An herbal formulation containing Zingiber officinale rhizomes and Allium sativum cloves can increase oral glucose tolerance in mice

Zingiber officinale rhizomes and Allium sativum cloves are two plant parts widely consumed in Bangladesh as spices, and which are readily available and affordable. The two plants also have ethnomedicinal uses, one of which uses being to treat diabetes. Since the plant parts are consumed in culinary dishes (which involves boiling or steaming), the objective of this study was to determine whether methanol extract of steamed Z. officinale rhizomes (MEZO) and steamed A. sativum cloves (MEAS) can both individually as well as more so in combination improve glucose tolerance in oral glucose tolerance tests (OGTT) in mice. Administration of either MEZO or MEAS to mice at a dose of 400 mg per kg body weight led to reductions in blood glucose levels in glucose-challenged mice by 37.0 and 34.9%, respectively. Administration of a combination of (MEZO + MEAS) at individual doses of 100, 200 and 400 mg each led to dose-dependent reductions in blood glucose levels, respectively, by 22.5, 30.4, and 42.2%. Thus, the two extracts demonstrated a synergistic effect when administered in combination. A standard antihyperglycemic drug, glibenclamide, when administered at a dose of 10mg per kg lowered blood glucose level by 43.9%. Thus, at the highest dose tested, the combination of the two extracts lowered blood glucose levels in a comparable manner to that of glibenclamide. Since the extracts were prepared from steamed plant parts, the results suggest that partaking of the two spices in culinary dishes can be effective in controlling blood glucose in diabetic patients. Correspondence to: Prof. Mohammed Rahmatullah, Dean, Faculty of Life Sciences, University of Development Alternative, Lalmatia, Dhaka-1207, Bangladesh, Phone: +880-1715032621, E-mail: rahamatm@hotmail.com


Introduction
Diabetes mellitus is a disorder primarily characterized by high glucose levels in blood and glucose intolerance. This disorder, which is rapidly increasing throughout the world, can lead to complications of various organs of the body including kidneys, eyes, and heart [1], which in turn can lead to untimely death. Allopathic medicine cannot cure this disorder; however, orally taken drugs are present to increase insulin production from the pancreas and to lower elevated blood glucose levels. Blood glucose level can also be controlled through regular insulin injections. The various oral drugs used to lower blood glucose include sulfonylureas, meglitinides, biguanides, thiazolidinediones, and alpha-glucosidase inhibitors. However, these drugs can have adverse effects ranging from hypoglycemia to gastrointestinal disorders and even increased risks of bladder cancer and non-fatal heart attacks. Also for countries like Bangladesh, the rural and the poorer sections of the population does not have affordability and availability to these medications. As a result, more affordable and readily available substitutes are necessary.
Zingiber officinale Rosc. (Zingiberaceae) and Allium sativum L. (Liliaceae) are two commonly cultivated and widely used spice plants of Bangladesh. Rhizomes of the first, known in English as ginger and locally as ada, and cloves of the second, known in English as garlic and locally as roshun, are used in a large number of dishes of Bangladesh, which includes vegetable, fish and meat dishes. The antidiabetic efficacy of Z. officinale rhizomes have been reviewed [2,3]. The efficacy of A. sativum cloves against diabetes and diabetes-induced complications have also been variously reported [4][5][6]. However, the available reports deal with raw rhizomes or cloves or extract there from.
Since the rhizomes or cloves are consumed following cooking (which involves boiling or steaming), it was of interest to determine if steamed plant parts still maintain their antihyperglycemic efficacies, for then cooked rhizomes or cloves as present in various culinary dishes can form an easy means to reduce elevated blood glucose levels. For the last few years we had been screening various medicinal plants including spices for their antihyperglycemic potential as determined through oral glucose tolerance tests or OGTT [7][8][9][10][11][12]. The objective of the present study was to evaluate whether methanolic extract of steamed rhizomes of Z. officinale and steamed cloves of A. sativum individually or in combination can improve glucose tolerance in glucose-loaded mice and as such demonstrate antihyperglycemic activity.

Plant material collection
Rhizomes of Z. officinale and cloves of A. sativum were collected from a local market in Dhaka city, Bangladesh in January and April 2016, respectively. The plant parts were steamed for 20 min and thoroughly dried separately and finely powdered prior to extraction with methanol.

Preparation of methanolic extracts
The plant parts were dried thoroughly following steaming for 20 min and finely powdered, and 100g of dried and powdered individual plant parts (rhizomes or cloves) were extracted with methanol (w:v ratio of 1:5). The final weight of the methanol extract of Z. officinale rhizome (MEZO) was 5.8g, and the final weight of A. sativum clove (MEAS) was 4.2g. The extracts were stored at -20 o C till use.

