1. Department of Zoology, Department of Zoology, University of Agriculture, Faisalabad, Pakistan; khaliqaminhas3@gmail.com
2. Associate Professor; Department of Zoology, Government College University, Lahore, Pakistan; andleeb.batool@gcu.edu.pk
3. Lecturer; Muhammad Nawaz Sharif University of Agriculture, Multan, Pakistan; ai394@cam.ac.uk

*             Correspondence: khaliqaminhas3@gmail.com

Keywords: albino mice, cinnamon, fenugreek, diabetes, herbal extract, allopathic extract

1. INTRODUCTION

Diabetes mellitus (DM) is a sickness that disturbs the body’s capability to make or use insulin. Panaceas formed insulin hormone that aids to transport glucose from the blood into the cells so they can break it down and use it for fuel. People cannot live without insulin [1]. Type 2 diabetes mellitus (T2DM) used to be named as adult onset diabetes and non-insulin dependent diabetes mellitus (NIDDM). According to IDF, T2DM is common form, accounting for 85 to 95% of cases in developed nations and an even higher percentage in developing nations [2]. The treatment of DM is major goal of the current era, it is the main issue around the globe and a number of efforts for an effective treatment are going on. Oral hypoglycemic drugs and insulin therapy are the first and important treatment for the DM but they have side effects and fail to alter the course of diabetic complications [3]. DM is an ailment which cannot be cured but can only be managed [4]. Diet and exercise are crucial in managing diabetes, especially type 2 diabetes. The U.S. Food and Drug Administration has accepted oral diabetes drugs to treat type 2 diabetes, these are: alpha-glucosidase inhibitors, biguanides, meglitinides, sulfonylureas, thiazolidinedione, a new group called DPP-4 inhibitors [5]. Biguanides reduces liver glucose production, utilization of glucose and insulin sensitivity. Stomach disorders and acidosis are the side effects of using biguanides [6]. Human insulin is a polypeptide, its molecular weight is about 6000 Da, based on two amino acid chains. Disulphide bridge link these chains. Normal human pancreas contains about 8-10 mg. of insulin. Insulin is inactivated by digestive enzyme so oral administration is not prescribed. Liver and kidney degrade about 80% of the inserted insulin. Normal human released about 40 units of insulin per day. The insulin dose used to control DM is different in different patients [7]. Numerous traditional plants and their derived compounds have been used in treatment of DM. These plants provide a possible source of antidiabetic drugs. The ethanolic extract of okra which contains, different hypoglycemic compounds i.e. isoquercitrin and quercetin 3-O-gentiobioside and which reduced insulin levels and improved glucose tolerance in obese mice. The extracts of Allium Sativumin is also used for the the treatment of diabetes mellitus [8]. Bitter melon is beneficial for patients with T2D. The components of bitter melon are: alkaloid, inorganic lipid, triterpene, proteid, steroid, and phenolic compounds are the effective ingredients of bitter melon are held responsible for its hypoglycemic effects [9]. Cinnamon ( Cinnamomum zeylanicum ) is a spice obtained from the inner bark of several tree species from the genus Cinnamomum. Cinnamon consists of a variety of viscous compounds, including cinnamaldehyde, cinnamate, cinnamic acid and numerous essential oils. A substance from cinnamon has been isolated and coined as “insulin potentiating factor”. Several polyphenols have been isolated from cinnamon. A study demonstrated that the aqueous extract of cinnamon containing polyphenols purified by high performance liquid chromatography showed insulin like activity [10]. Cinnamon is one of the oldest herbal treatments that has been stated in the Chinese medicine since as long as 4000 years ago. There have been several new studies on cinnamon focusing on improving the serum glucose and lipid levels in type 2 diabetic patients [11]. Cinnamon shows many pharmacological properties when orally taken, such as antidiabetic,antioxidants and antibacterial effects [12]. The most important elements of cinnamon are tras-cinnamaldenyde and cinnamaldehyde and which are present in essential oil. Cinnamon bark contains procyanidins and catechins, the compound of procyanidins include both procyanidin A-type and B-type linkages. These procyanidins extracted from cinnamon and berries also have antioxidant activities. Its other elements also have important activities, including antimicrobial, antifungal, antioxidant and antidiabetic [13]. Fenugreek (Trigonella foenum-graecum) is cultivated throughout the world as a semiarid crop. South Asian used its seeds and leaves as ingredients in their dishes for enhancing taste. In past herbal medicine are used to treat different ailments fenugreek is one of them. Fenugreek seeds have been used as traditional medicines not only in controlling blood sugar level but also in high cholesterol, inflammation and gastrointestinal ailments [14]. Fenugreek contains alkaloids mainly (trigonelline) and protein high in lysine and L-tryptophan. Its steroidal saponins and mucilaginous fiber are thought to account for many of the beneficial effects. Fenugreek fiber may help lower blood glucose level. Although it does not have any serious side effect but excessive used of fenugreek seed can cause intestinal distress and vomiting in humans [15]. Fenugreek is found all over South Asia and the fenugreek seeds are usually used as one of the major constituents of Indian spices. Administration of fenugreek seeds also improved glucose metabolism and normalized creatinine kinase activity in heart, skeletal muscle and liver of diabetic rats. It also reduced hepatic and renal glucose-6-phosphatase and fructose -1-6-bisphosphatase activity. This plant also shows antioxidant activit [16]. Alloxan is an organic compound based on a pyrimidine heterocyclic skeleton. This compound exists as the monohydrate and has a high affinity for water. The name is derived from allantoin, a defecated product of uric acid by the fetus into the allantois and oxaluric acid derived from oxalic acid and urea, found in urine. The original technique for Alloxan was by oxidation of uric acid by nitric acid [17]. Alloxan also has the ability to cause diabetes through its action on the pancreas to reduce glucose-mediated insulin secretion and induce selective necrosis of β-cells. It is possible to differ the severity of diabetes by using different doses of alloxan, which therefore makes it useful to study the effects of a milder diabetic condition. For animals including mice, rats, rabbits, and dogs, a single dose of aqueous alloxan (about 150 mg/kg) may be administered intravenously or intraperitoneally. A period of 7 to 12 days is allowed before testing for hyperglycemia [18]. The animal models of T2D can be obtained either naturally or induced by chemicals or dietary or surgical manipulations or by combination. In current years, large number of new genetically modified animal models including tissue-specific knockout mice transgenic and generalized knock-out has been used for the study of diabetes [19]. Alloxan and streptozotocin- induced adult diabetic animals are included in this group. Since the initial findings in 1943 of alloxan induced beta cell necrosis in rabbits, this compound has long been used for inducing experimental diabetes. Alloxan acts by selectively destroying the pancreatic beta islets leading to insulin deficiency, hyperglycemia and ketosis. In many rodent and non-rodent animals, diabetes is induced by using alloxan, it is most preferably used in case of rabbit because of the ineffectiveness of streptozotocin (STZ) in rabbits for induction of diabetes [20]. Among widely used herbs by diabetic patients is fenugreek (Trigonella foenum graecum). The seeds were commonly used as a spice in Middle Eastern countries and widely used in South Asia and Europe and has been shown in animals to have anti-diabetic properties [21]. The problem with herb use in diabetes, the doses and the way of intake are not well documented. Moreover, most patients used mixture of herbs either alone or in combination with antidiabetic treatments which has not yet been evaluated. The aim of the present study therefore, was to evaluate the effect of extract of cinnamon and fenugreek on patients with T2D and to compare it with allopathic drug glibenclamide. The objective of our study is to compare the effect of herbal (cinnamon and fenugreek extract) and synthetic drug (glibenclamide) on alloxan induced diabetic mice and to observe the efficiency of combination of cinnamon and fenugreek extract to control the blood glucose level.

