Republic of the Philippines

Department of Education
Region I
Division of Pangasinan I
Bayambang National High School
Bayambang, Pangasinan

Anti-hyperglycemic Activity of Paragis

(Eleusine indica) Ethanolic Leaf Extract on White Mice (Mus musculus)

A Science Investigatory Project

S. Y. 2020 – 2021

Name of Proponent:
Helena S. Solis

Research Consultant:
Doctor Elizabeth L. Tangangco
Doctor of Veterinary Medicine
Don Mariano Marcos Memorial State University
Bacnotan, La Union

Researchers Advisor:
Mr. Darwin F. Benitez
 INTRODUCTION
Background of the Study

Diabetes is expeditiously increasing at an alarming rate in Asian countries including the

Philippines, the prevalence of diabetes is increasing. Rapid urbanization with increasing reliance

on electronic gadgets and quiet living has contributed significantly to this epidemic. Diabetes

care in the Philippines is disadvantaged and challenged with respect to resources, government

support, and economics (Tan Gerry, 2015).

In 2017, approximately 425,000,000 adults (ranging 20-79 years) were living with a

killing disease known as Diabetes. An average of 79% of adults with diabetes were living in low

and middle income countries (International Diabetes Federation, 2019). According to World

Health Organization (WHO), Diabetes killed 1,600,000 people in 2016, making it the 7 th leading

cause of death in 2016. In year 2014, 8.5 % of adults aged 18 years and older had Diabetes. In

the Philippines, there were over 3,721,900 cases of Diabetes in 2017 (International Diabetes

Federation, 2019). Health professionals predicted that the total number of people worldwide with

Diabetes would reach 320,000,000 by 2025, but the number has already reached over

400,000,000 cases.

Recently, Paragis, also known as goosegrass went viral on social media sometime in

2017. There are only a handful of studies about Paragis, people from different cultures in the

Philippines have been using Paragis as a home remedy for certain ailments such as Anti-

inflammatory, Antibacterial, Anti-Convulsant, Antioxidant, Antiplasmodial, Cytotoxic, and

Anti- Diabetic (Eslit, 2018). Paragis (Eleusine indica) is a species of grass that belongs to the
 family Poaceae. Paragis is an annual, erect, tufted, glabrous grass with leaves that measures 10

centimeters to 30 centimeters long. Paragis can grow between 10 centimeters and 1 meter in

height. (Dr. Godofredo U. Stuart Jr.,2018).

This concept led the researcher to conduct a research study about Paragis and its effects

for Lowering of Blood Glucose Level. The objective of this study is to propose an alternative

medicine to counter the high cost and availability of the current medicines for many rural

population countries, with no side effects in enhancing the glucose metabolism and to prevent

further severe diseases. The researchers knew for a fact that there are scientifically approved

medicines, but the researchers aimed to find more affordable treatment in aiming the enhanced

glucose metabolism by lowering the blood glucose level using Paragis (Eleusine indica) leaf

ethanolic extract.

Conceptual Framework

Figure 1. Paradigm of the study

INPUT PROCESS OUTPUT

Phytochemical screening Present Phytochemical in
-test for Present Phytochemicals Paragis Leaf Ethanolic
Doses of Paragis (Eleusineindica) Plant Authentication Extract
Leaf Ethanolic Extract Acclimatization of mice Doses of Paragis
-3ml/kg (30%) -Naturally Illuminated room (Eleusineindica) Leaf
-3ml/kg (60%) - Adequate amount of food Ethanolic Extract
-3ml/kg (90%) -indefinite supply of water -3ml/kg (30%)
Positive Control Induction of Blood Glucose -3ml/kg (60%)
-500mg/kg Metformin Level -3ml/kg (90%)
Negative Control - Dexamethasone 10mg/kg Positive Control
-No treatment method Treatment -Metformin
Paragis (Eleusineindica) Leaf Negative Control
Test Subjects Extract -No treatment method
-White Mice (Mus musculus) -3ml/kg (30%), (60%), (90%) Depletion of Blood Glucose
Positive Control Level (BGL) Using Paragis
-Metformin (Eleusine indica) Leaf
Negative Control Ethanolic Extract on White
-No treatment method Mice (Mus musculus)
 As shown in Figure 1, the leaf samples will be brought to Lyceum Northwestern

University for the plant authentication and botanical identification to determine and verify its

specific species. A phytochemical analysis will be conducted to determine the significant

compounds present in the Paragis leaves.

