INTRODUCTION

Nature and Importance of the Study

Various medications for Diabetes Milletus have been discovered and have

become available over the course of the past few years. Drugs like Sulfonylurea,

Biguanides, Glinides, and Alpha-Glucosidase Inhibitors are only a few of the drug

treatments for Hyperglycemia or the elevated level of sugar glucose in the blood

that occurs in Diabetes Milletus.

Diabetes is a chronic disease that occurs either when the pancreas does

not produce enough insulin or when the body cannot effectively use the insulin it

produces. Insulin is a hormone that regulates blood sugar. Hyperglycemia, or

raised blood sugar, is a common effect of uncontrolled diabetes and over time

leads to serious damage to many of the body's systems, especially the nerves

and blood vessels.

According to the World Health Organization (WHO) in 2014, 9% of

adults 18 years and older had diabetes. In 2012 diabetes was the direct cause

of 1.5 million deaths. More than 80% of diabetes deaths occur in low- and

middle-income countries. The most common long-term complications

of diabetes include: damage to the large blood vessels of the heart, brain and

legs (macro vascular complications) damage to the small blood vessels,

causing problems in the eyes, kidneys, feet and nerves (micro vascular

complications).

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 As this situation urges, the demand for Anti-hyperglycemic treatments

in the society also increases. Consequently, people around the world began

to look for more Anti-hyperglycemic treatment sources preferably those which

are cheaper.

According to the Applied Science Reports of the Chemical Analysis

from the Department of Crop Production and Protection, Department of

Botany, Department of Biochemistry Obafemi Awolowo Ile-Ife University of

Nigeria, the Basella rubra exhibits 91.3% of antioxidants (3044mg/100g). Also

according to Stuart (2013), the parts of the Spinach Vine each has many

uses; recent study evaluated an aqueous extract from the leaves and stem of

the Basella rubra through Phytochemical Screening showed a rich source of

Phytonutrients, a few are Tannins, Saponins and Vitamin C, including

Enzymic and Nonenzymic antioxidants all of which are good nutrients for

decreasing the blood glucose levels to the point of normality. In view of this,

the researchers decided to use the Spinach Vine (Basella rubra) as a source

of Anti-hyperglycemic treatment. Extract the phytonutrients from the leaves

and stem of the Spinach Vine (Basella rubra) was used. This process allows

the utilization of the phytonutrients in the leaves and stem of the Spinach Vine

(Basella rubra) to determine its Anti-hyperglycemic ability.

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Objectives of the Study

This study aims to determine the potential Anti-hyperglycemic ability of

Spinach Vine (Basella rubra) leaves and stem extract to Albino Rats ( Rattus

albus). Specifically, this research intends to:

1. Produce an extract from Spinach Vine leaves and stem of

Spinach Vine (Basella rubra);

2. Determine the Anti-hyperglycemic ability of the stem extract and

leaves extract of Spinach Vine (Basella rubra).

Statement of Hypothesis

Ho : There is no significant difference on the Anti-hyperglycemic ability

of Spinach Vine (Basella rubra) leaves and stem extract in different dosage.

Significance of the Study

Diabetes is one of the major direct causes of death all around the world.

Injectable insulin that are used to keep blood glucose near-normal are highly

expensive, thus, low and middle-income denizens can barely afford such

medication. Thus, the researchers use the Spinach vine to produce an Anti-

hyperglycemic cure. It will be cheaper than the laboratory made Anti-

hyperglycemic or treatments because the subject is easy to find and has mass

production, making it affordable for low and middle-income denizens. It will also

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be safer since the substance to be used is the extract of the leaves and stem of

the Spinach Vine.

Scope and Limitation of the Study

This study focused only on seeking the potential Anti-hyperglycemic

ability from the leaves and stem of the Spinach Vine ( Basella rubra). This

study was conducted during the month of August 2016 at Gerona’s Farm,

Buac Gamay, Sogod Southern Leyte.

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 REVIEW OF RELATED LITERATURE

Basella or vine spinach is a popular tropical leafy-green vegetable

commonly grown as backyard plant in the home gardens. In the true sense, it is

different from English spinach ( Spinacea oleracea) in that the plant is a creeping

vine, and its leaves feature glossy, broad, deep green, thick, and mucilaginous.

Commonly found in the backyard gardens of many south Asian families, it is

gaining popularity in some of the tropical and temperate climates of America,

Australia and Europe for its succulent, nutritious greens, and tender stems.