2. LITERATURE REVIEW

2.1 Diabetes Mellitus

DM is included among the top leading cause of death in the world. It has been estimated that number of diabetes in India is expected to increase 57.2 million by the year 2025. T2DM accounts for 90 – 95% of diabetic cases, according to the U.S. National Institutes of Health (NIH), some of these patients have had pre-diabetes that went uncontrolled. Once considered a disease of middle and old age, T2DM is also becoming more common in youths as the frequency of childhood obesity grows Diabetes involves chronic levels of abnormally high glucose [22].

2.2 Alloxan Monohydrate

 Alloxan monohydrate has the ability to induce diabetes due to its action on the pancreatic cells. The different severity of diabetes can be insduced by using different doses of alloxan. So, it is useful in studying the effect in different diabetic conditions. The original method for Alloxan preparation was by oxidation of uric acid by nitric acid [23]. The mechanism of action of the drug has been well-known when injected intravenously, intraperitoneally or subcutaneously. Furthermore, the quantity of alloxan required for inducing diabetes depends on the animal species, route of administration and nutritional status [24]. Moreover, alloxan has been confirmed to be non-toxic to the human beta-cells, even in very high doses. The 1st phase appears after the first few minutes after alloxan injection includes temporary hypoglycemia that lasts for almost 30 minutes [25]. This little hypoglycemic response has been noted to be the result of a transient stimulation of insulin secretion that was confirmed by an increase of the plasma insulin concentration [26]. The 2nd phase appears after injection of alloxan leads to increase in blood glucose level after a period of one hour. Moreover; the plasma insulin concentration has been seen to decrease at the same time. This is the first hyperglycemic phase after the first contact of the pancreatic beta cells with the toxin. This hyperglycemic phase lasts for 2-4 hours which is followed by reduced plasma insulin concentrations. In 3rd phase, after 4-8 hours after the alloxan injection the glucose level drops which lasts for several hours [27]. The last and the 4th phase of the blood glucose response is the final lasting diabetic hyperglycemic phase during which complete degranulation and loss of the beta cells within 24-48 h after administration of the alloxan takes place. Surprisingly, the non-beta cells and other endocrine and non-endocrine islet cell types along with extra pancreatic parenchyma remain intact, providing the evidence of selective toxic action of alloxan [28].

