Pancreatic beta cell protection / regeneration with phytotherapy

Although currently available drugs are useful in controlling early onset complications of diabetes, serious late onset complications appear in a large number of patients. Considering the physiopathology of diabetes, preventing beta cell degeneration and stimulating the endogenous regeneration of islets will be essential approaches for the treatment of insulin-dependent diabetes mellitus. The current review focused on phytochemicals, the antidiabetic effect of which has been proved by pancreatic beta cell protection/regeneration. Among the hundreds of plants that have been investigated for diabetes, a small fraction has shown the regenerative property and was described in this paper. Processes of pancreatic beta cell degeneration and regeneration were described. Also, the proposed mechanisms for the protective/ regenerative effects of such phytochemicals and their potential side effects were discussed.


INTRODUCTION
Diabetes mellitus is still one of the most important causes of death and disability in both developed and developing countries.According to the report by World Health Organization (WHO, 2015), 9% of adults in the world suffer from diabetes and this disease will be the 7 th leading cause of death in 2030.Generally, diabetes is classified into two main types: type 1 diabetes (T1D) and type 2 diabetes (T2D), which were previously known as insulin-and non-insulin-dependent diabetes, respectively.T1D results from pancreatic beta cell degeneration and is characterized by lack of insulin production, while patients with T2D show a state of insulin resistance and usually relative insulin deficiency.Over time, diabetic patients with poor management undergo micro-and macro-vascular complications including nephropathy, retinopathy, neuropathy, and cardiovascular diseases (Deshpande, Harris-hayes, Schootman, 2008).
At present, only insulin and oral antihyperglycemic drugs are available for T1D management (Lorenzati et al., 2010).Although the currently available drugs are useful in controlling early onset complications of diabetes, serious late onset complications appear in a large number of patients (Tzoulaki et al., 2009).Considering the physiopathology of diabetes, preventing beta cell degeneration and stimulating the endogenous regeneration of islets will be essential approaches in treatment of diabetes.
Phytochemicals have always been an important source of remedies for human health problems.Numerous experimental and clinical studies have documented beneficial effects of phytotherapy for managing diabetes (Ghorbani, 2013a,b).Antidiabetic effect of phytochemicals is mediated through different mechanisms such as decreasing glucose absorption from intestine, inhibiting glucose production in the liver, increasing glucose uptake by tissues, enhancing insulin secretion from beta cells, and/or increasing pancreatic tissue regeneration (Asgary et al., 2008;Kamyab et al., 2010;Jelodar, Mohsen, Shahram, 2007;Shafiee-Nick et al., 2011;Shafiee-Nick et al., 2012a).The current review focused on phytochemicals, the antidiabetic action of which has been proved, at least in part, by pancreatic beta cell protection or regeneration (Table I).

Method of literature review
Review of the related literature was carried out in the databases of Google Scholar, Medline, and Scopus.The search terms included diabetes, islets, pancreas, plant, herb, and regeneration.Cross references of the related articles were also retrieved.Papers were included if they reported the effect of a plant or phytochemical agent on histopathological features of islets of Langerhans in diabetic animals.Two authors (A.G. and A.H.) independently searched the databases and extracted data from each article.

Pancreatic beta cell degeneration/regeneration
T1D is primarily considered as an autoimmune disease resulting from the reaction of immune effector cells with endogenous beta cell antigens.Gradual invasion of T-cells, macrophages, and the released cytokines and oxidative radicals lead to insulitis.The proinflammatory cytokines, especially tumor necrosis factor alpha, interferon gamma, and interleukine-1 beta activate intracellular pathways and lead to beta cell dysfunction, apoptosis, and necrosis.Because of poor intracellular antioxidant capacity, beta cells are vulnerable to oxidative stress.Therefore, oxidative molecules are important mediators of beta cell damage induced by both T1D insulitis and T2D glucotoxicity (Sharma et al., 2009).
Many of the advances in the pathogenesis of T1D have emerged from animal studies.Streptozotocin (STZ) or alloxan-induced diabetic rat or mouse is the most currently used animal model for diabetes.A single intraperitoneal or intravenous injection of STZ is well reported to induce insulin-dependent diabetes in rats (Ghorbani et al., 2010;Ghorbani et al., 2013;Shafiee-Nick et al., 2012b).The histologic feature of islets from the pancreas of diabetic animals is characterized by a severe decrease in the number of islets, inflammation, vacuolation of the islets, and degranulation of the beta cells (Adeyemi et al., 2008;Aralelimath, Bhise, 2012).In addition, regular arrangement of alpha and beta cells is disturbed and clumping of beta cells, pyknosis, and necrosis are seen in the islets (Singh et al., 2008).
The subject of pancreatic beta cell regeneration is one of the most controversial topics of T1D research and treatment.It is believed that beta cell can regenerate through the replication of pre-existing beta cells or neogenesis from stem cells and progenitor cells inside or outside the islets (Bouwens, Rooman, 2005).Meier et al. (2006) showed a direct piece of evidence for beta cell regeneration through beta cell replication in T1D patients.Neogenesis can be originated from different cell types within pancreas: alpha cells, delta cells, duct epithelium, acinar cells, and centroacinar cells.However, this process depends on extra-pancreatic activators including hormones, growth factors, and others (Bouwens, Rooman, 2005).In adults, turnover of beta cells is slow and reduces with increasing age.However, during body growth and after injury, beta cells can replicate to maintain glucose homeostasis (Avolio et al., 2013).Several approaches are currently being investigated to differentiate stem/progenitor cells into beta cells; but, none has been approved for use in diabetic patients yet.Therefore, further works on this issue are warranted.

