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Antidiabetic Activity of Aqueous Leaf Extracts of Guazuma ulmifolia Lamk., Ethanolic Extracts of Curcuma xanthorrhiza and their Combinations in Alloxan-induced Diabetic Mice



I.K. Adnyana, E. Yulinah, Yuliet and N.F. Kurniati
 
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ABSTRACT

Guazuma ulmifolia Lamk. and Curcuma xanthorrhiza are traditionally used to treat diabetes mellitus and are having potential in the development of drug for diabetes due to their antidiabetic activity. Therefore, this study aimed to evaluate the antidiabetic activity of aqueous leaf extracts of Guazuma ulmifolia Lamk. and ethanolic extracts of Curcuma xanthorrhiza and their combinations in alloxan-induced diabetic mice. Aqueous leaf extracts of Guazuma ulmifolia Lamk., ethanolic extracts of Curcuma xanthorrhiza and its combinations were administered orally to Swiss Webster mice. Alloxan was used to induce diabetes mellitus. The parameters studied included oral glucose tolerance test, blood glucose serum level of alloxan induced diabetic mice and histology of pancreatic cells. The results showed that in oral glucose tolerance test and alloxan-induced diabetic mice, plasma glucose level in both extracts studied and their combinations were significantly lower than positive control, suggesting a potential effect as antidiabetic. By the histology study, combination of G. ulmifolia at dose of 25 mg kg-1 b.wt. and C. xanthorrhiza at dose of 12.5 mg kg-1 b.wt. (the lowest combination) showed less damage of pancreatic cells. The results suggest that Guazuma ulmifolia Lamk. and Curcuma xanthorrhiza has an antidiabetic activity, probably by increasing insulin production, thereby justifying its traditional claim.

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  How to cite this article:

I.K. Adnyana, E. Yulinah, Yuliet and N.F. Kurniati, 2013. Antidiabetic Activity of Aqueous Leaf Extracts of Guazuma ulmifolia Lamk., Ethanolic Extracts of Curcuma xanthorrhiza and their Combinations in Alloxan-induced Diabetic Mice. Research Journal of Medicinal Plants, 7: 158-164.

DOI: 10.3923/rjmp.2013.158.164

URL: https://scialert.net/abstract/?doi=rjmp.2013.158.164
 
Received: March 15, 2013; Accepted: June 17, 2013; Published: December 30, 2013



INTRODUCTION

Diabetes mellitus is one of the common metabolic disorders in which a person has high blood glucose level. Diabetes mellitus with micro-and macrovascular complications causes significant morbidity and mortality. Diabetes mellitus is found to be one of the five leading cause of death in the world (Vats et al., 2004; Senthil Kumar et al., 2006). In modern drugs, side effects due to the used of insulin and oral hypoglycemic agents lead to dissatisfaction with the antidiabetic therapy (Sumana and Suryawashi, 2001). Therefore, a lot of patients nowadays use natural products to treat diabetes mellitus (Holman and Turne, 1991; Rao et al., 2001).

Guazuma ulmifolia Lamk., commonly known in Indonesia as ‘jati belanda’, is a small to medium sized tree, up to 30 m in height and 30-40 cm in diameter and distributed in Argentina, Bolivia, Brazil, the Caribbean, Central America, Colombia, Ecuador, Mexico, Paraguay and Peru. It has been cultivated in India for over 100 years and recently introduced to Indonesia. The leaves have been known to contain alkaloid, flavonoid, tannin and steroid/triterpenoid (Sukandar et al., 2012).

Curcuma xanthorrhiza, commonly known in Indonesia as ‘temulawak’, is a plant species that flourishes up to 1800 m above sea level in a tropical climate. Curcuma xanthorrhiza is distributed in Indonesia, Malaysia, Thailand, Philippines, China, Barbados, India, Japan, Korea, the United states and some countries in Europe. The rhizomes have been known to contain quinone, flavonoid and steroid/triterpenoid (Sukandar et al., 2012).

There are lot of studies had been done regarding the antidiabetic activity of G. ulmifolia and C. xanthorrhiza (Alarcon-Aguilara et al., 1998; Yasni et al., 1991; Alonso-castro and Salazar-Olivo, 2008). In this study, antidiabetic activity of Guazuma ulmifolia Lamk., Curcuma xanthorrhiza and combination of Guazuma ulmifolia Lamk. and Curcuma xanthorrhiza was evaluated in order to get the best diabetic treatment.

