Syzygium cumini (Duhat) bark: a lowering agent for lactic acid in Wistar

rats

Angela Dominique U. De Guzman1,2,3, Bianca G. Cruz1,2,3, Raymond Deriada C

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1,2,3
, Shaina Louise V. Ditablan1,2.3, Lourdes Jeanne F. Gonzales1,2,3, Kesh L
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Allysa B. Reforma1,2,3 E
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College of Medical Laboratory Science F
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Our Lady of Fatima University M
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Research Development and Innovation Center D
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In Partial Fulfillment
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of the requirements for the Degree A
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Bachelor of Science in Medical Laboratory Science (BSMLS) O
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January 2022 Y

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Chapter 1
 THE PROBLEM AND ITS BACKGROUND

1.0 Introduction
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Lactic acid is a physiological production, also commonly found in L
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disease states. The severity of the clinical course advances when there E

is a high production that is comorbid with decreased clearance. O

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Profound hemodynamic effects from severely elevated lactic acid levels
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can lead to death. Serum lactate levels can act as a marker and as a D
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therapeutic target. The higher the level of serum lactate and the longer C
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its normalization, the greater risk of death. Normal lactate levels are
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below two mmol/L, while hyperlactatemia has lactate levels between A
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two and four mmol/L. Four mmol or higher levels are severe lactate O
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levels. Lactic acidosis is also defined when the pH is less than or equal
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to 7.35 and lactatemia greater than two mmol/L with a less than or R
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equal to 42 mmHg partial pressure of carbon dioxide or PaCO2
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(Foucher & Tubben, 2021).
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According to MedicalNewsToday (2017), the most common causes E

of lactic acidosis are the following: cardiogenic shock, hypovolemic

shock, sepsis, severe heart failure, and severe trauma; while the
commonly affected people have the following underlying conditions as

other causes for lactic acidosis: kidney conditions, diabetes mellitus,

alcoholism, liver disease, short bowel movement, extreme physical

exercise, and those undergoing HIV treatments. The treatments include

the following: intravenous fluid (for better circulation), oxygen, positive

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pressure ventilation (for oxygen delivery to the lungs), vitamin therapy, O
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and hemodialysis with bicarbonate. Exercise-induced lactic acidosis
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can heal with rest and rehydration with water. Lactic acidosis can be a E

temporary and non-threatening condition for some people. It can also O

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have a complicated treatment if it is part of other underlying health
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conditions in which success will come through the management of its D
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primary cause. C
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Mild and intermediate lactate levels with sepsis have heightened A
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rates of 30-day in-hospital mortality. Lactic acidosis can reduce cardiac O
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contractility and vascular hyporesponsiveness to vasopressors by T
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different mechanisms. Yet, there is no definite causal connection R
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between lactic acidosis, and extreme situations are more of a
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precipitator than a direct cause regarding mortality. In conclusion, I
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lactic acidosis worsens the underlying comorbidities, along with an N
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impact on mortality. E
 The treatment for lactic acidosis requires the consideration of its

cause as a critical step in a patient's recovery. Such in the case of lactic

acidosis secondary to mesenteric ischemia, in this case, surgery may be

a necessity, or if the seizure activity causes convulsions that induce

lactic acidosis, then treating seizures is crucial. The treatment must

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suit a particular individual (Foucher & Tubben, 2021). However, O
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treatments for lactic acidosis induced by underlying diseases may not
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always be accessible for the patients due to costly medication/remedy, E

lack of supply, a lengthy time of treatment, or lack of access to O

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medicines due to their remote area.
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Since supplements for lactic acidosis may not always be available C
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and managing the primary causes of lactic acidosis may be expensive,
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an affordable alternative medication for lactic acidosis to better the A
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situation is beneficial. In this research, the researchers will test the O
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capabilities of Duhat bark as an agent to lower high lactic acid levels. T
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1.2 Statement of the Problem I
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This research will study the effectivity of Syzygium cumini (Duhat) E

bark as an agent to lower the lactic acid level in Wistar rats.

Specific Objectives:

1.2.1 What is the main phytochemical component and amount of Duhat

extract that can lower the lactic acid level in Wistar rats?