2.3 Glibenclamide

In 1920 the sulphonylureas (SUs) were initially prepared and have become necessary in the management of T2D. The glibenclamide involves in a direct secretion of pancreatic cells. When the concentration is lower, hypoglycemia can occur [29]. After oral administration Sulphonyleureas, is well absorbed reach the maximum concentration [30]. Due to Ingestion of food Glibenclamide have a very little effectiveness. So, it is not permissible to take medicine before 15-20 min of meal. Glibenclamide is an intermediate drug with very effective metabolites .50 % of these metabolites are eliminated by liver. Sulphonyleureas are highly bounded to the plasma proteins so they have ability to interact with protein bound drug, e.g. warfarin. The side effect of using Glibenclamide includes hyponatremia, acute porphyria, skin reaction, weight gain etc.

2.4 Traditional Herbal Medicine

Multiple organs are affected by diabetes mellitus which is a heterogenous metabolic disorder. It results in hyperglycemia, hyperlipidemia, hyper aminoacidemia, and hyperinsulinemia leading to decrease in insulin, secretion and insulin action. a number of medicinal plant have been identified and used for the cure of hyperglycemia, recently these (Pterocarpus marsupium, Momordia charanti, Syzigium cumini, Azadirachta indica, Allium cepa) plants have been scientifically used on diabetics, for their hypoglycemic property and have successfully proven effective [31]. Indian traditional medicines are formed by the combination of several medicinal herbs to accomplish effective results against the ailment. In ancient time of herbs, combined extracts of different plants are chosen rather than individual ones because they are more effective [32]. The phytochemical study of individual plants evaluated that the active ingredients are usually present in minute quantities that are insufficient to achieve the desirable effects. The   scientific studies have revealed that the use of combined medicinal plants may prove greater result as compared to individual use of the plant [33]. One of the main reasons for using polyherbal formulation is that they are effective even at low dose and safe at high dose thus possessing superior risk to benefit ratio. This benefit is not available with allopathic drug like sulphonyl ureas, glibenclamide.

2.5 Cinnamon as Medicine

Cinnamon is the bark of Cinnamoni cassia is also used as taste enhancer in food industry, as antioxidant and spicy agent. In recent years, several studies have reported that cinnamon extract has antidiabetic diabetic mice and T2D. [34] showed that cinnamon oils can improve effectiveness of insulin. A study reported that Cinnamon Extract (CE) increases glucose uptake in adipocyte [35]. Cinnamon is amongst the world’s oldest and most frequently consumed spices and is used as an herbal remedy [36]. The cinnamon of commerce is the dried inner stem-bark of a small evergreen tree 10-15 meters tall. It is native to tropical southern India and Srilanka. There are two types of cinnamon, common cinnamon (vernacular name: dalchini) or true cinnamon (Cinnamomum zeylanicum, C. verum) and cassia (Cinnamomum aromaticum). Cinnamon has been used for centuries, as flavor modifiers to make food more palatable. Its ingredients impart characteristic flavor and spicy aroma to food. The range of variation in the general composition of various cassia barks as observed by researchers is as follows: moisture, crude fiber, carbohydrate, protein, fixed oil, volatile oil, and cold alcohol extract  [37]. Cinnamaldehyde (more precisely trans-cinnamaldehyde or 3-phenyl-2- propenal) is the main constituent in cinnamon bark oil, whereas, that of leaf oil is eugenol [38]. Interest in cinnamon as a potentially useful treatment for type 2 diabetes began almost 20 years ago. In 1990, Khan et al. isolated an unidentified factor from cinnamon and termed it as insulin potentiating factor (IPF). They demonstrated that IPF may be involved in the alleviation of the signs and symptoms of diabetes, and other diseases related to insulin resistance [39]. Broadhurst et al. compared 49 herbs, spices and medicinal plant extracts for their insulin-like or insulin-potentiating action in an in vitro model [40]. Cinnamon extracts showed to improve insulin receptor function by activating the enzyme that causes insulin to bind to cells (insulin receptor- kinase) and inhibiting the enzyme that blocks this process (insulin-receptor phosphatase), leading to maximal phosphorylation of the insulin receptor, which is associated with increased insulin sensitivity [41].

