INTRODUCTION:

Diabetes mellitus (DM) is a metabolic disorder that is distinguished by hyperglycemia resulting from inadequacy of insulin action, insulin secretion from pancreatic β cells or a combination of both.¹ Chronic hyperglycemia caused by the insufficiency of insulin action leads to abnormalities in the carbohydrate, lipid, and protein metabolism.² Type-1 (Insulin dependent, deficient) diabetes is an autoimmune disorder, characterized by pancreatic β cell destruction with an absolute deficiency of insulin.³ Pathogenesis of Type-2 (Insulin resistant and concomitant deficient insulin secretion) diabetes is due to dysregulated glucose homeostasis, impaired insulin secretion and action.⁴

Main stray of treatment consisted of the plant based preparation for centuries and continues to be relevant in the era of modern medicine. Many people continue to depend on plant based remedies to treat pain, fever, anxiety, asthma, gastritis, diabetes and other diseases.⁵ Accordingly many plant based medicines are available in the market; most of them are prescribed by the practitioners of folklore medicines. However, in most of the cases, the claims made by the promoters of such products are not validated. We have come across two plant based products which claim to reduce blood glucose level and help manage diabetes.

Several animal models have been developed to screen the compounds for anti-diabetic activity.⁶Streptozotocin is used for the induction of both insulin dependent (40-70mg/kg body weight) and non-insulin dependent diabetes mellitus (30-40mg/kg body weight). STZ shows permanent or comparatively permanent hyperglycemia and occurs within 24 hours.⁷Alkylation of DNA is the main reason for the death of β cell in STZ induced animals.⁸

Figure 1: Structure of Streptozotocin

MATERIAL AND METHODS:

Chemicals and reagents:

Streptozotocin, Metformin and Dibasic sodium phosphate were procured from HIMEDIA. Dextrose and Citric acid monohydrate were procured from Loba Chemie Pvt Ltd, Mumbai. GOD/POD Kit was used for measuring the blood glucose levels (Aspen Laboratories). All other chemicals and reagents used were analytical grade unless otherwise indicated.

Instrumentation:

For obtaining the serum from blood collected, a research centrifuge (REMI C-24 PLUS) was utilized. The proposed work for measurement of blood glucose level was carried out on a Semi Auto analyzer (Rapid Star 21 Plus). All weighing was done using electronic balance.

Collection of plant material:

The product ADD1 (DIA ARECA) was a gift sample from the manufacturer. It is a marketed product containing areca nut, betel leaf extract, lime, neem, jamboo and edible grass which is labeled as “food supplement to manage diabetes naturally”. The preperation is in liquid from with advice to take 10ml thrice a day. The specific gravity was 1.03 and hence 1 ml weighed nearly 1g. The dose to be administered was decided on the basis of this data (Conversion of human dose into animal dose on the basis of surface area calculations).

The second formulation, ADD2 was a mixture of dried herbs and obtained from Ayurvedic store at Puttur. This was marketed with trade name “Asanadi Kashaya Choorna” and claimed to manage “madhumeha” and “raktha shodhaka” by SDP remedis and research centre. A combined extract of these herbs was prepared by aqueous extraction.

Extraction of plant material:

The dried herbs collected were grounded and powdered by using an electric grinder to reduce particle size. The powder was then passed through a sieve no. 20 to get a uniform particle size of all the ingredients.

Three sets of 100g of the powdered drug were taken and 700ml of distilled water was added to each of the beakers. They were boiled for 3 hours with occasional stirring. The aqueous extracts obtained were filtered using Whatmann filter paper no. 42. All three extracts were combined and the volume was reduced to 300ml. This volume contains 10 recommended human doses. The extract was stored under cold condition.

Experimental animals:

Albino Wistar rats of either sex, 6-8 weeks old, weighing (180g-200g) were obtained from the animal house of NGSM Institute of Pharmaceutical Sciences, Deralakatte, Mangaluru. The animals are grouped and housed in cages and maintained under standard laboratory conditions (temperature 25±2°) with dark and light cycle (12h/12h). Standard dry pellet diet with water ad libitum was given to the animals with free access. Animals were kept in the laboratory at least a week before experimental testing. Each experimental group of rats were chosen randomly and used only once. All experiments were carried out under the guidelines of the institutional animal ethical committee (IAEC) of NGSM Institute of Pharmaceutical Sciences, Deralakatte, Mangaluru IAEC NO. : 1781/PO/EreBi/S/2014/CPCSEA.

Selection of doses^9:

For the assessment of anti-diabetic activity of the two plants based formulations, dose levels were calculated using the following formula:

Animal Equivalent Dose (AED) for rat was calculated for on the basis of body surface area either by multiplying or dividing the given human dose (mg/kg body weight) by Km ratio (6.2 or 0.162).

AED (mg/kg) =Human dose (mg/kg body weight) x Km ratio (6.2)

Two dose levels (High dose and Low dose) were calculated for each formulation. The dose for each animal was then calculated according to their body weight.

