Effects of 22 traditional anti-diabetic medicinal plants on DPP-IV enzyme activity and glucose homeostasis in high-fat fed obese diabetic rats

Abstract The present study investigated the effects of hot water extracts of 22 medicinal plants used traditionally to treat diabetes on Dipeptidyl peptidase-IV (DPP-IV) activity both in vitro and in vivo in high-fat fed (HFF) obese-diabetic rats. Fluorometric assay was employed to determine the DPP-IV activity. For in vivo studies, HFF obese-diabetic rats were fasted for 6 h and blood was sampled at different times before and after the oral administration of the glucose alone (18 mmol/kg body weight) or with either of the four most active plant extracts (250 mg/5 ml/kg, body weight) or established DPP-IV inhibitors (10 μmol/5 ml/kg). DPP-IV inhibitors: sitagliptin, vildagliptin and diprotin A, decreased enzyme activity by a maximum of 95–99% (P<0.001). Among the 22 natural anti-diabetic plants tested, Anogeissus Latifolia exhibited the most significant (P<0.001) inhibitory activity (96 ± 1%) with IC50 and IC25 values of 754 and 590 μg/ml. Maximum inhibitory effects of other extracts: Aegle marmelos, Mangifera indica, Chloropsis cochinchinensis, Trigonella foenum-graecum and Azadirachta indica were (44 ±7%; 38 ± 4%; 31±1%; 28±2%; 27±2%, respectively). A maximum of 45% inhibition was observed with >25 μM concentrations of selected phytochemicals (rutin). A. latifolia, A. marmelos, T. foenum-graecum and M. indica extracts improved glucose tolerance, insulin release, reduced DPP-IV activity and increased circulating active GLP-1 in HFF obese-diabetic rats (P<0.05–0.001). These results suggest that ingestion of selected natural anti-diabetic plants, in particular A. latifolia, A. marmelos, T. foenum-graecum and M. indica can substantially inhibit DPP-IV and improve glucose homeostasis, thereby providing a useful therapeutic approach for the treatment of T2DM.


Introduction
Diabetes Mellitus has become a worldwide concern, manifesting as one of the most major health issues within the world's population. There are several forms of diabetes, including gestational diabetes, but Type 1 and Type 2 diabetes are by far the most prevalent. Type 2 diabetes (T2DM), most often associated with obesity, is a particularly widespread disease and many patients from all over the globe are afflicted by this condition. T2DM patients are characterized by impaired β-cell function and insulin secretion together with tissue insulin resistance [1]. Since the prevalence and associated complications of T2DM are so damaging, more effective therapies are being sought to either delay or prevent the progression of T2DM [2]. As so, plants are most reliable source as studies to date found that they contain series of potential phytogroups including alkaloids, glycosides, terpenoids, phenolic, flavonoids and plant-derived peptides, each of them has shown potential antidiabetic activity in different experiment [2]. Besides, the diet of patients with T2DM plays a vital role in helping to maintain blood glucose control involving such factors as energy density, carbohydrate content, dietary fiber and natural products that may directly or indirectly affect the [54,55] Dalbergia sissoo DC.
Having adequate bioactive insulin in the circulation is the key to control of glucose homeostasis as the hormone is unique in stimulating tissue glucose uptake and limiting hepatic glucose output. DPP-IV interferes with normal insulin action by degrading and therefore diminishing the insulinotropic and other β-cell actions of GLP-1 and GIP [4]. Natural resources are being explored to find new dietary ways to promote healthy blood glucose control including manipulation of the microbiome [7]. Over the years, many studies have revealed the anti-diabetic activity of plants used traditionally for the treatment of diabetes and defined their actions mediated via effects on the gastrointestinal processing of food and both the secretion and action of insulin [8][9][10]. More recently, dietary components including dairy, tuna, rice, salmon and amaranth have been found to exhibit DPP-IV inhibitory properties in vitro [11,12].
In the present work, 22 traditional medicinal plants with proven anti-diabetic activity were selected to assess their effects on DPP-IV enzyme activity in vitro (Tables 1 and 2). Furthermore, four of the most effective plants (A. latifolia, A. marmelos, T. foenum-graecum and M. indica) were selected to assess their acute effects on plasma DPP-IV activity, glucose-lowering and insulin-releasing properties in high fat fed obese-diabetic rats. Trigonella foenum graecum ↓ ↑ ND [22,98] Effects of plant: -↑, increase; ↓, decrease (beneficial effect on hyperglycemia); ND, effect not determined 1 Effects on hyperglycemia were demonstrated in mice or rats given streptozotocin or alloxan or high fat diet to induce diabetes. 2 Effects on insulin secretion were demonstrated in vitro using pancreatic β-cells or in vivo using blood plasma of rats or mice. Beneficial actions in vitro were dose-dependent and did not affect cellular viability at low concentrations. 3 Effects on glucose uptake and metabolism were demonstrated in vitro using isolated mouse abdominal muscle.