MATERIALS AND METHODS

Collection of plant material: The plant material of Guazuma ulmifolia leaf and Curcuma xanthorrhiza rhizome used for the investigation was obtain respectively from Bogor and Bandung, west java, Indonesia. The plants have been identified authentically by school of life sciences and technology, Institute of Technology Bandung, Indonesia.

Preparation of extracts: The leaves of G. ulmifolia and rhizomes of C. xanthorrhiza were collected and shadow dried. The shade-dried plants were subjected to pulverization to get coarse powder. The aqueous extract was prepared by boiling 1 kg of the powdered grains G. ulmifolia in 10 L of aquadestilate. Furthermore, the supernatant was collected and freeze dried. The ethanolic extract was prepared by cold maceration of 1 kg of the powdered grains of C. xanthorrhiza in 20 L of 95% ethanol by using maseration method. Furthermore, extract was evaporated by using rotavapor. Both the extracts were stored in the refrigerator at 2-8°C.

Animals: Healthy adult Swiss Webster mice (20-35 g) were obtained from school of pharmacy, Institute of Technology Bandung. Mice were maintained on normal mouse chow and tap water ad libitum.

Oral glucose tolerance test (OGTT): The oral glucose tolerance test (Shirwaikar et al., 2006) was performed in overnight fasted (18 h) normal rats. Mice divided into 8 groups, each consisting of 6 mice were administered sodium-CMC 1% per oral (p.o.), metaformin 195 mg kg-1 b.wt. p.o., G. ulmifolia aqueous extract 25 and 50 mg kg-1 b.wt. and C. xanthorrhiza ethanolic extract 25 and 50 mg kg-1 b.wt., respectively. Glucose (3 g kg-1) was fed 30 min after the administration of extracts. Blood was withdrawn from the vein tails at 0, 60, 90, 120, 150 min of glucose administration. Glucose levels were estimated using blood glucose test strips from Easy touch and a glucometer from One Touch Horizon, LifeScan Inc. Middle East.

Induction of non-insulin-dependent diabetes mellitus (NIDDM): NIDDM was induced in overnight fasted adult male Swiss Webster by a single intraperitoneal injection of 50 mg kg-1 b.wt. alloxan monohydrate. Hyperglycemia was confirmed by the elevated glucose levels determined at day 7. Animals with blood glucose level more than 200 mg dL-1 were considered as diabetic. Mice found with permanent NIDDM were used for the antidiabetic study. Metformin (195 mg kg-1) and Glibenclamide (0.65 mg kg-1) were used as the standard drug. Glucose levels were estimated using blood glucose test strips from Easy touch and a glucometer from One Touch Horizon, LifeScan Inc. Middle East.

Antidiabetic study: Animals were divided into 10 groups, each consisting of 6 mice. The extracts were administered for 28 days. Group 1: normal control mice administered saline (0,9% w/v) and 1% CMC sodium suspension; Group II: diabetic control mice administered 1% CMC-sodium suspension; Group III: Diabetic mice administered metformin (195 mg kg-1) daily for 28 days; Group IV: Diabetic mice administered glibenclamide (0.65 mg kg-1) daily for 28 days; Group V: Diabetic mice administered G. ulmifolia 50 mg kg-1 b.wt., Group VI: Diabetic mice administered C. xanthorrhiza 50 mg kg-1, Group VII: Diabetic mice administered combination of G. ulmifolia 25 mg kg-1 and C. xanthorrhiza 25 mg kg-1, Group VIII: Diabetic rats administered combination G. ulmifolia 50 mg kg-1 and C. xanthorrhiza 50 mg kg-1, Group IX: G. ulmifolia 50 mg kg-1 and C. xanthorrhiza 25 mg kg-1, G. ulmifolia 25 mg kg-1 and C. xanthorrhiza 12.5 mg kg-1. Glucose levels were estimated using blood glucose test strips from Easy touch and a glucometer from One Touch Horizon, LifeScan Inc. Middle East.

Histology: The whole pancreas from each animal was removed after sacrificing the animal and was collected in 10% formalin solution and immediately processed by the paraffin technique. Sections of 5 μm thickness were cut and stained using Gomori’s method (Gomori, 1941).

Statistical analysis: Data were statistically evaluated using one-way ANOVA by using SPSS version 17.0 software. The values were considered significant when p<0.05.

RESULTS

Glucose tolerance: The effects of extracts of Guazuma ulmifolia, Curcuma xanthorrhiza and their combinations on glucose tolerance test are shown in Fig. 1. Before administration of glucose, the blood plasma glucose levels were similar among groups.

