Le présent travail rend compte des études phytochimiques et biologiques d'extraits non volatils de quatre plantes utilisées au Bénin dans le traitement du diabète. La première partie passe en revue la bibliographie sur les plantes sujettes à notre étude. Dans cette partie, la systématique, l'importance en pharmacopée ainsi que les travaux déjà effectués sur ces plantes ont été présentés. La deuxième partie présente le mode d'extraction et les études chimiques des extraits et les résultats obtenus. La spectrophotométrie a permis de déterminer quelques grandes familles de composés présents dans les extraits : les polyphénols totaux, les flavonoïdes et les tanins tandis que la GC/MS et la LC/MS ont servi à mettre en exergue la présence de composés volatils et non volatils. La troisième partie décrit les tests biologiques in vitro et ex vitro effectués sur les extraits. Les extraits ont montré de manière générale des activités : inhibitrice de l' α-glucosidase, antioxydantes (DPPH, FRAP, ORAC), antimicrobiennes et l'une (Bridelia ferruginea) une activité cytotoxique sur les cellules cancéreuses (PA1, MCF7, PC3, DU-145), avec une efficacité variable d'une plante à une autre. La quatrième partie discute de manière générale des résultats issus des études phytochimiques et des tests biologiques. Parmi les quatre échantillons de plantes sélectionnées pour notre étude, seul l'extrait semi-éthanolique des racines de Ceiba pentandra a une faible teneur en familles de composés dosés et présente des activités biologiques (ci-dessus citées) faibles comparativement aux extraits de Bridelia ferruginea, de Pseudocedrela kotschyi et de Polygonum senegalensis. L'ensemble des résultats tant sur le plan chimique que biologique met en évidence les potentialités des extraits de plantes étudiées, pour une exploitation à des fins thérapeutiquesfutures. / The present work had reported on the phytochemical and biological studies of non-volatile extracts of four plants used in Benin for diabetes treatment. The first part reviewed the bibliography of investigated plants in our study. In this part, the systematic, the importance in the pharmacopoeia and the previous works done on these plants were presented. The second part has presented the extraction method and chemical studies of the extracts and results obtained. The spectrophotometry has permitted to identify some important families of compounds in the extracts: the total polyphenols, flavonoids and tannins whereas the GC / MS and LC/MS were used to highlight the presence of volatile and non-volatile compounds. The third part described the biological tests in vitro and ex vitro carried out on the extracts. The extracts showed in general activities: α-glucosidase inhibition, antioxidant (DPPH, FRAP, ORAC), antimicrobial, and one of them (Bridelia ferruginea) were cytotoxic on cancer cells (PA1, MCF7, PC3, DU-145), with a variable efficiency from one plant to another. The fourth part had discussed in general about the results obtained from phytochemical studies and biological tests. Among the four plants samples selected for our study, only the semi-alcoholic extract of Ceiba pentandra roots had a low-dosed compounds families and presented of this biological activities (cited below) low comparatively to Bridelia ferruginea, Pseudocedrela kotschyi and Polygonum senegalensis extracts. Both of the chemical and biological results highlight the potential of certain species for future exploitation of their non-volatile extract for therapeutic purposes.

