Diabetes and preparation of plant material Fresh and

Diabetes mellitus (DM)
is a group of metabolic disorders with elevated blood glucose levels or
hyperglycemia 1 and currently it is one of the most costly and burdensome
chronic diseases 2. The diabetic patient exhibits a higher risk in the
development of several chronic health complications including obesity,
atherosclerosis, dyslipidemia and renal failure worldwide 3. Commercial
available oral hypoglycemic drugs for diabetes mellitus produces severe side
effects including hypoglycemia, weight gain, gastrointestinal disturbances and
hepato-renal toxicity 4, 5.

Pancreatic ?-amylase
and intestinal ?-glucosidase plays important role during glucose metabolism,
but during diabetic condition the elevation of blood glucose is also due to the
action of pancreatic ?-amylase and intestinal ?-glucosidase. These hydrolyzing
enzymes can also be an effective therapeutic method to maintain the elevated
blood glucose level, hence inhibition of ?-amylase and ?-glucosidase can be a
key strategy in the control of diabetes mellitus 6.

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            As an alternative herbal products
and their derivatives act as a source of therapeutic agents in traditional
medicines with a great concern to the scientific community to evaluate and
isolated natural products in experimental studies. These herbal remedies are
apparently effective, produce minimal or no side effects in clinical
experiments and are of relatively low costs as compared to oral synthetic
hypoglycemic agents 7. It is a well-known fact that free radicals produced in the human system causes oxidative damage by release of reactive oxygen species (ROS) and reactive
nitrogen species (RNS) from activated neutrophil and macrophages. Hence produce damages to various organs inducing diseases likediabetes, Parkinson’s disease, heart disease,
arthritis, autism, cancer, Alzheimer’s dementia, cataracts and aging 8.

Annonacherimola a commonly used folk medicine against antiparasitic,
antitumor, treatment of intestinal diseases, anti- peroxidative and
cytoprotective, etc., 9,10. Research has proved that Annonacherimola has the capacity to
scavenge free radicles and based
on the above observation an effort was made to analyze its efficiency against oxidative
stress produced during diabetes, combating diabetic complication.

2. Materials and methods

2.1. Collection and preparation of
plant material

Fresh and young leaves of Annona cherimola were collected from
Nilgris district, TamilNadu and
authenticated. The leaves were thoroughly cleaned many times with distilled
water to remove dirt and contaminations, further shade dried at room
temperature. The shade dried leaves were coarsely powdered using electric
blender and stored in an air-tight container for further use and extraction 11.

2.2. Chemicals and solvents

All the chemicals, solvents were of analytical
grade, purchased from Fischer Inorganic and Aromatic Limited, Chennai, India. Alloxan
monohydrate was procured from SD Fine
Chem. Limited, Mumbai, India.

2.3. Mineral analysis

             Mineral contents like phosphorus, potassium, magnesium, manganese,
zinc, calcium, sulphur, copper, chromium, arsenic, nickel, aluminium, sodium,
ash was determined using Atomic adsorption spectrophotometer as per the
method suggested by the Association of Official Analytical Chemist 12.

2.4. Extraction and estimation of chlorophylls,
carotenoids and alpha amylase inhibition assay

            Chlorophyll-a, chlorophyll-b and
carotenoid content were analyzed using spectrophotometer method described by Arnon 13, alpha amylase inhibition assay was
determined by the protocol described earlier 14.

2.5. Experimental animal                    

           Adult male albino Wistar
rats weighing around 180-200g were procured from Animal House facility,
K.S.R.C.T.B.T and the experiment was conducted with approval from institutional
animal ethical board (KSRCT/BT/IAEC/2017/19). 
All the animals had free access to water and fed with standard feed
procured from Hindustan
Lever Ltd., Bangalore. India.

2.6. Induction of diabetes in rats

Single intraperitoneal injection of alloxan monohydrate (150 mg/kg b.w.)
was used to induce diabetes in overnight fasted rats. Blood Glucose level were
monitored after induction and diabetes was confirmed after 72 hrs. Using
One-touch select glucometer.  Fasting
blood glucose above 250 mg/dl in diabetic induced rats was alone taken for the
study 15.

2.7. Experimental
design

Rats were divided in to three groups,
six rats in each group. (i) diabetes induced rats treated with Annonacherimola(250 mg/kg b.w.) (ii) diabetes induced rats treated with Annonacherimola(500 mg/kg b.w.) (iii) diabetes induced rats treated with standard drug glibenclamide (10 mg/kg b.w.)
16.

All the experimental animals were maintained at ambient temperature
throughout the experimental period. Extracts and drug were supplemented using
an intragastric tube for 25 days.

Fasting blood glucose was
monitored for every week throughout the experiment.

2.8.
Infrared Thermal Imaging of Animals

Infrared camera (FLIR T420, FLIR Systems, Boston, MA,
USA) with wide temperature range of ?4 to 2192?F (?20 to 1700?C), FOV 25?× 19?, and thermal
sensitivity of 0.1?C was used in this study. As a baseline anterior to
posterior view of whole body, thermal images of all the rats were taken under
standard conditions at constant distance of (12 cm). Thermal images were taken
in normal rats, alloxan induced rats, during treatment and after treatment for
a period of five weeks

2.9 Sacrifice study

The animals were sacrificed by decapitation and the
blood was collected with and without anticoagulant. Tissue samples was instantly dissected out, washed,
dried and divided in to two parts one with buffered formalin and the other to
measure their antioxidant status.

2.10. Biochemical profiling and
antioxidant

Body weights
of all the experimental animals were monitored using digital weighing scale.
Blood glucose using GlucoChek glucose estimation kit (Aspen diagnostic (P) Ltd.
Delhi, India), Insulin was estimated using Radio immuno assay (RIA) kit
supplied by Linco research Inc, Stat diagnostic, Mumbai, India. C peptide was
estimated following the method of described earlier 17. Lipid profile was
estimated using standard kits purchased from TransAsia Bio Medical Limited, Mumbai,
India. Very low-density lipoprotein (VLDL-C) and low density lipoprotein (LDL-
C) 18.

Urea,
Uric acid and creatinine were estimated using standard reagent kits purchased
from Coral clinical systems, Goa, India. Alanine transaminase, alkaline phosphatase using standard kits
purchased from Transasia Bio Medical Limited, Mumbai, India.

SOD and catalase were assessed following the methods
described earlier 19, 20.

2.12. Statistical analysis

Values
are presented as means ± SEM. The statistical significance was evaluated by
one-way using the statistical software SPSS Version 17 (Origin Lab Corporation,
USA). The data were analyzed by one way analysis of variance (ANOVA) followed
by Tukey’s test.

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