglitazone (10 μM) was used as standard. Stems ethanol extract and its fractions (hexane and ethyl acetate), betulinic acid and stigmasterol were found safe at their highest concentration (100 μg/mL) by inhibiting cells well below their IC50 values viz. 18.60, 35.27, 21.40, 28.86 and 33.06%, respectively. Both betulinic acid and stigmasterol at the highest safe concentration (100 μg/mL) significantly (p <0.05) induced adipogenesis like insulin, enhanced adipogenesis like rosiglitazone and exhibited glucose uptake activity. The present study demonstrates that both betulinic acid and stigmasterol possess an in vitro antidiabetic potential. However, in vivo antiglycemic study on these compounds and their chemical analogs are still warranted to ensure their therapeutic potential as safe antidiabetic agents

Isolation and identification of flavonoids from Tetracera indica Merr. (Dilleniaceae) leaves

Leaves of Tetracera indica are used in traditional medicine for the treatment of diabetes mellitus in Malaysia. This study was aimed at investigating the phytochemical constituents present in T. indica leaves which may be responsible for the antidiabetic effect of the plant’s leaves. Crude methanolic extract from the leaves of T. indica was separated and purified by column chromatography of silica gel, ODS and sephadex LH-20. The structures of the isolated compounds were identified and elucidated by UV, 1H-NMR, 13C-NMR and MS spectroscopic techniques. Four flavonoids were isolated from the leaves of T. indica, and these compounds were identified as follows: 5,7-dihydroxy-8-methoxyflavone (wogonin), 5,7,8-trihydroxyflavone (norwogonin), quercetin and 5-Hydroxy-7-methoxyflavone (tectochrysin). The three compounds i.e. norwogonin, quercetin and tectochrysin are being reported for the first time from T. indica leaves. Keywords: Tetracera indica, flavonoids, diabetes mellitu

Potential insulin-like and insulin-sensitizing activity on 3T3-L1 adipocytes by terpeniods isolated from Tetracera indica

Aerial parts of Tetracera indica Merr. (Dilleniaceae) are traditionally used to treat diabetes in Malaysia. T. indica has been reported to contain terpenoids namely stigmasterol and betulinic acid as major constituents. Hence, the aim of this study was to evaluate in vitro antidiabetic potential of these two major terpenoids on 3T3-L1 pre-adipocytes and adipocytes. Cytotoxicity test was performed through MTT assay on 3T3-L1 pre-adipocytes to determine the safe dose for further in vitro antidiabetic evaluation. 2-NBDG glucose uptake test on mature 3T3-L1 adipocytes with the isolated terpenoids was carried out to confirm their antidiabetic effect. Insulin and rosiglitazone were taken as control and positive control, however, untreated cells with normal media (DMEM) was taken as negative control, respectively. Ethanol extract and its ethyl acetate fraction displayed the most significant antidiabetic effect on 3T3-L1 pre-adipocytes by showing their good ability to induce adipogenesis. Upon repeated silica gel column chromatography, ethyl acetate fraction afforded stigmasterol and betulinic acid whose structures were elucidated through 1H-and 13C-NMR spectroscopy. Both compounds showed significant glucose uptake activity during fluorescence glucose uptake test by 2-NBDG (fluorescent glucose analogue) on mature adipocytes by displaying higher 2-NBDG uptake than insulin at their highest concentrations. Furthermore, both terpenoids showed more or less similar 2-NBDG uptake like rosiglitazone. This clearly proves their insulin-sensitizing ability. Our study suggests strong antidiabetic potential of isolated terpenoids in terms of insulin-like and insulin-sensitizing abilities

Antidiabetic characterization and mechanism of action of wogonin in rats

This research deals with the in vivo, in vitro, and in situ evaluations of antidiabetic effect of wogonin to understand its mechanism of action as an antdiabetic agent. Wogonin was isolated from the leaves extracts of T. indica. Its thirteen chemical analogues (including semisynthetic derivatives) were also taken into account for structure activity relationship study and their structures were characterized by IR, UV, 1D and 2D NMR and MS spectral analysis. The results of animal study, cell culture antidiabetic (insulin secretion, adipogenesis, glucose uptake), and protein expression showed that the methyl ether group at position C-8 is responsible for wogonin’s antidiabetic property via β-cells of islet of langerhans’ recovery as well as through glucose uptake mechanism which indicated by up regulation of GLUT 4 and C/EBP-α. The mechanism could be enhanced by the addition of acetate group at C-5 and C-7. SAR study revealed that the total number and the configuration of hydroxyl groups play a vital role in regulating antidiabetic and antioxidant activities. Presence of C-2-C-3 double bond and C-4 ketonic group were found to be two essential structural features in the bioactivity of wogonin especially for antidiabetic property. For in situ experiments, the molecular docking study, quercetin showed the highest score among the chemical analogues of wogonin revealing the importance of hydroxyl groups. Results conclude that wogonin exerts antidiabetic effect through different mechanisms of action. In vivo study, it displayed antidiabetic effect through regenerating beta cells of pancreas, thereby found to increase the level of insulin in body. However, in vitro experiments, it showed effect like insulin and rosiglitazone by inducing adipogenesis and glucose uptake. It also showed α-glucosidase, dipeptyl peptidase-4 inhibitory effects, however, at high concentration. In vivo and in vitro results demonstrate that wogonin may exert its antidiabetic effect through multiple modes of action. SAR study showed that by removing its methoxy group and through addition of hydroxyl groups to the different positions of wogonin could increase its antidiabetic effect and decrease its toxicity