1.2.2 What is the demographic profile of the Wistar rats in the experimental

and control group in terms of: C

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a. Sex L
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b. Age E
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c. Weight
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1.2.3 What is the baseline lactic acid level of the experimental and control M
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group before treatment? D
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1.2.4 What are the lactic acid levels of the experimental and control groups? L

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1.2.5 What are the lactic acid levels of the experimental group upon treatment
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of the different concentrations of Duhat extract and the control group upon A
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treatment of commercial tannin? O
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1.2.5.1 100 mg Y

1.2.5.2 200 mg S
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1.2.5.3 300 mg I
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1.2.6 Is there a significant difference in the lactic acid level of the

experimental group before and after treatment compared to the control group?
1.2.7 Is the Syzygium cumini (Duhat) bark extract effective as a lowering agent

for lactic acid level in Wistar rats?

1.3 Hypothesis

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There is no significant difference in the lactic acid level of the L
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experimental group before and after treatment as compared to the control G
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group.
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The Syzygium cumini (Duhat) bark extract is not effective as a lowering M

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agent for lactic acid level in Wistar rats. D
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1.4 Significance of the Study A
B
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The outcome of this study will benefit the following: T
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Patients with lactic acidosis. This study will be beneficial for patients R
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with drug-induced lactic acidosis due to the discovery of a new S
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treatment option that uses a more natural approach. I
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Doctors. The doctors will be given treatment recommendations in N
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addressing patients experiencing lactic acidosis.

Farmers. Farmers of Syzygium cumini (Duhat) plant (including vendors)

will yield more income due to a higher demand. Additionally, the bark
that is usually of no use to consumers will now be utilized in the field of

medicine.

Advocates for natural remedies. This study is in favor of those who

advocate and opt for a more natural regimen. It will also help them

promote Duhat tree as a lowering agent for lactic acidosis with factual
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information. O
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The Future Researchers. The results and findings of this research will
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serve as reference for future researchers once they conduct similar E

studies. This will contribute to the realm of related literature in the O

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academe.
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1.5 Scope and Delimitation of the Study
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This study will only focus on the effectiveness of Syzygium cumini O
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(Duhat) bark as a lowering agent for increased level of lactic acid in T
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Wistar rats. The bark is the only plant that will be utilized due to its R
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abundance in tannin content. This also serves as the plant’s protection
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from bacteria and fungi according to the United States Department of I
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Agriculture. N
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The model organism of choice will be male Wistar rats, aged

between 8-12 weeks and about 180-200 grams based on the study of
Novita Sari, H., Rina, A. (2020). At this age range, many of their

developmental processes are ongoing. Older counterparts possess a

higher risk of diseases while the female rats have hormonal fluctuation

tendencies – both of which are not suitable to utilize in the experiment.

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The lactate blood test will be the sole test utilized in this O
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experiment and it will be performed with the aid of a veterinarian.
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Currently, veterinary clinics use automated tools and equipment for a E

faster, more efficient procedure in monitoring lactic acid level in O

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animals.
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1.6. Definition of Terms
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Biguanides. These are a class of oral type 2 diabetes medications that O
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operate by limiting glucose synthesis in the liver, enhancing insulin T
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sensitivity, and lowering the amount of sugar absorbed by the R
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intestines.
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Buformin. Like phenformin and metformin, stops cancer from growing N
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and spreading. The ability of these medications to disrupt the Warburg E

effect and convert the cytosolic glycolysis characteristic of cancer cells

to normal pyruvate oxidation by the mitochondria gives them

anticancer properties.

Decoction. Decoction is a method of extracting herbal or plant

chemicals by boiling the material, which can include stems, roots,

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bark, and rhizomes, to dissolve the chemicals. O
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Diabetes Mellitus. Diabetes mellitus is a condition in which the body E

does not create adequate insulin or does not appropriately respond to O

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insulin, resulting in excessively high blood sugar (glucose) levels.
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Urination and thirst are increased, and people may lose weight even if D
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they are not attempting to do so. C
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Distilled Water. Distilled water will be used as solvent in the process of A
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extraction in replacement of alcohol. O
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T
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Infusion. The process of extracting chemical compounds or tastes from R
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plant material in a solvent such as water, oil, or alcohol by leaving the
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material to remain suspended in the solvent for an extended period of I
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time (a process often called steeping). The resulting liquid is known as N
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an infusion. E
Lactate blood test. Lactic acid testing is a blood test that determines

how much lactic acid is produced in the body. Muscle tissue and red

blood cells produce the majority of it. Carbohydrate breaks down into

water and carbon dioxide when the body's oxygen level is normal.

