Chaenomeles Sinensis: A Potent α-and β-Glucosidase Inhibitor

Problem statement: Glycosidase inhibitors are vital sources for the tr ea ment of diabetes type II with a special importance in pharmacology, food industry and biotechnology, since for diabetes control, different diets and drugs, especially herb al medicines are recommended in this era. Approach: While screening for the potent natural glycosidase inhibitors, we found the fruits of Chaenomeles sinensis (C. sinensis), as the most effective glycosidase inhibitor. The crude 80% methanolic extract of the fruits and its n-hexane, methylene chloride, et hyl acetate, n-butanol and aqueous fractions were further investigated for α-glucosidase, β-glucosidase, α-galactosidase and β-galactosidase enzyme inhibition activities. Results: All the C. sinensis extracts showed remarkable α-glucosidase and β-glucosidase inhibitory activities (at a concentrat ion of 5 μg 210 μL reaction) ranging from 82-99 and 5-85%, respectively. Among all the inhibition s tudies, n-butanol fraction demonstrated the highest (99%) α-glucosidase inhibitory activity, whereas minor α-galactosidase (18-35%) and β-galactosidase (10-34%) inhibitions were examined in all the fract ions of C. sinensis. Conclusion: C. sinensis fruits may prove as potent natural anti-diabetic source wi th noteworthy α-glucosidase and β-glucosidase inhibitions, because the inhibition of these enzyme s provide a strong biochemical basis for the management of type II diabetes by controlling gluco se absorption. These results provide intense rationale for further animal and clinical studies.


INTRODUCTION
Diabetes Mellitus (DM) is a major chronic life-threatening disorder, in which homeostasis of carbohydrate and lipid metabolism is improperly regulated by the pancreatic hormone, insulin; resulting in an increased blood glucose level. DM is a serious metabolic disease that has a significant impact on the health, quality of life and life expectancy of patients, as well as on the health care system. As per WHO, at least 171 million people worldwide have diabetes, which is likely to be more than double by 2030 and around 3.2 million deaths every year are attributable to complications of diabetes; six deaths every minute [1] .
From the basic two types of DM, one being insulin dependent (type I) and another non-insulin dependent (type II), later is the most prevalent form and is developing with as increase in obesity and aging in the general population [2] . In recent years, there has been renewed interest in the DM treatment using herbal drugs as they are generally non-toxic and World Health Organization has also recommended the evaluation of the effectiveness of plants in condition where we lack safe modern drugs. Plant derivatives with hypoglycaemic properties have been used in folk medicine from very ancient time [3] . Despite the introduction of hypoglycaemic agents from natural and synthetic sources, diabetes and its secondary complications continue to be a major medical problem to people [4] . Medicinal plants used to treat hyperglycemic conditions are of considerable interest to ethno botanical community as they are recognized to contain valuable medicinal properties in different parts of the plant. So, traditional herbal therapies by indigenous systems of medicine for treating this ailment are being intensively explored.
In continuing this investigation, we assessed the various enzyme (such as, α-glucosidase, β-glucosidase, α-galactosidase and β-galactosidase) inhibition activities on Korean herbal plants, as these enzymes are involved in a variety of biochemical processes related to metabolic disorders and diseases, such as diabetes, viral or bacterial infections, lysosomal storage disorders and cancer. Therefore, an exhausted attempt has been given on the design of efficient glycosidase inhibitors for many promising applications [5][6][7][8] . In our screening, we found C. sinensis as the most promising candidate for further scrutiny.
The plant Chaenomeles sinensis (Thouin) Koehne (also called as Pseudocydonia sinensis (Thouin) CK Schneid, common name: Chinese-quince, ma gua) belonging to Rosaceae family is mainly distributed in Korea, China and Japan. Its fruits have been shown to have higher amounts of phenolics. This high phenolics content can be expected to act as a factor for improvement in health [9] . Its fruit is used as traditional Chinese medicine to treat throat diseases. It also reported antiinfluenza, antioxidant, antiulcerative, antiviral, antihemolytic, antipruritic and antibacterial (against Streptococcus pyogenes (which is a causative agent of tonsillitis and pharyngitis) activities [9][10][11] .
To our knowledge, there are no prior reports discussed on the anti-diabetic activity of C. sinensis. Therefore, the objective of this study was to investigate the anti-diabetic potential of the crude extract of C. sinensis and its various fractions by confirming the in vitro enzyme inhibition studies on glycosidase.

Plant material:
The dried and matured fruits of C. sinensis were obtained from "Korean Collection of Herbal Extracts" a Biotech company in Korea. A collection of voucher specimen is available with the company (Korea Collection of Herbal Extracts, 2000).

Extraction:
The dried and ripe fruits were chopped into small pieces and pulverized into a fine powder. The powdered C. sinensis fruits (2 Kg, dry weight) were kept for extensive decoction in 80% methanol for 2 months at room temperature. The extract was then concentrated using rotary vacuum evaporator at 20-30°C to obtain the dried crude extract (210 g).
Enzyme assays: The enzyme inhibition activities for α-glucosidase, β-glucosidase, α-galactosidase and β-galactosidase were evaluated according to the method previously reported by Shibano et al. [12] with minor modifications. The enzymatic hydrolysis of substrate was monitored by the amount of o-nitrophenol released in the reaction mixture at 410 nm using microplate reader. All experiments were carried out in triplicates.
Statistical analyses: All assays were performed at least three times with triplicate samples. The inhibition rates for all the assays were calculated as a percentage of control (buffer containing MeOH) without inhibitor. All results are expressed as mean±SD.

DISCUSSION
Glycosidase inhibitors are the important tools for studying the mechanisms of action of glycosidases and are also prospective therapeutic agents for some degenerative diseases, including diabetes, viral attachment and cancer [2,7,8] . As synthetic glycosidase inhibitors have many side/adverse effects, herbal medicines are being the center point for the researchers in the current era.
Therefore, in this study, for the first time, the C. sinensis crude extract (in 80% methanol) and its fractions were investigated for glycosidase inhibition activities. The present enzyme inhibition studies demonstrated that, the C. sinensis fruit extract is a potent novel α-glucosidase inhibitor with moderate β-glucosidase inhibition and lower αand β-galactosidase inhibitions. The α-glucosidase inhibition may be related to the high phenolic content and antioxidant property of the fruits [9,10] .
Therefore based on this in vitro study, we recommend that, the C. sinensis fruits may have beneficial effects in managing type II diabetes mellitus.

CONCLUSION
The results of the in vitro enzyme inhibition studies emphasize the potent effect of the C. sinensis fruit extract on diabetes treatment. Based on this, further pre-clinical and clinical studies can be pursued.