Allium sativum
Garlic (Allium sativum) has been long used as an herbal remedy in treating different diseases because of its antioxidant, antihypertensive, and anticancer effects (Afshari et al., 2006;Ashraf et al., 2013;Benkeblia, 2005;Hajzadeh et al., 2006).It is also one of the best-researched medicinal plants in terms of managing diabetes.Clinical trials have shown that the consumption of garlic decreases Pancreatic beta cell protection/regeneration with phytotherapy 3     fasting blood glucose (FBG) and lipids in diabetic patients (Ghorbani, 2013a, b).According to the animal studies, it may also induce a protective/regenerative effect on pancreatic beta cells.Albajali et al. (2011) reported that the aqueous extract of A. sativum increased the diameter of pancreatic islets in STZ-induced diabetic rats.In another study, intraperitoneal injection of A. sativum oil decreased beta cell degeneration and level of anti-islet antibodies in the animals with T1D (Osman et al., 2012).Alashkham et al. (2013) also showed regenerative action of this oil in the pancreatic islets of diabetic rats.Yet, in another study, there was no significant difference between the histopathology of the pancreas of garlic treated and untreated diabetic rats (Jelodar et al., 2005).Allicin is the major active component of A. sativum and a precursor of many secondary compounds formed in crushed garlic preparations or aged garlic.This compound has been proposed to be responsible for the health promotion benefits of A. sativum and protective actions in pancreatic islets (Osman et al., 2012).

Anastatica hierochuntica
Anastatica hierochuntica from Brassicaceae family is commonly called "Kaff Maryam" or "Rose of Jericho" and is one of the folk medicinal plants, which is widely used in Arab countries (Daur, 2012;Shaban, Al-Azzawie, Mohamme, 2011).Experimental studies have demonestrated that aqueous and methanolic extracts of A. hierochuntica have antioxidative, hypoglycemic, and hypolipidemic effects in diabetic rats (Rahmy, El-Ridi, 2002;Shaban, Al-Azzawie, Mohamme, 2011).This hypoglycemic action may be due to its beneficial effects on pancreatic beta cells.Rahmy, El-Ridi (2002) reported that A. hierochuntica increased the number of beta cells in pancreatic islets of diabetic rats.Similarly, Shaban, Al-Azzawie, Mohamme (2011) showed that this plant enhanced volume density of islets and percentage of beta cells in diabetic rats.

Annona muricata
Annona muricata from Annonaceae family (commonly called Soursop) is a small tree, all parts of which are used in natural medicine in the tropical areas in South and North America and west of Africa (Adewole, Caxton-Martins, 2006).According to the animal studies on STZ-induced diabetic rats, the administration of extract of A. muricata leaves can increase the number of islets and beta cells in pancreas (Adewole, Caxton-Martins, 2006;Adeyemi et al., 2008).

Azadirachta indica
Neem (Azadirachta indica), a tree in the mahogany family, is among the well-known medicinal plants in Africa.Using a clinical study, its hypoglycemic and hypolipidemic effects among diabetic patients have been shown in recent years (Kochhar, Sharma, Schdeva, 2009;Kumari, 2010).Two animal studies have demonstrated that A. indica has also a regenerative effect on the islets of Langerhans.According to the report by Akinola, Caxton-Martins, Dini (2010), oral feeding of ethanolic extract of A. indica leaves increased beta cell density and decreased oxidative stress in the pancreas of STZ-induced diabetic rats.Ebong et al. (2006) also showed regenerative action of this extract in alloxan-induced diabetic rats.

