Biotechnology Journal

DOI 10.1002/biot.200900207

Biotechnol. J. 2009, 4, 17041711

Review

Plant-based corosolic acid: Future anti-diabetic drug?

Ganapathy Sivakumar1, Daniel R Vail1, Vipin Nair1, Fabricio Medina-Bolivar1 and Jackson O. Lay Jr.2
1 Arkansas 2 Arkansas

Biosciences Institute, Arkansas State University, Jonesboro, AR, USA Statewide Mass Spectrometry Facility, University of Arkansas, Fayetteville, AR, USA

Diabetes is one of the nations most prevalent, debilitating and costly diseases. For diabetes, frequent insulin treatment is very expensive and may increase anti-insulin antibody production, which may cause unwanted side effects. Corosolic acid may also have some efficacy in the treatment of diabetes, but without induction of anti-insulin antibodies. Recently, corosolic acid from Lagerstroemia speciosa L. leaf extracts has been reported to act via an indirect mechanism (unlike insulin) in animal experiments. The insulin-complementary anti-diabetic therapeutic value observed in these Japanese preliminary clinical trials has led to renewed interest in the biosynthesis of this compound. So far, there has been no clear evidence for a corosolic acid biosynthetic pathway in plants. This article provides possible roles of corosolic acid and hypothetical information on the biosynthetic pathway in plants.

Received 25 August 2009 Revised 9 October 2009 Accepted 28 October 2009

Keywords: Anti-diabetic Banaba Corosolic acid Lagerstroemia speciosa Terpenoids

1 Introduction
Currently, diabetes is one of the most serious global diseases, affecting an estimated 246 million people worldwide as of February 2008. International Diabetes Federation predicts that number may increase to 380 million by the year 2025. In 2006, the global diabetes drug market, estimated at over $21 billion, was a top force in drug spending growth. Nearly 21 million people in the United States have diabetes and the cost associated with this disease in the US in 2007 was $174 billion [1]. About 400 000 Americans are on dialysis therapy, and chronic kidney disease caused by diabetes will reach half a million individuals by 2010 and may exceed one million by 2018 [2]. Without urgent action, within the next two decades the number of adults living with type-2 diabetes in developing countries is projected to increase by 31% [3]. Today, almost 9.7% of women in the US have diabetes, thus it will affect

Correspondence: Ganapathy Sivakumar, Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR 72401, USA E-mail: sivakumar@libero.it Abbreviation: IPP, isopentenyl diphosphate

both mothers and unborn children. Furthermore, it will cause pregnancy complications such as miscarriage and birth defects. Gestational diabetes affects about 4% of all pregnant women and about 135 000 cases of gestational diabetes are reported in the US each year. In the US approximately 90% of people having type-2 diabetes are overweight [4]. Diabetes is caused by defects in insulin production, insulin secretion, and insulin signaling [5]. Insulin is one of the most important hormones that participate actively in protein anabolism during growth phase. There are two types of diabetes mellitus: type-1 (insulin dependent) and type-2 (noninsulin dependent). With type-1 diabetes, the pancreas no longer makes insulin, which can lead to heart attack, blindness, nerve damage, blood vessel disease and kidney damage. In addition, cells are destroyed by an autoimmune attack [6]. Type-2 diabetes is one of the leading causes of mortality and develops as the consequence of relative insulin insufficiency. Thus, high blood glucose levels cause the aforementioned symptoms as well as severe apnea. Many complex factors, including genetic and physiological changes, can lead to both types of diabetes. Both types of diabetes are also involved in multiple other mechanisms including non-enzy-