Antioxidant-rich plant foods can inhibit starch and lipid digestion that are relevant to the management of type-II diabetes. Our objective was to compare the three phenolic substances (total phenolic, total flavonoids, and condensed tannin content) in crude, semi-purified extracts from eight types of foods (purified by XAD-7 column), five fractions (semi-purified extracts fractionated by Sephadex LH-20 column) from black legumes, and to compare their antioxidant capacities. The IC50 values of these crude extracts, semi-purified extracts and fractions against alpha-amylase, alpha-glucosidase and lipase were measured. Results showed that Fraction V from black soybean had the lowest IC50 value (0.25 mg/mL) against alpha-amylase; Fraction V from black bean had the lowest IC50 value (0.25 micro gram/mL) against alpha-glucosidase; Fraction IV of black bean had the lowest IC50 value (76 micro gram/mL) against lipase; myricetin showed the lowest IC50 value against alpha-amylase, alpha-glucosidase and lipase.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24247号 / 農博第2526号 / 新制||農||1094(附属図書館) / 学位論文||R4||N5418(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 井上 和生, 教授 谷 史人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

T2DM is a chronic disease characterized by postprandial hyperglycemia. One of the therapeutic approaches to attenuate hyperglycemia is to inhibit intestinal ɑ-glucosidase enzyme and/or suppress glucose transporters that regulate intestinal glucose transporters such as SGLT1 & GLUT2. Berries rich in polyphenol antioxidants have various health benefits. Although the antidiabetic effects of various berry extracts or berry mixture in pre-clinical and clinical studies, the underlying pathways at the molecular level is still unclear. In this study, we investigated antioxidant and antidiabetic effects of selected berry extracts by determining free radical scavenging activates, Caco-2 intestinal ɑ-glucosidase activity, glucose uptake and the gene expression of ɑ-glucosidase and glucose transporters in Caco-2 cells. Total phenolic contents of berry extracts varied from 28.55 ± 0.06 to 56.15 ± 1.08 gallic acid equivalent (GAE μg/mL) and correlated with antioxidant capacities. Both cranberry extract (CBE) and blackberry extract (BBE) at 200 μg/mL concentration significantly decreased glucose uptake in Caco-2 cells. While mRNA expression and activity of ɑ-glucosidase were inhibited by CBE and BBE, mRNA expression of SGLT1 and GLUT2 was only inhibited by CBE. Moreover, CBE and BBE significantly decreased glucose uptake in the presence of sucrose and AS. Our data suggest that CBE and BBE have different molecular mechanisms in suppressing hyperglycemia and their effects are mediated by inhibiting carbohydrate digestion and absorption.

In order to improve the production of acarbose, the fermentation medium of acarbose-producing strain Actinoplanes sp. KCTC 9161 – L14 mutant was optimized in this internship. Fractional factorial design was employ to investigate the influences of glucose, maltose and corn power on acarbose production (by a-glucosidase inhibitory ability). Two significant factors: glucose and maltose have significant and positive effects on acarbose amount. In addition, a model was obtained from the regression results of fractional factorial experiment. Other success, we demonstrated that chromatography by active charcoal column can used to purify acarbose from fermentation broth. Acarbose amount in purification solution was 191.5 g/L and an acarbose - purification process was inducted. / Nhằm mục đích nâng cao khả năng sinh tổng hợp hoạt chất acarbose từ chủng đột biến Actinoplanes sp. KCTC 9161-L14, môi trường lên men của chủng dùng để sản xuất acarbose đã được tối ưu hóa. Một phần mềm thiết kế đã được thiết lập để khảo sát ảnh hưởng của glucose, maltose và bột ngô đến khả năng sản xuất acarbose (thông qua hoạt tính ức chế a-glucosidase). Kết quả đã cho thấy, hai yếu tố glucose và maltose có ý nghĩa quan trọng và ảnh hưởng trực tiếp đến khả năng sinh tổng hợp acarbose. Một phương trình đã được hình thành từ kết quả tối ưu. Bên cạnh đó, chúng tôi đã chứng minh được cột sắc ký sử dụng than hoạt tính có thể tinh sạch acarbose từ dịch lên men. Hàm lượng acarbose trong dung dịch tinh sạch đạt 191,5 g/l và một quy trình tinh sạch acarbose được đề xuất.

In order to improve the production of acarbose, the fermentation medium of acarbose-producing strain Actinoplanes sp. KCTC 9161 – L14 mutant was optimized in this internship. Fractional factorial design was employ to investigate the influences of glucose, maltose and corn power on acarbose production (by a-glucosidase inhibitory ability). Two significant factors: glucose and maltose have significant and positive effects on acarbose amount. In addition, a model was obtained from the regression results of fractional factorial experiment. Other success, we demonstrated that chromatography by active charcoal column can used to purify acarbose from fermentation broth. Acarbose amount in purification solution was 191.5 g/L and an acarbose - purification process was inducted. / Nhằm mục đích nâng cao khả năng sinh tổng hợp hoạt chất acarbose từ chủng đột biến Actinoplanes sp. KCTC 9161-L14, môi trường lên men của chủng dùng để sản xuất acarbose đã được tối ưu hóa. Một phần mềm thiết kế đã được thiết lập để khảo sát ảnh hưởng của glucose, maltose và bột ngô đến khả năng sản xuất acarbose (thông qua hoạt tính ức chế a-glucosidase). Kết quả đã cho thấy, hai yếu tố glucose và maltose có ý nghĩa quan trọng và ảnh hưởng trực tiếp đến khả năng sinh tổng hợp acarbose. Một phương trình đã được hình thành từ kết quả tối ưu. Bên cạnh đó, chúng tôi đã chứng minh được cột sắc ký sử dụng than hoạt tính có thể tinh sạch acarbose từ dịch lên men. Hàm lượng acarbose trong dung dịch tinh sạch đạt 191,5 g/l và một quy trình tinh sạch acarbose được đề xuất.