ADD1 dose calculation:

The recommended human dose was 30ml per day. This was converted to animal dose by assuming the average weight of 60kgs for the human. The animal dose levels thus selected were 3.1ml/kg (low dose) and 6.2ml/kg (high dose).

ADD2 dose calculation:

The recommended human dose was 30ml per day (Equivalent for 30g of the herbs). This was converted to animal dose by assuming the average weight of 60kgs for the human. The animal dose levels thus selected were 3.1ml/kg (low dose) and 6.2ml/kg (high dose).

Experimental induction of diabetes:

Streptozotocin solution (20mg/ml) was freshly prepared in 0.1M of citrate buffer (pH 4.5). This solution was administered via intraperitoneal route in the volume of 2 ml/kg body weight and rats were given dextrose solution (5%) instead of water for 48h. Diabetes was then confirmed by measuring the fasting blood glucose level (>300mg/dl) after a period of 48 hours.¹⁰

Experimental design:

Animals were divided into seven groups consisting of six rats in each group and then kept in separate labelled cages. Group I: Normal control (distilled water)

Group II: Disease control (Streptozotocin)

Group III: Diabetic animals+ Standard drug (Metformin)

Group IV: Diabetic animals + ADD2 (dose level 1)

Group V: Diabetic animals + ADD2 (dose level 2)

Group VI: Diabetic animals + ADD1 (dose level 1)

Group VII: Diabetic animals + ADD1 (dose level 2)

The above treatment was given to each group of animals for a period of 21 days. Blood samples were withdrawn from retro-orbital sinus of the rats using capillaries on the days 1st, 7th, 14th and 21st. The blood samples were centrifuged at 3000rpm for 5 minutes and serum was obtained. Serum glucose levels were determined by GOD/POD method using a corresponding kit from Aspen Laboratories and the pink colour intensity of quinoneimine complex formed after the treatment with the GOD/POD reagents was estimated in a semi auto analyzer at 505nm.

RESULTS:

Acute toxicity study:

Acute toxicity study was carried out in accordance with OECD 425 guidelines. The test compounds ADD1 and ADD2 were administered to two nulliparous/non-pregnant Wistar rats of the age between 9-10 weeks at a dose of 2000mg/kg body weight by oral route. These animals were observed for toxic manifestations and behavioral changes for 24h. There was no death or symptoms of toxicity. The dose was repeated with four more animals in each group and was observed. All the animals did not show any toxic symptoms.

The study was repeated with 5000mg/kg body weight with three animals for both the formulations. This was necessary because the dose recommended for anti-diabetic study on the basis of human dose data was 6 ml/kg body weight. Here also we found no toxic effect. Thus, proving that both the formulations are safe up to 5000mg/kg. None of the toxicity indicators as per Irwin’s table was positive.

Quantitative determination of blood glucose level by (GOD/POD) method:

Blood glucose level in serum was determined by GOD/POD method procedure¹⁰ using a reagent kit. In brief, to 10μl of serum, standard and distilled water (blank) into 3 Eppendorf tubes, 1000μl of the enzyme was added, mixed and kept at 37°C for 10 minutes. The color of standard and serum developed was read at 505 nm against reagent blank.

Anti-diabetic study of formulation ADD1 and ADD2:

The study was carried out with the two different dose levels of 3.1ml/kg body weight and 6.2ml/kg body weight. As stated earlier, this dose was arrived at by converting the recommended human dose of 30ml per day into animal dose.

The estimation of blood glucose level was analyzed on 1^st, 7^th, 14^th and 21^st day. Significant activity was observed with both the doses on the 21st day as compared to the diabetic animals.

However, the reduction in blood glucose level was not significant on 7th and 14th day with lower dose of ADD1 as compared to lower dose of ADD2. The result was moderately significant with the higher dose. Higher dose of ADD2 was significantly more as compared to higher dose of ADD1. We found some inconsistency in the higher dose readings of ADD1.

The level of significance was analyzed at P value < 0.05, P value < 0.01 and P value <0.001.

From the data obtained we can say that the formulation ADD2 is more statistically significant when compared to that of the formulation ADD1.

Table 1. Effect of Formulation ADD1 in STZ induced diabetic rats.

Groups	Dose	Blood Glucose Level (mg/dl)
Groups	Dose	Day 1	Day 7	Day 14	Day 21
I	Distilled water	122.4±5.32	118.6±4.22	119.5±3.00	118.5±3.40
II	STZ(40mg/kg)	374.3±28.34	482.3±11.48	461.2±23.62	448.8±15.58
III	Metformin 500mg/kg+ STZ(40mg/kg)	581.6±29.55	215.1±26.62	185.5±41.34	134.0±8.52
IV	ADD1(3.1ml/200g)+ STZ(40mg/kg)	430.3±17.92	457.0±17.74	412.0±17.28	296.7±23.65***
V	ADD1(6.2ml/200g)+ STZ(40mg/kg)	528.6±32.74	341.8±23.94**	303.7±46.51*	244.6±19.67***

Effect of Formulation ADD1 on blood glucose level

Values are given as mean± S.E.M, n=6 in each group.