Berberine
Berberine is an isoquinoline alkaloid which is present in the root, rhizome, and stem bark of many plants, such as Berberis vulgaris, Coptidis rhizoma, Hydrastis canadensis, Mahonia aquifolium, and Mohonia nervosa (Ye et al., 2009).Berberine has been demonstrated to have beneficial effects on hyperglycemia and dyslipidemia in diabetic patients (Yin et al., 2008).Regarding its protective/regenerative effect on pancreas, Tang et al. (2006) demonstrated that oral administration of berberine decreased the levels of FBG and serum lipids and restored the damage of pancreas tissue in alloxan-induced diabetic rats.Another study reported that berberine increased insulin sensitization, insulin secretion, and beta cell regeneration in STZ-and high carbohydrate/fat dietinduced diabetic rats (Zhou et al., 2009).

Crocus sativus
Saffron (Crocus sativus) is a perennial stemless herb of the Iridaceae family which is widely cultivated in Iran and also in some other countries such as India.Crocin (crocetin glycoside) and safranal (C 10 H 14 O) are the main constituents of C. sativus and responsible for the pharmacological effects of this plant.Safranal has been reported to have a number of medicinal attributes including antioxidative and cytoprotective effects (Alinejad, Ghorbani, Sadeghnia, 2013;Sadeghnia et al., 2013).Safranal, C. sativus, and crocin show antihyperglycemic, hypolipidemic, and blood insulin elevating effects in insulin-dependent diabetic animals (Kianbakht, Hajiaghaee, 2011;Mohajeri, Mousavi, Doustar, 2009;Samarghandian et al., 2013).In addition, C. sativus has beneficial effects on the histological structure of pancreas in alloxan-induced diabetic rats (Elgazar, Rezq, Bukhari, 2013).Recovery of beta cells and increase of insulin immunoreactivity are of beneficial effects induced by C. sativus in the islets of Langerhans of diabetic rats (Mohajeri, Mousavi, Doustar, 2009).

Curcumin
Curcumin (diferuloylmethane) is an active ingredient of Curcuma longa, a spice employed as a flavoring and coloring supplement in foods.Evidence has suggested that it has potent antioxidant and cytoprotective effects (Alinejad, Ghorbani, Sadeghnia, 2013;Park et al., 2008).In diabetic rats, curcumin inhibits lymphocytes infiltration in the islets of Langerhans and keeps the number of islets and beta cells (Chanpoo, Petchpiboonthai, 2010;Abdel Aziz et al., 2013).These effects are accompanied by decreased FBG and increased serum insulin and C-peptide (Abdel Aziz et al., 2013).

Gymnema sylvestre
Accumulating evidence has demonstrated that G. sylvestre (commonly known as Gurmar in India) improves glycemic control in both T1D and T2D.Clinical trials have demonstrated that the administration of this herb decreases FBG, PPBG, and HbA1c in diabetic patients (Ghorbani, 2013b).The potential ability of G. sylvestre in regenerating pancreatic beta cells has been tested in recent years.It has been shown that aqueous extract of G. sylvestre leaves increases serum insulin level in streptozotocin treated rats.This effect is accompanied by a double increase in the number of beta cells (Shanmugasundaram et al., 1990).Regenerative effect of G. sylvestre has been also observed following long-term treatment of diabetic rats using a standardized dry extract of leaves (Aralelimath, Bhise, 2012).Gymnemic acid is considered to be the active ingredient responsible for the regenerative action of G. sylvestre on beta cells (Ahmed, Rao, Rao, 2010).

Juglans regia
Juglans regia (walnut) has been widely used in the traditional medicine of Asian countries as a remedy for various ailments.Its leaves have shown a significant hypoglycemic effect in diabetic animals.Increase of hepatic glycogenolysis, decrease of gluconeogenesis, inhibition of glucose absorption from the intestine, and enhancing serum insulin are the proposed mechanisms for hypoglycemic effect of J. regia (Asgary et al., 2008;Kamyab et al., 2010;Jelodar, Mohsen, Shahram, 2007).Increase of insulin level can be mediated through regenerative actions of J. regia on pancreatic islets.It has been shown that density of islets, percent of beta cells, and islets size significantly increase in pancreatic tissue of diabetic rats receiving walnut leaf (Jelodar, Mohsen, Shahram, 2007).Regenerative property of J. regia is accompanied by increase in the serum insulin level (Asgary et al., 2008;Javidanpour et al., 2012).Yet, in contrast to leaf, treatment with the shell septum of J. regia has no effects on the pancreatic structure of diabetic rats (Dehghani, Mashhoody, Panjehshahin, 2012).