Plant materials and preparation of extract
Twenty-two plants used traditionally to treat diabetes were purchased to assess their ability to inhibit DPP-IV enzyme activity and improve glycemic control. The plants selected and their traditional and pharmacological actions are given in Tables 1 and 2 Table 3. All plant components (Tables 1-3) were dried and grounded to obtain a fine powder. About 1 g of each dried powder was infused using 40 ml of boiled water. Aqueous extracts were chosen based on traditional use and prior studies of plants selected. The infusion was left for 15 min before being filtered through Whatman no. 1 filter paper. After that, the filtrates were dried under a vacuum (Savant Speedvac; New York, U.S.A.) to produce plant extract that was used to perform DPP-IV inhibitory experiments. For this purpose, the dried extract was dissolved in a 100 mM Tris-HCl buffer at an initial concentration of 5 mg/ml.

Determination of DPP-IV inhibitory activity in vitro
A fluorometric method was used to determine the DPP-IV inhibitory activity of plant extracts based on that described previously [6,13]. For in vitro studies, a 100 mM Tris-HCl buffer was prepared and adjusted to pH 8.0 using 100 mM Tris-base. Reactions were performed in 96-well black-walled, clear-bottomed microplates (Premier Scientific Ltd, Belfast, U.K.) using 8 mU/ml of DPP-IV enzyme and 200 μM of fluorescent substrate (Gly-Pro-AMC) with or without plant extract, known DPP-IV inhibitor or selected phytochemicals. These included caffeine, catechin, epicatechin, Glycyrrhiza glabra L.

Determination of the acute effects of plant extracts on DPP-IV activity in vivo
High fat-fed rats were used to study DPP-IV inhibitory activity of four medicinal plants (A. latifolia, A. marmelos T. foenum-graecum and M. indica) in vivo. Sitagliptin and vidagliptin were used in comparison as positive controls. Animals were fasted for approximately 6 h prior to experimentation. Blood samples were collected from the cut tip of the tail from conscious rats before and after oral administration of glucose with or without plant extract (250 mg/5 ml/kg) or an established DPP-IV inhibitor (10 μmol/5 ml/kg) at 0, 30, 60, 120, 180, 240, 360 and 480 min. Samples were collected in chilled fluoride/heparin-coated micro centrifuge tubes followed by centrifugation at 12000 rpm for 5 min. Plasma was stored at −20 • C for measurement of insulin, DPP-IV activity and active GLP-1 . An Ascencia Contour glucose meter (Bayer, Newbury, U.K.) was used to measure blood glucose and insulin was determined by dextran-charcoal radioimmunoassay [14]. Active GLP-1 (7-36) was determined in the plasma samples collected at 60 min using a specific GLP-1 (Active) ELISA Kit (EGLP-35K, Merck Millipore, Dorset, U.K.).

Statistical analysis
Statistical analysis tests were performed by using Graph Pad-Prism 5. The results are represented as mean + − SEM. Data were analyzed using by unpaired Student's t test (nonparametric, with two-tailed P values) and one-way ANOVA with repeated measures was used and adjusted using Bonferroni correction. P value of < 0.05 was considered significant.

