INTRODUCTION:

Diabetes mellitus is metabolic disorder characterized by chronic hyperglycemia, polyuria, polydypsia, polyphagia, emaciation and weakness due to disturbance in carbohydrate, fat, protein metabolism associated with absolute or relative deficiency in insulin secretion and/or insulin action [1].

At least 171 million people worldwide have diabetes, this figure is likely to be more than double by 2030 and around 3.2 million deaths every year are attributable to complications of diabetes at the rate of six deaths every minute [2]. In 2011, India had 62.4 million people with type 2 diabetes, compared with 50.8 million in 2010, according to the International Diabetes Federation (IDF) and the Madras Diabetes Research Foundation (MDRF). As the economy started growing, so did the incidence of diabetes. The nationwide prevalence of diabetes in India now tops 9%, and is as high as 20% in the relatively prosperous southern cities. By 2030, the IDF predicts, India will have 100 million people with diabetes [3, 4].

The economic burden of diabetes mellitus, together with some disenchantment about allopathic treatments for this disorder, has led to a growing interest in potentially safer and gentler management options that are presented by alternative medicine. The World Health Organization (WHO) has also recommended the evaluation of traditional medicinal plants effectiveness in conditions where safety of modern drugs is vital issue. This has leaded to anti-diabetic natural products research which produce minimal or no side effects [5, 7].

Herbal drugs constitute a part in all traditional systems of medicine. It is a triumph of popular therapeutic diversity. Herbal medicine also referred to as botanical medicine, is defined as the use of plant or parts of plant to prevent or treat illness. Oral hypoglycemic agents like sulphonylureas and biguanides are still the major players in the management of the disease but there is growing interest in herbal remedies due to secondary failure rates and accompanying side effects with oral hypoglycemic agents [7, 8]. Herbal medicines have been the highly esteemed source of medicine throughout human history. They are widely used today indicating that herbs are a growing part of modern, high-tech medicine [6]. The medicinal plants, besides having natural therapeutic values against various diseases and considerable works have been done on these plants to treat diabetes mellitus, describes that the anti-diabetic activity of medicinal plants is due to the presence of phenolic compounds, flavonoids, terpenoids, coumarins and other constituents which show reduction in blood glucose levels [9].

Aegle marmelos shows the control on blood glucose level by the mechanism of regeneration of islets of pancreas with increased functioning of β cells [11] while Withania coagulans shows the control on blood glucose level by secretion of insulin from existing β cells and restoration of glycogen content in liver and muscles [12]. The two herbal plants show different modes of action leading to their anti-diabetic activity. Hence in present study, it was envisaged to study the potential synergistic combination for ant diabetic activity using alloxan-induced diabetic pre-clinical animal model.

MATERIALS AND METHODS:

Plant Profiles:

Aegle marmelos [10, 21, 23, 25, 26]:

Botanical name Aegle marmelos

Family Rutaceae

Common Names Bael, Bilva, bel, sadaphal and shriphal and held sacred

Plant part used: Leaves

Figure 1 Photograph of Aegle marmelos leaves

Morphology:

A. marmelos is a slow-growing, medium sized, 25 to 30 feet tall tree. There are sharp, axial one inch long spikes on this tree. The leaflets are oval or lancet shaped, 4-10 cm long, 2-5 cm wide. Leaves composed of 3 to 5 leaflets in it.

Chemical constituents:

Alkaloids, cardiac glycosides, terpenoids, saponins, tannins, flavonoids and steroids. Leaf part contains Skimmianine, Aeglin, Rutin, γ-sitosterole, β-sitosterol, Flavone, Lupeol, Cineol, Citral, Glycoside, O-isopentenyl, Hallordiol, Mameline, Citronellal, Cuuminaldehyde phenylethyle cinnamamides, Euginol, Marmesinin, Aegelin, Glycoside.

Traditional use:

A.marmelos is traditionally used to treat diabetes complications and others like jaundice, constipation, chronic diarrhea, dysentery, stomachache, stomachic, tumors, ulcers and upper respiratory tract infections.

Withania coagulans [15-17, 25]

Botanical Name Withania coagulans

Family Name Solanaceae

Common Name Vegetable rennet, paneer ke phool

Part Used Fruits

Figure 2 Photographs of Withania coagulans fruits and leaves

Morphology:

Shrub branched, 2-3m in length. Lamina oval to oblong, 1-6 cm length, 0.3-2.6 cm width, Fruit berry, globose, 1.5-1 cm long, 0.7-1 cm width, Sepals covers the fruit and ended into crown like structure. Seeds oval to rounded, yellowish brown, 41-59 in number, 0.1-0.3cm long, 0.2-0.3cm wide, dotted.

