Pharmacological Evaluation of Antidiabetic Activity of Vitex Negundo Linn. Leaves Extract and its Zinc Oxide Nanoparticles

Diabetes mellitus (DM) is a metabolic disorder characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both.1 The level of hyperglycaemia associated diabetes increases the risk of microvascular damage (retinopathy, nephropathy and neuropathy). It is associated with reduced life expectancy, significant morbidity due to the related microvascular complications, increased risk of macrovascular complications (ischaemic heart disease, stroke and peripheral vascular disease), and diminished quality of life. 2 Glucose blood levels are maintained by insulin which is a hormone released from the pancreas. When these level increases, insulin is produced from the pancreas and maintained the level of glucose. In diabetic patients, the production of insulin is absent or less which causes hyperglycemia3

1.1 Types of Diabetes Mellitus:

There are mainly two major types of diabetes mellitus.

Type 1: It is also called as Insulin dependent Diabetes Mellitus (IDDM). It is due to failure of body for insulin production. It is childhood disease so it is called as juvenile onset diabetes mellitus 4

Type 2: It is also called as Non – Insulin Dependent diabetes Mellitus (NIDDM). In this type of cells are unable for insulin usage. The other name for this type is adult onset diabetes mellitus5

1.2 Nanotechnology in Herbal Medicine

The goal of the applied science and technology discipline of nanotechnology is to create tools and dosage forms that are between 1 and 100 nm in size. The use of nanotechnology in biological disease treatment, diagnosis, monitoring, and control6 Recently, systems have been called nanomedicine. Lipids, polysaccharides, and synthetic biodegradable polymers are among the safe components used to create the nanocarriers.7 The creation of instruments and dosage forms with sizes ranging from 1 to 100 nm is the aim of the applied science and technology field of nanotechnology. Treatment, diagnosis, monitoring, and management of biological diseases using nanotechnology better distribution of tissue macrophages, prolonged administration, defense against chemical and physical deterioration, etc. Therefore, there may be a future for improving the activity and resolving issues with plant medicines using nanoscale drug delivery systems for herbal medications. In order to combat more chronic diseases including cancer, diabetes, asthma, and others, it is imperative that nanocarriers be incorporated into the regular medical system as an NDDS 8.

MATERIAL AND METHOD

2.2 Collection and authentication of Vitex Negunda Linn.

Vitex Negunda Linn. plant were collected from Thiruvalla, Kerala. The plant material was identified and authenticated by Mrs. A. M. Gaharwar, Assistant Professor of Vasantrao Naik College of Agricultural Biotechnology, Yavatmal.

2.3 Experimental Animals

Healthy male Sprague dawley rats approximately 8 weeks of age weighing about 200to 210 gm used for the pharmacological screening. The animals were purchased from NIN Hyderabad. The animals were housed in polyoropylene cages with wire and meshtop and husk bedding and maintained under the standard environmental conditions (25 ± 20 C, relative humidity 5 % light dark cycle for 12 hours each) and the fed withthe standard pellet diet and water were used for the entire study. Animal study protocol was duly approved by IAEC and P. Wadhwani college of Pharmacy, Yavatmal with research project No.650/PO/Re/S/2002/CPCSEA/2025/14. The rats were treated according to the rule and regulation of IAEC and CPCSEA.

2.3 Procurement of diagnostics kits and chemicals

Diagnostics kits for estimation of glucose,urea, creatinine, triglyceride and cholesterol were obtained from Ambika diagnostics. Streptozotocin was obtained from M.P. Biomedical, Glibenclamide was generously obtained from Cipla Pvt Ltd, Mumbai. All other chemicals used were of laboratory grade.

METHODS:

Extraction

The leaves of Vitex Negunda Linn. were collected, dried in shade and coarsely powdered. The powdered leaves were defatted with petroleum ether and then subjected to maceration with methanol.9

2.5 Phytochemical Screening10

Test for Carbohydrates

Dissolve 2 gm extract in 5 ml distilled water and filter it. The filtrates were used to test for the presence of carbohydrates.