Diabetes mellitus is a growing threat to human health. Current pharmacological agents cause undesirable side-effects. Herbal remedies offer the potential for alternative treatment strategies that may prove more cost-effective and devoid of the undesirable side-effects. The purpose of this study was to evaluate the in vitro anti-diabetic activity of aqueous and methanol extracts of Sclerocarya birrea (A. Rich.) Hochst. (Anacardiaceae) and Ziziphus mucronata Willd. (Rhamnaceae), which are traditionally used for the treatment of diabetes mellitus in southern Africa. Polyphenolic contents of extracts were quantified using the aluminium trichloride and Folin-Ciocalteau methods. The capacity of individual extracts to scavenge both the 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 2,2-diphenyl-1-picrylhydrazyl radicals was used as a measure of antioxidant activity. The inhibitory activities of the crude extracts of both plants on the enzymes, α-amylase and α-glucosidase, were determined using colorimetric assays. The effects of the crude extracts on cell viability was assessed in C2C12 myotubes, HepG2 hepatocarcinoma cells, 3T3-L1 adipocytes and RIN-m5F pancreatic β-islet cells, using the Sulforhodamine B assay. Fluorescence detection was used to investigate the effects of the crude extracts on glucose uptake in C2C12, HepG2 and 3T3-L1 cells. Insulin secretion was assessed in RIN-m5F cells, using ELISA. Crude extracts of both plants contained flavonoids and phenols, but flavonoid content was predominantly higher. All the extracts displayed antioxidant activity, with the methanol extract of S. birrea possessing the most potent free radical scavenging ability (IC50 = 2.16 μg/ml). Aqueous and methanol extracts of S. birrea displayed significantly (p < 0.05) greater inhibition of α-amylase, than the positive control, acarbose. Only the methanol extract of Z. mucronata inhibited α-amylase activity. Furthermore, crude extracts of both plants also displayed potent α-glucosidase inhibitory activity. Most of the crude extracts had low toxicity, where concentrations of 100 μg/ml of crude extract of the plants did not induce 50% cell death. iv Although no significant increase in insulin secretion from cultured RIN-m5F cells was noted, the crude extracts of both plants significantly (p < 0.05) increased glucose uptake in C2C12, HepG2 and 3T3-L1 cells, with efficacy significantly (p < 0.05) higher than the positive control, insulin. From the results, the plant extracts appear to exert their hypoglycaemic effects independently of insulin, via an extra-pancreatic mechanism, possibly involving interactions with the different receptors. An additive hypoglycaemic effect originates from the inhibition of both α-amylase and α-glucosidase. The findings of the present study provide evidence that S. birrea and Z. mucronata possess in vitro anti-diabetic activity. Further investigations are required to elucidate the mechanism(s) of action of the crude extracts using more targeted in vitro assays. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Pharmacology / unrestricted

Research was conducted to investigate the effect of phenolic compounds derived from inherently rich antioxidant grape extracts (GE) on α-glucosidase inhibitory activity in vitro and in vivo blood glucose control, oxidative stress, and inflammation associated with obesity-induced type 2 diabetes. Because intestinal α-glucosidase plays a key role in the digestion and absorption of complex carbohydrates, the inhibition of this enzyme is a metabolic target for managing diabetes by improving post-prandial blood glucose control. Initially, red Norton wine grape (Vitis aestivalis) and pomace extracts were evaluated and determined to have notable phenolic content and antioxidant properties. Next, grape skin (GSE) and pomace extract (GPE) were tested and both had in vitro yeast and mammalian α-glucosidase inhibitory activity. The GSE was 32-times more potent at inhibiting yeast α-glucosidase than acarbose, a commercial oral hypoglycemic agent. From HPLC and LC-MS analysis, three phenolics from the GSE (resveratrol, ellagic acid, and catechin) were identified as