Chemical Constituents:

Withanolide: coagulin C, 17b-hydroxywithanolide K, withanolide F, (17S,20S,22R)-14a,15a,17b,20b-tetrahydroxy-1-oxowitha 2, 5, 24 trienolide, coagulin L and (14R,17S,20S,22R)-14,17,20-trihydroxy-3b-(O-b-D-glucopyranosyl)-1-oxowitha-5,24-dienolid. Fruits contain high concentration of minerals magnesium and calcium.

Traditional Use:

Antimicrobial, anti-inflammatory, hepatoprotective, anti- hyperglycemic, antitumor, cardiovascular, immunosuppressive, free radical scavenging and central nervous system depressant activities of the plant have been reported.

Collection and Authentication of Plant material

The fresh leaves of Aegle marmelos were collected from the local area of Nagpur district. While the fruits of Withania coagulans were purchased from local market of Nagpur district. The plant specimens were dried and their herbarium sheets were prepared. It was authenticated at Department of Botany, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, Maharashtra, India.

Preparation of extracts

The aqueous extract of leaves of Aegle marmelos

The leaves were thoroughly washed with water and dried in shade. The air dried leaves were grounded in coarse material and defatted using petroleum ether. The defatted material then soaked in water for 8 hours and stirred occasionally [10]. After soaking, the mixture was filtered using Whatmann #1 filter paper. The filtrate was concentrated upto 500 mL on rotavapor (Cole-Parmer) under reduced pressure. The concentrated crude extract was lyophilized (DELVAC LYODEL) into powder.

The aqueous extract of fruits of Withania coagulans

The whole fruits were mechanically crushed and extracted with distilled water at room temperature up to 48 hours. The extract was filtered and concentrated in the rotary evaporator (Cole-Parmer) under reduced pressure to obtain semisolid material, which was then lyophilized (DELVAC LYODEL) for powder formation.

Evaluation of extracts

Characteristics of extracts

The aqueous extracts of Aegle marmelos and Withania coagulans were evaluated for its physical state, colour, odour, taste and percent yield.

Phytochemical screening of extracts

The aqueous extracts of Aegle marmelos and Withania coagulans were subjected to preliminary phytochemical testing for the detection of major phytoconstituents phenols, tannins, steroids, alkaloids, glycosides and flavonoids [14].

Anti-diabetic activity studies

All experimental protocols were approved by Institutional Animal Ethical Committee (CPCSEA-IAEC/Proposal No.15/22/1/2013) of Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, Maharashtra, India. Both the extracts and their different combination were subjected to anti-diabetic study.

Animal species

Swiss albino mice (25-30 g) of either sex were used. The animals were kept in house with control temperature 37 ±2 °C with a ratio of 12 hrs day: 12 hrs night, dark - light cycle.

High fat diet

All the mice except normal group were fed with high fatty forage composed of general forage (60%), sucrose (10%), dextrose (10%), corn starch (10%) and Vegetable oil (10%) for four weeks [20].

Blood glucose determination

The blood glucose level was measured using glucometer. The blood was collected from the tip of the tail vein by pricking of the overnight fasted mice and the blood glucose was measured using ACCU-CHEK Active glucometer according to manufacturer’s instruction (Roche Diagnostic Pvt. Ltd., India) [18].

Preparation of alloxan induced type 2 diabetes model in mice

After 4 weeks of dietary manipulation, alloxan (50 mg/Kg body weight) was given by intraperitoneal (i.p) injection once every other day for 3 times to establish type 2 diabetes mellitus model. The alloxan injected i.p., within 3-4 minutes after dissolving it in normal saline. Mice in the control group were administered with equivalent amount of normal saline. Fasting blood glucose was measured third day after the final injection. The diabetic state was assessed by measuring blood glucose level with glucometer. Fasting blood glucose was checked regularly till stable hyperglycemia was achieved. The mice exhibiting blood glucose level more than 250 mg/dL were included in study as a stable hyperglycemic diabetic animal model. Once the stable hyperglycemia was achieved, the mice belonging to respective group received the saline, standard, individual extract and combined extracts dose [13, 14].

Evaluation of anti-diabetic activity of plants extracts

The animals were divided in six groups and each group was having six animals.

