A Systematic Review of in vitro Studies Conducted on Effect of Herbal Products on Secretion of Insulin from Langerhans Islets

- Purpose: Diabetes mellitus is the most important health problem that its prevalence is increasing. Diabetes is characterized by defects in insulin secretion, insulin action or both. Recent studies provided evidences that loss of functional β -cell mass through apoptosis is central to the development of diabetes. The management of diabetes without any side effects is still a challenge to the medical system. Recently, there has been a special interest to herbal medicine in care and management of diabetes due to their natural origin and less side effects. The current systematic review focuses on main component of antidiabetic plants with directly effect on insulin secretion of pancreas. Methods: All in vitro studies which assessed the potential effect of, main components, multi herbal, whole plant, or extract of the plants directly on pancreatic insulin secretion published from 2001 to November 2011 were included. Exclusion criteria were clinical trial studies that did not assess insulin secretion, and review articles, or letters to the editor. Results: The majority of these studies showed that the improvement of β -cell function and insulin secretion is possible with antioxidant compounds. Suppression of oxidative stress, cytokine-induced impairment, suppression of nuclear factor κ B a key regulator of endothelial activation, activation of uncoupling protein 2 (UCP2), insulin-like activity and increasing intracellular calcium, were among the most important indicated pathways. Conclusions: By considering the role of oxidative stress in pathogenesis of β -cell dysfunction, antioxidant compounds could be helpful in management of diabetes and its complications.


INTRODUCTION
It is estimated that diabetes mellitus (DM) affects more than 366 million people worldwide and it is expected that the figure reach a staggering 552 million by 2030 (1).DM is a multifactorial disease characterized by chronic hyperglycemia due to defects in insulin secretion, insulin action or both, resulting from a deficit in β-cell mass (2,3).Several mechanisms have been proposed for β-cell destruction, including damage from inflammatory cytokines, circulatory free fatty acids, and hyperglycemia (4,5).The management of DM without any side effects is still a challenge to the medical system.Although, the synthetic products are widely used in clinical settings for more than 50 years, they are associated with various undesirable side effects such as hypoglycemia.In the last few years, there has been a growing interest to herbal medicine in care and management of diabetes both in developing and developed countries, due to their natural origin and less side effects (6)(7)(8)(9).
Wide array of plants are demonstrated to have antidiabetic activity.Biological actions of these plants are related to chemical composition of the plant products.Herbal products that are rich in phenolic compounds, alkaloids, flavonoids, terpenoids, coumarins, and glycosides usually show positive effects (6).On the other hand, many conventional drugs for treatment of diabetes such as metformin are secretagogues and have plants origin (10).The World Health Organization expert committee on diabetes has listed as one of its recommendations that traditional medical plants as methods of treatment of diabetes should be further investigated (11).
Insulin is the most important peptide hormone that is secreted from the islet β-cells of langerhans in response to hyperglycemia but in a complex process.The first step in this process is an increase in production of adenosine triphosphate (ATP) from adenosine diphosphate which leads to an increased ATP/ADP ratio in the cytoplasm with subsequent closing of ATP-sensitive potassium channels (12).Depolarization of plasma membrane could activate the voltage-dependent Ca 2+ channels and hence Ca 2+ influx.The increase in intracellular concentration of Ca 2+ triggers the insulin secretion (13).These pathways can be exhibited in diabetes and results in abnormal pattern of insulin secretion (13).Some studies provided evidences that loss of functional βcell mass through apoptosis is central to the development of both type 1 (14) and type 2 diabetes (15).Proliferation of islet β-cells is a very important component of β-cell adaptation to increased apoptosis and insulin resistance.Similarly by the strategy to induce β-cell proliferation and preserving functional β-cell mass, it would be possible to prevent the onset of diabetes (14,(16)(17)(18)(19)(20).Approaches to achieve this objective are stimulation of insulin secretion and inhibition of βcell apoptosis (21).
Many medicinal plants modulate the expression, synthesis and degradation of insulin.Induction of insulin release is the main mechanism of action for some antidiabetic plants (22); however increase in islet number and size as well as producing the antioxidative effects could be accounted as anti-diabetic mechanism of action of some other herbal medicine (7).Besides, antidiabetic effects of these compounds, the other benefit of such insulin releasers are looked in islet transplantation which is the final step in management of diabetic type 1 and sometimes progressive type 2 patients.The bottle neck in the islets transplantation is to keep them survived and functional during isolation before transplantation.The belief is that direct treatment of insulin producing cells or pancreatic islets with different herbal products improves their viability and function.Therefore, this could be a novel approach for improving the outcome of islet transplantation.The current systematic review is a novel work that focused on main components of plants with antidiabetic effects acting directly on insulin secretion of pancreatic islet cells.