Isolation of antidiabetic compounds from tetracera Indica

Mempelas paya (Tetracera indica) is a rain forest climber of Malaysia which is traditionally used to treat various ailments including diabetes and its related infirmities. The objective of this research was to explore an in vitro antidiabetic activity of the stems of T. indica as well as to isolate the active principles responsible for its in vitro antidiabetic effect. In vitro antidiabetic evaluation was done on 3T3-L1 pre-adipocytes and adipocytes. Two flavones (wogonin and norwogonin) were isolated from the stems ethanol extract of T. indica and subjected to cytotoxicity test against 3T3-L1 adipocytes with regard to evaluate their antidiabetic potential. Both flavones were isolated and purified through silica gel and sephadex LH-20 column chromatography and their structures were elucidated through 1H-and 13C-NMR spectroscopy. Cytotoxicity test was performed through MTT assay on 3T3-L1 pre-adipocytes to determine the safe dose for further in vitro antidiabetic evaluation. The stems ethanol extract, fractions and both flavones were further subjected to adipogenesis to investigate insulin like activity or insulin sensitizing activity for the purpose of evaluating antidiabetic activity. Both flavones were introduced to the cells in three different safe concentrations as well as in different adipogenic cocktails. The adipogenic cocktails were modified by the compounds and rosiglitazone in the presence or absence of insulin. Results showed that both flavones induced adipogenesis like insulin and enhanced adipogenesis like rosiglitazone significantly. Furthermore, both flavones as well as rosiglitazone as positive control were subjected to fluorescence glucose uptake test by 2-NBDG (fluorescent glucose analog) on mature adipocytes. Results suggested significant glucose uptake activity by both flavones. It is suggested that further in depth study on both flavones might help to discover new safe antidiabetic agents. Keywords: Tetracera indica Merr., Dilleniaceae, Flavones, 3T3-L1 Adipocyte, Adipogenesi

Phytochemical and in vitro antidiabetic investigations of stems of Tetracera Indica Merr., a Malaysian traditional medicinal plant

Tetracera indica Merr. (Family: DILLENIACEAE) is a large, woody, rain forest climber of Malaysia which is commonly known as Mempelas paya or sand paper plant. Different parts of T. indica are traditionally used to treat various ailments including diabetes. Four flavonoids (MHQ1-Wogonin, MHQ2-Norwogonin, MHQ3-Quercetin, MHQ4-Techtochrysin) and two terpenoids (MHQ5-Stigmasterol, MHQ6-Betulinic acid) were isolated from the stems ethanol extract of T. indica and subjected to cytotoxicity test against 3T3-L1 adipocytes with regard to evaluate their antidiabetic potential. All the compounds were isolated and purified through silica gel and sephadex LH-20 column chromatography and recrystallization with ethanol and their structures were elucidated through 1D and 2D 1H- and 13C-NMR spectroscopy and their spectral data were compared with the previously reported data of the same compounds. Cytotoxicity test was performed by MTT assay on 3T3-L1 pre-adipocytes to determine the safe dose of all isolated compounds. MHQ1 and MHQ4 showed IC50 below 100 µg/ml whereas rest of the compounds found to be safe up to 100 µg/ml. MHQ1, MHQ2, MHQ3 and MHQ4 were further subjected to adipogenesis to investigate insulin like activity or insulin sensitizing activity for the purpose of evaluating antidiabetic activity. All compounds were introduced to the cells in different safe concentrations as well as in different adipogenic cocktails. The adipogenic cocktails were modified by the compounds and rosiglitazone in the presence or absence of insulin. Results showed that MHQ-1, MHQ-2 and MHQ-4 induced adipogenesis like insulin and enhanced adipogenesis like rosiglitazone significantly. Furthermore, MHQ1 and MHQ2 as well as rosiglitazone as positive control were subjected to fluorescence glucose uptake test by 2-NBDG (fluorescent glucose analog) on mature adipocytes. Results suggested significant glucose uptake activity by MHQ1 and MHQ2. It is suggested that further study on the isolated compounds might help to discover a new safe compound with strong antidiabetic activity