Acute effects of plants extract on glucose tolerance and plasma insulin in high fat-fed rats
Four plants (A. latifolia, A. marmelos, T. foenum-graecum and M. indica), the most potent in inhibiting in vitro DPP IV enzyme activity, were selected for evaluation of effects on DPP IV activity and oral glucose tolerance in high-fat fed rats. Hot water extracts (250 mg/5 ml/kg) substantially improved the glycemic excursion from 30 to 240 min and increased plasma insulin from 30 to 120 min as compared with oral administration of glucose alone (P<0.05-0.001; Figure 3A,C). Established DPP-IV inhibitors (sitagliptin and vildagliptin) also improved glucose tolerance and insulin release following oral administration (P<0.05-0.001; Figure 3B,D).

Discussion
DPP-IV inhibitors are used in the treatment of T2DM based on their ability to extend postprandial levels of circulating plasma GLP-1 and GIP, thereby improving insulin secretion and helping to maintain good blood glucose control. Since their introduction to the clinic [15], this drug class has proven to be effective and highly popular. Weight reduction and glycaemic control are inferior to the related family of GLP-1 mimetics but DPP-IV inhibitors have the advantage of being orally active, thereby avoiding the need for daily injections and increasing patient compliance. In certain developing countries, the limited availability and cost of these and other modern medicines, such as metformin, sulphonylureas, thiazolidenediones, SGLT2 inhibitors and insulin formulations, have resulted in increasing attention being paid to traditional plant medicines with reputed anti-diabetic activity for treatment of T2DM [3,8,10,[16][17][18]. A considerable number of plants have been used traditionally for the treatment for diabetes and its complications but only a limited number have been subjected to scientific scrutiny and fewer still scrutinized for the mechanisms responsible for their anti-diabetic effects [9,16,19]. In the present study, we have examined 22 medicinal plants with proven glucose-lowering ability ( Table 2) to assess whether part of their mode of action relates to an ability to inhibit DPP-IV. Hot water extracts of every plant studied exhibited some degree of DPP-IV inhibition in vitro ranging from 9 to 96%, but the most substantial effects were observed (in descending order) with A. latifolia (bark), A. marmelos (leaves), M. indica (seeds), T. foenum-graecum (seeds), C. cochinchinensis (bark), and A indica (seeds). These plants inhibited DPP-IV by 27-96% with IC 25 values of 446-4720 μg/ml. Although considerably less effective than pure preparations of sitagliptin and vildagliptin, these observations suggest that a component of the anti-diabetic actions of these plants may be due to inhibition of DPP-IV. This adds to the results of previous studies which have highlighted gastrointestinal effects of anti-diabetic plants and their ability to enhance insulin secretion and/or action [20][21][22][23]. Based on these in vitro results, the four most active plants were selected from the 22 initially screened for in vivo evaluation of effects on oral glucose tolerance, insulin secretion and plasma DPP-IV activity using high-fat fed rats. This included A. latifolia, A. marmelos, T. foenum-graecum and M. indica. The first two were particularly effective in inhibiting DPP-IV in vitro with up to 44-96% inhibition and IC 50 values of 754-790 μg/ml. This compares with almost total inhibition of DPP-IV by sitagliptin and vildagliptin with IC 25 and IC 50 values of 2.04 × 10 −3 to 2.43 × 10 −3 μg/ml and 2.04 × 10 −2 to 1.70×10 −2 μg/ml, respectively. As expected, administration of either sitagliptin or vildagliptin orally to high fat fed obese-diabetic rats, together with glucose, induced a remarkable improvement in glucose tolerance and glucose-stimulated insulin secretion. This was associated with a 70-72% decrease in plasma DPP-IV activity known to result in strong augmentation of the stimulatory insulin-releasing effects of the incretin hormones GLP-1 and GIP. Consistent with this, circulating concentrations of active GLP-1 (7-36) were increased by 81-89% at 60 min following administration of these DPP-IV inhibitors. Extracts of A. latifolia, A. marmelos, T. foenum-graecum and M. indica also significantly inhibited DPP-IV enzyme activity and increased active GLP-1 (7-36) but by lesser extents of 12-18% and 32-45%, respectively. Interestingly, the glucose lowering actions of the four plant extracts were very similar to the DPP-IV inhibitors despite a smaller plasma insulin response. This indicates that other factors such as a delayed glucose absorption make a major contribution to the acute anti-hyperglycaemic activity of these plants in vivo [9,23]. Further long-term studies are required to determine how their effects compare with other plant-derived substances that exhibit anti-diabetic properties, such as metformin.