METHODS
To obtain all related studies, Google Scholar, PubMed, Web of Science, and Scopus databases from 2001 to November 2011 were searched.The search terms used were as follows: "insulin secretion", "insulin-secreting cell", "insulin release", " islet" and "herbal or natural product" and their synonym in Persian databases of IranMedex, and Magiran.We found many in vitro studies which focused on antidiabetic plants, but they did not assess the effect on insulin secretion.Therefore all available in vitro studies which assessed the potential effect of, main components, multi herbal, whole plant, or extract of the plants directly on insulin secretion of isolated islet cells (or insulinsecreting cell lines such as RIN, HIT, β-TC, MIN6, INS-1) of pancreas were included.Exclusion criteria were clinical trials that did not assess insulin secretion, and review articles, or letters to the editor.Thesis and other unpublished data were not included.The title and abstract of all of search results were examined to eliminate the duplication.Also, the reference lists of articles were reviewed for additional relevant studies.

RESULTS
The number of initial search results and included studies are shown in the Figure 1.Forty-nine articles were selected as our final research database .The summary of these studies are shown in Table 1.These studies showed that herbal products can increase insulin secretion by affecting different steps of this process.Suppression of oxidative stress, cytokine-induced impairment, suppression of nuclear factor κB (NF-κB); NF-κB is a key regulator of endothelial activation; uncoupling protein 2 (UCP2) activation, and increasing intracellular Ca 2+ , are among the most important indicated pathways .

In vitro studies on insulin secretion
The majority of experimental studies published between 2001 and 2011 were carried out on rats or mice.In addition, the most frequently drugs used for induction of diabetes were streptozotocin (STZ) and alloxan.

Figure 1. Flow diagram of the study selection process
It was established that these models are useful for study of multiple aspects of diabetes (73,74).The cytotoxic action of these diabetogenic agents is mediated by reactive oxygen species (ROS) with some differences in their mechanism of action.By formation of superoxide radicals, alloxan can stimulate massive increase in cytosolic calcium concentration which leads to destruction of β-cells of pancreas (75).STZ enters the β-cell via glucose transporter 2 (GLUT2) and causes DNA alkylation.In addition, by activation of poly adenosine diphosphate ribosylation it causes nitric oxide (NO) release and necrosis of pancreatic β-cells (74).