Vine spinach belongs to the Basellaceae family, and has two chief

cultivars, Basella alba, which features green-stems and deep-green leaves, and

Basella rubra with purplish-stems and deep-green leaves with pink veins.

In a study conducted through Phytochemical screening, the Basella rubra

cultivar are an excellent source of numerous essential phytonutrients like

Saponins, Tannins, Flavonoids, Terpenoids and reducing sugars, thus having an

excellent potential in reducing blood glucose.

A 1 1/2-cup serving of cooked alugbati has only 15 calories per serving,

around a half gram of fat and 1.4 grams of dietary fiber. It also has 75

micrograms of folate, or folic acid, per serving. This provides 12.5 percent to 19

5
percent of the recommended dietary allowance of folate. Also known as vitamin

B-9, folate is needed for healthy brain function and is important for maintaining

overall mental and emotional health. It is needed to produce DNA and, as a B

vitamin, helps with the breakdown and use of carbohydrates by your body.

Alugbati is also rich in a number of antioxidants, namely lutein and talinum,

according to a 2008 study published by the University of the Philippines at Los

Banos. Antioxidants protect your body’s cells from potential damage that can be

caused by free radicals and exposure to environmental toxins

(http://www.livestrong.com/article/517905-health-benefits-of-alugbati/).

The plant is a perennial vine and grown as annual or biennial pot-herb. It

prefers hot humid climate and moist, fertile, well-drained soil to flourish. Although,

its seeds can be sown directly for planting, usually thick cuttings about the length

of 20 cm are preferred for easy propagation, and fast growth. Being a vine, the

plant requires trellising for its spread at a faster rate. It bears white or white-pink

color tiny flowers depending upon the species and deep-purple to black color

berries.

Basella rubra features pink or purplish stems and pink color veins running

in the leaves. In either case, leaves and terminal tender, 8-12 inches stems are

ready for harvesting about 35 to 45 days after planting (http://www.nutrition-and-

you.com/basella.html).

Malabar Spinach is very nutritious and helpful in curing malnutrition. The

leaves contain Vitamin E, Vitamin K, flavonoids, saponins, - Carotene, water

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soluble polysaccharides, bioflavonoids, essential amino acids (arginine, leucine,

isoleucine, lysine, threonine and tryptophan) and minerals (rich in calcium and

iron compounds and contains a low percentage of soluble oxalates). This herb

works as medicine due to presence of numerous biologically active compounds

such as carbohydrates, proteins, enzymes, fats and oils, vitamins, alkaloids,

quinines, terpenoids, flavonoids, carotenoids, sterols, simple phenolic glycosides,

tannins, saponins, polyphenols

(http://www.bimbima.com/health/post/2015/02/13/medicinal-plant-malabar-

spinach-basella-alba-rubra.aspx).

The leaves as well as stems of Malabar spinach contain exceptionally high

levels of vitamin A. Only 100 grams of fresh vine spinach leaves provide us with

8000 IU (international unit) of the recommended daily allowance of this nutrient.

It is interesting to note that the vitamin C content of Basella or vine

spinach is higher compared to the English spinach. A serving of 100 grams of

fresh Malabar spinach offers 102 mg of vitamin C, which is almost equal to our

recommended daily allowance (RDA) of this vitamin. Similar to the English

spinach, even Malabar spinach is a wonderful natural source of iron.

Malabar spinach also encloses substantial amounts of several B-complex

vitamins, including vitamin B6 (pyridoxine), folate and riboflavin. A serving of 100

grams of fresh vine spinach leaves supplies us with 140 µg or 35 percent daily

recommended allowance of folate for an adult human.

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 Aside from the above mentioned properties of Malabar spinach, these

greens are also an excellent resource of several essential minerals like calcium,

manganese, magnesium, potassium and copper. Similar to the English spinach,

consuming Malabar spinach on a regular basis also helps to avoid anemia

caused by iron deficiency and osteoporosis. In addition, incorporating Malabar

spinach into your diet may also help to protect you from serious conditions like

cardiovascular disease and colon cancer

(http://www.herbs2000.com/herbs/herbs_malabar_spinach.htm).