active inhibitory compounds. The acute administration of GPE (400 mg/kg bw) to mice reduced postprandial blood glucose level by 35% following an oral glucose tolerance test compared to the control. The daily supplementation (250 mg/kg bw) of GSE and GPE for 12-weeks to mice affected fasting blood glucose levels, oxidative stress, and inflammatory biomarkers associated with obesity and type 2 diabetes. At the end of the study, the GSE group gained significantly (P < 0.05) more weight (24.6 g) than the control, high fat, or GPE groups (11.2, 20.2, 19.6 g, respectively). Both GSE and GPE groups had lower fasting blood glucose levels (119.3 and 134.2 mg/dL, respectively) compared to the high fat group (144.6 mg/dL). The 12-week supplementation of GSE was associated with a higher plasma oxygen radical absorbance capacity (ORAC), lower liver lipid peroxidation as measure by TBARS, and lower levels of inflammation as measured by plasma C-reactive protein compared to the high fat group. In conclusion, our collective observations from these studies provide insight into the potential effects of antioxidant rich grape extracts on diabetes-related biomarkers through a dual mechanism of antioxidant protection and specific inhibition of intestinal α-glucosidases. / Ph. D.

Fermentation and roasting of cocoa (Theobroma cacao) decrease levels of polyphenolic flavanol compounds. However, it is largely unknown how these changes in polyphenol levels caused by processing affect cocoa's anti-diabetic and anti-obesity bioactivities, such as inhibition of certain enzymes in the body. Polyphenol profiles, protein-binding abilities, presence of compounds termed oxidative polymers, and abilities to inhibit α-glucosidase, pancreatic α-amylase, lipase, and dipeptidyl peptidase-IV (DPP4) in vitro were compared between unfermented bean (UB), fermented bean (FB), unfermented liquor (UL), and fermented liquor (FL) cocoa extracts. Overall, there were significant decreases (p<0.05) in total polyphenols, flavanols, and anthocyanins between the two sets of unfermented and fermented cocoa extracts (CEs). All CEs effectively inhibited α-glucosidase (lowest IC50 = 90.0 ug/mL for UL) and moderately inhibited α-amylase (lowest IC50=183 ug/mL for FL), lipase (lowest IC25=65.5 ug/mL for FB), and DPP4 (lowest IC25=1585 ug/mL for FB) in dose-dependent manners. Fermentation and roasting of the samples affected inhibition of each enzyme differently (both processes enhanced α-amylase inhibition). Improved α-glucosidase and α-amylase inhibitions were correlated with presence of different classifications of oxidative polymers, suggesting that these compounds could be contributing to the bioactivities observed. Some α-glucosidase inhibition might be due to non-specific protein-binding. Improved DPP4 inhibition was strongly correlated to increased CE degree of polymerization. In conclusion, potential enzyme inhibition activities of cocoa were not necessarily negatively affected by the large polyphenol losses that occur during fermentation and roasting. Additionally, it is possible that more complex compounds could be present in cocoa that contribute to its potential anti-diabetic and anti-obesity bioactivities. / Master of Science in Life Sciences

Type 2 diabetes mellitus (T2DM) is an increasing global health concern, currently affecting an estimated 382 million individuals. There is no cure for T2DM and the search for new and improved treatments is ongoing. Presently, various pharmacological regimens are available to treat T2DM, but with varied success. Thousands of traditional herbs are also used to treat T2DM, but mainly without scientific validation. The aim of this study was to assess the polyphenolic content, antioxidant capacity, as well as in vitro toxicity and hypoglycaemic activity of a commercial ‘antidiabetic’ tea mixture (Diabetea) and its individual constituents: Achillea millefolium L. (Yarrow), Agathosma betulina Bartl. & Wendl. (Buchu), Salvia officinalis L. (Sage), Taraxacum officinalis L. (Dandelion), Thymus vulgaris L. (Thyme), Trigonella foenum-graecum L. (Fenugreek) and Urtica urens L. (Nettle). All herbs were tested as crude