After extracting the leaf extracts from the Paragis leaves, its physical and chemical

attributes will be identified. On the other hand, chemical characterization which includes

screening for alkaloids, flavonoids, saponins, and tannin will be conducted to identify if they are

present in the chemical constituents of the Paragis(Eleusineindica) leaves.

In the experiment, the different concentrations per treatment (Trial 1 – 90%, Trial 2 –

60%, Trial 3 – 90%) of the Paragis leaf extract will be prepared and subjected to Anti-diabetic

test to examine the Lowering of Blood Glucose Level using Paragis leaf ethanolic extract.

Various concentrations of the paragis leaf extract will be administered orally to the mice and will

be compared to the positive control (Metformin) and negative control (no treatment given). Forty

(40) mice will be used as test subjects in the study. To test the Anti-diabetic effect of Paragis leaf

extract, the researchers will collect the blood samples from the mice and will analyze it to

determine the initial blood glucose level if they will have the same level prior to the

experimentation. Furthermore, the mice will be acclimatize for fourteen (14) days provided with

sufficient amount of food, indefinite supply of water and will be stored in a naturally illuminated

room (12 hours day time/night time cycle).

Statement of the Problem

The main objective of this study is to determine the hypoglycemic effect of E. indica leaf

ethanolic extract in Dexamethasone -induced diabetes in experimental rats. Specifically, this

study sought to answer the following questions:

1) What are the phytochemicals present in E. indica ethanolic leaf extract?

2) What are the different concentrations of E. indica ethanolic leaf extract used for the

treatment of Dexamethasone -induced diabetes in experimental rats?

3) Is there a significant difference in the mean blood glucose levels of diabetic rats after

treatment of E. indica ethanolic leaf extract and the positive control, Metformin?

4) Is there a significant relationship between the different concentrations of E. indica

ethanolic leaf extract and the mean blood glucose levels of diabetic rats?

Null Hypotheses

There is no significant difference in the mean blood glucose levels of diabetic rats treated

with different concentrations of E. indica ethanolic leaf extract and the positive control,

Metformin.

Significance of the Study

Generally, Diabetes Mellitus can really harm and affect people in terms of health. Thus,

the researchers came up with the study, “Anti-hyperglycemic Activity of Paragis (Eleusine
indica) Leaf Ethanolic Extract on White Mice (Mus musculus)” which aims to treat

Hyperglycemia in an inexpensive and safe way.

This study will contribute in finding of an alternative natural product that treats

Hyperglycemia.

The Paragis (Eleusine indica) helps in reducing the blood Glucose Level (BGL) that

reduces the number of people diagnosed with Diabetes mellitus that will cause lowering of

Diabetes death rate. This study may counter the high cost and the availability of the current

medicines for the rural populations, since Diabetes is one of the leading causes of mortality in

the Philippines (DOH, 2017)

This study may serve as guide to conduct more intensive and innovative information that

will help in reducing Diabetes patients. It serves as the template to make development in

Diabetes and Paragis.

Scope and Limitation

This study focus on the effectiveness of Paragis (Eleusineindica) leaf ethanolic extract in

enhancing glucose metabolism by lowering the blood glucose level of white mice.

A phytochemical analysis was conducted in LORMA Colleges in San Fernando La Union

and will determine the active compounds present in Paragis (Eleusineindica). A Ethanolic

Extraction process will be employed to extract necessary compounds from Paragis leaves.

This study focus only on the effect of the different concentration/dosage of Paragis

Ethanolic leaf extracts against Hyperglycemia. The ethanolic leaf extracts will be administered

using oral gavage at different concentrations as follows: 30%, 60%, 90%. During the conduct of
 the experiment, it will be observed if the effects of the Paragis ethanolic extracts on different

concentrations in white mice reduce blood glucose level. The experimentation set-ups for each

trial will be compared with a positive control specifically, Metformin, and negative control

where in mice will not receive any treatment. The study will be conducted for six (6) days in

Don Mariano Marcos Memorial State University, Bacnotan, La Union with a laboratory suited

the environment needed for the white mice. In the Hyperglycemic model, Forty (40) 3 months

old male and female white mice will be divided into five groups. A total of forty (40) white mice

will be used for this study.