Group I-Normal; received vehicle [(normal saline per oral (p.o.)]

Group II- Untreated; received vehicle (normal saline p.o.)

Group III-Standard (Glibenclamide 5 mg/Kg)

Group IV-Aegle marmelos (AM) (300 mg/kg)

Group V- Withania coagulans (WC) (400 mg/Kg)

Group VI- Aegle marmelos+ Withania coagulans (37.5+50 mg/Kg) (AM+ WC1)

Group VII- Aegle marmelos+ Withania coagulans (75+100 mg/Kg) (AM+WC2)

Group VIII- Aegle marmelos+ Withania coagulans (150+200 mg/Kg) (AM+WC3)

Group IX- Aegle marmelos+ Withania coagulans (300+400 mg/Kg) (AM+ WC 4)

The doses of individual and combined extracts were dissolved in distilled water and given to mice per oral.

Acute anti-diabetic study

All the mice were kept on fasting overnight and were given doses orally as mentioned above. After dose administration, anti-diabetic activity was assessed by withdrawing blood samples at FBG, 2, 4, 6 and 24hrs respectively.

Sub-chronic anti-diabetic study

The study was conducted up to 21 days. Animals were administered drug p.o. for 21 days and FBG levels (overnight) and body weight were measured on 0, 1, 7, 14 and 21^stday.

RESULTS AND DISCUSSION

Authentication of plant material

The specimen voucher number for authentication of Aegle maemelos was 9800 and that of Withania coagulans was 9801.

Figure 3 Herbarium sheet for Aegle marmelos and Withania coagulans.

Characteristics of aqueous extracts of leaves of Aegle marmelos and fruits of Withania coagulans

The physical state, color, odor, taste and percent yield of all three extracts are shown in table 2.

Table 2 Characteristics of extracts of Aegle marmelos and Withania coagulans

Sr. No	Characteristics	*Aegle* *marmelos*	*Withania* *coagulans*
1	Physical state	Solid	Solid
2	Colour	Dark brown	Dark brown
3	Odour	Aromatic	Aromatic
4	Taste	Bitter	Bitter

Physicochemical parameters of extracts

The physicochemical parameter like loss on drying (LOD), pH, Total ash value, acid insoluble ash value was determined for aqueous extracts of Aegle marmelos and Withania coagulans are shown in table 3.

Table 3 Physicochemical parameters of extracts

Sr. No	Name of plant	Loss on drying	pH	Total ash content	Acid insoluble ash
1.	Aegle marmelos	4.29 %w/w	8.18	3.8 %w/w	2.05 %w/w
2.	Withania coagulans	2.19 %w/w	5.9	11.37 %w/w	3.21 %w/w

Phytochemical screening

The following phytoconstituents were found to present in the aqueous extract of Aegle marmelos and Withania coagulans respectively.

Table 4 Phytochemical screening of extracts of Aegle marmelos and Withania coagulans

Sr. No.	Chemical constitutents	*Aegle* *marmelos*	*Withania* *coagulans*
1.	Carbohydrates	+	+
2.	Alkaloids	+	+
3.	Glycoside	+	+
4.	Flavonoids	+	+
5.	Tannins	+	+
6.	Saponin	+	+
7.	Steroid	+	+

Evaluation of anti-diabetic activity of plants extract

An increase in blood glucose level by 2.5 times was observed in diabetes induced animals as compared to normal control group (figure 4). The mice exhibiting blood glucose level more than 250 mg/dL were included in study. All the groups were given the treatment and results obtained were analysed by two way ANOVA followed by post hoc Bonferroni’s multiple comparison test.

Acute anti-diabetic study

Results of acute anti-diabetic studies are shown in table 5.

Table 5 Results for acute anti-diabetic studies

Sr. no.	Treatment group	Blood Glucose Levels (mg/dL)
Sr. no.	Treatment group	*FBG (mg/dL)**	2hrs	4 hrs	6 hrs	24 hrs
1.	Normal	103.66±0.91	106.16±0.47	108.83±0.65	111.66±1.08	120.16±0.83
2.	Untreated	256.50±1.17	256.83±1.40	261.33±1.85	262.66±1.83	256.50±2.64
3.	Standard	258.66±1.60	178.66±0.84	175.16±1.37	179.16±1.27	178.50±2.45
4.	AM	254.16±1.27	253.16±1.49	250.66±1.33	252.00±1.73	251.33±1.33
5.	WC	255.16±2.27	252.00±1.82	250.00±2.03	247.16±0.94	245.83±0.70
6.	AM+WC 1	261.83±1.66	259.16±0.87	251.83±0.94	214.66±1.20	234.66±1.62
7.	AM+WC 2	261.83±2.54	235.50±1.14	215.83±1.47	210.66±1.81	213.00±0.85
8.	AM+WC 3	265.50±1.14	232.33±2.21	222.66±1.25	215.33±1.22	203.00±1.59
9.	AM+WC 4	257.33±1.08	251.66±0.91	244.83±1.53	239.50±1.23	202.66±2.076

