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Hypoglycemic effect of Dita (Alstonia scholaris) leaf ethanolic extract on alloxan-induced diabetes

in Sprague Dawley rats

Bauag, Marlon Jr. D.

ABSTRACT

This study was aimed at determining the hypoglycemic effect of the leaf
ethanolic extract of Dita (Alstonia scholaris) in female Sprague Dawley rats with
alloxan-induced diabetes. It was carried out using a randomized complete block
research design, utilizing 25 test animals which were clustered into five groups with
five members each. The first three groups were the treatment groups – administered
with varying doses of the plant’s leaf ethanolic extract namely, 40 mL/kg (Treatment
1), 60 mL/kg (Treatment 2), and 80 mL/kg (Treatment 3) – and the last two groups
were the positive and negative control groups – administered with commercially
available metformin, 40 mL/kg, and distilled water, respectively. The treatments and
controls were administered orally through gastric lavage. Blood samples were
obtained from the test animals 12 hours after the administration of treatments and
controls through tail snip technique and the glucose concentration measured using a
glucometer to determine their hypoglycemic effects, then the results of which were
gathered and analyzed. Based on the statistical treatment of the data using paired t-
test, Treatment 2 and 3, when administered to the test animals at a concentration of
60 mL/kg and 80 mL/kg body weight, have a hypoglycemic effect that is comparable
with that of the positive control.

KEYWORDS Alloxan-induced diabetes, Dita, Ethanolic extract, Hypoglycemic effect, Sprague


Dawley rats

INTRODUCTION

According to the World Health Organization (2004), more than 150 million people throughout
the world suffered from diabetes and in 2010, there were already 285 million with diabetes which is
equivalent to 6.4% of the world population. The prevalence of the disease is predicted to increase by
two-fold in 20 years, where the total number of individuals with diabetes would be 439 million, or
7.7% of the world population, by the year 2030 (Shaw et. al., 2010).
There had been many treatments developed in the past decades for diabetes with the same
underlying goal of maintaining adequate blood glucose concentration (Donner, 2006; Jain & Saraf,
2010; & Klonoff, 2010). Unfortunately, these could produce various side effects, such as weight gain,
lactic acidosis, vitamin B12 deficiency, edema, bone fractures, gastrointestinal problems, and
pancreatitis (Loke et. al., 2009; & Klonoff, 2010)
Hence, medicinal plants were being looked upon once again for the treatment of diabetes
because of their natural origin and lesser side effects (Modak et. al., 2007). Dita (Alstonia scholaris) is
easily found in Southeast Asia and its bark contains some medicinal properties. However, there were
little information and only a few studies were conducted on the medicinal properties of its leaves.
Therefore, this study was conducted to determine the hypoglycemic effects of Dita’s leaf ethanolic
extracts using various doses on alloxan-induced diabetes in Sprague Dawley rats.

MATERIALS AND METHODS

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Research Method

This study utilized the randomized complete block experimental research design. 25 female
Sprague Dawley rats were injected intraperitoneally with alloxan 120 mg/kg body weight to induce
diabetes. After 12 days, blood samples were obtained through tail snipping. The rats were clustered
into five equal groups – Treatments 1, 2, and 3, Positive Control Group, and Negative Control Group.
Treatment 1 was administered with 40 mL/kg Dita leaf ethanolic extract; Treatment 2 with 60 mL/kg
leaf ethanolic extract; and Group 3 with 80 mL/kg leaf ethanolic extract. The positive control group
was administered with metformin, 40 mL/kg, and the negative control group was administered with
distilled water. After 12 hours, blood samples were again collected from the test animals to determine
the effects of the different treatments and controls to their blood glucose concentration.

Materials

This study made use of the following materials – aluminum foil, beaker, cotton, glass funnel,
Erlenmeyer flask, hypodermic needle and syringe, scissors, and stirring rod.

Equipment/Apparatus and Utensils

This study made use of the following equipment/apparatus and utensils – glucometer,
refrigerator, stomach tube or intubation cannula, stopwatch, water bath, and weighing scale.