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Lactic Acidosis. Lactic acidosis is a type of metabolic acidosis that O
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occurs when a person's body cannot respond to variations in lactic acid
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production or utilization. Lactic acidosis affects the ability of the liver E

(and, in certain cases, the kidneys) to eliminate excess acid from the O
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body.
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Maceration. The process of maceration is the breakdown and softening C
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of various substances. The procedure is most commonly used in food
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preparation when softening and flavoring seed by soaking it in sugar, A
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alcohol, or other flavored liquids. O
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Percolation. Percolation is a method of extracting all of the soluble R
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elements from comminuted plant material by extracting the crude
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medicine with a new solvent. I
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Phenformin. Phenformin is a lipid-soluble medication that causes E

lactic acidosis by penetrating the mitochondrial membrane and

blocking mitochondrial oxidative phosphorylation as well as

gluconeogenesis.

Syzygium cumini (Duhat). Syzygium cumini, also known as Duhat or

Jambolan, is a flowering plant in the Myrtaceae family that is prized for

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its seed, timber, and decorative value. O
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Water bath - A water bath is a piece of laboratory equipment that keeps E

samples at a steady temperature for an extended length of time. To O

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prevent ignition, a water bath is preferred over an open flame while
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heating combustible compounds. D
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Wistar rats. The Wistar rat is a genetically diverse stock that is
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employed in medical and biological research in a wide range of sectors. A
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It's good for geriatric studies because of its extended lifespan and high O
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rate of spontaneous tumors. It's an albino type that's easy to handle T
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but takes longer to learn than the Long Evans rat. R
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Chapter 2 O
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REVIEW OF RELATED LITERATURE
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This chapter reviewed the associated literature and studies that C
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gave direction to the study. Included also is the conceptual framework
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that was the basis of this study. E

2.0 Conceptual Framework

 Listed on the first box are the primary materials that the

researchers will utilize in the experiment. The Syzygium cumini (Duhat)

bark will be tested for its ability to lower lactic acid level in male Wistar

rats. Commercial tannin will act as a control to base off the rate of
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effectivity of Syzygium cumini (Duhat) bark extract. Phenformin will be O
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used as the drug for inducement of lactic acid to the Wistar rats to
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achieve a uniform level of lactic acid across all subject groups. E

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The second box shows the step-by-step procedure of the
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experiment. It begins with modifying the Syzygium cumini (Duhat) bark D
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into its concentrated powder form. Next to this would be the grouping C
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of Wistar rats (four groups) based on the dosage of the extract and a
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separate control. Prior to inducement, the baseline lactic acid level shall A
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be tested by the veterinarian. After this, inducement of lactic acid level O
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will be performed using the mentioned drug along with strenuous T
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activities. The last step is administering the concentrated powder of R
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varying dosage to each corresponding group. In totality, the lactic acid
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level shall be monitored before and after inducement as well as after I
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treatment. Results will then be compared. N
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The last box consists of the expected outcomes of the experiment.

This study shall yield a concentrated powder of Syzygium cumini

 (Duhat) bark extract as end-product. Comparative results of the

(induced) lactic acid level before and after the treatment will also be an

outcome of this study. These results will be the basis in determining

the effectivity of Syzygium cumini (Duhat) bark in addressing lactic

acidosis caused by elevated lactic acid.