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matic glycation [7], increased aldose reductase activity [8], and involvement of inducible nitric oxide synthase [9]. More recently, microRNAs were shown to regulate insulin production, insulin secretion and insulin action [5]. In type-2 diabetes patients the intestinal endocrine L cells produce more glucagon-like peptide-1 during insulin resistance for compensation. Thus, the activation of wingless-type (Wnt) signaling pathway and the cross-talk between Wnt and insulin signaling pathways stimulates expression of cancer genes [10]. Popular synthetic anti-diabetic drugs such as glibenclamide, metformin and pioglitazone have many side-effects [11]. Furthermore, these drugs are hypoglycemic (blood glucose level reducing) and, to different extents, adipogenic (promoting weight gain). Unwanted weight gain during therapy is problematic because obesity is one of the causative agents of type-2 diabetes [12]. Moreover, these drugs are temporary treatments, and so are not intended for long-term treatment or as a permanent cure for diabetes. Many medicinal plants play an important role in the treatment of diabetes, especially in underdeveloped and developing countries where there are limitations on presently available therapeutic options for diabetes such as oral hypoglycemic agents and insulin [13]. In addition, frequent insulin treatment is very expensive and may increase anti-insulin antibody production which causes many side effects. Herbal medicines used for many years in different cultures around the world are in some ways ideal therapeutics and may even represent possible permanent cures for diabetes [14, 15]. Until now, more than 400 plant species have been scientifically claimed to have anti-hyperglycemic activity [16, 17]. The reason for a recent effort in introducing new plant-derived molecules into the drug development pipeline is obvious, and understanding of the molecular targets of natural products is important [18, 19]. Enrichment of bioactive molecules from natural prod-

Figure 1. Lagerstroemia speciosa seedlings.

ucts can be achieved using the enzymes catalyzing their biosynthesis [20]. The chemical synthesis of bioactive terpenoids are not safe, are expensive, and unable to produce pure enantiomerical forms [21]. Moreover, the chemical synthesis of bioactive molecules require several highly toxic substances, and the final products from these reactions may still contain toxic residues and require further purification.

Corosolic acid

To prevent diabetes mellitus without side effects requires potent natural stereoisomers of anti-diabetic molecules from plant sources. Plants contain several hypoglycemic and hypolipidemic constituents that have been the object of clinical trials to demonstrate their beneficial action in diabetes treatment [22]. The Lagerstroemia speciosa L. (banaba) plant (Fig. 1) belonging to the family Lythraceae is commonly used in Ayurveda medicine and is an important medicinal crop in Asian

Figure 2. Negative-ion, APCI-MS spectrum of corosolic acid from Lagerstroemia speciosa leaf extracts.

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Table 1. Chemical components of Lagerstroemia speciosa

Table 2. Corosolic acid biosynthesizing/accumulating plant species

Molecules Castalagin Corosolic acid Ellagitannins Ellagic acid Flosin B Gemin D Grandinin Hippophaenin A Lagerstroemin Lagerstannins A, B and C Maslinic acid Reginin A Valoneic acid dilactone Vescalagin

Reference [59] [24] [60] [60] [61] [59] [59] [59] [60, 62] [59] [63] [60] [61] [59]

Plant species Actinidia arguta Agrimonia pilosa Campsis grandiflora Camptotheca acuminata Centella asiatica Crataegus pinnatifida Datisca cannabina Eriobotrta japonica Glechoma longituba Lagerstroemia speciosa Myricaria elegans Perilla frutescens Platostoma africanum Potentilla chinesis Prunus dulcis Rubus biflorus Serissa serissoides Symplocos paniculata Tiarella polyphylla Ugni molinae Weigela subsessilis Youngia koidzumiana

Reference [64] [65] [66] [67] [68] [69] [70] [36] [71] [24] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83]

countries. In addition, this plant is of significant commercial interest on the international pharmaceutical markets and is called queen crape-myrtle. Anti-diabetic, anti-obesity, antioxidant, insulin-like glucose uptake activity [2327] and hypoglycemic [2832] effects of L. speciosa leaf extracts have been reported, although banaba leaf extract has not been evaluated yet by the FDA. The principal component of banaba leaves is corosolic acid (2-hydroxyursolic acid, molecular formula C30H48O4, molecular weight 472.70) (Fig. 2), an ursan type triterpene which has recently attracted much attention due to its anti-diabetic activities [33]. The chemical compounds of L. speciosa are listed in Table 1. Corosolic acid biosynthesis/accumulating plant species are listed in Table 2. Due to the insulin-like properties of corosolic acid, it is known as a phyto-insulin and is also commonly referred to as botanical insulin [34]. Currently, corosolic acid is very expensive, costing approximately $43 per milligram.