Procedure

A. Aqueous Extraction of the Dita Leaf

Fresh Dita leaves were air-dried, cut into small pieces, and extracted with ethanol for
three days. The solvent was removed completely by evaporation at +50C. The preparation was
placed in a refrigerator for preservation purposes.

B. Phytochemical Analysis of the Dita Leaf

The phytochemical analysis of the Dita leaf was performed using the protocol of
Guevarra et. al. (2005). It was tested for the presence of flavonoids, saponins, and tannins.

C. Acute Oral Toxicity Test

This study employed acute oral toxicity as an initial step to assess and evaluate the
toxic characteristics of the leaf extracts of Dita in Sprague Dawley rats. Acute oral toxicity was
performed to establish a dosage regimen in this study and to provide initial information on
the mode of toxic action of the leaf ethanolic extract of Dita in the test animals. To carry out
this test, this study employed the OECD Guideline for Testing of Chemicals 401 (OECD, 1987).

D. Preparation and Intraperitoneal Administration of Alloxan

The alloxan solution was prepared by adding 120 mg of alloxan to 5 ml distilled water.
This will be used to chemically induce diabetes to the test animals pursuant to the Standard
Procedure on Diabetes Induction in Mice by the Institutional Animal Care and Use Committee
(IACUC) (2014). After 12 hours, the concentration of blood glucose was obtained following the
Standard Procedure for Glucose Monitoring of Blood.

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E. Administration of the Dita Leaf Extracts

The test animals were segregated into five groups with five animals per group. The
first, second, and third groups were treated with varying doses of Dita leaf ethanolic extracts
which were 40 mL/kg, 60 mL/kg, and 80 mL/kg, respectively. The fourth group was treated
with metformin, 40 mL/kg, and served as the positive control group and the fifth group with
distilled water and served as the negative control group. The administration of the treatments
and controls was performed through oral gastric lavage.

Statistical Treatment

The Paired t-test was used in this study to determine if there was a difference in the
hypoglycemic effects between the varying doses of Dita (A. scholaris) leaf ethanolic extract. It was as
well used to determine if there was a difference in the hypoglycemic effects between the varying
doses of the ethanolic extract of the plant’s leaf when compared to that of the positive control. The
level of confidence utilized in this study was 99% with a margin of error of 0.01.

RESULTS AND DISCUSSION

Table 1. Phytochemical components of Dita (A. scholaris) leaf ethanolic extract


Secondary Metabolite Result
Flavonoids +
Saponins +
Tannins +

Table 1 shows the presence of secondary metabolites namely, flavonoids, saponins, and
tannins, in the leaf ethanolic extract of Dita (A. scholaris).

Table 2. Mean concentration of blood glucose in Sprague Dawley rats before and 12-hours after the
administration of treatments and controls
12-Hours After Administration
Before Administration (mg/dL)
Treatment (mg/dL)
(n=5)
(n=5)
Treatment 1: 40 mL/kg 332.2 89.6
Treatment 2: 60 mL/kg 196.6 53.2
Treatment 3: 80 mL/kg 567.6 93.4
Positive Control: Metformin, 40
189.8 106.0
mL/kg
Negative Control: Distilled water 194.6 221.2

Table 2 shows the mean concentration of glucose in Sprague Dawley rats before and 12-hours
after the administration of the different treatments and controls. The result shows that the Dita leaf
ethanolic extracts were able to lower the concentration of blood glucose in otherwise hyperglycemic
test animals.

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Table 3.1. Results of the paired t-test on the difference between the mean blood glucose concentration of
Sprague Dawley rats before and 12-hours after the administration of treatments and controls
Before Administration After Administration
Treatment Paired t-test
(mg/dL) (mg/dL)
Treatment 1: 40 mL/kg 332.2 89.6 0.000101762*
Treatment 2: 60 mL/kg 196.6 53.2 5.37534E-07*
Treatment 3: 80 mL/kg 567.6 93.4 1.02476E-07*
Positive Control:
189.8 106.0 7.25853E-08*
Metformin, 40 mL/kg
Negative Control:
194.6 221.2 0.062681647ns
Distilled water
*=Significant at 0.01

Table 3.1 shows the results of the paired t-test on the difference between the mean blood
glucose concentration of the test animals before and 12-hours after the administration of the different
doses of the Dita leaf ethanolic extract and the controls. It shows that all the various treatments of
the leaf ethanolic extract of the plant are able to significantly reduce the concentration of blood
glucose in the test animals.