* Represents statistical significance vs. diabetic control (P < 0.05).

** Represents statistical significance vs. diabetic control (P < 0.01).

*** Represents statistical significance vs. diabetic control (P < 0.001).

Figure 2: Effect of formulation ADD1 on blood glucose level of STZ induced diabetic rats

Table 2. Effect of Formulation ADD2 in STZ induced diabetic rats.

Groups	Dose	Blood Glucose Level (mg/dl)
Groups	Dose	Day 1	Day 7	Day 14	Day 21
I	Distilled water	122.4±5.32	118.6±4.22	119.5±3.00	118.5±3.40
II	STZ(40mg/kg)	374.3±28.34	482.3±11.48	461.2±23.62	448.8±15.58
III	Metformin 500mg/kg+ STZ(40mg/kg)	581.6±29.55	215.1±26.62	185.5±41.34	134.0±8.52
IV	ADD2(3.1ml/200g)+ STZ(40mg/kg)	545.6±15.36	363.4±27.24*	268.4±26.51**	312.4±20.74**
V	ADD2(6.2ml/200g)+ STZ(40mg/kg)	468.4±65.40	336.4±24.51**	216.9±39.22***	154.0±27.10***

Effect of formulation ADD2 on blood glucose level

Values are given as mean± S.E.M, n=6 in each group.

* Represents statistical significance vs. diabetic control (P < 0.05).

** Represents statistical significance vs. diabetic control (P < 0.01).

*** Represents statistical significance vs. diabetic control (P < 0.001).

Figure 3: Effect of formulation ADD2 on blood glucose level of STZ induced diabetic rats.

Statistical analysis:

Results of biochemical estimation were reported as mean ± S.E.M. The total variation present in data was analyzed by one way analysis of variance (ANOVA) using SPSS. A value of p<0.05 was considered to indicate a signiﬁcant difference between groups

DISCUSSION:

The present study was done to evaluate the anti-diabetic activity of two marketed herbal formulations ADD1 and ADD2. This study was conducted using in vivo low dose streptozotocin induced diabetic model. The parameter that was used for the assessment of anti-diabetic activity was blood glucose levels of treated, normal and diabetic rats. Metformin was used as a standard drug for the comparison of efficacy.

Acute oral toxicity study of formulation ADD1 and aqueous extract of formulation ADD2 were conducted and did not show any toxic symptoms or death up to 2000mg/kg. The dose selection was made by using the existing human dose which is based on the body surface area. Two dose levels were selected for both the formulations. ADD1 and ADD2 were administered at the doses 3.1ml/kg and 6.2ml/kg body weight.

ADD1 is a formulation containing the extract of areca catechu and some other plant materials. The study results were in line with the assumption that the extract will have anti-diabetic effect. However, it was not as potent as standard drug metformin. It is possible to have a significant action but prolonged use may be necessary as in the case of many plant based formulations.

It is interesting to note that the composition of ADD1 is similar to the paan, a arecanut – betle leave- slaked lime chewing preperation popular in Indian subcontinent. Paan is consumed for its carminative, digestant and antacid activity. The result of the current suggests that it may have antidiabetic action as well.

The second formulation ADD2, is an extract of 13 herbs which are extensively used in Ayurvedic formulations. The blood glucose lowering effect of the ADD2 extract was quite significant and was better than ADD1. The two formulations ADD1 and ADD2 were compared and we have found that ADD2 has more significant potential activity than formulation ADD1 in both the dose levels.

The limitation of the present study is not working out the mechanism of action of the formulation. It is possible that the phytoconstituents in the formulations act by helping β-cell regeneration, increasing insulin sensitivity or by both.

The second limitation is that the anti-diabetic effect of the extracts was investigated. We have not identified any specific molecule in either of the formulations. However, it may be entirely possible that the formulations are efficacious because of all the ingredients present in them. Isolation of compounds may abolish the activity of these formulations.

CONCLUSION:

The present study was conducted to evaluate the anti-diabetic activity of two plant based formulations ADD1 and ADD2 using Streptozotocin induced diabetic model.

The blood glucose lowering effect of ADD1 was not very drastic; still the formulation has potential anti-diabetic effect whereas ADD2 showed significant anti-diabetic activity in STZ induced diabetic model justifying the claim made in the label of the marketed product.

In conclusion, the near absence of any adverse effects noticed in the animals under study groups or which was used for acute toxicity study confirms the safety and non-toxicity of both the products studied. The result justifies the usage of these plant based products for diabetes. It is also possible that they have other beneficial effects, especially ADD2 which contains 13 herbs.

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of N.G.S.M. Institute of Pharmaceutical Sciences, Mangaluru for the facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 02.07.2019 Modified on 10.08.2019

Research J. Pharm. and Tech 2020; 13(2):664-668.

DOI: 10.5958/0974-360X.2020.00127.4