METHODOLOGY
Materials and Method

Test Animals

The test for the Diabetic property of Paragis (Eleusine indica) ethanolic leaf extract will

carry out 40 healthy white mice (Mus Musculus). The experimental mice that were used are

three months old, weighing approximately 20-30 grams. The mice were acquired from a Bureau

of Animal Industry (BAI) certified seller in Virgen Milagrosa University Foundation, San Carlos

City, Pangasinan. For acclimatization the experimental mice were placed in a Polyethylene made

cage with a temperature of controlled and naturally illuminated room with adequate food and

indefinite supply of water.

Ethical Consideration

Finding treatments and preventions is the main concern and focus of research in the field

of medicine. Preclinical was conducted with the use of animals that have characteristics similar

to humans to ensure and secure the welfare of the procedure or the new product.
 In order to prevent undue sufferings, Research protocol was reviewed by Bureau of

Animal Industry. A thorough literature review was made beforehand to prevent variable

duplication.

The animals have access to veterinary care. All personnel who work with the animals

should be sufficiently trained in both the experimental procedure and ethical handling of the

animals. All the researchers were trained and have knowledge on how to handle the mice

securely and properly. The researchers used only the required number of mice needed to acquire

data throughout the experiment. The researchers also ensured that the tests given or undergone

by the animals are only regarded to the study.

The mice that were used received obligated care, food, and shelter throughout the

experiment.

The researchers fully understood and adhered to the rules and guidelines for the usage of

animal testing.

Test Animal Disposal

After the conducting of experiment, wastes were removed and disposed from the facility to

ensure the sanitary of the workplace and to prevent the contamination of the work areas with

waste materials. The animals used were painlessly and mercilessly killed immediately. The

animals were properly disposed by incineration, supervised by the Certified Veterinary doctors

of Don Mariano Marcos Memorial State University.

Inducing of Blood Glucose in Mice

The mice were injected with dexamethasone (10mg/kg, i.p.). Five days after the injection,

the mice with fasting blood glucose higher than 180mg/dl will be used for the experiments. 8
mice were used in each experiment. The animals was used once only in all of experiments. The

food and water were removed from cages 12hr before testing.

Preparation of Paragis Extract

Fresh Paragis Leaves (Eleusine indica) were collected in Bayambang, Pangasinan. Dried

Paragis leaves (about 100g) were submitted to extraction in LORMA Colleges with 300ml

ethanol (80%) in a Soxhlet Apparatus for 72hr. After extraction, the solvent was filtered and

then evaporated by Rotavapor. The obtained Paragis ethanolic extract was stored at -20℃ until

being used.

Treatment Administration

Paragis extract was suspended in distilled water and administrated through oral gavage to

administer the extract directly to the animals’ stomach. The volume of administered extract is

1ml for each mice.

Experimental Design

In the experiment, 40 white mice were used. The mice were divided into 5 groups. 8 mice

were in each group.

Negative Control Group: High Blood Glucose control white mice were administered 1ml

of distilled water.

Positive Control Group: High Blood Glucose control mice were administered Metformin

through oral gavage (600ug/kg body wt.).

Treatment 1 Group: High Blood Glucose control mice were administered 30%

concentration of Paragis Ethanolic extract (3ml/hd/mice) using an oral gavage.

Treatment 2 Group: High Blood Glucose control mice were administered 60%

concentration of Paragis Ethanolic extract (3ml/hd/mice) using an oral gavage.

 Treatment 3 Group: High Blood Glucose control mice were administered 90%

concentration of Paragis Ethanolic extract (3ml/hd/mice) using an oral gavage.

Statistical Analysis

Analysis of Variance (ANOVA) with a level of 0.05 was used to determine the

significant difference between the 5 groups in terms of blood glucose level.

 RESULTS AND DISCUSSION

This chapter overseen the presentation, analysis, and interpretation of the data gathered

by the researchers. The researchers used five set-ups; the first group was given distilled water

which serves as the negative control. The second group was induced with positive control, which

is the commercial product (metformin). The third group was induced with 30% of ethanolic leaf

extract of Paragis (Eleusine indica). The fourth group was induced with 60% of ethanolic leaf

extract of Paragis (Eleusine indica). And for the fifth group was induced with 90% of ethanolic

leaf extract of Paragis (Eleusine indica).

Table 1. The mean counts of blood glucose level induction.