extracts, prepared using hot water (HW) and dichloromethane (DCM). The total polyphenolic content of each extract was determined using the Folin-Ciocalteau and aluminium trichloride methods. The non-cellular antioxidant activity was assessed using 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods. The cell-based antioxidant activity was measured against p-chloranil-induced generation of reactive oxygen species (ROS) in Ea.hy926 cells, using the fluorescent dye, 2',7'-dichlorfluorescein-diacetate (DCFH-DA). The effect of each extract on the viability of C2C12 myotubes, Ea.hy926 endothelial cells and human lymphocytes (HL) was determined using sulforhodamine B (SRB). The in vitro hypoglycaemic activity was assessed against α-amylase and α-glucosidase activity using 3,5-dinitrosalicylic acid (DNSA) and p-nitrophenyl-α-D-glucopyranoside (p-NPG), respectively. The type of inhibition exerted on these enzymes was determined using the Michaelis-Menten enzyme kinetics model, expressed as mixed, competitive, non-competitive and uncompetitive. Glucose uptake activity was measured using the 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) fluorescent analogue. T. vulgaris and S. officinalis had the highest amount of polyphenols of all extracts tested. The HW extracts of T. vulgaris and S. officinalis showed significant (p < 0.05) cell-free antioxidant activity and cell-based radical scavenging activity. In addition, U. urens (HW) also limited cell-based ROS generation (p < 0.05). The Diabetea extracts presented with poor antioxidant activities, of which some had a pro-oxidant effect on Ea.hy926 cells. The positive linear relationship between antioxidant activity and polyphenolic content was shown to be dependent on the solvent type used. All of the DCM extracts had low antioxidant activity and polyphenolic content. None of the extracts produced < 50% cell density at the concentrations tested (1.3 - 20 μg/mℓ). In general, the DCM extracts showed a greater decrease in cell density than the HW extracts. The Ea.hy926 cells were the least affected by the extracts in terms of decreased cell density. The DCM extract of U. urens inhibited α-amylase activity in a mixed manner, which was comparable to the percentage inhibition exerted by the commercial drug, acarbose. Both the HW and DCM extracts of U. urens caused a significant (p < 0.05) increase in glucose uptake into C2C12 myotubes. The HW extract of T. vulgaris had a significant (p < 0.05) inhibitory activity against α-glucosidase (mixed). It also caused the uptake of glucose into C2C12 myotubes, which was significantly (p <0.05) more active than insulin. S. officinalis (DCM extract) also inhibited α-glucosidase activity (p < 0.05) in a mixed manner. Its HW extract displayed potent hypoglycaemic potential by causing glucose uptake into C2C12 myotubes, which was more significant (p < 0.05) than the activity of the positive control, insulin. The DCM extract of A. betulina was active against α-glucosidase (non-competitive), which was comparable to the activity of acarbose. Its HW extract also showed a significant (p < 0.05) glucose uptake activity. Furthermore, the DCM extracts of T. officinalis, A. millefolium, Diabetea and HW extracts of T. foenum-graecum and T. officinalis also caused a significant (p < 0.05) increase in glucose uptake into C2C12 myotubes. This study provides evidence for the antidiabetic potential of T. vulgaris and S. officianlis, in terms of antioxidant capacity and potential to prevent of post-prandial hyperglycaemia and alleviate hyperglycaemia by mimicking the action of insulin. In addition, the organic preparation of U. urens is also a potent α-amylase inhibitor. All herbs tested in this study exerted some form of in vitro antidiabetic activity. The Diabetea mixture, as a traditional preparation, did not have a significant antidiabetic capacity. In vitro observations from this study do not support the use of Diabetea as an antidiabetic preparation and reveal that some of the individual extracts prove more efficacious than the herb mixture. / Dissertation (MSc)--University of Pretoria, 2014. / lk2014 / Pharmacology / MSc / Unrestricted