2.6 Fenugreek as Medicine

Fenugreek is known for its pleasantly bitter, slightly sweet seeds. The seeds are available in any form whether whole or ground form is used to flavor many foods mostly curry powders, teas and spice blend. Fenugreek seed has yellow embryo and a central hard and which is surrounded by a corneous and comparatively large layer of white and semi-transparent endosperm. The chemical composition of fenugreek (such as seeds, husk and cotyledons) showed that endosperm had the highest (4.63 g/100 g) saponin and (43.8 g/100 g) protein content. The seeds of fenugreek contain about 0.1–0.9% of diosgenin and are extracted commercially. Seeds also contain the saponin (fenugrin B). Several coumarin compounds of fenugreek seeds have been found to contain a number of alkaloids (e.g., large amount of trigonelline is degraded to nicotinic acid and related pyridines during roasting [41]. The polyphenol compounds, such as rhaponticin and isovitexin are the major bioactive compounds in fenugreek seeds. Dietary fiber from fenugreek blunts glucose after a meal. Fenugreek seeds contain, and the gum is composed of galactose and mannose. The latter compounds are associated with reduced glycemic effect. The hypoglycemic effect of fenugreek has been especially documented in humans and animals with type 1 and type 2 diabetes mellitus [42].

3. MATERIALS AND METHODS

3.1 Selection of Medicinal Plants

Cinnamon (Cinnamomum Zeylanicum) sticks and fenugreek (Trigonella foenum-graecum) seeds used for the preparation of herbal extract was purchased from a local herb store in Lahore. These herbs were identified from the botany department of GC University Lahore.

3.2 Preparation of Herbal Extracts

3.2.1 Cinnamon Extract

The cinnamon sticks were cleaned, washed with distilled water, dried in shadow, and powdered using a mechanical grinder.10 g of powder was extracted in 100 ml double distilled water with revolving evaporator in vacuum state till the volume of water reduced to 50mL. The supernatant was then filtered through Whatman paper no. 1 (size include 10 mm to 500 mm diameter circles and 460 mm x 570 mm sheets) to obtain the cinnamon water extract. The final concentration was 0.2 g/ml (Sheng et al., 2008). This extract was renewed after every week.

3.2.2 Fenugreek Extract

The fenugreek seeds were washed by distilled water, dried in shadow, and powdered by using a mechanical grinder. For the aqueous extract 50g of fenugreek powder was mixed with 100 ml of water and placed on water bath for about 2 hours at 88°C. The extract was filtered capron silica cloth. The filtrate was stored in dark bottle in refrigerator at 4°C. The final concentration was 250mg/ml (Jamal et al., 2010). This extract was renewed after every week

3.3 Animal Modeling

The swiss albino mice used for experiment were kept in animal house of Government college university Lahore. The mice of age 1 month and weight 25-30g were housed at a constant temperature (25°C) under a 12-hour light-dark cycle and were provided with standard mice food and water (Hariri et al., 2011). The mice were kept in standard cages of size 8” X 11”X 10”. The average daily consumption of feed (PicoLab mouse diet) and water for an adult 25 g mouse was 3-5 g and 4 ml respectively. Both male and female mice were used.

Table 1 Components and percentage% of the diet given to mice

Components	Percentage %
Protein ≥	23
Fat ≥	6.5
Fiber ≤	4.5
Moisture ≤	12
Ash ≤	8
Vitamin E ≥	50

 

Albino mice were acclimatized for 7 days before experimental manipulation. The weight and blood sugar level of every mice was measure before the induction of diabetes.

Figure 1: Swiss Albino mice in a cage of size 8” X 11”X 10”

3.4 Diabetes Induction

The protocol defined by was used to induce diabetes in albino mice. The albino mice were starved overnight and their fasting blood glucose level was measured. Alloxan monohydrate (98%) of company Alfa Aesar was dissolved in injection water and injected intraperitonially (200mg/kg body weight) After giving injection the mice were provided with 10% sugar solution to prevent them from sudden hypoglycemic state. After 7 days the blood glucose level of albino mice was measured, the low dose of alloxan monohydrate (150 mg/kg body weight) was re-administered to those albino mice which became failed to undergo hyperglycemia [43]. Albino mice with blood glucose level more than 150 mg/dl were considered as diabetic and were used for further experiment.

3.5 Experimental Design

Albino mice were tagged by coloring their tail with differently colored permanent markers. The experiment was of 21 days and the weight and blood glucose level was checked after every 7 days. The experimental mice were divided into four groups.

Figure 2: Experimental Design

 Group 1: -ve Control Group (non-diabetic, untreated): Three non-diabetic mice are included in this group.

Group 2: +ve Control Group (diabetic, untreated): Five albino mice having diabetes which was induced by intraperitoneal injection of alloxan monohydrate (200 mg/kg) were placed in this group. These diabetic mice were left untreated.

Group 3: Experimental Group 1 (diabetic, treated with herbal extract): Six diabetic albino mice were included in this group. They were treated with the herbal extract mixture which is form by mixing cinnamon and fenugreek extract in a ratio 2:1. The aqueous extract 200mg/kg bodyweight was administered with gavage orally on daily basis.