Image for - Antidiabetic Activity of Aqueous Leaf Extracts of Guazuma ulmifolia 
  Lamk., Ethanolic Extracts of Curcuma xanthorrhiza and their Combinations 
  in Alloxan-induced Diabetic Mice
Fig. 1: Effect of aqueous extract of G. ulmifolia, C. xanthorrhiza and its combinations on plasma glucose concentration by using glucose tolerance test, Mice were administered glucose solution orally then plasma glucose level was measured at 60, 90, 120 and 150 min. Bars represent Mean±SD of 6 mice. *p<0.05, significant difference between control group and extract treated group

Table 1: Plasma glucose concentration in diabetic mice induced by single administration of alloxan
Image for - Antidiabetic Activity of Aqueous Leaf Extracts of Guazuma ulmifolia 
  Lamk., Ethanolic Extracts of Curcuma xanthorrhiza and their Combinations 
  in Alloxan-induced Diabetic Mice
Values are given as Mean±SD for groups of 6 mice each, * p<0.05, Extract treated groups were compared with the diabetic control

At 60 min after glucose administration, metformin could attenuate the increase of plasma glucose concentration significantly compared to control group. Similar result was observed as well in group of G. ulmifolia extract 50 mg kg-1, C. xanthorrhiza 50 mg kg-1 and combination of G. ulmifolia extract 25 mg kg-1-C. xanthorrhiza 25 mg kg-1. At 90 min, all treatment groups were significantly lower compared to control group. Furthermore, there was no significance observed in plasma glucose level at minutes 120 and 150 at all groups studied.

G. ulmifolia, C. xanthorrhiza and their combination extracts can reduce plasma glucose level in diabetic mice induced by single administration of alloxan: Upon single administration of alloxan, plasma glucose levels increased significantly compared to normal group. In this study, metformin and glibenclamide are used as reference drugs. Upon administration of alloxan, metformin could reduce plasma glucose significantly at 60 and 120 min compared to time 0, while glibenclamide could reduce plasma glucose significantly only at 120 min. Combination of G. ulmifolia 25 mg kg-1-C. xanthorrhiza 25 mg kg-1 could not reduce plasma blood glucose at all time point. However, combination of G. ulmifolia 50 mg kg-1-C. xanthorrhiza 25 mg kg-1 and combination of G. ulmifolia 25 mg kg-1-C. xanthorrhiza 12.5 mg kg-1 both could reduce significantly the plasma glucose concentration at 60 and 120 min compared to baseline (0 min), while others combination could reduced significantly the plasma glucose concentration only at 120 min (Table 1).

G. ulmifolia, C. xanthorrhiza and their combination extracts can reduce plasma glucose level in diabetic mice induced by multiple administrations of alloxan: After observing the ability of the extracts and its combination in reducing plasma glucose in diabetic mice induced by single administration of alloxan, the study was proceeded into more damage β-pancrease by repeatedly injecting alloxan (Table 2, 3). The data showed that normal group had significantly lower plasma glucose concentration compared to diabetic control group, suggesting the model of diabetic mice had been achieved (Table 2). Metformin and glibenclamide reduced plasma glucose level significantly in all days studied with the highest reduction was observed at day 28 (Metformin, 70.50±5.25; Glibenclamide, 49.20±14.81).

Table 2: Plasma glucose concentration at day 0 in diabetic mice induced by multiple administrations of alloxan
Image for - Antidiabetic Activity of Aqueous Leaf Extracts of Guazuma ulmifolia 
  Lamk., Ethanolic Extracts of Curcuma xanthorrhiza and their Combinations 
  in Alloxan-induced Diabetic Mice
Values are given as Mean±SD for groups of 6 mice each, *p<0.05, Extract treated groups were compared with the normal group

Table 3: Reduction of plasma glucose concentration in alloxan-induced diabetic mice after being treated by drug reference or extract studied
Image for - Antidiabetic Activity of Aqueous Leaf Extracts of Guazuma ulmifolia 
  Lamk., Ethanolic Extracts of Curcuma xanthorrhiza and their Combinations 
  in Alloxan-induced Diabetic Mice
Values are given as Mean±SD for groups of 6 mice each, *p<0.05, Extract treated groups were compared with the diabetic control

Furthermore, the extracts studied and their combinations showed various pattern in reducing plasma glucose level (Table 3). However, at day 28, all extracts studied and their combinations could reduce the blood plasma glucose significantly compared to diabetic group, where combination of G. ulmifolia 50 mg kg-1 b.wt.-C. xanthorrhiza 25 mg kg-1 b.wt. showed the highest downregulation (61.62±14.61) of plasma glucose level.