Just like other plants, the Spinach Vine has many uses, in the medical

field as well, each part of the plant has different uses, the roots are employed as

rubefacient, the sap is applied to acne eruptions to reduce inflammation,

decoction of leaves are used for its mild laxative effects, mucilaginous liquids

obtained from leaves and tender stalks are used for headaches, in other

countries, the root is used for removal of after birth and stomach pains. Other

uses of the Spinach Vine is for cosmetics, the fruit is used by women as rouge

for cheeks and lips, with the anthocyanin content, it makes for a natural food

colorant. Fruit provides dark violet color as food colorant. In the veterinary field,

ground leaves are rubbed on the human hand to introduce the preparation of the

animal vagina every morning for the treatment of sterility.

Since Malabar spinach is a versatile green leafy vegetable, many people

in East Asian culture hold it in high esteem, especially because of it high

phytonutrient contents. This vegetable contains very less calories as well as fats.

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On the other hand, it is packed with unbelievably high vitamin, mineral as well as

antioxidant contents (www.herbs2000.com/herbs/herbs_malabar_spinach.htm).

METHODOLOGY

The process followed in the conduct of this study is shown in Figure 2.

Gathering of Spinach Vine leaves and stem

Cleaning and weighing of Spinach Vine leaves and Spinach Vine

stem

Extraction & Filtering of Spinach Vine leaves and Spinach

Vine stem

Preparation of Experimental Set-up

Actual Experimentation

Anti-hyperglycemic Testing

Data Gathering

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 Data Analysis

Figure 1. Process Flow of the Research Conduct

Preliminary Testing

A preliminary testing was performed to determine the ideal time of waiting

for the extract to take effect and to determine dosage of the extracts.

Collection, Preparation and Extraction of Spinach Vine Leaves and Stem

The Spinach Vine (Basella rubra) was gathered from the locality of the

researcher, specifically at Brgy. Nahulid, Libagon Southern Leyte. It was washed

thoroughly to remove the dirt and other impurities that were attached to the

leaves and stem. It was tallied with 45-50 leaves and 45-50 6-inch long stems of

Spinach Vine (Basella rubra). The production of extracts was done at the

laboratory of Sogod National High School. Using a blender, extracts were

produced and filtered using a sterile strainer and cloth. The extracts were then

placed in a sterile reagent bottle for storage and were placed in a cooler to

prevent contamination.

Profile of the Experimental Subjects

According to the Foundation for Biomedical Research (FBR), 95% of

laboratory animals are mice or rats. Albino Rats (Rattus albus) were used in this

study. They are small and can be easily housed and maintained. Also, Albino Rats

(Rattus albus) are generally mild-tempered and docile, making them easy for the

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researchers to handle. They are used in medical trials because they are almost

identical genetically. This characteristic can help make uniform medical trials

results (National Human Genome Research Institute). In addition, their biological,

genetic and behavior characteristics closely resemble those of humans and

many conditions can be replicated according to the National Institute of Health

(NIH) Office Laboratory Animal Welfare. As a minimum requirement, the rats

used in experiments are of the same purebred species. The profile of the

experimental subjects is presented in Table 1.

Table 1

Profiling Experimental subjects Albino Rats (Rattus albus)

Subject Weight Age Heart Rate Body

Temperature
Range Average

9 Male 250-260 g 254.67g 1 month 332 beats per 36.80-37.60C

Albino Rats minute

Experimental Design

This study used Completely Randomized Design (CRD) design. There

were three treatments used, T0 is the control while T1 and T2 are with 1cc

leaves extract and stem extract, respectively, are the experimental treatments.

The experimental design and treatments are shown in Table 2.

Table 2

Dosage of Administered Spinach Vine Extract

Dosage (cc)

R1 R2 R3

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 Treatment

T0 (no extract))

T1 (leaves extract) 1 cc 1 cc 1 cc

T2 (stem extract) 1 cc 1 cc 1 cc

Blood Glucose Testing

Nine (9) white male Albino Rats (Rattus albus), with the same age, weight

and gender were used in this research. A fasting blood sugar test was

performed. A fasting of eight (8) hours was done before collecting blood samples

to determine the blood glucose levels of the rats. The experimental subjects have

reached normal blood glucose levels; the minimum is 112.98 mg/dl and the

maximum is 123.6 mg/dl.