Group 4: Experimental Group 2 (diabetic, treated with glibenclamide): Six albino mice were included in this group. They were treated with glibenclamide (by Sanofi Aventis Pakistan, brand name Daonil) which was commercially available in the market. The dilute solution of glibenclamide (600μg/kg body weight) was prepared by mixing it with distilled water and administered to the mice with the help of gavage on daily basis.

3.6 Determination of Blood Sugar Level

Evo check GS700 glucometer (pharm Evo) was used to measure the blood glucose level of albino mice. Tail of an albino mouse was sterilized with a cotton swab to make the tail vein visible. After that it was   punctured with the help of a lancet and the drop of blood was placed on specific point of glucometer strip and sugar level was noted. Albino mice with sugar level less than 120mg/dl are normal. The glucose level was checked after 7 days.

3.7 Blood Collection

The experimental mice were sacrificed after 21 days of experimentation. They were anesthetized by using ketamine HCL of company global pharmaceuticals. Ketamine mixed with sterilized injection water in the ratio of 1:2 and 0.1ml was given to each animal intraperitonially before the collection of blood. The blood glucose level of each mice was measured. Three ml blood was collected from each mouse heart by cardiac puncture using 5cc disposable syringe. After collection the blood was stored in EDTA gel coated tubes.

Figure 3: Anesthetize the mice using ketamine by injecting intraperitonially

Figure 4: Blood collections by cardiac puncture

3.8 Serum Preparation

 After collection of the blood, it is allowed to clot by leaving it undisturbed at room temperature for 15–30 minutes in gel coated tubes. Remove the clot by centrifuging at 1,000–3,000 x g for 10 minutes in a refrigerated centrifuge. The resulting supernatant is serum. Following centrifugation, it is important to immediately transfer the liquid component (serum) into a clean tube using a pipette. The samples should be maintained at 2–8°C and then further processed.

Figure 5: Serum separations after centrifugation

3.9 Serological Studies

For the blood chemistry analysis, several serological parameters were studied. Different liver function test (LFTs). Renal function test (RFTs) and lipid profile tests were performed to observe changes in serological parameters.

3.10 Renal Function Test

Renal tests are performed during study to observe the changes in the renal function.

3.11 Serum Creatinine

Principle of Test

In the alkaline solution creatinine reacts with the picric acid and formed the colored complex. The rate of formation of colored complex is measured and is directly proportional to the amount of creatinine present in the sample.

                                             Alkaline solution

Creatinine + picric acid                                          creatinine ─ picric acid complex

Test Procedure

Wavelength ———————————– 500nm (Hg 480-Hg 520)

Temperature ———————————– 30 °C or 37 °C

Optic path ————————————– 1cm light path

Gently mixed and it was incubated at 30 °C for 1 minute. Change in the optical density

(Δ OD/min) during next 2 minutes

Calculation (in mg/dl): Δ OD sample/Δ OD standard □ 2.0mmol/l.

 

3.12 Serum Urea

Principle of the Test

Urea is converted into ammonia in the presence of urease. Ammonia is than linked with α- ketoglutarate in the presence of glutamate dehydrogenase (GLDH) with the subsequent conversion of NADH to NAD. The rate of NADH conversion is directly proportional to urea concentration in the sample. Enzymatic determination according to the following reactions

                                 urease

Urea + H2O                            2NH3 + CO2

                                                                       GLDH

NH + 2 α – ketoglutarate + 2NADH                                 2glutamate + 2 NADH +2H2O

 

Test Procedure

Wavelength————————- 340 nm

Temperature————————- 30 °C or 37 °C

Optical path————————– 1 cm light path

Gently mixed and measure the change in the optical density (OD/min) during next 1 minute.

Calculations mmol/l: Δ OD standard X 8.35 (mmol/l).

3.13 Histology of Kidney

Microtomy is a process by which tissue can be sectioned and attached to a surface for future microscopic analysis. The aim of our study was histology of kidney. Different steps were involved in this process.

3.14 Removal of Kidney from Albino Mice

After anaesthizing the albino mice was sacrificed at 21 day and kidneys were to perform Microtomy by using Paraffin method.

Figure 6: Removal of kidney from dissected albino mice for histological studies

3.15 Fixation

Liver was excised and cut into smaller pieces and dissolved in 10% buffer formalin solution. Fixation is necessary to prevent autolysis and bacterial invasion.

Types of Fixative

• Formaldehyde
• Acetic acid
• Ethanol
• Picric acid
• Methanol
• Potassium dichromate
• Chromic acid
• Potassium permanganate

3.16 Tissue Processing

Tissue processing was performed according to the procedure normally applied by the Romanian Sanitary Veterinary and for Food Safety Direction, which involves the use of a Tissue Processor TPC15 and TBS88 Paraffin Embedding System. All samples were transferred into a cassette and immersed in multiple baths of progressively concentrated ethanol (to dehydrate the tissue), followed by toluene and finally hot paraffin.