*FBG: Fasting blood glucose level; A: B Aegle marmelos; WC: Withania coagluans

Figure 4 Graphical representation of acute anti-diabetic study

Table 6 Results of sub chronic anti-diabetic study

Sr. No.	Treatment	Fasting Blood Glucose (mg/dL)
Sr. No.	Treatment	Day 1	Day 2	Day 7	Day 14	Day 21
1.	Normal	103.66 ±0.91	120.16 ±0.83	117.00 ±4.22	107.66 ±3.39	105.50 ±3.54
2.	Untreated	256.50 ±1.17	256.50 ±2.64	261.16 ±6.48	265.66 ±2.76	256.00 ±2.87
3.	Standard	258.66 ±1.60	178.50 ±2.45	170.33 ±1.57	163.00 ±1.57	157.50 ±2.69
4.	AM	254.11 ±1.27	251.33 ±1.33	251.66 ±4.86	169.90 ±2.85	160.66 ±2.01
5.	WC	255.16 ±2.27	245.83 ±0.70	236.50 ±1.25	160.00 ±2.51	150.00 ±2.50
6.	AM+WC 1	261.83 ±1.66	234.66 ±1.66	211.83 ±2.38	187.33 ±0.98	170.50 ±1.87
7.	AM+WC 2	261.83 ±2.54	213.00 ±0.85	150.00 ±1.43	136.66 ±0.98	130.16 ±2.07
8.	AM+WC 3	265.50 ±1.47	203.00 ±1.59	155.50 ±3.73	141.16 ±1.66	129.50 ±2.55
9.	AM+WC 4	257.33 ±1.08	202.66 ±2.07	156.66 ±2.47	137.33 ±2.09	126.16 ±2.73

AM: Aegle marmelos; WC: Withania coagulans

Statistical analysis: Values expressed as Mean± SEM, n=6; *P<0.05,***P<0.001 when compared with Untreated (control) ^###P<0.001 when compared with standard control.

The subchronic antidiabetic study was conducted for 21 days and blood glucose level was tested on 7^th, 14^th and 21^st day. Table 6 mention the impact of the most effective dose of the extract on fasting blood glucose of diabetic mice, The group B+WC2 reduced the fasting blood glucose level by 42.72, 47.81, 50.29% on 7^th, 14^th and 21^st day of treatment respectively. Glibenclamide reduced fasting blood glucose by only 33.15, 36.99, 39.11% on 7^th, 14^th and 21^st day of treatment respectively. The combination AM+WC2 has shown significant fall in fasting blood glucose and hence, it was considered as most effective combination.

CONCLUSION:

Oral antihyperglycemic agents are useful in the treatment of diabetes mellitus but their use is restricted by the pharmacokinetic properties, secondary failure rates and accompanying side effects. Plant drugs are considered to be lesser toxic with little or lower side effects than synthetic drugs. Aegle marmelos (Bael) and Withania coagulans (Panir dodi) are the medicinal plants having the treasure of natural medicines which will be used in the treatment of diabetes mellitus. They were evaluated for anti-diabetic activity in type 2 diabetes model. The synergistic effect of these extracts was studied using different combinations of both the extract. The combination AM+WC 2 (75+100 mg/Kg) was found to be more effective than standard anti-diabetic drug glibenclamide, individual extract and their combinations.

ACKNOWLEDGEMENT:

The authors would like to thank University Grant Commission (UGC), New Delhi for the financial assistance in terms of Junior Research Fellowship (JRF) to the first two authors.

The authors would also like to thank, Head of the Department of Pharmaceutical Sciences, R.T.M Nagpur University, Nagpur, India for constant encouragement and support for the work.

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Received on 22.03.2014 Modified on 23.04.2014

Res. J. Pharmacology & P’dynamics. 6(2): April- June 2014; Page 101-107