Effect on insulin secretion by suppression of oxidative stress
We found that the potential antidiabetic activity of the published in vitro studies can affect different steps of insulin secretion.Because many of plants that were included in our study had more than a single active component, the observed hypoglycemic behaviors may be related to the combined synergistic actions.Some of them showed antidiabetic activity by improving oxidative metabolisms.Broussonitia Kazinoki (23), Saururus Chinensis Baill (24), American Ginseng (42), Commiphora Mukul (43), Germinated Fenugreek (51), Rhizoma Coptidis (57), and Curcuma Longa (58), Pueraria Lobata (62) are the examples of presence of antioxidant activity in the medicinal plants.Flavonoids Rhizoma Coptidis (57), and Curcuma Longa (58), Pueraria Lobata (62) posse hypoglycemic as well as antioxidant properties.Quercetin is an important flavonoid that increases insulin secretion by enhancing hepatic glucokinase activity (76) or changing intracellular calcium concentration (77).Quercetin in combination with apigenin and luteolin as flavonoids increased the viability of β-cells, insulin secretion, and cytokineinduced cytotoxicity resistance, and decreased NOsynthase (iNOS) and NF-κB activation (55).Flavonoids can also prevent cytokine-induced β-cell damage by declining NF-κB signaling (78)(79)(80).In addition, the flavonoids have phosphodiesterase inhibitor (PDEI) activities (81).Another main plant compounds with PDEI activity are alkaloids, saponins, lignans, and coumarins.Some benefits of PDEI activity especially in diabetic patients include anti-inflammatory and antioxidant effects (81), and improvement of isolated islet cells function (82).Genistein and soy isoflavonoids could significantly increase the insulin secretion by enhancing kcl-stimulated insulin secretion, increasing intracellular calcium concentration, and inhibiting extracellular signal-related kinase-1/2 (ERK-1/2), Janus kinase/signal transducer and activator of transcription pathway (JAK/STAT) or NF-κB activation (32,56).Activated ERK1/2 plays a pivotal role in environmentally-stimulated cellular responses, including cellular proliferation, growth, and differentiation.It is observed that genistein induces a rapid ERK1/2 phosphorylation, which may be necessary for growth factors to drive β-cell proliferation (83).Puerarin, an isoflavone, could increase insulin secretion by decreasing free radical production and increasing catalase (CAT) and superoxide dismutase (SOD) activities (62).Curcumin is a polyphenolic compound that is an inhibitor of NF-κB.Various biological activities for curcumin such as anti inflammatory and antioxidant make it helpful in increasing area and numbers of islets and secretion of insulin (40,58).
An association has been shown between oxidative stress and occurrence of several diseases, such as cardiovascular, diabetes, and metabolic syndrome (84)(85)(86).Oxidative stress is a phenomenon associated with the action of free radicals and reactive metabolites in the organism (87).It has been shown that both types of antioxidants, enzymatic (SOD, CAT, GPx) and nonenzymatic (vitamin C, vitamin E, zinc, uric acid and selenium), act against oxidants (87).Free radicals are derived from basic radical molecules such as superoxide anion radical or NO (88).When NO is produced by catalytic action of iNOS, it can cause a damage to proteins, lipids and DNA either directly or after reaction with superoxide (89).Taking together, it is evident that increased production of free radicals has a central role in development of diabetes complications (9).The NF-κB is activated by free fatty acids, inflammatory cytokines, and the receptor for advanced glycation end products (RAGE) (90)(91)(92).Studies in cultured endothelial cells and experimental animals have shown an association between activation of NF-κB, development of an inflammatory phenotype, insulin resistance, and impaired bioactivity of NO (93,94).By considering the fact that there is a strong association between oxidative stress and diabetes, the use of antioxidants should be helpful for management of diabetes (95)(96)(97)(98)(99).In the recent years, the positive antioxidant effects of some antidiabetic herbal products are established.Some of these medicinal herbs include species of Satureja (100), Urtica (101), Teucrium (102).As islet cell transplantation procedure is faced with oxidative stress, some studies with herbal products are investigated to assess their antidiabetic effects on isolated islet cells and showed positive effects.These substances include Setarud (IMOD); a mixture of Rosa canina, Tanacetumvulgare and Urtica dioica comprising selenium and urea treated by pulsed electromagnetic field of high frequency (25), specific PDEIs; milrinon, rolipram, sildenafil (82), calcium channel blockers, autonomic nervous system blockers and free radical scavengers; nanoparticles of cerium (103).

Effect on insulin secretion by insulin like activity
Adipose tissue enhances lipotoxicity by increasing intracellular lipid levels and also insulin resistance (104).So, adipose tissue as a key link between obesity and diabetes was assessed for the effects of natural products on glucose uptake.On the other hand, the classic target tissues of insulin which include hepatocytes, adipose tissue and skeletal muscle play important roles in homeostasis of glucose upon glucose uptake.Sarcopoterium Spinosum (31), Rooibos (33), Nigella Sativa L (36), Cichorium Intybus (48), Momordica Charantia (63) are the examples of natural products with insulinlike effects.This effect is attributed to some of antidiabetic compounds such as chlorogenic acid and caffeic acid (48).Chlorogenic acid by inhibiting glucose-6-phosphatase (G6P) in microsomes of liver suppresses gluconeogenesis and glycogenolysis and consequently reduces the hyperglycemia.In addition, G6P inhibition leads to increase glucose transport and its utilization.Finally it can stimulate insulin secretion through increased production of ATP (105).