Chemicals and drugs
Glibenclamide and glucose were obtained from Square Pharmaceuticals Ltd., Bangladesh. All other chemicals were of analytical grade.

Animals
Thirty-Five Swiss albino mice, which weighed between 15-18g were used in the present study. The animals were obtained from International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B). The animals were acclimatized for three days (72 hours) prior to actual experiments. The study was carried out over a period of 4 days (3 day-night period of acclimatization and one further day to conduct the actual experiment). No mice died during this period. The study was conducted following approval by the Institutional Animal Ethical Committee of University of Development Alternative, Dhaka, Bangladesh.

Oral glucose tolerance tests (OGTT) for evaluation of antihyperglycemic activity
Oral glucose tolerance tests were carried out as per the procedure previously described by Joy and Kuttan (1999) [13] with minor modifications. Briefly, fasted mice were grouped into seven groups of five mice each. The various groups received different treatments like Group 1 received vehicle (1% Tween 20 in water, 10ml/kg body weight) and served as control, Group 2 received standard drug (glibenclamide, 10 mg/kg body weight). Group 3 received methanol extract of Z. officinale rhizome (MEZO) at a dose of 400 mg per kg body weight. Group 4 received methanol extract of A. sativum clove (MEAS) at a dose of 400mg per kg body weight. Groups 5-7 received (MEZO + MEAS) at doses of 100, 200, and 400mg each (that is 100mg of MEZO + 100 mg of MEAS combined) per kg body weight, respectively. All substances were orally administered. Following a period of one hour, all mice were orally administered 2g glucose/kg of body weight. Blood samples were collected 120 minutes after the glucose administration through puncturing heart. Blood glucose levels were measured by glucose oxidase method [14]. The percent lowering of blood glucose levels were calculated according to the formula described below.

Percent lowering of blood glucose level = (1 -W e /W c ) X 100,
where W e and W c represents the blood glucose concentration in glibenclamide or various extracts administered mice (Groups 2-7), and control mice (Group 1), respectively.

Statistical analysis
Experimental values are expressed as mean ± SEM. Independent Sample t-test was carried out for statistical comparison. Statistical significance was indicated by a p value < 0.05 in all cases [15].

Results
Administration of either MEZO or MEAS to mice at a dose of 400mg per kg body weight led to reductions in blood glucose levels in glucose-challenged mice by 37.0 and 34.9%, respectively. Administration of a combination of (MEZO + MEAS) at individual doses of 100 (that is 100 mg each of MEZO + MEAS), 200 and 400 mg each led to dosedependent reductions in blood glucose levels, respectively, by 22.5, 30.4, and 42.2%. Thus, the two extracts demonstrated a synergistic effect when administered in combination. A standard antihyperglycemic drug, glibenclamide, when administered at a dose of 10mg per kg lowered blood glucose level by 43.9%. Thus, at the highest dose tested, the combination of the two extracts lowered blood glucose levels in a comparable manner to that of glibenclamide. The results are shown in Table 1 and strongly indicate that steaming does not reduce the oral glucose tolerance effect of rhizomes of Z. officinale or cloves of A. sativum. In fact, both extracts possessed substantial antihyperglycemic effects and the combination was more or less effective as glibenclamide at the highest dose (400 mg each) of the two extracts.

Discussion
Ginger extract and gingerols present in rhizomes has been shown to increase glucose uptake in skeletal muscle cells, which can account for the antihyperglycemic activity of Z. officinale [16]. It is interesting that methanolic extract of steamed ginger also demonstrated improved oral glucose tolerance, that is, antihyperglycemic effects. As such, cooked ginger (added to cuisines) can also form the basis of dietary regimen to control blood glucose. Interestingly, the same conclusion has also been reached by an earlier study [17]. The hypoglycemic effect of garlic (A. sativum) has been attributed to its sulfur-containing compounds [18]. It is quite possible that such compounds like allicin may either be resistant to breakdown during heating or may form other more active antihyperglycemic compounds during the steaming (heating) process. The identifications of relevant bio-active components and their mechanism of action needs further research and are currently being undertaken in our laboratory. The fact, however, remains that the two spices are readily available, affordable and consumed in Bangladesh by all sections of the people. As such, they can form a suitable substitute for costlier and difficult to obtain allopathic drugs for blood glucose control in diabetic and glucose metabolism impaired persons.

Authorship and contributorship
MS, SR, NR, IJT, and SH collected the plant materials, prepared the extracts and did the experiments. MR guided the work and wrote the manuscript. All authors read the manuscript and made necessary changes to it where applicable. The final version has been approved by all the authors connected with the research work.