Table 3.2. Results of paired t-test on the difference of the hypoglycemic effects between the different doses of
Dita (A. scholaris) leaf ethanolic extract in Sprague Dawley rats
Paired t-test
Treatment 1 vs Treatment 2 0.002995242*
Treatment 1 vs Treatment 3 0.371621696ns
Treatment 2 vs Treatment 3 0.00106602*
*=Significant at 0.01

Table 3.2 shows the results of the paired t-test on the difference of the hypoglycemic effects
between the different doses of the leaf ethanolic extract of Dita in the test animals. It shows that the
difference in the results obtained between Treatment 1 and 2 is statistically significant, which means
that Treatment 1 is more effective in reducing the concentration of glucose in the blood of the test
animals than Treatment 2. Likewise, the difference in the results obtained between Treatment 2 and
3 is statistically significant, which means that Treatment 3 is more effective in reducing the
concentration of glucose in the blood of the test animals than Treatment 2. However, the difference
in the results between Treatment 1 and 3 is statistically not significant, which suggests that Treatment
1 and 3 have the same effect in terms of their ability to reduce the glucose concentration in the blood
of the test animals.

Table 4. Results of paired t-test on the difference of the hypoglycemic effects between the different doses of
Dita (A. scholaris) leaf ethanolic extract in Sprague Dawley rats when compared to the positive control
Paired t-test
Treatment 1 0.016650005 ns
Treatment 2 *
0.000336118
Treatment 3 0.021033549ns
*=Significant at 0.01

Table 4 shows the results of the paired t-test on the difference of the hypoglycemic effects
between the different doses of the leaf ethanolic extract of Dita in the test animals when compared
to that of the positive control. It shows that Treatment 2 and 3 are as effective as the positive control
in lowering the glucose concentration in the blood of Sprague Dawley rats.

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CONCLUSION

Based on the findings of this study, it can be concluded that the ethanolic extract of the leaves
of Dita (A. scholaris) has the ability to lower the concentration of glucose in Sprague Dawley rats with
alloxan-induced diabetes. All the different doses of the ethanolic extract of the leaves of the plant can
significantly reduce the blood glucose level in the test animals. However, only Treatment 2 and 3,
when administered to the test animal in a dose of 60 mL/kg and 80 mL/kg body weight, respectively,
through oral gastric lavage, have the hypoglycemic effects which were comparable to that of the
positive control. The hypoglycemic effects of the ethanolic extract of the plant can be attributable to
the presence of secondary metabolites, particularly flavonoids, which is known to possess an anti-
hyperglycemic activity.

As cited by The world health organization incidence of diabetes mellitus worldwide has now
escalated From 108 million in 1980 to 422 million in 2014.
The Philippine center for disease and control education foundation diagnosed that there are
already 6 million Filipinos with dm and will continue to increase.

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LIST OF REFERENCES

A.Mamun, S. Islam, A.K. Alam, M.A. Rahman, M. Rashid Effects of ethanolic extract of Hibiscus rosa-
sinensis leaves on alloxan-induced diabetes with dyslipidemia in rats Bangl Pharm J, 16 (2013),
pp. 27-31

A.C. Powers Diabetes mellitus (16th ed)D.L. Kasper (Ed.), Harrison's Principles of Internal Medicine,
vol II, McGraw-Hill Companies. Inc., New York, NY (2005), pp. 2152-2180

A.K. Nadkarni. K.M. Nadkarni’s Indian Materia Medica, Vol. I, Popular Prakashan, Bombay, 80- 83
(1976).