These results suggest that many plants used traditionally to treat diabetes have orally available constituents that inhibit DPP-IV, thereby contributing to their spectrum of actions which in the case of some might be significant [24,25]. Indeed, additional in vitro and in vivo studies on individual plant extracts have reported that other plant species inhibit DPP-IV activity [26,27]. These observations suggest that these plant extracts exert part of their insulinotropic effects via inhibition of DPP-IV with resultant preservation of active forms of GLP-1 (7-36) and GIP (1-42) released from intestinal enteroendocrine cells by feeding. Overall, our results together with these previous studies indicate that net effects on insulin secreting cells reflect combination of direct actions of glucose and other nutrients compounded by potentiating effects of incretin hormones that are favored by the concurrent inhibition of DPP-IV. Further extensive studies will be required to measure active and total forms of GLP-1 and GIP following administration of the plants studied but the few reports in the literature suggest that some plants with reputed anti-diabetic activity increase circulating GLP-1 [28]. The extent to which this may reflect enhanced secretion as opposed to decreased degradation by DPP-IV is unknown.
Although the present study describes DPP-IV lowering activity of many medicinal plants with anti-diabetic actions, few precise details are known about the nature of the phytochemicals that are absorbed and subsequently inhibit DPP-IV. All 22 plants examined inhibited DPP-IV activity in vitro to some extent suggesting that such chemicals are commonly encountered in the plant kingdom. Studies to date suggest that these include alkaloids, glycosides, terpenoids, phenolic, flavonoids as well as protein hydrolysates and peptides [27,2]. These may act at the molecular level by binding to the active site of the enzyme, thereby inhibiting interactions with natural substrates. Small peptides may also serve as competitive substrates as is the case with diprotin-A. Indeed, we demonstrated DPP-IV inhibitory action for caffeine, catechin, epicatechin, gallic acid, isoquercitrin, quercetin and rutin which may realistically contribute to the observed effects as they have been reported to be present in plants at levels of up to 2-30% by weight, not accounting for losses during our extraction procedure [29]. Rutin is the most abundant of these phytochemicals and was also shown to be the most effective inhibitor of DPP-IV, with an action broadly similar to that observed with the established anti-diabetic drug, nateglinide. The active plant constituents might, like this meglitinide, serve as effective prandial blood glucose regulators stemming partly from their ability to preserve active forms of GLP-1 and GIP released by feeding [6].  In the light of the DPP-IV inhibitory actions of the 22 plants tested plus the selected phytochemicals, it is notable that flavonoids and their metabolites have been reported to exhibit anti-diabetic activities. An inverse relationship has been suggested also between flavonoid intake and T2DM risk [30]. Phytochemicals other than those tested, such as anthocyanin, aspalathin, chrysin, eriodictyol, hispidulin, kaempferol, lepidoside, mangiferin, naringenin, naringin, procyanidin, rhamnoside, terpenoids, vitexin and Lens culinaris extracts have been shown also to exhibit DPP-IV inhibitory activity [27,31]. In addition, a number of other plants (such as A. catechu, M. indica and A. marmelos) have been reported to contain potential phenolic compounds that exert antioxidant effects and DPP-IV inhibitory activity [32].

Conclusions
In conclusion, these findings indicate that a substantial proportion of plants used traditionally for the treatment of diabetes exhibit DPP-IV inhibitory activity which may contribute to their multiple glucose lowering actions. Such medicinal plants could provide an accessible therapy for diabetes particularly in populations without easy access to the recognized drugs. More work is required for isolation, identification and characterization of agents responsible for inhibition of DPP-IV but there is a good chance that multiple phytochemicals are involved in mediating such effects [33].

Data Availability
All data are included in the manuscripts and the identified participant information (PA) is included in the Author Contribution section for the data collections.