Role of corosolic acid

Although the current pharmacological and clinical data are still of a relatively preliminary nature, corosolic acid treatment in humans led to a decrease in post challenge plasma glucose levels [35]. Interestingly, corosolic acid suppresses the differentiation and down-regulates the expression of peroxisome proliferator-activated receptor (PPAR-) as well as enhancer binding protein (C/EBP-) mRNA in 3T3-L1 adipocytes. Also, it promotes [3H]glucose uptake [36]. This suggests that unlike most other anti-diabetic drugs, corosolic acid, without increasing adiposity, reduces blood

glucose. Moreover, in a KK-Ay mouse study, corosolic acid treatment reduced blood glucose and lowered plasma levels, indicating improved glucose metabolism by reducing insulin resistance [37]. The mechanism for this decrease in insulin resistance on treatment with corosolic acid involved an increase in mouse muscle GLUT4 (a glucose transporter) translocation from the low-density microsomal membrane [38]. These results indicate that corosolic acid can improve hyperinsulinemia in type-2 diabetes. In addition, after an oral administration of sucrose in a ddY mouse study, treatment with corosolic acid (1 mg/kg body weight) improved hyperglycemia and significantly reduced the hydrolysis of sucrose in the small intestine. In other words, corosolic acid inhibited increasing disaccharide concentrations in the small intestine [39]. More recently, it was demonstrated that, in CHO/hIR cells, corosolic acid stimulates glucose uptake via enhancement of insulin receptor phosphorylation by inhibiting certain protein tyrosine phosphatases enzymes [40]. In addition, an anti-diabetic action of corosolic acid was shown in rat liver where it increased glycolysis and decreased glycogenolysis in hepatocytes [33]. In a clinical study, a standardized treatment with 1% corosolic acid (called Glucosol), administered as a 32 or 48 mg daily dose, caused significant reductions in glucose levels in the blood of type-2 diabetics over 2 weeks. In a similar study it was further

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shown that a soft-gel capsule formulation of Glucosol resulted in the greatest reduction of blood glucose levels (30% reduction, compared to 20% observed with dry-powder filled hard gelatin capsule) [23]. This study suggests that the maximum dose before a reaching a plateau was not achieved, and that doses greater than 48 mg per day may further decrease blood glucose levels. Klein et al. [24] reported that corosolic acid is not the primary inducer of glucose uptake in KKAy/Ta Jcl mouse cells. They found that when tannins were removed from the banaba leaf extracts, glucose uptake was not affected on treatment. Additionally, increasing concentrations of pure corosolic acid did not lead to an increase in glucose uptake. They concluded that, unlike insulin, any anti-diabetic properties of corosolic acid were due to an indirect mechanism. These results correlate with a study that demonstrated that the gallotannin penta-O-galloyl-glucopyranose (PGG) was able to lower glucose levels in the blood and increase glucose tolerance [41].

Chemistry of corosolic acid

The biosynthesis of terpenoids in plants includes two independent isopentenyl diphosphate (IPP) generating pathways, the mevalonate and nonmevalonate pathways [42, 43]. In the mevalonate pathway, which occurs in the cytosol, IPP is biosynthesized from acetyl CoA and then isomerized to dimethylallyl diphosphate (DMAPP) [44]. This pathway is generally used to supply precursors for sesquiterpenes (C15), triterpenes (C30) and sterols. In the non-mevalonate pathway, localized in plastids, IPP and DMAPP are biosynthesized from pyruvate and D-glyceraldehyde 3-phosphate [45, 46]. The monoterpenes (C10), diterpenes (C20), tetraterpenes (C40) are synthesized from nonmevalonate pathway IPP Although the interaction . between these pathways is still not fully clear, cross talk between these different IPP biosynthetic pathways has been reported in Arabidopsis thaliana [47]. Recently, a successful terpenoid pathway engineering strategy demonstrated a more than thousand-fold increase of sesquiterpene patchoulol production for therapeutic applications [21]. So far, in plants, no clear reports on corosolic acid biosynthetic pathways have been published. There are a few reports on the chemical synthesis of corosolic acid from ursolic acid [4850]. In Lagerstroemia speciosa, ursolic acid (C30) is converted to corosolic acid [51]. The steps from acetyl-CoA to IPP are common for many metabolic pathways (Fig. 3).Although corosolic acid is biosynthesized in