Induction of Honey

The researchers separated the rats in different cages inducted with the

same amount of honey, and were observed for twenty-four (24) hours. A fasting

blood sugar test was performed. Fasting was done eight (8) hours to follow the

standards and guidelines of blood sample testing before collecting blood samples

again to confirm Hyperglycemia. Twenty (20) minutes after the blood collection,

1 cc of extract was pumped to a 1ml Insulin Syringe with a 31mm gauge. The

researchers injected the extracts with the guidance of the Professional

Veterinarian and waited 3 hours for the extract to take effect. Blood testing was

again performed to determine the Anti-hyperglycemic ability of the Spinach Vine

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(Basella rubra). This process was based and modified from Akinnuga, et al

(----------------)

Observation of Behavior

After the conduct, the researchers made arrangements with the caretaker

to perform the daily routine of the rats and observe variables such as;

1. time the Albino Rats (Rattus albus) drink water and eat food;

2. amount of water and food consume; and

3. physical activity

After four (4) weeks of observation, through constant visit and interview of

the care taker, observation reports were submitted to the Professional

Veterinarian. From the data, it was concluded that the rats did not have any

abnormal reactions, nor abnormal physical activities.

Statistical Treatment of Data

Data were analyzed using descriptive statistics. Mean was used to

determine the average weight of the experimental animals and mean glucose

level for each treatment. Then Analysis of Variance (ANOVA) was used to

determine the significance of the difference of the blood glucose levels across

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treatments. ANOVA was done for the data before the injection and also after

injection.

ANALYSIS, RESULTS AND DISCUSSION

Extract from Leaves and Stems of Spinach Vine (Basella rubra)

The Spinach Vine (Basella rubra) leaves and stem have produced an extract.
A soapy, aqueous extract was collected, prepared and used in this study.

Anti-Hyperglycemic Testing of Spinach Vine to Rats

The blood glucose levels were determined before induction of honey,

before the injection of Spinach Vine (Basella rubra) extracts and after the injection of
Spinach Vine (Basella rubra).
Table 3

Comparative Analysis of the Blood Glucose Levels before Induction of

Honey, before and after Injection of the Spinach Vine (Basella rubra)
Extracts
Blood Glucose Level Blood Glucose Levels Blood Glucose Levels MEA
Before Induction of Before Injection of After Injection of N
Honey (mg/dl) MEA MEA
N Spinach Vine (Basella N Spinach Vine (Basella
Treatme rubra) Extract (mg/dl) rubra) Extract (mg/dl)
nt
R1 R2 R3 R1 R2 R3 R1 R2 R3
T0 (no 120.7 119.5 114.6 118.3 149.0 140.0 146.0 145.0 140. 138. 138.1 139.0
extract) 8 6 7 4 2 4 2 3 8 2 3
T1 116.0 123.6 119.8 119.8 141.0 139.4 140.8 140.4 100. 104. 101.8 102.1
(Leaves 2 2 1 8 3 3 2
Extracts)
T2 (Stem 120.0 112.9 117.2 116.7 139.2 143.6 139.6 140.8 115. 113. 114.1 114.2
Extracts) 2 8 4 5 5 2 4 1 0

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Note: 100 mg/dl-125 mg/dl is considered the range for normal Blood Glucose Level if obtained from a fasting
blood sugar test (American Diabetes Association)

The blood glucose levels before induction of honey was determined. As

shown, the mean among the T0, T1 and T2 were almost the same (µ=118.34,

µ=119.81, and µ=116.75, respectively). Then after 24 hours with no food and no

water, and 8-hour fasting, the glucose level was again determined. In this case,

blood glucose levels increased in all treatments. This confirmed the occurrence

of Hyperglycemia (excess of sugar in the blood). There was an increase of

26.69(mg/dl) for T0, 20.62(mg/dl) for T1, and 24.07(mg/dl) for T2.

Table 4

Mean Increase in Blood Glucose Levels before and after Injection of the
Spinach Vine (Basella rubra) Extracts
MEAN Blood Glucose MEAN Blood Glucose Levels
Levels After Injection of Spinach Change in Glucose Level
Before Injection of Spinach Vine (Basella rubra) Extract
Treatment Vine (Basella rubra) Extract (mg/dl) Increase Percent
(mg/dl) Increase
(mg/dl)
T0 (no extract) 145.03 139.03 6.00 4.14%

T1 (Leaves 140.43 102.1 38.33 27.29%

Extracts)
T2 (Stem 140.82 114.20 26.62 18.90%
Extracts)
Note: 100 mg/dl-125 mg/dl is considered the range for normal Blood Glucose Level if obtained from a fasting
blood sugar test (American Diabetes Association)

After injecting Spinach Vine (Basella rubra) extract to Albino Rats (Rattus

albus), the blood glucose levels across all treatments decreased. It is evidently

shown in Table 4 that blood glucose levels of Albino Rats (Rattus albus)

decreased by 6 mg/dl which is 4.14%. But T1 registered the highest decrease

which is 38.33mg/dl, equivalent to 27.29%. Then T2 was second with 26.62

15
mg/dl increase or 18.90%, compared to the level before the injection of Spinach

Vine (Basella rubra) extract to Albino Rats (Rattus albus).