Momordica charantia
Momordica charantia (Karela, Ampalaya, bitter melon) has acquired a reputation for the management of diabetes.It has passed several animal and clinical studies and its beneficial effects on blood glucose and lipids have been shown in diabetic patients since 36 years ago (Ghorbani, 2013b).Effect of M. charantia fruit on pancreatic histopathological changes has been determined by at least five experimental studies.In adult diabetic animals, the results indicate that M. charantia increases the number of beta cells per islets and leads to the neoformation of islets from the pre-existing islet cells (Ahmed et al., 1998;Singh, Gupta, 2007b;Singh et al., 2008).In addition, the hypoglycemic effect of M. charantia remains after the cease of treatment (Singh et al., 2008).In neonatal diabetic rats, administration of aqueous or ethanolic extract of M. charantia fruit alleviates pancreatic damage and induces the renewal of pancreatic beta cells (Abdollahi et al., 2011;Hafizur, Kabir, Chishti, 2011).

Nigella sativa
N. sativa (black seed) has been used for centuries as a natural remedy for various ailments.Hypoglycemic and hypolipidemic effects of black seed have been reported in diabetic patients (Ghorbani, 2013a).Kanter et al. (2003) investigated the effect of N. sativa volatile oil on histopathology of pancreatic beta cells in diabetic rats and found that N. sativa treatment decreased the elevated serum glucose, increased the insulin concentration, and partially regenerated pancreatic beta cells in these animals.Afterward, they showed that the beneficial effect of N. sativa on the number of beta cells was accompanied by decreasing lipid peroxidation and increasing antioxidant enzyme activity (Kanter et al., 2004).In another experiment, it was represented that the administration of N. sativa oil to STZ-induced diabetic rats increased the diameter of islets of Langerhans (Albajali et al., 2011).The protective effect of N. sativa against beta cell destruction is attributed to its active constituent, thymoquinone.Treatment with thymoquinone inhibits STZ-induced islet degeneration and necrosis and leads to the partial regeneration of the islet and beta cells of diabetic animals (Abdelmeguid et al., 2010;Kanter, 2009;Sankaranarayanan, Pari, 2011).

Vernonia amygdalina
Vernonia amygdalina, also called bitter leaf, is a vegetable from compositae family and is commonly used in west of Africa for treating various diseases including diabetes (Atangwho et al., 2007).Pre-treatment with the aqueous extract of V. amygdalina protects the islets of Langerhans against alloxan-induced pancreatic degeneration (Sunday et al., 2012).Also, administration of V. amygdalina induces regeneration of the islet cells and decreases FBG in diabetic rats (Atangwho et al., 2007).