A.P. Macabeo, K. Krohn, D. Gehle, R.W. Read, J.J. Brophy and G.A. Cordell. Indole alkaloids from the
leaves of Philippine Alstonia scholaris. Phytochemistry 66(10): 1158 – 62 (2005)

Antidiabetic and Antihyperlipidemic Effect of Alstonia scholaris Linn Bark in Streptozotocin Induced
Diabetic Rats Deepti Bandawane*, Archana Juvekar , Manasi Juvekar Indian Journal of
Pharmaceutical Education ResearchP.No,114-120

Antihyperlipidemic agent: An in vivo on rats http://www.ncbi.nlm.nih.gov/pubmed/21060897

Asian Pac J Trop Biomed, 2 (2012), pp. 320-330 Alstonia scholaris


http://tropical.theferns.info/viewtropical.php?id=Alstonia+scholaris

Atta-Ur-Rahman, M. Asif, Ghazala, J. Fatima and K.A. Alvi. Scholaricine, an alkaloid from Alstonia
scholaris. Phytochemistry 24(11): 2771-73 (1985).

Benzie, I.F.; Wachtel-Galor, S. Herbal Medicine. In Herbal Medicine: Biomolecular and Clinical Aspects,
2nd ed.;

Blood glucose level and lipid profile of alloxan-induced hyperglycemic rats treated with single and
combinatorial herbal formulations
http://www.sciencedirect.com/science/article/pii/S2225411014000601

Chalut, D.S. Toxicological risks of herbal remedies. Paediatr. Child Health 1999, 4, 536–538

CRC Press-Taylor & Francis: Boca Raton, FL, USA, 2011.

D.K. Patel, S.K. Prasad, R. Kumar, S. Hemalatha An overview on antidiabetic medicinal plants having
insulin mimetic property

Dey, A. Alstonia scholaris R.Br. (Apocynaceae): Phytochemistry and pharmacology: A concise review.
J. Appl.

Ekor, M. The Growing Use of Herbal Medicines: Issues Relating to Adverse Reactions and Challenges
in evaluation of Alstonia scholaris: Anti-inflammatory and analgesic effects. J.
Ethnopharmacol. 2010, 129, 174–181.

Page 6 of 7
M. Jung, M. Park, H.C. Lee, Y.H. Kang, E.S. Kang, S.K. Kim Anti-diabetic agents from medicinal plants
Curr Med Chem, 13 (2006), pp. 1203-1218

Monitoring Safety. Front. Pharmacol. 2014, 4. [CrossRef] [PubMed]

N.X. Dung, P.H. Ngoc, D.D. Rang, N.T. Nhan, N. Klinkb and P. Leclercq. Chemical composition of the
volatile concentrate from the flowers of Vietnamese Alstonia scholaris (L.) R.Br. Apocynaceae.
Journal of Essential Oil Research 13(6): 424-26 (2001).

P. Versha, B. Ghosh, B. Anroop and M. Ramanjit. Antimicrobial activity of Alstonia Pharm. Sci. 2011, 1,
51–57.

Pharmacological activities of Alstonia scholaris linn. (Apocynaceae) - Pharmacognosy Reviews


Phytochemical and antibacterial activity of leaves of Alstonia scholaris R.Br.
https://www.researchgate.net/publication/264043850_Phytochemical_and_antibacterial_activ
ity_of_leaves_of_Alstonia_scholaris_RBr

PHYTOCHEMICAL STUDY ON THE LEAVES OF ALSTONIA SCHOLARIS AND THEIR EFFECTS ON


PATHOGENIC ORGANISMS
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3331187/scholaris leaf extracts. Indian
drugs 40(7): 412- 13 (2003).

Shang, J.H.; Cai, X.H.; Feng, T.; Zhao, Y.L.; Wang, J.K.; Zhang, L.Y.; Yan, M.; Luo, X.D. Pharmacological

Tatsuo Yamauchi, Fumiko Abe, Rong-Fu Chen, Gen-Ichiro Nonaka, Thawatchai Santisuk and William G.
Padolina. Alkaloids from leaves of Alstonia scholaris in Taiwan, Thailand, Indonesia and the
Philippines. Phytochemistry 29(11): 3547-52 (1990).

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