According to the American Diabetes Association, blood glucose level is

normal if it falls between 100mg/dl to 125mg/dl. In this case, Albino Rats (Rattus

albus) in T0 were still in hyperglycemia (µ=139.03mg/dl). Results further

confirmed that Spinach Vine (Basella rubra) extract has anti-hypergyclemic ability.

Leaves extract of Spinach Vine (Basella rubra) has higher anti-hyperglycemic ability

than the stem extract. But those have this important potential of this plant.

Table 5

Test for Significant of Difference in Glucose Levels before and after

Injection of Spinach Vine (Basella rubra) Extracts

Period MS p. value Interpretation

Before Injection 20.131 0.197 Difference is not significant.

After Injection 1153.334 0.000 Difference is highly

significant

ANOVA results between group comparison

2634.170 0.001 Difference is highly

significant

Note: Basis of the p-value is 0.05

It is evident from the table above that before injection of the extracts there

was no significant difference (p-value=0.197) on the blood glucose levels. All

were still Hyperglycemic (µ>125mg/dl). After the injection of Spinach Vine

16
(Basella rubra) there was a significant difference (p-value 0.000) detected. This

was due to the big decrease from the previous Hyperglycemic glucose levels to

the normal glucose levels. Thus, the null hypothesis was rejected.

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 SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATIONS

Summary of Findings

After the injection of Spinach Vine (Basella rubra), T0 (no extract) had an

average blood glucose level of 139.03 mg/dl. Then T1 (leaves extract) was 102.1

mg/dl and 114.20mg/dl for T2 (stem extract). It is found out that Spinach Vine

(Basella rubra) extract is a very good potential as anti-hyperglycemic.

Conclusion

Based from the results and findings of the study, it was concluded that the

leaves and stems of the Spinach Vine (Basella rubra) significantly decreased

blood glucose levels. This extract has very good potential to normalize blood

glucose levels of Hyperglycemic rats.

Recommendation

1. A combination of the leaves and stem extract is also recommended to be

tested.

2. A chemical analysis of the leaves extract and stem extract are worthy to

consider.

3. Other plants with the same characteristics may also be tried for this same

purpose.

4. The shelf life of the produced extracts from the Spinach Vine (Basella

rubra) leaves and stem need to be determined.

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5. The use of Spinach Vine (Basella rubra) in the formulation of drug that

decreased blood glucose levels is highly recommended. Then

commercialization of the product can be the next priority.

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 BIBLIOGRAPHY

Electronic Sources

(http://www.livestrong.com/article/517905-health-benefits-of-alugbati/)

(http://www.nutrition-and-you.com/basella.html)

(http://www.herbs2000.com/herbs/herbs_malabar_spinach.htm)

(www.herbs2000.com/herbs/herbs_malabar_spinach.htm)

(http://www.bimbima.com/health/post/2015/02/13/medicinal-plant-malabar-

spinach-basella-alba-rubra.aspx)

(http://www.mayoclinic.org/diseases-conditions/diabetes/basics/tests-diagnosis/

con-20033091)

(www.sturtxchange.com/alugbati.html)

(www.diabetesselfmanagement.com/what-is-a-normal-blood-sugar-level/)

Books

Philippine Medicinal Plants (Stuart 2013)

IACUC Standard Procedures; Glucose Monitoring of Blood in Rats and Mice

(The Institutional Animal Care and Use Committee 2015)

Applied Science Reports; Comparative Studies of Chemical Analysis of two

Species of Basella (Oloyede F.M 2013)

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APPENDICES

21
 Appendix A

Preparation of the Materials Sterilization of the Reagent Bottles

Extraction of the Leaves Extraction of the Stem

Gathering of the Albino Rats Separation and Induction of Honey

22
Gathering of Blood Samples and Submission to the Glucometer

Preparation for Injection

Observation of Behavior

23