Extract combination of G. ulmifolia 25 mg kg-1 b.wt. and C. xanthorrhiza 12.5 mg kg-1 b.wt. protects pancreatic cells from being damage due to alloxan administration: To understand further on the mechanism behind its antidiabetic activity, pancreatic cells were stained (Fig. 2). At day 1 of treatment, alloxan treated group had pancreatic cell damage. For the reference drugs, only glibenclamide showed a protective effect on pancreatic cell. Among all extract combinations studied, the lowest combination, G. ulmifolia 25 mg kg-1 b.wt. and C. xanthorrhiza 12.5 mg kg-1 b.wt., showed less damage of pancreatic cells.

Image for - Antidiabetic Activity of Aqueous Leaf Extracts of Guazuma ulmifolia 
  Lamk., Ethanolic Extracts of Curcuma xanthorrhiza and their Combinations 
  in Alloxan-induced Diabetic Mice
Fig. 2(a-j): Histopathological studies of pancreas at day 28 after treatment, (a): Normal group, (b): Control group, (c): Metformin group, (d): Glibenclamide group, (e): Guazuma ulmifolia 50 mg kg-1 b.wt., (f): Curcuma xanthorrhiza 50 mg kg-1 b.wt., (g): Guazuma ulmifolia 25 mg kg-1 b.wt.-Curcuma xanthorrhiza 25 mg kg-1 b.wt., (h): Guazuma ulmifolia 50 mg kg-1 b.wt.-Curcuma xanthorrhiza 50 mg kg-1 b.wt., (i): Guazuma ulmifolia 50 mg kg-1 b.wt.-Curcuma xanthorrhiza 25

DISCUSSION

The present study is the preliminary assessment of the antidiabetic activity of the aqueous extracts of Guazuma ulmifolia, ethanolic extract of Curcuma xanthorrhiza and their combinations. G. ulmifolia and C. xanthorrhiza extract have been studied for their antidiabetic activity (Alarcon-Aguilara et al., 1998; Yasni et al., 1991; Alonso-castro and Salazar-Olivo, 2008). There is no study has been done on antidiabetic activity of combination of G. ulmifolia and C. xanthorrhiza. In this study, the extracts and their combinations showed an antidiabetic activity in various pattern. Combination of both extracts, Guazuma ulmifolia Lamk. 25 mg kg-1 b.wt. and Curcuma xanthorrhiza 12.5 mg kg-1 b.wt., with lower dose compared to the extracts given separately (50 mg kg-1 b.wt. each) has similar antidiabetic activity.

Diabetic mice were induced by i.v. injecting alloxan. Alloxan induces diabetes by pancreatic cell damage mediated through generation of cytotoxic oxygen free radicals which cause DNA fragmentation of DNA pancreatic cells (Shankar et al., 2007; Szkudelski, 2001). The damage of pancreatic cell results in lowering the production of insulin, leading to hyperglycemia.

In glucose tolerance test, G. ulmifolia, C. xanthorrhiza and their combinations could reduce plasma glucose level significantly compared to control group. Furthermore, the study was continued by damaging the pancreatic cell and found that the both extracts and its combination still can reduce the plasma glucose level. Hence, the possible mechanism by which all extracts studied bring about its action may be by potentiating secretion of insulin from the existing beta cells or/and by increasing sensitivity of insulin receptor. To understand further the mechanism behind this, the pancreatic cell was stained. pancreatic cell treated with extracts was less damage compared to diabetic control group (Fig. 2). It suggests that the extract treated group is having more insulin production compared to diabetic control group. However, determine insulin serum level may be important to do in the future for confirmation.

CONCLUSION

All extracts studied can reduce plasma glucose level in various pattern. Combination of the extracts with lower dose (Guazuma ulmifolia 25 mg kg-1 b.wt.-Curcuma xanthorrhiza 12.5 mg kg-1 b.wt. ) than its single extracts given (Guazuma ulmifolia 50 mg kg-1 b.wt. and Curcuma xanthorrhiza 50 mg kg-1 b.wt.) gives similar effect, strengthening the advantage of combining the extracts. Furthermore, combination of these two extracts may reduce the side effect due to its low dose needed for anti-diabetic treatment. The important fact regarding this combination is less pancreatic beta-cells damage upon alloxan administration which may offer a new hope for diabetic patients in the future. Further investigation needed for study is to determine the exact compound(s) responsible for antidiabetic effect.

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