Molish test

A few drops of the extract was treated with molish reagent (Alpha – napthol in alcohol) and a drop of concentrated sulphuric acid was added. Purple color was obtained. This confirms the presence of Carbohydates.11

Test for Alkaloids Hager’s test

Filtrate was treated with saturated aqueous solution of picric acid. Presence of alkaoids were confirmed by the formation of yellow coloured precipitate.12

Test for saponin Foam test

Small quantity of the extract was shaken with 2 ml of water. Persistance of foam produced for 10 min indicated the presence of saponins.

Test for tannin

Take 0.5 gm of the dried powdered plant. Boil 0.5 gm sample in 20 ml of water in a test tube. Filter the above mixture. Add few drops of 0.1% ferric chloride. Development of brownish green color or blue-black coloration indicates presence of tannin.13

Test for terpenoids Salkowski Test:

Mix 2 ml of chloroform to extract solution carefully added con. H2SO4 to form a layer. A reddish brown colouration of the interface indicated the presence of terpenoids.14

Test for Glycosides Killer Killani Test

To 50 mg of each extracts, 2 ml of glacial acetic acid, 1 ml Fecl3 solution were added, heated and the cooled. This was transferred to a test tube containing 2 ml conc.H2SO4 and observed.

Test for steroids Salkowski Test

To 100 mg of each extracts, 2 ml of CHCl3, 2 ml of conc. H2SO4 was added, mixed thoroughly and both the layers were observed for colour.15

Test for phenol Ferric chloride test

Treat the extract with 3-4 drops of ferric chloride solution. Formulation of bluish black colour indicated the presence of phenols.

Test for Amino acid Ninhydrin Test

Add ninhydrin reagent to the test solution and boiled for few minutes. Formation of blue colour indicated the presence of amino acids.16

Test for flavonoids Ferric chloride test

Add a few drops of ferric chloride solution to the extract solution. Development of intense green colour indicates the presence of flavonoids.

Lead acetate test

Treat the extract with few drops of lead acetate solution. Formation of yellow precipitate indicate presence of flavonoid.17

Anthraquinone

1 ml of filtrate + 10 ml Benzene filter and 5 ml of ammonia to the filtrate and shake well gives pinkish colour.16

2.5 Synthesis of Zinc Oxide Nanoparticles

This synthesis method involved dissolving 11.5 g of zinc sulfate in 40 mL of water and thoroughly mixing the mixture using a magnetic stirrer. Using a sterile injection, 10 mL of vitex negundo linn. leaf extract was injected to this combination drop by drop. For at least ten minutes, the resultant liquid was constantly mixed. Drop by drop, sodium hydroxide solution (2M) was added to the solution above. The mixture was then repeatedly cleaned with distilled water, centrifuged for 15 minutes at 3000 rpm, and dried in a hot air oven at 60 0 C.A meticulously dried zinc oxide Nano powder was obtained. To produce the anticipated fine zinc oxide nanoparticles, the powder was ground into a fine powder using a mortar and pestle. Likewise, zinc oxide nanoparticles were prepared using 11.8 g of zinc nitrate and 8.8 g of zinc acetate dihydrate. To conduct the additional optical and structural characterizations, the zinc oxide nanoparticles were transferred into a sterile vial17.

2.6 Experimental Designs

The animals were grouped into seven following groups as follows (n=6)

Group-I (Normal Control Group): - Animals were treated with normal saline solution. Group-II (Negative Control): - Animals were treated with STZ (60 mg/kg)

Group-III (STZ + MEVN): - Diabetes rats were treated with methanolic extract of Vitex Negunda Linn.  (400mg/kg)

Group IV (STZ + NPMEVN): - Diabetes rats were treated with nanoparticles of extract of V i t e x N e g u n d a L i n n. (300 mg/kg)

Group V (STZ + Glibenclamide):- Diabetes rats were treated with standard drug (5mg/kg)

Dose Selection

Dose of Vitex Negunda Linn. was selected as per the dose mentioned in the research article by Most. Fatimatuz Zahura Falguni. (Most. Fatimatuz Zahura Falguni et. al. 2017).