3.17 Embedding

 During the embedding process, additional paraffin was added to create a paraffin block which allowed the sectioning of the tissues into very thin slices (3-5μm) using a microtome (microtom LEICA RM 2155). The sections were deparaffinized and rehydrated.

3.18 Staining

Staining of processed histology samples, the microtome slices were hematoxylin – eosin – methylene blue stained using a TST3 Tissue Stainer. Hematoxylin and methylene blue were used to stain nuclei blue, while eosin was used to stain the cytoplasm and extracellular matrix in varying degrees of pink.

3.19 Microscopic Analysis

All histological findings were performed using computer-based image analysis to assess organ damage. Microscopic images were captured by using a trinocular microscope (IRMECO-GmbH model IM-910 ,21439 Schwarzenbek/ Germany displayed on a computer via scope Tek® (scope photo 3.0).

 3.20 Statistical Analysis

The data was presented as Mean ± S.E.M. We used the analysis of variance (ANOVA) and Tukey’s multiple comparison test as post hoc was applied for inter group comparison. In all cases, a p value of less than 0.05 was considered to be significant. Statistical analysis was done by using SPSS version 11.5 software.

4. ANALYSIS AND RESULTS

Hypoglycemic activity of herbal extract (cinnamon and fenugreek) was observed in 6 swiss albino mice with age 1 month and having 29 g mean body weight. The hypoglycemic effect of herbal extract was compared with synthetic drug i.e. glibenclamide. Alloxan monohydrate (200mg/kg) was injected intraperitoneally in mice for the induction of diabetes. This dose causes the rise in blood sugar level higher than 150 mg/dl from 0 to 21 days, this is the clear indication of diabetes in group II (positive control) as shown in Table 4.2. The symptoms shown by diabetic mice were thinning of body hair, timed eyes, weakness, sluggishness and shivering.

Biochemical Results

4.1 Effect of Herbal and Synthetic Drug on Body Weight of Mice

There was a slight increase in body weight of mice from 26.00±1.00 to 27.06±0.35 grams at 0 and 21 days respectively in group I (-ve control). There was significant decrease (P< 0.05) in body weight in group II as compared to group I. However, mice treated with herbal extract (cinnamon and fenugreek) showed a comparable weight gain from 28.33±0.21 to 32.58±0.33 grams. The group IV mice treated with glibenclamide also showed a normal weight gain from 26.66±0.88 to 30.66±0.76 just like group III mice which is towards normal as shown in Table 1

Table 1: Body weight of control (-ve/+ve) mice and treated (herbal and glibenclamide) mice

	Body weight	in grams	of mice
No. of days	Control		Treated
	-ve control group   (Normal)	+ve control group   (Diabetic)	Herbal     (200mg/kg)	Glibenclamide     (600µg/kg)
0	26.00±1.00a	27.00±0.89a	28.33±0.21a	26.66±0.88a
7	26.00±0.57a	27.96±0.82a	28.51±0.35b	27.50±0.85b
14	26.66±0.66a	26.40±0.83a	30.46±0.34b	28.66±0.76b
21	27.06±0.35a	24.42±0.73ac	32.58±0.33bd	30.66±0.76ab

Values represents means of triplicate with Mean±S.E.M. Mean with different superscript differ significantly at P < 0.05. Similar alphabets represent statistically significant values

Figure 7: Body weight of control (-ve/+ve) mice and treated (herbal and glibenclamide) mice

4.2 Effect of Herbal and Synthetic Drug on Sugar Level of Mice

There was no increase in sugar level of mice from 91.33±1.86 to 88.33±4.41 mg/dl at 0^th and 21^st days respectively in group I (-ve control), whereas in group II (+ve control) the increase in sugar level occurred from 98.4±1.86 to 212.0±12.41 mg/dl at 0^th and 21^st day respectively during the same period of time. There was significant increase (P< 0.05) in sugar level in group II as compared to group I which confirmed that diabetes had induced. However, mice treated with herbal extract (cinnamon and fenugreek) showed a comparable decrease in sugar level. With the passage of time at 21^st day the sugal level was reduced from 169.2±6.64 to 120.0±10.0 mg/dl. The group IV mice treated with glibenclamide also showed decrease in blood sugar level which from 165.83±5.23 to 110.0±9.66 mg/dl on 21^st day just like group III mice which is towards normal as shown in Table 2.