the cytoplasm, reports indicate cross talk between mevalonate and non-mevalonate pathways. The first two steps from the C30 pathway involving production of squalene and oxidosqualene, catalyzed by squalene synthase and oxidosqualene synthase, respectively, are well described in the literature [52].The third and forth steps involving production of -amyrin (ursane type) and ursolic acid may be catalyzed by -amyrin synthase and -amyrin oxidase (Fig. 3), although -amyrin synthase has not yet been reported. There are many reports on the functional characterization of -amyrin synthases and some that discuss -amyrin as a small percentage of the reaction products or even the mutagenesis of -amyrin synthase to give a new enzyme that produces more of the alpha products. In cultured cells, -amyrins have been observed to be the major product of the oxidosqualene cyclase OEA [53]. For the next steps, there are probably two P450s that catalyze the oxidation reactions. The order of these may be reversed. The first one would generate an acid (ursolic acid). CYP71av1, a P450 involved in artemisinin biosynthesis, provides an example for this type of reaction [54].The final step enzyme ursolate hydroylase, also a P450, introduces a stereo-specific hydroxyl into the corosolic acid A ring (Fig. 3). Again, there is a precedence for this type of P450 catalyzed reaction. The existence of this hypothetical corosolic acid biosynthetic pathway needs to be tested and proven. In addition, there is important stereochemistry associated with corosolic acid and the other derivatives. Wen et al. [48, 50] found that the two stereoisomers of corosolic acid, in which the A ring 2- and 3-hydroxyl groups are on the same side, had a decreased glycogen phosphorylase inhibitory effect. However, other modifications to the A ring produced several corosolic acid derivatives that had an increased potency.

Conclusion

To produce plant-based, cost-effective corosolic acid and bioactive derivates for global treatment of diabetes, new approaches will be needed. One option is a high-tech in vitro tissue culture platform using a bioreactor system. There is an international patent [55] demonstrating corosolic acid accumulation in a plant cell culture. In addition, liquid suspension cultures of cells derived from calli of Eriobotrya japonica and Lagerstroemia speciosa show great promise as a production system for corosolic acid. Corosolic acid yields have been shown to increase 25 times in suspension-cultured cells compared to those of the natural leaf for

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Figure 3. Possible biosynthetic route for corosolic acid in plants (MVA, mevalonic acid; MEP, non-mevalonic acid).

E. japonica and 18.3 times compared to those in a callus. The results are even more pronounced for suspension-cultured cells of L. speciosa, in which corosolic acid productivity increased 56 times compared to that in the cells of the natural leaf. Advanced root culture technology is more stable and less complex for commercial-scale large-volume production of small molecules than cell culture. Adventitious root cultures have recently been used on an industrial scale for biopharmaceutical production [56], establishing the feasibility of a root culture system as a vigorous commercial platform [57]. In South Korea, there are three companies commercially producing plant-based small molecules from ginseng root culture technology using 10 00020 000-L bioreactors [58]. There are some Japanese companies that are commercially producing corosolic acid from Lagerstroemia speciosa leaves. If it is possible to increase corosolic acid to commercial production levels in plant tissues in vitro, this bioproduction platform could be precisely

regulated to reproducibly synthesize natural bioactive isomer corosolic acid for use as a therapeutic agent.

This research was partially funded by the Arkansas Biosciences Institute grant 262147, National Science Foundation (grant # EPS-0701890), Arkansas ASSET Initiative and Arkansas Science & Technology Authority. Also we thank Professor Joe Chappell for critical suggestions with respect to the biosynthetic pathway and Dr. Barbara S. Neal, National Tropical Botanical Garden, Kalaheo, Hawaii as well as Drs. Alan W. Meerow, Tomas Ayala-Silva & Wilhelmina C. Wasik, USDA-ARS-SHRS, National Germplasm Repository, Miami, Florida for providing the Lagerstroemia speciosa seed material. The authors have declared no conflict of interest.

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