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Figure 1 shows an overview of the process of this study. O
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 Syzygium cumini  Extraction of  Syzygium cumini M

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(Duhat) bark Syzygium cumini (Duhat) bark D
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 Wistar rats (Duhat) bark extract: a lactic C
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 Commercial  Grouping of acid lowering L

Wistar rats agent L

tannin (Pine bark
A
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extract) Group A  Research data on O
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 Phenformin Group B the capabilities of A
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Group C Syzygium cumini O
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Control (Duhat) bark in Y

 Baseline test lowering lactic S

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 Inducement acid level in I
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 Treatment Wistar rats.
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 Discovery of the

effectiveness of

Syzygium cumini

(Duhat) bark

extract in treating

lactic acidosis.
 Figure 1: Conceptual Framework
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2.1 Related Studies and Literatures E

(Note: studies that has a connection to the major topics in your O

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research e.g. - Research problem – uric acid, diabetes, MRSA, biofilm
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formation D
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- Research materials – banana plant, mice or rats, bacteria C
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- Procedure or process – biofilm inhibition assay, Kirby Bauer
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2.2 Justification of the Study O
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This study aims to prove the potentials of Syzygium cumini R
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(Duhat) seeds and bark in the treatment of lactic acidosis. Although
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there have been researches about the capabilities of this plant as I
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antidiabetic, there is no knowledge about its possible capacities to N
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lower lactic acid levels. The vast works of literature found in the E

database contain reports of how Jambolan extracts have

pharmacological actions. Hence, further study of the active compounds

found in S. cumini can benefit us.

On the other hand, lactic acidosis starts when a person either

produces too much or utilizes too little lactic acid, in which case, the
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body fails to adapt to such changes. According to DeFronzo et al. O
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(2016), despite metformin as a drug of choice for type 2 diabetes
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mellitus, some patients disagree to accept metformin as a medication E

because of the risk of lactic acidosis. The anion gap's presence in a O

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diabetic patient, if associated with compromised renal function, should
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make clinicians consider metformin as a factor to contribute to it. Out D
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of proportion to the patient's clinical performance, the consideration is C
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significant in patients with severe anion gaps linked to lactic acidosis
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(Blough et al., 2015). A
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The researchers will determine if the seeds and bark of Syzygium O
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cumini can control the lactic acid levels through details gathered by T
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observations and results indicated by the use of a blood test on Wistar R
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rats with induced lactic acidosis. Sometimes lactic acidosis presents
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urgent medical care where it requires the treatment of symptoms; I
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therefore, the researchers value the development of a new cost-effective N
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medication for lactic acidosis, especially for lactic acidosis cases E

induced by pharmaceutical drugs. The data collected in this project

benefits the patients with lactic acidosis, people looking for a cheap
medication for lactic acidosis, the medical community, the future

researchers who will conduct studies related to the variables utilized,

and the people with curiosity or demand for this study.

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Chapter 3 O
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RESEARCH METHODOLOGY D
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3.0 Research Design
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This study will utilize a true experimental research design and O
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will employ a mixed method approach (quantitative & qualitative). T
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More specifically, this study will rely on statistical analysis to test the R
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hypothesis with the use of a variable that can be manipulated, a control
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group, and a random distribution of the test subject. Experimental I
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research also allows the researchers to manipulate and control the N
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environment of the subject to avoid major interferences on the findings. E

The primary independent variable is the Syzygium cumini (Duhat) bark

that will be turned into a concentrated powder form. This variable is

not affected by other factors in the experiment. On the other hand, the

primary dependent variable will be the lactic acid level of male Wistar

rats: post-treatment. The quantitative data from the results will vary

according to the dosage (of extract) administered to each corresponding

group. The numerical data also includes the optimum concentration

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effective for lowering lactic acid level. Moreover, the change in lactic O
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acid level after treatment as compared to the baseline level will be part
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of the quantitative data. On the other hand, the qualitative data will E

conclude and define the numerical results. The effectiveness of the O

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extract based on the results comes first. Also included is the
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phytochemical components of Syzygium cumini (Duhat) bark extract D
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that is able to lower the lactic acid level. Description of the end-product C
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from the bark extract also adds to the qualitative data. Pretest and
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posttest results will be monitored accordingly and a professional A
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statistician will be consulted to thoroughly evaluate the data obtained O
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from the observations. All the aforementioned variables will approve or T
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disprove the hypothesis about the Syzygium cumini (Duhat) bark R
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extract as a lowering agent of lactic acid.
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3.1 Locale of the Study N
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The laboratory experiment will be conducted on the second

semester of academic year 2021-2022. The researchers will perform the

experiment at the laboratory premises of Our Lady of Fatima University

- Antipolo campus. It provides the researchers enough and safe space,

making it conducive for laboratory procedures. Majority of the tools and

equipment needed are also available and accessible in the said

institution. It is also a convenient place for the researchers to gather

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considering their academic affiliation to the OLFU-Antipolo campus. O
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3.2 Test Sample of Population O