           Table 2 Effect of herbal and synthetic drug on sugar level of mice

Blood sugar level (mg/dl
No.of  days	Control		Treated
	-ve control group (Normal)	+ve control group (Diabetic)	Herbal extract   (200mg/kg)	Glibenclamide   (600µg/kg)
0	91.33±1.86a	98.4±3.89a	94.5±5.31a	95.33±3.89a
7	81.67±4.41a	160.0±10.37b	169.2±6.64b	165.83±5.23b
14	93.33±3.33a	164.0±9.14b	173.33±4.77b	164.5±6.65b
21	88.33±4.41a	212.0±12.41bc	120.0±10.0ad	110.0±9.66ad

Means with different superscripts differ significantly at P <0.05. Similar alphabets represent statistically non-significant values.

4.3 Effect of Herbal and Synthetic Drug on Serum Creatinine

Figure 8: Effect of herbal and synthetic drug on sugar level of mice

Serum creatinine level remained from 0.70±0.056 to 0.80±0.058 mg/dl from 0^th and 21^st day in group I. Whereas, in group II there was significant increase (P <0.05) in serum creatinine level occurred from 0.72±0.058 to 2.88±0.27 mg/dl at 0^th and 21^st day respectively. Group III Mice treated with herbal extract showed value of   0.78±0.048 to 2.48±0.28 mg/dl from 0^th to 21^st day respectively. Group IV mice treated with glibenclamide showed a value of 0.716±0.477 to 1.78±0.18 mg/dl on 21^st day which is towards normal range.

Table 3: Effect of herbal and synthetic drug on serum creatinine (mg/dl) level in control (-ve/+ve) and treated group of mice

No. Of days	Serum	Creatinine  (mg/dl)
	Control		Treated
	-ve control group (Normal)	+ve control group (Diabetic)	Herbal extract (200mg/kg)	Glibenclamide (600µg/kg)
0	0.70±0.056a	0.72±0.058a	0.78±0.048a	0.716±0.477a
21	0.80±0.058a	2.88±0.27bc	2.48±0.28bcd	1.78±0.18bcd

 

Means with different superscripts differ significantly at P <0.05. Similar alphabets represent statistically non-significant values

4.4 Effect of Herbal and Synthetic Drug on Serum Urea

Serum urea level remained same from 12.33±1.45 to 12.67±1.76 mg/dl from 0^th and 21^st day in group I. Whereas, in group II there was significant increase (P <0.05) in serum urea level occurred from 13.2±1.02 to 34.4±1.47 mg/dl at 0^th and 21^st day respectively. Group III Mice treated with herbal extract showed value of   14.83±0.91 to 24.5±0.85 mg/dl from 0^th to 21^st day respectively. Group IV mice treated with glibenclamide showed a value of 13.67±0.67 to 26.0±0.93 mg/dl on 21^st day which is towards normal range.

Table 4: Effect of herbal and synthetic drug on serum urea mg/dl in control (- ve/+ve) and treated groups of mice

		Serum urea	mg/dl
No. Of days	Control		Treated
	-ve control group (Normal)	+ve control group (Diabetic)	Herbal extract (200mg/kg)	Glibenclamide (600µg/kg)
0	12.33±1.45a	13.2±1.02a	14.83±0.91a	13.67±0.67a
21	12.67±1.76a	34.4±1.47b	24.5±0.85bcd	26.0±0.93bc

Means with different superscripts differ significantly at P <0.05. Similar alphabets represent statistically non-significant values

4.5 Histological Results:

4.5.1 Kidney

Photomicrograph of kidney section taken from control group was without any pathological abnormality. There was normal appearance of glomeruli in kidney of normal mice given in Figure 4.5

Figure 9: Photograph of a kidney section from –ve control (group I) without any abnormality. Kidney of normal mice showed normal appearance of the glomeruli (→). H & E X400.

4.5.2 Effect of alloxan on kidney of mice

Section of kidney taken from alloxan induced diabetic mice showed marked degeneration of glomeruli atrophies and severe vacuolations represented in Figure 10.

Figure 10: Photograph of a kidney section from +ve control (diabetic) showing marked degeneration of the glomeruli and severe vacuolations (→). H&E staining

4.5.3 Effect of Herbal Extract on Diabetic Mice Kidney

Kidney section from group 3 treated with dose of 200mg/kg B.W of herbal extract showed pathological improvements with normal appearance of glomeruli as shown in Figure 11.

Figure 11: Photograph of a kidney section from treated group with Herbal extract showing pathological improvement with normal appearance of the glomeruli (→)

            4.5.4 Effect of Glibenclamide on Mice Kidney

The histology of mice treated with glibenclamide of dose 600µg/kg B.W showed a normal appearance. The micrograph was showing normal hepatocytes and normal microvasculature.

Figure 12: Photograph of a kidney section from treated group with Glibenclamide showing pathological normality with normal appearance of the glomeruli (→).

