Download Group III: - Leaves Extract of Populus deltoides (Low dose).

Evaluation of Antidiabetic Activity of Leaves Extract of
Populus deltoides
M. Pharm Dissertation Protocol Submitted to
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA,
BANGALORE
By
RAJEEV JHA B.Pharm,
Under the Guidance
Mrs. NAGALAKSHMI N.C. M.Pharm,
Asso.Professor
Dept. of Pharmacology
MALLIGE COLLEGE OF PHARMACY
#71 SILVEPURA, BANGALORE-560090
1
Annexure – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
Mr. Rajeev Jha
Name and Address of the
01
Candidate
S/o Gunanand Jha,
V.D.C:-Pipara-5,District:-Mahottary,Zone:Janakpur, Nepal
E-mail:- [email protected]
Mallige College Of Pharmacy
#71, Silvepura,
02
Name of the Institution
Chikkabanavara Post
Bangalore- 90
03
Course of the Study Branch
M.Pharm (Pharmacology)
04
Date of Admission to course
27/06/2012
05
Title of the Topic
Evaluation of Antidiabetic activity of Leaves
Extract of Populus deltoides
Brief resume of the intended work
6.1. Need for the Study
Enclosure-I
06
6.2. Review of the Literature
Enclosure – II
6.3. Objective of the Study
Enclosure – III
Materials and Methods
07
7.1. Source of data
Enclosure – IV
7.2. Methods of collection of data
Enclosure – V
7.3. Does the study require any
Investigations on animals?
Enclosure – VI
If yes give details
08
7.4. Has ethical clearance been
obtained from your institution
in case of 7.3.
Yes
List of References
Enclosure – VII
2
09
Signature of the Candidate
(Rajeev Jha)
10
Remarks of the Guide
Name and Designation of
(in Block Letters)
11.1. Guide
The present research work is original and not
published in any of the journals with best of my
knowledge upon extensive literature review. This
work will be carried out in the Pharmacology
laboratory by Mr. Rajeev Jha under my
supervision.
Mrs. NAGALAKSHMI N.C. M.Pharm,
Asso.Professor
Dept. of Pharmacology
Mallige College Of Pharmacy,
Bangalore, Karnataka.
11.2.Signature
11.3.Co-Guide (if any)
11
11.4.Signature
11.5. Head of the Department
Dr. SHIVAKUMAR SWAMY M. Pharm., Ph. D.,
Principal & HOD
Mallige College Of Pharmacy,
Bangalore, Karnataka
11.6.Signature
12
Remarks of the Principal
The present study is permitted to perform in the
Pharmacology laboratory of our institution and
the study protocol has been approved by IAEC.
12.1. Signature
(Dr. Shivakumar Swamy)
3
Enclosure-I
06. Brief resume of the intended work:
6.1. NEED FOR THE STUDY:
Introduction:
Insulin is the main hormone controlling intermediary metabolism. Its most striking acute
effect is to lower blood glucose. Reduced secretion of insulin often coupled with reduced
sensitivity to its action, insulin resistance which is closely related to obesity, causes diabetes
mellitus. This disease recognized since ancient times, is named for the production of copius
volumes of sugary urine.1
Glucose is the obligatory source of energy for the adult brain and physiological control of
blood glucose reflects the need to maintain adequate fuel supplies in the face of intermittent
food intake and variable metabolic demands. More fuel is made available by feeding than is
required immediately, and excess calories are stored as glycogen or fat and these are need to be
mobilised during fasting in a regulated manner. And the most important regulatory hormone is
insulin.1
Diabetes mellitus is a chronic metabolic disorder which occurs due to genetic and/or
acquired deficiency in production of insulin (which is a regulatory hormone of carbohydrates,
proteins and fat metabolism) by pancreas resulting in hyperglycemia which in turn damages the
many of body system in particular the blood vessels and nerves.2Type 1 diabetes mellitus is
characterized by loss of the insulin-producing beta cells of the islets of Langerhans in the
pancreas, leading to insulin deficiency. This type can be further classified as immune-mediated
or idiopathic. The majority of type 1 diabetes is the immune-mediated nature, in which beta cell
loss is a T-cell-mediated autoimmune attack.3
Both type 1 diabetes and type 2 diabetes can present with excessive thirst, excessive
urination, fatigue, weakness, weight loss, and blurred vision. Type 1 diabetes is also known as
insulin-dependent diabetes mellitus. There is severe deficiency or absence of insulin secretion
due to destruction of β-islet cells of the pancreas. There is evidence of an autoimmune
mechanism involving auto antibodies that destroy the β-cells. It develops quickly, often in
children, and symptoms can be striking. If not treated promptly can quickly result in critical
conditions, such as diabetic ketoacidosis, shock and death. Left untreated, all types of diabetes
can lead to serious long-term complications, such as kidney failure, blindness, serious skin
infections, gangrene, neuropathy, peripheral, birth defects, and stroke.4-5Patients with diabetes
4
have an increased incidence of atherosclerotic cardiovascular, peripheral arterial and
cerebrovascular disease. Hypertension and abnormalities of lipoprotein metabolism are often
found in people with diabetes.4
Quercetin has been found to be an inhibitor of the enzyme aldose reductase, which plays a
role in converting glucose (sugar) to sorbitol (a sugar alcohol) in the body. People with diabetes
develop secondary problems, such as neuropathy, retinopathy, diabetic cataracts, and
nephropathy because of sorbitol buildup in the body.6
The International Diabetes federation projects that by 2030 there will be 552 million
people with diabetes on a global scale. In the United States, the Centers for Disease Control
calculate that 25.8 million people (or 8.3% of the population) have diabetes and nearly 2 million
Americans develop diabetes each year. Thus, the burden of diabetes is enormous in terms of the
magnitude of the population affected.7Estimates for 2010 indicate that 285 million adults have
diabetes in the seven regions of the IDF. It is believed that by 2025, more than 75% of the world
population with diabetes will reside in developing countries and the countries with the largest
populations of adults with diabetes will include: India, China and the United States. India has
50.8 millions of people with diabetes in 2010 and expected to increase by 87 million by 2030.8
Natural compounds with Antidiabetic activity, in descending frequency of occurrence,
include complex carbohydrates, alkaloids, glycopeptides, terpenoids, peptides and amines,
steroids, flavonoids, lipids, coumarins, sulfur compounds, inorganic ions and others.9Herbal
medicines are considered to be less toxic and fewer side effects in comparison to synthetic
drugs.10. For many patients, traditional medicine sometime offers gentler means to manage such
diseases, to improve the quality of life of persons living with chronic diseases, as well as for the
ageing population.11 Antidiabetic plants have often been used by practitioners of herbal medicine
in treating individuals with non-insulin-dependent (type 2) diabetes. However, the use of such
herbs by type 1 (insulin-dependent) diabetics can be hazardous and requires that such patients
carefully monitor their blood sugar to prevent hypoglycemic and hyperglycemic episodes.
Populus deltoides is an Indian folk medicinal plant containing flavanoids in leaves used
in the treatment of rheumatism,gout,scurvy,bloodpurifier,anticancer and pain. It is also used as
diuretic, uric acid eliminator,antiinfectious in case of urinary problems, thinner of bronchial
secretions and tonic.12Recent studies on diabetes claims that the flavonoids possess antidiabetic
activity13-14 .
However the literature reveals no scientific data on antihyperglycemic effect of Populus
deltoides. In view of this, the present study is taken up to investigate the possible Antidiabetic
activity of Populus deltoides leaves extract in diabetic rats.
5
ENCLOSURE: II
6.2. REVIEW OF LITERATURE
Populus deltoides, the eastern cottonwood, is a cottonwood poplar native to North
America, growing throughout the eastern, central, and southwestern United States, the
southernmost part of eastern Canada, and northeastern Mexico.15A native tree of USA introduced
in India around 1950,is widely grown in all over northern India as an agroforestry tree.16
Populus deltoides is a large tree growing to 20–40 meters (67–130 feet) tall and with a
trunk up to 1.8 meters (5.9 ft) diameter, one of the largest North American hard woody
trees.17The leaves are large, deltoid (triangular), 4–10 cm (1.6–3.9 inches) long and 4–11 cm (1.6–
4.3 inches) broad with a truncated (flattened) base and a petiole 3–12 cm (1.2–4.7 inches) long.
The leaves is very coarsely toothed, the teeth are curved and gland tipped, and the petiole is flat;
they are dark green in the summer and turn yellow in the fall (but many cottonwoods in dry
locations drop their leaves early from the combination of drought and leaves rust, making their
fall color dull or absent). Due to the flat stem of the leaves, the leaves has the tendency to shake
from even the slightest breeze.17
Scientific classification:-
Kingdom:Subkingdom:Phylum:Division:Class:Order:Family:Genus:Species:-
Plantae
Tracheobionta
Anthophyta
Magnoliophyta
Dicotyledons
Salicales
Salicaceae
Populus
deltoides
Chemical Composition
Leaves contains:-
Salicortin,Salicin,Salicyl alcohol,Pyrocatechin,1-o-p-cumaroyl-b-D-glycoside,Populoside,asalicyloyl salicin,Chrysin-7-glucoside,Deltoidin,Tremulacin,Benzcatechin,Quercetin-3,3’dimethyethes,pyrocatechol,w-salicylol- salicin.18
Bark contains:-
The bark contains salicortin, salicin, salicyl alcohol, pyrocatechin, a-salicyloylsalicin,
grandidentatin, grandidentoside, populoside, trichocarposide, and 6-methyldihydroquercetin.18
Folk Medicine:The bark tincture of Populus deltoides has been used to treat rheumatism, gout, and scurvy
and infections of the chest, kidneys, and stomach. The buds have been used as a vulnerary and
pectoral. In Europe, the fresh flowers are steeped in cold water to purify the blood. Used by
Amerindians as a folk cancer remedy. Buds are used for earache, bronchitis, or cough. The
decocted rotten leaves used as an herbal bath for general body pain and the buds in poultices for
hip or lung pain. The roots are chewed to apply as a hemostat. The decocted buds is used for colds
6
and respiratory problems; heart ailments, sprains, and strains, and the root for backache, female
problems, metrorrhea, and weakness. 19
A bioassay-guided fractionation of the ethyl acetate extract from the twigs of the hybrid
poplar 'Neva', Populus nigra × Populus deltoides, led to the isolation of three flavonoids. These
compounds were further screened for their antimicrobial activity against plant pathogens,
including three bacteria (Pseudomonas lachrymans, Ralstonia solanacearum and Xanthomonas
vesicatoria) and one fungus (Magnaporthe oryzae). Compounds showed significant antibacterial
activity.20 Several reports suggested that Hot-water extraction of the Populus deltoides leaves led
to the identification of phenolic glycosides.21
Recently experiments conducted at Dehradun and two bioassay studies with aqueous
Extract of partially decomposed leaves of Populus deltoides showed the allelopathic effect on
wheat and retarded the germination and growth of wheat.22
Antioxidant Enzyme isoforms on Gels in Two Poplar Clones Differing in Sensitivity after
exposure to ozone. The effect of acute ozone fumigation on isozyme patterns of superoxide
dismutase , peroxidase and ascorbate peroxidase in mature and young leaves of two poplar clones,
contrasting in O3-sensitivity was analysed. Untreated leaves of both the O3-sensitive clone
Eridano of Populus deltoides×P.maximowiczii and the O3-resistant clone I-214 of P.euramericana
showed four distinct SOD isoforms. And concluded that the hybrids antioxidant enzyme activity
is developmentally regulated and greatly affected by acute O3 stress treatments and the different
enzymes activity displayed by the two poplar clones, especially for POD and APX isoformes,
could partly explain their distinct O3-sensitivity.23
Effects of pure and mixed plantations of Populus deltoides with Alnus glotinosa on growth
and soil properties were studied by planting them in five proportions. After 13 years, the effects of
species interactions on tree growth and nutrient concentration in live and senescent leaves and soil
properties were assessed. Within the framework of this experiment, it appeared that production
was maximized when these two species were grown together in the relative proportions of 30%
Populus deltoides and 70% Alnus glutinosa.24
Asymmetrical natural hybridization between Populus deltoides and P. balsamifera
investigated the direction of natural hybridization between two sympatric forest tree species
Populus
deltoides
and
Populus
balsamifera
using species-specific single nucleotide
polymorphism markers in both the nuclear and chloroplast genomes. All natural hybrid
individuals, identified from morphological traits, had nuclear alleles corresponding to both
parental species, while the chloroplast genotypes showed similarity to P. deltoides, indicating
asymmetrical hybridization with P. deltoides as the maternal and P. balsamifera as the paternal
7
donor species. This observed asymmetrical hybridization may be attributable to cytonuclear
interactions.25
A high affinity pyruvate decarboxylase is present in cottonwood leaves veins and petioles.
Leaves of Populus deltoides emit acetaldehyde under various conditions, and discovered that
leaves veins and petioles contain the enzyme pyruvate decarboxylase ,which produces
acetaldehyde as a product. We have purified leaves vein PDC 143-fold, and it appears to act as a
high affinity PDC, operating under the semi-aerobic conditions occurring in vascular bundles. The
conventional explanation for this is that ethanol formed in the roots is transported to the leaves
where it is converted to acetaldehyde by the alcohol dehydrogenase found in the leaves. It is
possible that acetaldehyde could also be formed in leaves by action of pyruvate decarboxylase
(PDC), an enzyme with an uncertain metabolic role, which has been detected, but not
characterized, in cottonwood leaves. Leaves PDC is present in leaves veins and petioles, as well
as in non-vein tissues. Veins and petioles contained measurable pyruvate concentrations in the
range of 2mM.26
8
ENCLOSURE: III
6.3.
OBJECTIVES OF THE STUDY
 Collection, authentication and extraction of leaves of Populus deltoides.
 Qualitative estimation of phytoconstituents.

To determine the acute toxicity of crude extract.
 To Evaluate the Antidiabetic activity in experimental model.
-Normohypoglycemic model in rats
-Alloxan induced diabetes in rats
 To confirm the therapeutic efficacy of crude extract by estimating biochemical parameters
and
histology.
9
ENCLOSURE: IV
07. MATERIALS AND METHODS
7.1. SOURCE OF DATA:
The research work is aimed to generate data from experiments to be conducted at
pharmacology laboratory of our institution.
Animals
Adult male wistar rats and Swiss albino mice will be used for this purpose. The animal
will be obtained from animal house of Mallige College of Pharmacy. Animal clearance will be
obtained from institutional animal ethical committee for experimental purpose. They will be
maintained under laboratory condition with controlled environment of temperature, humidity as
per committee for the purpose of control and supervision of experiments on animal (CPCSEA)
guidelines. They will be provided with standard diet and water ad libitum.
Drugs
Glipizide received as a gift sample from manufacturers will be used in this study, all other
chemicals used will be of analytical grade.
The experiments involve the following steps:

Collection and authentication of the leaves of Populus deltoides.

Extraction of leaves of Populus deltoides.

Qualitative analysis of phytoconstituents and determination of acute toxicity fixed dose
method (OECD guide line no. 420)
27
of CPCSEA will be adapted to perform acute
toxicity of the Extract.

Evaluation of the extract of leaves of Populus deltoides for the Antidiabetic activity in
experimental animals.
10
ENCLOSURE-V
7.2. METHOD OF COLLECTION OF DATA:
1. Collection of raw material:
Leaves of Populus deltoides will be collected from the gardens of Dehradun, Uttrakhand.
The sample will be identified and authenticated by the botanist. Fresh leaves will be cleaned and
shade dried at room temperature.
2.
Extraction of Populus deltoides:
The powdered materials will be extracted with ethyl alcohol by Soxhlet’s extraction
method. The Extract will be concentrated using rotary flash evaporator and percentage yield of the
same will be recorded. Finally the extract will be used for qualitative phytochemical analysis and
to evaluate Antidiabetic activity.
3.Qualitative Phytochemical Analysis:
The crude Extract thus obtained will be subjected for preliminary phytochemical analysis
using standard procedures described in the literature.
4.Screening of Antidiabetic activity:28
A. Effect of alcoholic extract in normoglycemic rats (NG)
Male albino strain rats weighing (160–200 g), aged 8-14 weeks older are to be equally
divided into four groups of six rats each .Animals in the control group will receive normal saline
(orally). Group II-standard group will be administered reference drug Glipizide 5 mg/kg p.o, The
test group of animals will be treated with the extract of Populus deltoides leaves at a therapeutic
dose of (Low dose) and (High dose)mg/kg b.w. Blood samples will be collected at 0 day, 7th day
and 14th day, 21st day, 28
th
day after the administration. Blood samples will be collected from
tail vein and blood glucose will be estimated. 29
11
Group I: -
Control group
Group II
Standard group – Glipizide (5 mg/kg, p.o)
Group III: -
Leaves Extract of Populus deltoides (Low dose).
Group IV: -
Leaves Extract of Populus deltoides (High dose).
Data to be recorded:-
Effect of extract on Normoglycemia:
Group
Treatment
Fasting blood glucose level
0 days
7th day
14th day
21st day
28th day
I
II
III
IV
B.Effect of the Populus deltoides Leaves Extract on Alloxan Induced Diabetic
Rats
Male Wistar rats (150-200g) will be made diabetic by a single i.p injection of 120mg/kg
body weight of Alloxan monohydrate in sterile normal saline. The rats will be maintained on 5 %
glucose solution for next 24h to prevent Alloxan induced hypoglycemia. Five days later blood
samples will be drawn from retro orbital pouch and glucose levels will be determined to confirm
the development of diabetes (>300mg/dl). The animals with blood glucose level of 250-400 mg/dl
will be selected for the study.
12
The diabetic rats will be selected and divided into four groups, each containing six animals.
Group I-
Diabetic control
Group II-
Diabetic animals will be treated daily with Glipizide (5 mg/kg,p.o) 30
Group III-
Diabetic animals will be treated daily with Populus deltoides leaves
Extract(Low dose).
Group IV-
Diabetic animals will be treated daily with Populus deltoides leaves Extract
(High dose).
Data to be recorded:Effect of Extract on diabetic rats:
Group
Treatment
Fasting blood glucose level
0 day
7th day
14th day
21st day
28th day
I
II
III
IV
13
C. Evaluation of effect of Extract on biochemical parameters and
histopathological changes.
1. Blood analysis
The parameters like cholesterol, HDL-cholesterol, triglycerides, LDL-C and VLDL will be
determined in serum using Auto analyzer. Liver profile parameters like total protein, albumin,
alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT),
and bilirubin will be determined in serum using Auto analyzer. Blood urea nitrogen, Creatinine
and uric acid will be determined using auto analyzer. Atherogenic index will be calculated by
using the following formula. 31
Total cholesterol − HDL-C
Atherogenic index =
X 100
HDL-C
2. Organ weight Analysis
At the end of the 28th day Brain, Spleen, Pancreas, Heart, Liver and Kidney will be
carefully dissected out and organs of all the animals will be examined macroscopically. The
positions, shape, sizes and colors of the internal organs will visually observe for signs of gross
lesions.
3. Histopathological investigation:Tissue Processing:
Pancreas, Heart, Liver and kidney tissues will be placed in 10% formalin (diluted to 10%
with 20 mm phosphate buffer pH 7.4) for 1 hr to rectify shrinkage due to high concentration of
formalin. The tissues will be dehydrated by ascending grades of isopropyl alcohol by immersing
in 80% isopropanol overnight and 100% isopropyl alcohol for 1 hour. The dehydrated tissues will
be cleared in two changes of xylene, 1 hour each. The wax impregnated tissues will be embedded
in paraffin blocks using the same grade wax. The paraffin blocks will be mounted and cut with
rotary microtome at 3 micron thickness. The sections will be floated on a tissue floatation bath at
40 °C and taken on glass slides and smeared with equal parts of egg albumin and glycerol. The
sections will be melted in an incubator at 60 °C and after 5 min the sections will be allowed to
cool.
14
Tissue Staining
The sections will be deparaffinised by immersing in xylene for 10 min in horizontal
staining jar. The deparaffinised sections will be washed in 100% isopropyl alcohol and stained in
Ehrlich’s hematoxylin for 8 min in horizontal staining jar. After staining in hematoxylin, the
sections will be washed in tap water and dipped in acid alcohol to remove excess stain (8.3% HCl
in 70% alcohol). The sections will be placed in running tap water for 10 min for blueing (slow
alkalization). The sections will be counter stained in 1% aqueous eosin (1 gm. in 100 ml tap
water) for 1 min and the excess stain will be washed in tap water and the sections will be allowed
to dry. Complete dehydration of stained sections will ensure by placing the sections in the
incubator at 60°C for 5 min. When the sections will be cooled, they will be mounted in DPX
mount having the optical index of glass (the sections will be wetted in xylene and inverted on to
the mount and placed on the cover slip). The architecture will be observed low power objective
under microscope. The cell injury and over aspects will be observed under high power dry
objective. Light microscopic examination of the sections will be carried out and micrographs will
be produced using Vanox-T Olympus photographing microscope. The histopathological
examinations will be reviewed by the pathologist.
4. Statistical Analysis:
The data obtained from the study will be subjected for statistical analysis using One-way
ANOVA followed by Turkey Kramer Multiple Comparison Test to assess the statistical
significance of the results.
5. Work plan details:
Total duration for the completion of proposed research work may be ten months.
1. Collection of plant materials including authentication
- One month.
2. Duration of experimentation on animals including
- Five months.
preparation of crude Extract.
3. Literature collection
- Two months.
4. Dissertation writing and communication
- Two months.
of research papers.
15
ENCLOSURE-VI
7.3 Does the study require any investigation or interventions to be conducted
on patients or other humans and animals? If so please describe briefly.
The proposed study requires the investigation on albino rats of either sex (Wistar Strain)
weighing 150 - 200 gm for the Antidiabetic activity. Whereas albino mice of Swiss Strain will
be utilized for the acute toxicity study.
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
The present study protocol is approved from Institutional Animal Ethics Committee.
16
Enclosure VII
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Picard S, Chenault J, Augustin S, Venot C. Isolation of a New Phenolic Compound from Leaves of
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Seyed Abdollah Mousavi Koupar, Seyed Mohsen Hosseini, Masoud Tabari, Alireza Modirrahmati,
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Hamzeh, Mona, Sawchyn, Christina, Périnet, Pierre, et al. Asymmetrical natural hybridization
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18