Proposed mechanisms for protective/regenerative effect of phytochemicals
In this review, the protective effects of several plants on drug-induced b-cells destruction, increasing islets size, and beta cell population were presented.Although these pieces of evidence did not fully reveal the involved molecular mechanisms, decreasing apoptosis, increasing cells' antioxidant capacity, and immunomodulation wee the postulated mechanisms of action.
Beta cell apoptosis and replication rates, islet size, and islet neogenesis are the major determinants of pancreatic endocrine capability for insulin secretion and glucose homostasis (Montanya, Tellez, 2009).Changing the balance of beta cell replication and apoptosis alters the length of beta cell cycle which contributes to the islet size and insulin release.Decreasing apoptosis results in the enhancement of beta cell viability and increase in insulin production.
Intrinsic and extrinsic pathways are considered as two general routes for the activation of apoptosis.The former is activated by stress factors including growth factor deprivation, cell cycle disturbance, and DNA damage, which lead to mitochondrial release of cytochrome c and subsequent stimulation of caspase-9.The latter begins with cell death receptors and the associated activation of caspase-8.Finally, both pathways stimulate effector caspases (3, 6, and 7) which target the substrates that promote DNA fragmentation and cell death (Sharma et al., 2009;Forouzanfar et al., 2013).
It has been well documented that oxidative stress plays an important role in beta cell dysfunction and apoptosis (Yang et al., 2011).Because of poor antioxidant capacity, beta cells are vulnerable to the oxidative stress induced by both T1D insulitis and T2D glucotoxicity (Sharma et al., 2009).Therefore, drugs and phytochemicals that improve glycemia and/or oxidative stress ameliorate or prevent islet lesions.In this regard, protective effect of some phytochemicals on pancreas has been found to be mediated through their antioxidant effects.Zhou et al. (2009) reported that treatment with berberine restored the reduced superoxide dismutase activity and increased lipid peroxidation of pancreas of diabetic animals to the near control level.These antioxidant effects of berberine, therefore, mediate its anti-apoptotic action against beta cell apoptosis in insulin-resistant animal models and against palmitate-induced lipoapoptosis in HIT-T15 insulin producing cells (Gao, Zhao, Li, 2011;Wu, Lu, Dong, 2011).Similarly, the beta cell protective effect of N. sativa can be attributed to the antioxidant properties of this plant, which increases superoxide dismutase activity, inhibites lipid peroxidation, and decreases the generation of reactive oxygen species (ROS) in pancreas tissue (Abdelmeguid et al., 2010).Also, reduction of lipid peroxidation in the pancreas of diabetic rats treated with A. indica suggests the beneficial potential of this plant in the amelioration of ROS-induced pancreatic islet lesions (Akinola, Caxton-Martins, Dini, 2010).In addition to the direct evidence for pancreas, protective/regenerative effects of the phytochemicals on other tissues may support their beneficial actions on pancreatic structure of diabetic rats.For example, hepatoprotective property of Allicin (Vimal, Devaki, 2004), N. sativa (Al-Ghasham et al., 2008), and A. indica (Oyewole, 2011;Ezz-Din et al., 2011) and nephroprotective action of N. sativa (Al-Ghasham et al., 2008) andA. indica (Ezz-Din et al., 2011) have been demonstrated.Also, berberine can promote axonal regeneration in the injured nerves of rats' peripheral nervous system (Han, Heo, Kwon, 2012).
Immunomodulatory action and stimulation of proliferation and differentiation of progenitor cells may be also among the mechanisms involved in beta cell protective/regenerative effects of some phytochemicals (Abiramasundari, Sumalatha, Sreepriya, 2012;Ghazanfari, Hassan, Ebrahimi, 2002).

CONCLUSION
Destruction of pancreatic islets is the major determinant for the onset of hyperglycemia and development of complications in insulin-dependent diabetic patients.
Preventing beta cell degeneration, stimulating endogenous regeneration of islets, and islet transplantation will be of essential approaches for T1D management.At present, the limited supply of donor islets prevents tissue transplantation from being used in the patients.Therefore, development of phytochemical products with beta cell regenerative property can be a promising option for the patients who have lost their mass of functional islet cells.Among the hundreds of plants that have been investigated for diabetes, a small fraction has shown the regenerative property, which was described in this paper.For most of these herbs, however, the number of studies supporting their beneficial effects on pancreas is not enough.Only A. sativum, A. indica, berberine, C. sativus, G. sylvestre, J. regia, M. charantia, and N. sativa had more than one piece of evidence for their regenerative property so that their consumption may decrease insulin dependence on diabetic patients.The exact mechanism responsible for the protective/regenerative effects of phytochemicals on pancreatic islets is yet to be elucidated.However, antioxidant property of phytochemicals may in part mediate their protective action against pancreatic beta cell apoptosis.Regardless of the molecular mechanisms, it seems that patients at the earliest stages of diabetes can be treated with these plants to delay or prevent the full destruction of pancreatic islets.Also, construction of polyherbal compounds through the combination of these phytochemicals may yield more potent regenerative agents for beta cells.Upcoming clinical trials on this topic are particularly warranted.

TABLE I -
Phytochemicals with protective/regenerative effects on pancreatic beta cells.

Plants Animal model Treatment Effects on pancreas Other effects Reference
Verma et al., 2010sVerma et al., 2010

TABLE I -
Phytochemicals with protective/regenerative effects on pancreatic beta cells.(cont.)

TABLE I -
Phytochemicals with protective/regenerative effects on pancreatic beta cells.(cont.)

TABLE I -
Phytochemicals with protective/regenerative effects on pancreatic beta cells.(cont.)