5. DISCUSSION

The field of tradition medicine and drugs are getting rapid popularity from the last few days both in developing and developed countries because of their natural origin and less side effects. Different medicinal plants are used to derive organic medicines [47]. Medicines prepared from herbs have no side effects and rarely have milder side effects. However, the allopathic medicines have unconcerned side effects. It is important to note that two organic medicines extracted from plant extract have been approved by the FAD for the treatment of human ailments [48]. Sulphonylureas is really popular for the treatment in a T2D patient who has failed on non-pharmacological measures and is non-obese [49]. They can be used in combination with other classes of antidiabetic drugs. Side-effects of taking sulphonylureas include lower blood glucose level, increase in body weight, skin irritations, acute porphyria and, rarely hyponatremia [50]. The present study evaluated the hypoglycemic activity of cinnamon and fenugreek and compared it with the hypoglycemic activity of allopathic drug glibenclamide. In this study, we induced diabetes in Swiss albino mice by using alloxan monohydrate (200 mg/kg). Diabetes induced by alloxan monohydrate is categorized by hyperglycemia due to decreased level of insulin. The increase in blood glucose level was observed on 7th day after injection of alloxan. The surgical method of diabetes induction is associated with high mortality [51]. The present study evaluates the decrease in body weight of group II (+ve control) from 27.00±0.89 to 24.42±0.73 grams at 0th and 21st day respectively However, group III mice treated with herbal extract (cinnamon and fenugreek) showed a comparable weight gain from 28.33±0.21 to 32.58±0.33 grams. The group IV mice treated with glibenclamide also showed a normal weight gain from 26.66±0.88 to 30.66±0.76 grams. A study suggests that glibenclamide treatment significantly increased the body weight of STZ-induced diabetic rats [52]. In present study diabetic mice treated with herbal extract (cinnamon 200mg/kg and fenugreek 250mg/kg) showed decrease in sugar level. With the passage of time at 21st day the sugar level was reduced from 169.2±6.64 to 120.0±10.0 mg/dl. A recent study reported that diabetic mice treated with the herbal extract (cinnamon 200mg/kg and fenugreek 250 mg/kg) for 21 days displayed a significant glucose lowering effect for the period of 0-6 week [53]. There are numerous studies reported that cinnamon has antihyperglycemic and antihyperlipidemic effects on diabetic animals and for T2D [54]. The experimental mice treated with herbal extract (cinnamon and fenugreek) showed a comparable decrease in sugar level. With the passage of time at 21st day the sugar level was reduced from 169.2±6.64 to 120.0±10.0 mg/dl. Our study showed an agreement with the previous study which proposed that consumption of a cinnamon and fenugreek mixture has considerable effects in reduction of blood sugar levels in patients with T2D [55-79]. The results of our biochemical tests showed that serum creatinine level remained from 0.70±0.056 to 0.80±0.058 mg/dl from 0th and 21st day in group I. Whereas, in group II there was significant increase (P <0.05) in serum creatinine level occurred from 0.72±0.058 to 2.88±0.27 mg/dl at 0th and 21st day respectively. Group III Mice treated with herbal extract showed value of   0.78±0.048 to 2.48±0.28 mg/dl from 0th to 21st day respectively. Group IV mice treated with glibenclamide showed a value of 0.716±0.477 to 1.78±0.18 mg/dl on 21st day which is towards normal range. Administration of different doses of Fenugreek seed extract and Cinnamon bark extract decreased serum urea and creatinine levels in diabetic rats as compared to untreated diabetic rats and that effects are dependent on doses [80-114]. The antidiabetic effect of fenugreek ethanolic extract (Trigonella-foenum graecum L) was investigated in normal and streptozotocin-induced diabetic rats. Fenugreek extract when orally taken (0.1, 0.25, and 0.5 g/kg body weight) for 14 days cause a significant decrease on the level of glucose, urea, creatinine. Treatment of fenugreek extract alleviated body weight loss in diabetic rats. Administrations of the extract significantly decreased serum glucose, urea, creatinine, levels, whereas it increased serum insulin in diabetic rats. Serum creatinine and serum urea are excreted exclusively through the kidney. Therefore, damage to the kidney will make the kidney inefficient to excrete both and causes its accumulation in the blood. Therefore, high level of creatinine and urea will cause kidney damage. Histological slides of diabetic kidney demonstrated the distorted nephrons in mice. The mice of treated groups have not shown any distorted nephrons in their histology of kidney. The study suggested that the herbal extract is effective for diabetes; it is also effective in protecting against kidney damage.

6. CONCLUSION

Commercially produced drugs are mostly derived from plants and form the majority of today’s modern medicine. Therefore, many herbs have shown to have antidiabetic property by regulating insulin secretion, insulin sensitivity. In order to improve the glycemic control of the patients, it is suggested that ingesting mixture of cinnamon and fenugreek extract has significant effects in the regulation of blood glucose. Despite the difference in hypoglycemic effect the herbal extract can be used as an effective alternative against diabetes with optimized dose for humans after experiment under observation.

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