Effect on insulin secretion through increasing viability and proliferation of β-cells
It is well established that replacing β-cells by islet transplantation has the potential to cure type 1 DM and on the other hand the efficacy of islet transplantation depends upon number and state of functional islet cells (106).Apoptosis affects the initial stage of islet transplantation which yields non-functional cells.Human pancreas contains an average one million islet cells (107) and in a good isolation process, a total of 500,000 islets with more than 80% viability can be obtained.Since the viability of islets is affected by numerous factors in the early or late period of post transplantation (108), isolated procedure would be given lower yield (109,110).Thus, islet yield and its post-transplant survival remain major issues.During initial stage of islet transplantation, islet cells are avascular and suspected to hypoxic ischemia condition which is produced by oxidative stress (96).Astragalus Membranaceus Bge (38), Codonnopsis Pilosula Nannf (38), Lycium Chinense Mill (38), Green Tea (48), Coptidis Rhizoma (65) are natural products with suppression effect on apoptosis.Some medicinal plants such as Nigella Sativa L (36) can increase insulin secretion by including proliferation of islet cells.In our study, we found other antidiabetic plants that show their effects by increasing islet cell viability.Sarcopoterium Spinosum (31), Cornus Officinalis Sieb.et Zucc (39), Germinated Fenugreek (51), Rhizoma Coptidis (57), Curcuma Longa (58) are the examples of plants with this effect.All of these plants have antioxidant effects and the observed antidiabetic effects may be related to combination of these mechanisms.

Effect on insulin secretion through inhibition UCP2
UCP2 as a mitochondrial carrier protein is expressed in islets of pancreas and has negative effect on glucose-stimulated insulin secretion (113).
Animal (114,115) and human (116,117) studies have shown that increased UCP2 expression in islets can lead to β-cell dysfunction and development of type 2 diabetes mellitus.Thus, UCP2 deficiency can prevent β-cell dysfunction.The KYQRF formula (28) which is a combination of several medicinal plants, and Gardenia Jasmine Ides Ellis (64) markedly suppresses UCP2.
In this review, however, we had some limitations.We focused and included only the studies with in vitro analysis.Unfortunately, in vitro studies do not reflect all the aspect of in vivo application of new treatments, as most in vitro models consider single cell types, metabolic pathway or enzyme involvement.This greatly reduces the possibility of identifying the antidiabetic plant extracts or compounds (118).Another disadvantage of in vitro studies is that only acute or immediate effects are measured, whilst effects that may only appear after chronic exposure to the antidiabetic compounds are overlooked (118).Hence, there is need to carry out in vivo studies on these antidiabetic plants.
The major mode of action of medicinal plants with antidiabetic activity is to increase insulin secretion.Some mechanisms of actions were related to their effects on the activity of pancreatic β-cells or the insulin-like activity of the plant extracts, or directly stimulation of insulin secretion or suppression of oxidative stress.All of these actions may be responsible for the reduction and or abolition of diabetic complications.As many of the studied plants showed more than one effective mechanism in increasing islet insulin secretion, the observed hypoglycemic behaviors may be due to a combination of synergistic mechanisms.However, the benefits of antioxidants in management of diabetes could not be ignored (119) In vitro studies in assessment of herbal and natural products on insulin secretion of pancreatic islets Dose-dependent increase in GSIS at 0. 1-5 mg/ml of HR II and HR IV vs. control, increase in GSIS at 5 mg/ml HR III vs. control Keys: T2DM, type 2 diabetes mellitus; NO, nitric oxide; iNOS, inducible form of NO synthase; NF-κB, nuclear factor kB; PARP, poly ADP-ribose polymerase-1; JAK/STAT, Janus kinase /signal transducer and activator of transcription pathway; GSIS, glucose stimulated insulin secretion; ROS, reactive oxygen species; BW, body weight; UCP2, uncoupling protein 2; DMSO,dimethyl sulfoxide; PTK, protein tyrosine kinase; ATP, adenosine triphosphate; BG, blood glucose; ERK ½, extracellular signal-related kinase-1/2; ADP, adenosine diphosphate; KATP, potassium channel adenosine triphosphate ; STZ, streptozotocin; PEPCK, phosphor enolpyruvate carboxy kinase; Hsp 70,heat shock protein 70; HO-1,hemeoxygenase-1; PGE2, prostaglandin E 2 ; COX-2, cyclooxygenase-2; Socs-3, suppressor of cytokine signaling-3; IRS2, insulin receptor substrate-2; PDX-1, pancreas duodenum homeobox-1; GK, glucokinase; HNF4α, hepatic nuclear factor 4 alpha; MDA, malondialdehyde; GR, glutathione reductase; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; LDH, lactate dehydrogenase; MAPK, mitogen-activated protein kinase; SNAP, S-nitroso-N-acetylpenicillamine.