Table
Group Before inducing After 3 hrs of After 5 days of
1 dexamethasone inducing inducing shows
(mg/dL) dexamethasone dexamethasone
the (mg/dL) (mg/dL) mean
G1 (Negative 50 62 102
count control) of
G2 (Postive 55 62 96
blood control)
G3 (30%) 54 59 97

G4 (60%) 55 61 98

G5 (90%) 65 79 109

glucose level observed during the inducing of blood glucose level. All the 5 groups have

increased of blood glucose level after 3 hours inducing. After 5 days of inducing, all the 5

treatments blood glucose level also increased.

 Table 2. The mean count of total blood glucose level of White Mice using 5 treatments.
Treatments Before After 1 hour of After 2 hours of
treatment treatment
treatment (mg/dL) (mg/dL)

Table (mg/dL) 2
T1 (3mg/dL) 97 87 83
shows the
T2 (6mg/dL) 98 90 80
mean count
T3 (9mg/dL) 109 86 78
of blood
T4 (Positive 96 89 81
control)
T5 (Negative 102 139 152
control)
glucose level observed during testing. Among 5 treatments, T1, T2, T3 and T4 have the most

significant decrease of blood glucose level while the negative control had increased in blood

glucose level.

The decrease of blood glucose level can be attribute to phytochemical present in Paragis

(Eleusine indica) specifically flavanoids and tannins as cited by Pharmaceutical Crops 2014.

Graph 1. Mean difference in the blood glucose level.

 1.6
Mean Difference before and after the administration of plant sample mand positive control
1.4

1.2
Mean Difference (mg/dL)

0.8

0.6

0.4

0.2

0
T1 T2 T3 T4 T5

Graph 1 shows the mean difference in the blood glucose level between the administration

of dexamethasone and plant sample, positive control and negative control.

It can be depicted from the graph that the most evident decrease of blood glucose level

are Treatment 1, 2, 3 and 4. T3 (90% of plant extract) showed a 0.31mg/dL or 31% of decrease

of blood glucose level, T2 (60% of plant extract) showed a 0.18mg/dL or 18% of decrease of

blood glucose level, T1 (30% of plant extract) showed a 0.14mg/dL or 14% and T4 (positive

control) showed a 0.15mg/dL or 15% of decrease of blood glucose level. On the other hand, T5

(negative control) showed that they have no effect on the decrease of blood glucose level of

White Mice.
 Table 3. Significant difference in the blood glucose level.

P-
SV SS df MS F value F crit
4580.07 127.165 2.22E- 3.05556
BG 18320.3 4 5 4 11 8
36.0166
WG 540.25 15 7

18860.5
Total 5 19        

Table 3 shows the result of the analysis of variants among the 5 treatments. F value of

127.1654 with a F crit of 3.055568 shows that the F value is greater than F crit it means that the

means of the 5 treatments are not equal. It shows that the result is highly significant.

T-test (see Appendix E) shows that between T1 and T2, the latter has the higher value of

decrease in the blood glucose level. Between T1 and T3, the latter has the higher value of

decrease in the blood glucose level. Between T1 and T4, the latter has the higher value of

decrease in the blood glucose level. Between T1 and T5, the former has the higher value of

decrease in the blood glucose level.

Between T2 and T3, the latter has the higher value of decrease in the blood glucose level.

Between T2 and T4, the latter has the higher value of decrease in the blood glucose level.

Between T2 and T5, the former has the higher value of decrease in the blood glucose level.

Between T3 and T4, the former has the higher value of decrease in the blood glucose

level. Between T3 and T5, the former has the higher value of decrease in the blood glucose level.

Between T4 and T5, the former has the higher value of decrease in the blood glucose level.
 Among all the group of treatments, treatment 5 or negative control significantly has lesser

value of decrease in blood glucose level which attributed to the phytochemical present in Paragis

(Eleusine indica) ethanolic extract.

The result indicated that treatment 3 or 90% of ethanolic leaf extract is the most effective

than the other treatment, it is efficient based on decrease of blood glucose level of White Mice

(Mus musculus).

CONCLUSION

The researchers found out that Paragis (Eleusine indica) ethanolic leaf extract has a

significant effect in depletion of the blood glucose level. Therefore, the following null

hypotheses are rejected stating that the Paragis (Eleusine indica) ethanolic leaf extract will not be

an effective in depletion of blood glucose level on White Mice (Mus musculus). There is no

significant difference between before and after treatment of blood glucose level, there is no

significant difference between the six groups of the blood glucose level