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ASSIGNED TO BIANCA
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D
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3.3 Ethical Consideration C
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This section presents the assessment on the utilization of Wistar A
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rats with considerations to ethics. It has been significant to use O
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laboratory animals to gain knowledge to improve the people, animals, T
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or the environment, so the researchers must also consider the moral R
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status of the animals as experimental subjects. The researchers follow
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the guidelines prepared by the National Committee for Research Ethics I
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in Science and Technology (NENT). N
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Respect for animals' dignity

 The primary methods of the researchers to induce lactic

acidosis are through the administration of medication that can cause

lactic acidosis and with the utilization of rat wheels for the Wistar rats

to undergo a strenuous activity that may lead to a transient lactic

acidosis. The researchers will not introduce acute medical diseases to

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induce lactic acidosis. The researchers will provide care for the O
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acclimation of the laboratory rats through humane handling from
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purchasing with a permit to housing, providing sufficient water and E

food, extracting fluid or blood by a healthcare professional with as little O

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recurrence as needed to ensure less pain, and through giving a
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treatment. D
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Responsibility for considering options (Replace)
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The researchers had concluded the lack of other options A
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other than the utilization of rats as test samples to investigate the O
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potential of Syzygium cumini in the lowering of high lactic acid levels. T
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To accomplish this study is necessary to obtain for a certain amount of R
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time.
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The principle of proportionality (balance between suffering and benefit) N
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The researchers will ensure a process tolerable for Wistar E

rats to experience as test samples. To obtain the principle of

proportionality, the value of the knowledge the public can obtain from
this study will not cause more harm to the test samples as much as

other methods can do, such as introducing an acute disease to get

results.

Responsibility to consider the reduction of the number of animals

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(Reduce) O
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The researchers will use nine Wistar rats for the
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experimental group and three Wistar rats for the control group. The E

researchers also consider obtaining a few spare Wistar rats in case of O

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emergency, like the death of one of the samples. This number of rats is
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the minimum number considered thoroughly by the researchers. D
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Responsibility for the minimization of the risk of suffering and the
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improvement of animal welfare (Refine) A
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The researchers will minimize the risk of suffering and O
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provide good animal welfare through enough food and water, a well- T
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ventilated place to avoid abnormal cold or heat, and the allowance to R
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behave naturally. The researchers will also recognize the conditions of
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the test samples BEFORE and AFTER the experiment, such as I
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transportation, breeding, anesthetizing, etc. In short, the researchers N
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will consider the need for a time before and after the adaptation of the E

rats.
Responsibility for maintaining biological diversity

The researchers will not use animals that can threaten

biological diversity or endangered animals.

Responsibility when intervening in a habitat

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The researchers will ensure not to disrupt the natural O
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behavior of their laboratory animals, which may also help get better
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results. E

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Responsibility to the opening and sharing of data and material
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The researchers offer the research findings to be public D
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knowledge to avoid the repetition of the experiment. It can also help C
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reduce the utilization of the animals for the experiments.
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Requirement of expertise on animals O
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The researchers seek the assistance of a veterinarian and T
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animal caretakers for the optimum results of the activity. R
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Requirement of due care I
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The researchers are familiar and will comply with the N
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guidelines stated. E
3.4 Materials and Methods

3.4.1 Materials

3.4.2 Procedures
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3.5 Collection of Data
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Through experimentation, the researchers will collect data on the O

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demographics of Wistar rats in the experimental and control groups
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based on their age, sex, and weight. The researchers will also collect D
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information from journals and other studies about the phytochemical C
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components of Syzygium cumini that are related to reducing lactic acid
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levels. The researchers will measure lactic acid levels three times to A
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determine the baseline level upon inducement and after treatment. O
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3.7 Data Analysis R
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