2.7 Induction of Diabetes in rats by Streptozotocin

The animals were fasted overnight and diabetes was induced by intraperitoneal injection of STZ at dose of 60 mg/kg. (S.K. Prasad, Alka Kulshreshtha et, al.) STZ was first weighed individually for each animal according to the body weight and solubilized with 0.1 M Sodium citrate buffer pH 4.5. It was then injected. 5 % dextrose was provided for 24 hours to inhibit hypoglycemia. Administration of single dose of STZ in rats results in hyperglycemia within 72 hours.

Sample Collection

Blood samples were collected by Retro orbital plexus puncture method and blood glucose levels, urea, triglycerides, cholesterol and creatinine was estimated by semi- automated electronic bioanalyser17

2.7 Estimation of Biochemical Parameters

1) Blood Glucose Principle:

Glucose is oxidized into the gluconic acid and Hydrogen peroxide. The hydrogen peroxide further reacts with phenol and 4- amino antipyrine by the catalytic action of peroxidase to form a red coloured quinoamione dye complex. (Trinder P. and LevinsonS):

•  The serum sample was seperated from blood sample of rats by centrifugation.

The supernants clear serum sample is used for the testing is given below.

• Test sample was prepared by taking 1 ml of enzyme reagent and 10 µl of serum sample.

• The blank sample was prepared by taking 1 ml of enzyme reagent.

• The standard sample was prepared by taking 1 ml of enzyme reagent and 10 µl of standard.

• All samples were mixed and incubated at 370C for 10 min and the absorbance of standard and test were read against distilled water blank at 505 nm18

Formula:

2) Cholesterol:

                         Abs. of test – Abs. of blank

Glucose =                                                              × 100

                         Abs. of Std – Abs. of blank

Indicator of liver function, biliary function, intestinal absorption, propensity towards coronary artery disease and adrenal disease. Decreased level of HDL cholesterol is associate with increased   risk   of   coronary   artery   disease   and   other   atherosclerotic   complications.19 On addition of precipitating reagent to the serum lipoprotein precipitant out and the HDL fractions remains in the supernant, which is determine by above reactions.

Procedure:

• The serum sample was seperated from blood sample of rats by centrifugation.

• The supernants clear serum sample is used for the testing is given below.

• Test sample was prepared by taking 1 ml of enzyme reagent and 0.01 ml of serum sample.

• The blank sample was prepared by taking 1 ml of enzyme reagent.

• The standard sample was prepared by taking 1 ml of enzyme reagent and 0.01 ml of standard.

• All samples were mixed and incubated at 370C for 10 min and the absorbance of standard and test were read against distilled water blank at 505 nm using green filter.

Formula:

                               Abs. of test – Abs. of blank

Total cholesterol =                                                 × 200

                               Abs. of Std. – Abs. of blank

3) Triglycerides

Elevated level of triglyceride in the blood have identified as risk factors related to coronary heart disease. Elevated level of triglyceride are found in atherosclerosis, diabetes mellitus, bilary obstruction and other metabolic disorder associate with endocrine disturbance

Procedure:

• The serum sample was seperated from blood sample of rats by centrifugation.

The supernants clear serum sample is used for the testing is given below:

• Test sample was prepared by taking 1 ml of enzyme reagent and 10 µl of serumsample.

• The blank sample was prepared by taking 1 ml of enzyme reagent.

• The standard sample was prepared by taking 1 ml of enzyme reagent and 10 µl of standard.

• All samples were mixed and incubated at 370 C for 10 min and the absorbance of standard and test were read against distilled water blank at 546 nm using green filter.21

Formula: