Hypoglycemic effect of Hibiscus rosa sinensis L.

flower extract
in Alloxan induced hyperglycemic rats
--------------------------------------------------------

A Research Paper Presented to the Faculty of Davao Medical School Foundation, Inc.
College of Medicine, Medical School
Drive, Bajada, Davao City
_______________________
In Partial Fulfillment of the Requirements
In Pharmacology
_______________________

Submitted by:
Nadera, Milliza Jane
Nasiluan, Joriel Neco
Nawal, Norhaiah
Naparan, Fiel
Ninal, Kryscel Czarinah
Nuval, Clarence
Ong, April Joy
Orongan, Rachel
Pacatang, Christine
Pacatang, Jenelle
Palaca, Deo Paolo
Pama, Yvonne
Pantojan, Marie
Parada, Irvin
Santos, Jose Rigel
Group 5 Med 2B

Chapter 1
Introduction
Background of the Study
People who are diagnosed with having Diabetes type 2 can achieve and maintain a
normal blood sugar level through diet and exercise but some, despite the change in lifestyle, still
need further interventions. Drug therapy is an effective mean of lowering the blood sugar level
granted that the patient is compliant. One of the drugs prescribed first when patient is diagnosed
with diabetes type 2 is Metformin (Glucophage, Glumetza). This drug improves the sensitivity of
the body tissue to insulin so that the body may take in insulin more effectively. Another drug
with the similar action as Metformin is Thiazolidinediones but this drug is linked to serious side
effects such as weight gain, increased risk of heart failure and fractures. Sulfonylureas and
Meglitinides act by helping the body secrete more insulin but unlike the former, the drug action
of the latter is much faster, they do not stay in the body very long and they have decrease risk of
producing complications. Possible side effects of Sulfonylureas are low blood sugar and weight
gain. As for the Meglitinides, side effects include low blood sugar levels. DPP-4 inhibitors and
GLP-1 receptor agonists are being used also to lower down blood sugar levels but are not as
effective. SGLT2 inhibitors are the newest diabetes drug in the market. They prevent
reabsorption of glucose in the blood by the kidneys and facilitating excretion of the glucose
through the urine. Another effective therapy but is quite invasive is Insulin therapy. This
intervention is used as the last resort to control blood sugar levels.
Drug interventions are popular mediums nowadays to lower down blood sugar levels but
before the discovery of hypoglycaemic drugs, insulin injections, and other artificial methods

used to lower down blood glucose, healers in the past (and some still practice it

today)

relied

on the use of herbs. In the modern times such as today, a lot of medicinal plants were made
subject for experiments with the goal of discovering the hidden benefits they possess. Among the
possible benefits, researchers were relentless to discover what plants have hypoglycaemic
effects.
Hibiscus rosa sinensis or also commonly known here in the Philippines as Gumamela
have a wide range of beneficial effects and included with its effects is hypoglycaemia. This study
aims to measure the hypoglycaemic effect of Hibiscus rosa sinensi.
Objectives of the study
The study aims to determine the hypoglycemic effect of Hibiscus rosa sinensis flower extract
containing flavonoid to the blood sugar of laboratory induced diabetes albino mice.

Specifically, the study aims:

1. To extract flavonoid from the Hibiscus rosa sinensis flower
2. To determine if the flavonoid of Hibiscus rosa sinensis flower has hypoglycemic effect
3. To determine the baseline blood sugar of the lab induced diabetic mice
4. To determine the blood sugar of the mice after the experiment using the flavonoid and a
hypoglycemic drug
5. To determine if there is significant difference between the hypoglycemic effect of flavonoid
and the hypoglycemic drug

Hypothesis

There is a significant relationship between the gumamela plant extract to the blood glucose levels
of a rat
Null Hypothesis
There is no significant relationship between the gumamela plant extract to the blood glucose
levels of a rat
Significance of the Study
This research is significant to the following:
1

Society. This study aims to provide the society with a possible solution to diabetes that is

natural, widely available and has no toxic effect.

Researchers. This study allows the researcher to understand and experience the phases of
making medicine. Allowing them to apply their knowledge in pharmacology and use their

knowledge in developing a naturally occurring cure for diabetes.

Future Researchers. This study will be a good start for the quest for a natural cure for
diabetes that could be used to the next phase of drug development.
Scope and Limitations
This study was limited within the school laboratory of DMSF. The subjects are albino rats

which were weighed and measured prior to the experiment. Only the flower of the gumamela
will be used in the study. The study will focused on the effect of gumamela extract in lowering
the subjects blood glucose levels within a 1 month observation period.
THEORETICAL FRAMEWORK
Flavonoids beneficial effects in relation to diabetes mellitus involves its capacity to
avoid glucose absorption or to improve glucose tolerance through its stimulation of glucose

uptake in peripheral tissues, regulation of the expression of the rate-limiting enzymes involve in
carbohydrate metabolism pathway and acting as insulin secretagogues or mimetics, probably, by
influencing the pleiotropic mechanisms of insulin signaling (Cazarolli, 2008).
Several studies have proven the effect of flavonoids on blood glucose levels in mice. A
study by Zhou in 2009 concluded that flavonoids extracted from lotus should be evaluated as a
candidate for future studies on diabetes mellitus. The effect of flavonoids hesperidin and naringin
was investigated to significantly increase the glucokinase mRNA level in type-2 diabetic mice
(Jung, 2005).

CONCEPTUAL FRAMEWORK
Independent Variable
Gumamela Flower Extract

Dependent Variable
Blood glucose levels

Chapter 2
Review of Related Literature
Hibiscus rosa-sinensis Linn.
Common Name: Gumamela, China Rose
Botanical Name: Hibiscus rosa-sinensis L.
Kingdom: Plantae
Subkingdom: Tracheobionta (Vascular plants)
Super division: Spermatophyta (Seed plants)
Division: Magnoliophyta (Flowering plants)

Class: Magnoliopsida (Dicotyledons)

Subclass: Dilleniidae
Order: Malvales
Family: Malvaceae
Genus: Hibiscus
Species: Hibiscus rosa-sinensis

Plant Description
Hibiscus rosa-sinensis are native to tropical and subtropical regions such as Asia. With its
wide ranging colors, it is used as an ornamental plant grown in almost all houses. Different parts
of the plant have been studied; these include the roots, leaves and flowers. The roots are
cylindrical measuring approximately 5-15cm in length and 2cm in diameter, with a sweet and
mucilaginous taste. The leaves are also mucilaginous, shaped as ovate or ovate-lanceolate. The
flowers are pentamerous with corolla consisting of five petals, usually red in color and about 3
inches long (Kumar, 2012). Variations in color are widespread.

Constituents
The edible portion of the flower (61.6%) was reported to have the following nutrient
composition (per 100 g): moisture 89.8%, nitrogen 0.064%, fat 0.36%, crude fiber 1.56%,
calcium 4.04mg, phosphorous 26.68mg, iron 1.69mg, thiamin 0.031mg, riboflavin 0.048mg,
niacin 0.61mg, and ascorbic acid 4.16mg.
Petals of Hibiscus rosa sinensis were reported to contain quercetin-3-di-O-B-Dglucoside;

quercetin-3-7-di-O-B-D-glucoside;

quercetin-3-O-B-D-sophorotrioside;

and

kaempferol and kaempferol-3-O-B-D-glucoside. The major anthrocyanin contained in the red

flowers was cyaniding-3-sophoroside. Red-petalled varieties were found to have more number of
anthrocyanin bands compared to that observed in yellow yellow orange varieties. The varieties
in the different colored groups differed in the quantitative distribution of anthrocyanins,
leucoanthrocyanins, flavonol and carotenoids. Flavonoid aglycones found in the flowers (per g
fresh tissues) included quercetin 7 mg, and cyaniding 36 mg.
The flowers were also reported to contain the following flavones: quercetin-3,5diglucoside; quercetin-3,7-diglucoside; cyaniding-3,5-diglucoside, and cyaniding-3-sophoroside
from the deep yellow and white flowers and from ivoery white flowers is kaempferol-3xylosylglucoside.
Five compounds were extracted from the chloroform extract of the flower, n-nonacosa13-one, n-triacontane, n-dotetracontane, n-nonacosan-4-ol-18-one, and n-hentriacontan-4-one10-ol; and five compounds were isolated from the hydroalcoholic flower extract, n-docosane,
henicos-11-ene-9-one, stigmast-5-ene-3B, 4a-diol, stigmast-5-ene-3B-benozyloxy-12B-ol, and npentacos-4-en-3-one-18, 23-diol.
The following chemicals were isolated from the ethanol extract of the flowers:
hexadecanoic acid, hexanedioic acid and squalene. Polyphenolic compound isolated from the
flowers included quercetin-7-O-galactoside; kaempferol-7-O-16-O-p-hydroxybenzoyl-B-Dglucosyl-(1->6)-B-D-glucopyranoside, and scutellarein-6-O-a-L-rhamnopyranoside-8-C-B-Dglucopyranoside. The flowers were reported to contain cyclopeptide alkaloids as well (Lim).
Leaves and stems contain -sitosterol, stigmasterol, taraxeryl acetate and three
cyclopropane compounds and their derivatives. Flowers contain cyanidin diglucoside, flavonoids
and vitamins, thiamine, riboflavin, niacin and ascorbic acid (Ghani, 2003). Quercetin-3-

diglucoside, 3,7-diglucoside, cyanidin- 3,5-diglucoside and cyanidin-3-sophoroside-5- glucoside

have been isolated from deep yellow flowers; all above compounds and kaempferol-3xylosylglucoside have been isolated from ovary white flowers (Rastogi & Mehrotra, 1993).
The phytochemical analysis of Hibiscus rosasinensis leaves extract has a positive
response for the presence of alkaloids, flavonoids, tannins, saponins, steroids, and terpenoids.
Phytochemicals are naturally occurring biologically active chemical compounds in plants.
Phytochemicals are known to have protective and disease preventing properties particularly free
radical mediated diseases such as diabetes, cancer etc. Certain phytochemicals are almost
structurally identical to insulin and act as an insulin mimic that helps in the remedy of diabetes.
Most plants with antidiabetic properties have been found to contain secondary metabolites such
as glycosides, alkaloids and flavonoids33 . It has been shown that many plants efficient
antioxidant properties owing to their phenolic constituents. It has been reported that Hibiscus
rosasinensis leaves contain bioactive compouds such as anthocyanins, and flavonoids alkaloids
and vitamins. Hence, compounds which can scavenge the excess of free radicals formed or that
inhibit their production are of wide therapeutic value. The presence of biologically active
ingredients in the extract may account for the pharmacological activity (Anandhi, 2013).

Uses
It is considered as an emollient, emmenagogue, anodyne, expectorant, refrigerant and can act as
anti-infectious, anthelmintic, anti-inflammatory, diuretic, antipyretic, hypotensive, and
antispasmodic.

Flavonoid

Flavonoids beneficial effects in relation to diabetes mellitus involves its capacity to

avoid glucose absorption or to improve glucose tolerance through its stimulation of glucose
uptake in peripheral tissues, regulation of the expression of the rate-limiting enzymes involve in
carbohydrate metabolism pathway and acting as insulin secretagogues or mimetics, probably, by
influencing the pleiotropic mechanisms of insulin signaling (Cazarolli, 2008).
Several studies have proven the effect of flavonoids on blood glucose levels in mice. A
study by Zhou in 2009 concluded that flavonoids extracted from lotus should be evaluated as a
candidate for future studies on diabetes mellitus. The effect of flavonoids hesperidin and naringin
was investigated to significantly increase the glucokinase mRNA level in type-2 diabetic mice
(Jung, 2005).
Diabetes Mellitus
Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from
defects in insulin secretion, insulin action, or both. The chronic hyperglycemia of diabetes is
associated with long-term damage, dysfunction, and failure of various organs, especially the
eyes, kidneys, nerves, heart, and blood vessels (American Diabetes Association, 2004).

Criteria for the Diagnosis of Diabetes Mellitus

Glibenclamide
Mechanism of Action
The drug works by binding to and activating the ATP-sensitive potassium channels
(KATP) inhibitory regulatory subunit sulfonylurea receptor 1 (SUR1) (Serrano-Martin et al,
2006) in pancreatic beta cells. This inhibition causes cell membrane depolarization, opening
voltage-dependent calcium channels. This results in an increase in intracellular calcium in the
beta cell and subsequent stimulation of insulin release. Its action in diabetic patients is therefore
dependent upon the presence of functioning -cells in the islets of Langerhans.

After a cerebral ischemic insult, the bloodbrain barrier is broken and glibenclamide can
reach the central nervous system. Glibenclamide has been shown to bind more efficiently to the
ischemic hemisphere. Moreover, under ischemic conditions SUR1, the regulatory subunit of the
KATP- and the NCCa-ATP-channels, is expressed in neurons, astrocytes, oligodendrocytes,
endothelial cells (Simard et al, 2012) and by reactive microglia (Ortega et al, 2012).

Dose, Route, & Mode of Administration

Oral route, 5mg/kg of body weight
For substances being tested for safety, oral dosing mimics the most commonly used mode
of administration of substances to humans. When placing substances directly into the mouth, it is
important to ensure that tablets or gelatin capsules containing test material are placed far back in
the mouth and that the animal swallows, to ensure receipt of the full dose. The number and size
of capsules or tablets administered should be proportional to the size of the animal being dosed,

to minimize regurgitation. Gavage (esophageal or gastric) is often used in research settings,

instead of mixing substances in water or food, to ensure precise and accurate dosing of animals.
Using the smallest volume possible is recommended for the oral route of administration,
optimally 5 mL/kg for all species (Brown et al, 2000).
Related Studies
In a study by Sachdewa A. et al (2001), 250 mg/kg Hibiscus rosa sinensis alcoholic leaf
extract administered orally everyday for seven days showed significant improvements in the
study animals ability to use the external glucose load, with an average of 33% blood glucose
lowering effect. In another study (Sachdewa, 2000), the same administration significantly
improve glucose tolerance of rats.
In an article published in an Indian national newspaper, Narendran (2013) states that
there is an antidiabetic properties of hibiscus rosa sinensis. The Department of Biochemistry of
the University of Kerala induced diabetes in rats using a drug. The rats were given extract of the
flower petals and observed. The effect of the petal extract was compared with the effect of a
standard drug, metformin, used for controlling diabetes. It was found that not only was hibiscus
extract effective as an anti-diabetic agent but the overall effect was much better than metformin
treatment. The histopathological analysis of the hearts of the animals also showed that the petal
extract had a protective effect.
Non-obese diabetic mice were randomly divided into 6 groups and were subjected to the
effects of Hibiscus rosa sinensis leaves on the blood glucose. The experiment was conducted for
four weeks. Serum glucose levels were estimated using a glucometer every week in every group.
The result showed decrease in blood glucose levels in the group that used the extract of Hibiscus
rosa sinensis. (Moqbel, F.,et. al, 2011)

A study conducted by Sundarrajan et. al in 2011 aimed to determine the antidiabetic

effect of Hibiscus cannabinus leaf extracts on Albino wistar rats of male sex weighing 200-250
gms. Streptozotocin was injected intra-peritonially to induce the increase in blood glucose
levels. The experimental design were composed of 4 groups, each group contained 6 mice.
Group 1 was the negative control. Group 2 was the diabetic control group. Group 3 was
composed of diabetic rats receiving methanolic extract of Hibiscus cannabinus. Group 4
contained diabetic reats administered with Glibenclamide. Blood extraction was done on the
orbital sinus of the eye on the 5th day, 15th day and 20th day post induction. Blood glucose was
measured by GOD-POD kit method. The statistical tools used were ANOVA followed by
Dunnets t test. P<0.05 were considered significant. It was eventually found out that methanolic
extract from Hibiscus cannabinus leaf manifested hypoglycemic activities.
A study conducted by Kumar et. al in 2010 sought to find the hypolipidemic activity of
Hibiscus tiliaceus (L.) flowers extracts. Streptozotocin was used to induce the increase in blood
glucose levels. The wister rats used weighed 150-250g. After induction of hyperglycemia, the
rats were divided into 5 groups with 6 rats per group. The extract was administered once a day
for 21 days. Group 1 was the normal and healthy group. Group 2 was the diabetic control group.
Group 3 was composed of diabetic rats receiving 250 mg of the plant extract. Group 4 was
composed of diabetic rats receiving 500 mg of the plant extract. Group 5 contained diabetic reats
administered with Glibenclamide. The statistical tool used was Student's t-test in order to
determine significance between the groups. A p < 0.05 was considered to be significant.
A study done by Mandade &

Sreenivas in 2011 aimed to determine the antidiabetic

effects of the Ethanolic Extract of Hibiscus rosasinensis L. on Streptozotocin-Induced Diabetic

Rats. Possible Morphologic Changes in the Liver and Kidney were also observed. The aerial

part of Hibiscus rosa sinensis was used in creating the aqueous-ethanol extract. Phytochemical
studies were done on the plant extract in order to check if sterols, carbohydrates and glycosides
were present. The animals used in this study are Wistar albino rats of either sex weighing
between 60-80 g. Streptozotocin was used to induce hyperglycemia. The extract was
administered the rats for 15 days. After administration, the livers and kidneys of the rats were
taken in order to be examined by a pathologist. The study concluded that H. rosa sinensis aerial
extract was able to lower the blood glucose of the test animals. An increase in the plasma insulin
and C-peptide levels were also observed. It was also concluded that the extract can protect liver
and Kidney from damage due to diabetes.
A study by Sharma et. al in 2007 looked into the hypoglycemic and hypolipidemic effect
of Aegle marmelos (L.) leaf extract on Streptozotocin induced diabetic mice. The leaves were
lyophilized into powder. The animals used in the study were 15 matured albino mice five to six
weeks of age, weighing 35 g. Streptozotocin was used to induce hyperglycemia. The study found
out that the Aegle marmelos leaf extract could have hypoglycemic, anti-hyperglycemic and
hypolipidemic properties.
A study conducted by Mishra et. al in 2013 aimed to compare the antidiabetic profile of
Ayurvedic herbo-mineral formulation and its constituents on normal and streptozotocin-induced
diabetic rats. The composition of the Ayurvedic polyherbo-mineral preparation are: Gymnema
sylvestre, Momordica charantia, Syzygium cumini, Azadirachta indica, Withania somnifera,
Hibiscus rosa-sinensis, Ocimum sanctum, Pterocarpus marsupium and Coccinia indica and a
herbomineral substance Shilajeet. Streptozotocin was used to induce the hyperglycemia.
Results of the study showed that the levels of blood glucose and glycosylated hemoglobin
(HbA1c) were lowered .

Selvaraj et. al in 2013 conducted a study about how fractions of Tinospora cordifolia
stem extract can demonstrate insulin secretion in Diabetes Induced Wistar Rats. Streptozotocin
was used to induce hyperglycemia. Two fractions of the alcoholic extract were used, the Ethyl
acetate soluble fraction (F1) and basic fraction (F4). Results showed that both fractions showed
significant hypoglycemic effect. But among the 2 fractions, the F4 was the one that was able to
enhance insulin secretion.
A study done by Delaviz et. al in 2011 evaluated the Anti-diabetic eff ects of an alcoholic
extract of Juglans regia in an animal model. Four groups of animals were employed. Normal diet
was given to the group 1 animals. Streptozotocin was administered to groups 2, 3 and 4. Groups
3 and 4 were treated with the J. regia leaf extract in dosages of 200 and 400 mg/kg body weight
for 28 days. The results showed that there were significant decreases in in blood glucose,
glycosylated hemoglobin, LDL, triglyceride, and total cholesterol. It was also noted that there
was a significant increase in the insulin and HDL levels.
Dr. Richard A. Anderson and his colleagues at the Human Nutrition Research Center of
the U.S. Department of Agriculture screened extracts of a number of commonly consumed plants
to see how well they could mimic the effects of insulin, a protein hormone that is responsible for
regulating our blood sugar levels. From a selection of 49 culinary and medicinal plants, they
found in laboratory tests that cinnamon was far more effective than any other plant in fulfilling
insulins appointed role. Dr. Andersons group established that the active component in cinnamon
responsible

for

its

insulin-like

activity

is

water-soluble

chemical

compound

called methylhydroxychalcone polymer, or MHCP. They found that MHCP was highly effective,
providing essentially the same biological activity as insulin itself. It was effective not only in
increasing the uptake of glucose (blood sugar) by cells, but also of stimulating the synthesis of

glycogen, a polymeric form of glucose that is stored primarily in the liver and muscle tissues for
use at times of peak energy demand, such as exercise. And MHCP turned out to be synergistic
with insulin in these actions, providing a net effect greater than the sum of its parts.
A study was conducted on G. sylvestre leaves extract. The study focused on the
hypoglycemic effets of G. sylvestre . The present study demonstrates the effect of G.
sylvestre leaves extract (18 mg/kg body weight) on several etiological factors of STZ-diabetic
rats including hyperglycemia, hypoinsulinemia, hyperlipidemia and oxidative stress at the same
time. This study showed a significant decrease in plasma glucose level (20.20%) in diabetic rats
treated with G. sylvestre leaves extract (18 mg/kg body weight) compared to that of untreated
diabetic rats. Significant increase in diabetic rats insulin level post treated with G.
sylvestre leaves extract confirmed the result. Moreover, glucose level might be decreased in
treated diabetic rats as a result of decreasing gluconeogenesis that was indicated by low levels of
ALT and AST in treated diabetic rats compared to untreated diabetic rats
Rajasekaran et al in 2006 studied the oral administration of Aloe vera gel extract at a
dose of 300 mg/kg body weight per day to STZ-induced diabetic rats for a period of 21 days.
They found that it resulted in a significant reduction in fasting blood glucose, hepatic
transaminases (AST and ALT), plasma and tissue (liver) cholesterol, triglycerides, free fatty
acids and phospholipids and a significant improvement in plasma insulin. In addition, the
decreased plasma levels of high-density lipoprotein-cholesterol and increased plasma levels of
low-density lipoprotein- and very low-density lipoprotein-cholesterol in diabetic rats were
restored to near normal levels following treatment with the extract. Furthermore, they analyzed
the fatty acid composition of the liver and kidney and found that the altered fatty acid

composition in the liver and kidney of diabetic rats was restored following treatment with the
extract. They recommended use of Aloe vera as an anti-diabetic agent.

Chapter 3
Methodology
Extraction of Plant Sample:
1. The chopped sample was placed in a 500mL Erlenmeyer flask weighing 500 g.
2. Sufficient 95% ethanol is poured in. This is done to macerate the solution.
3. The mixture is left for 48 hours.
4. The product was filtered and discarded the remaining plant materials.
5. The extract is subjected to rotatory evaporization.
6. The concentrated extract was obtained.
Administration of Extract:
All rats are induced with Alloxan. The rats will be grouped into three comprising of six rats:
Group 1: Negative control, diabetic mice with water.
Group 2: Diabetic mice treated with Hibiscus rosasinensis corolla ethanol extract (250 mg/kg).
Group 3: Positive control, diabetic mice treated with Glibenclamide (5mg/kg).
Before giving the extract and the drug, each mouse must be weigh to calculate the right dosage
and to observe any untoward effects.
Method of Administration of Hibiscus rosasinensis
1. Prepare Hibiscus rosa sinensis extract using an empty syringe with the calculated dose of
250mg/kg.
2. Hold the mouse properly and place the nozzle of the syringe into its mouth carefully and
slowly allowing the mouse to swallow.

3. Repeat steps 1 and 2 to the other mouse using different syringe.

4. Observe the mice for 30mins for any untoward effect.
5. Record.

Method of Administration of Glibenclamide

1. Disperse the glibenclamide tablet in water.
2. Prepare the dissolved drug using an empty syringe with the calculated dose of 5mg/kg.
3. Hold the mouse properly and place the nozzle of the syringe into its mouth carefully and
slowly allowing the mouse to swallow.
4. Repeat steps 1 to 3 to other mouse using different syringe.
5. Observe the mouse for 30mins for any adverse effect.
6. Record.
Alloxan- Inducer of hyperglycemia
Alloxan (2,4,5,6-tetraoxypyrimidine; 5,6-dioxyuracil) has been commonly utilized to induce
hyperglycemia to animal models such as rats, mice, and rabbits. According to Szkudelski , T
(2001), one of the actions of alloxan on the pancreas is the rapid uptake by insulin-secreting
cells.
Dosage
According to Szkudelski, T (2001), the dosage that should be given rat models to induce
hyperglycemia depends on the rats weight, nutritional status and diet. The most frequent dose
used to induce diabetes in rats intravenously is 65mg/kg b.w. While intraperitoneally or

subcutaneously, the effective dose must be 2-3 times higher or it may be insufficient if given
below 150mg/kg.(Szkudelski, T, 2001)
Route of administration
Alloxan can be effective when given parenterally: intravenously, intraperitoneally, or
subcutaneously. (Szkudelski, T ,2001)
Mechanism of action
Alloxan-induced diabetes has been commonly used for experimental model of insulin dependent
diabetes mellitus. In an article written by Rohilla, A and Ali, S (2001), they explained the
mechanism of action alloxan on rat models. It is demonstrated that alloxan can induce a sudden
rise of insulin-secreting cells with or without glucose. After a release of insulin, it will be
followed by a complete inhibition of the islets to respond to high levels of glucose. One of the
most important actions of alloxan in the pancreas is the rapid uptake by pancreatic beta cells that
gave an important feature determining alloxan diabetogenicity. Reduction process follows due to
the presence of different reducing agents such as reduced glutathione (GSH), cysteine, ascorbate
and protein-bound sulfhydryl (-SH) groups. In the sugar binding site of glucokinase, alloxan
reacts with two-SH groups resulting in the formation of disulfide bond and inactivation of the
enzyme. The main product of alloxan reduction is dialuric acid that is then re-oxidized back to
alloxan to establish a redox cycle for the generation of reactive oxygen species (ROS) and
superoxide radicals. These superioxide radicals release ferric ions from ferritin and reduce them
to ferrous and ferric ions. Also, to yield hydrogen peroxide, superoxide under go dismutation ,in
the presence of superoxide dismutase. As a result in the presence of ferrous and H2O2 according

to Fenton reaction, highly reactive hydroxyl radical are formed. In addition, one of the effect of
the ROS to the DNA of pancreatic cells is the fragmentation in the beta cells, that causes DNA
damage and stimulates ADP-ribosylation, a process in DNA repair. (Rohilla, A and Ali, S, 2001)
Blood extraction
In measuring the glucose level of the mice, the study will utilize the method of choice which is
obtaining blood from the tip of the tail. Sampling from the tail tip is a simple procedure that can
be carried out in any laboratory. As proposed by Mouse Metabolic Phenotyping Center (MMPC)
Consortium, standard procedures were made for assessing metabolic diseases of mice
particularly diabetes and obesity.
Collection of blood requires 12 mm of tissue cut from the tail tip distal to the bone with sharp
scissors or a scalpel, and then blood is obtained by direct flow or by gently massaging the tail
and collecting the blood in a capillary tube or other container. Following the initial cut, a 2-hour
recovery period is recommended prior to obtaining the first test sample. Subsequent samples are
obtained by gently removing the scab and repeating the massaging procedure. Vein is easily
accessed and moderate quantities of blood can be obtained from this procedure. Assessment of
glucose metabolism should not be performed on anesthetized mice due to its hyperglycaemic
effect.
Multiple samples will be taken and a maximum of the blood volume to be drawn is no more than
1.5 % of the total blood volume of the mice in 24 hours. A baseline pre-procedure fasting glucose
level of mice, an induced state of hyperglycemia, and a post-procedure glucose level will be
measured.

Testing for blood sugar

Glucose concentration can be obtained from whole-blood samples or from plasma or serum
samples. Ideally, glucose levels should be measured from plasma samples.
The study will utilize commercially available instrument such as plasma glucose analyzer or
enzymatic spectrophotometric or fluorometric assay following completion of glucose
measurement level. If not available, whole-blood glucose monitors will be used. It is more
commonly used and has the advantage of requiring only small blood volume. The machine
follows enzymatic type of assay.
References :
Department of Laboratory Animal Resources. University of Toledo. (2011). .Guidelines for
blood collection: Rodents and Rabbits. pp 3-5
Ayala, J., Samuel, V., Wasserman, D., Et.al., (2010). Standard operating procedures for
describing and performing metabolic tests of glucose homeostasis in mice
Rats weighing 200-230 g were used in one study. Another study utilised rats weighing 175-250
g. Rats that weighed 250-300 g were also used in one study. Those that weigh around 150-250 g
may also be utilised. One study stated the use of rats weighing 200-240 g, while another stated
the use of those weighing 140-160 g.
Rats are fed normal pellet diets and had access to water ad libitum. The pelleted feed is supplied
as regular, breeder, certified, irradiated, or autoclavable. They eat 10-30 g/day, and drink 20-50
mL/day of water.

Rats share 90% of their genomes with humans. Almost all disease-linked human genes have
equivalent genes within their genomes, making them a suitable research tool. Male albino rats,
specifically Wistar rats, may be used in this study as they are usually the species being utilised
when conducting invasive experiments. They are also the first rat strain developed to serve as
model animals, making them ideal for the study. Sprague-Dawley rats were also derived from
this strain, and thus may also be used if Wistar rats are not available. Its main advantage is it
calmness and ease of handling. Males are preferred over females due to the fact that female rats
are smaller and considerably more active, and therefore may be a little difficult to handle
compared to that of males.
Rats are preferably kept steel cages. Cages made with wood are unacceptable; the urine of rats
may soak in to the wood, causing ammonia buildup, which may lead to respiratory disease, or
worse, exacerbate it. They may also chew through the wood, allowing them to escape. Wood
may also have splinters, making it unsafe for the rats. Cage floors should be solid, not wired.
Wired floors may cause chilling even in a warm room, and this may impact the rats in a
biologically abnormally manner and also cause unnecessary discomfort or pain. They tend to pile
up in heaps when resting on grids or wired floors, while those on solid flooring spread out quite
comfortably on the bedding. If solid PVC floors are chosen however, the claws of the rats must
be clipped to avoid considerable discomfort, given that the surface is not abrasive. Dust-free
woodchip beddings are also recommended. Polypropylene cages may also be used.
Rat rooms are usually maintained at 30-70% relative humidity and a temperature of 18-26 degree
Celsius, with a 12-hour light/dark cycle.

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