CHAPTER 1

THE PROBLEM AND ITS BACKGROUND

1.1 INTRODUCTION

Peperomia Pellucida (L.) is an herbaceous plant found in man south American

and Asia countries. The species developed during rainy period (often in the spring)

and thrives. In loose, humid soils under the shade of tress. 1-4 it grows in moist

habitat and is found throughout the major parts of India. According to the study

(Journal of pharmaceutical and allied Sciences a (3, 1637-1652,2012) Peperomia

Pellucida showed mild toxicity in certain organs and it will be necessary to fully

establish it safety profile as this may likely unlock its mechanism of action as an

anti-inflammatory agent. It also demonstrates anti-microbial activity against

clinical strains of selected microorganisms, thereby justifying its usage in tradition

medicine and not cause death or toxicological symptoms in mice, extract leaf

showed mild to moderate congestions and infiltration of chronic inflammatory

cells.

Peperomia Pellucida (L.) is consumed as Vegetables and used in

Cameroonian traditional medicine for the management of diseases and for fracture

healing. The plants has a strong mustard- like odor and may cause asthma-like

symptoms patient with known hypersensitivity reaction to the plants species.

There is innumerable adverse effect caused by these allopathic medicine

indicating the need to identify alternatives with fewer side effect. In the search for

new drug treatment, metabolites derived from plants used. In folk medicines it is

used to prepare infusions and decoction to treat gastric ulcers as well as gout

arthritiss, wound, high blood cholesterol and skin related problems however

1
scientific studies, the gastro protector constituents of Peperomia pellucida have

not yet been identified. Humans and mice share many common genetic features

and by examining the physiology, anatomy and metabolism of a mouse, scientists

can gain a valuable insight into how humans function. Now scientists use mice to

simulate human genetic disorders? in order to study their development and test

new therapies. As a scientific tool, mice have helped to speed up the progress of

research and enabled the development of important new drugs. Mice are cost

effective because they are cheap and easy to look after

Pansit-pansitan, scientifically known as Peperomia pellucida, is a medicinal

plant valued for its anti-gout properties which help to lower the uric acid in the

blood. P. pellucida has been used as a food item as well as a medicinal herb. The

plant is used as medicine for treating different types of various ailments or

diseases such as abdominal pain, abscesses, acne, boils colic, fatigue, gout,

headaches, renal disorders, and rheumatic joint pain. P. pellucida (Olasiman ihalas)

was one of the ten clinically tested medicinal plants endorsed by the Department

of Health. (Queiroz et al. (2020)

The present study with aim of gaining more knowledge as it plays a significant

role in treating some health problems and some inhabitant rely on this as

traditional medicine for their primary health care needs. In addition, this study

also aims to be beneficial not only for the researchers but also to all the people

who are on the field of studying science that this study open ups to new ideas for

them and most importantly it becomes be of help in the community as whole.

We researchers conducted this study to determine if Peperomia pellucida boil

leaf be of help for the female white mice to lessen their uric acid. We aim for the

effectiveness of Peperomia pellucida boil leaf as a means of treatment for

2
 therapeutical illness and if this become successful be of use for some

pharmaceutical treatment.

1.2 CONCEPTUAL FRAMEWORK

INDEPENDENT VARIABLE DEPENDENT VARIABLE

Peperomia pellucida (Olasiman Effect on the Uric acid of

ihalas) concetrations (250 Mice
mg/ml, 150mg/ml and 50mg/ml)

Figure 1. Conceptual Framework of the study

The above figure shows the conceptual framework of the study showing the

Independent and dependent variables P. pellucida (Olasiman ihalas) concetrations

(250ml, 150ml, 50ml) is the independent variable, while Effect on the Uric Acid of

Mice is the dependent variable.

1.3 STATEMENT OF THE PROBLEM

Plants have played a significant role in maintaining human health (Craig, 1999)

and improving the quality of human life for thousands of years and have served

humans well as valuable components of food and medicines (Winston, 1999). The

plant P. pellucida (Olasiman ihalas) will be tested for its anti-hyperuricemic effects.

Uric acid is a purine derivative and an oxidative metabolic product of purine

nucleotides in humans and other carnivorous animals. The researcher aims to study

the effect of P. pellucida (Olasiman ihalas) boiled on the uric acid of the female white

mice.

3
Specifically, this study sought to answer the following questions:

1. What is the baseline of the uric acid level of female white mice before

administration of the P. pellucida (Olasiman ihalas) boil leaf??

2. What is the uric acid level of female white mice after administration of

Pyrazinamide.

3. What is the uric acid level of female white mice after administration of P.

pellucida (Olasiman ihalas) boiled leaf?

4. Is there a significant decrease in the uric acid after the administration of P.

pellucida (Olasiman ihalas) boiled leaf?

5. Is there a significant difference of uric acid decrease across different

concetration of P. pellucida?

1.4 OBJECTIVES OF THE STUDY

The objective of this study is to determine the effect of the P. pellucida

(Olasiman ihalas) boiled leaf on the uric acid of female white mice.

Specifically, this study aims to,

1. Determine the effect of Peperomia Pellucida (Olasiman Ihalas) boiled leaf

in the uric acid level of the female white mice.

2. To evaluate the significant difference between the different concentrations

of P. pellucida (Olasiman ihalas) (250 mg/ml, 150mg/ml and 50mg/ml) in

terms of the uric acid level of female white mice.

4
1.5 HYPOTHESIS OF THE STUDY

NULL HYPOTHESIS:

Ho1: There is no effect of Peperomia Pellucida (Olasiman Ihalas) boiled leaf in the

uric acid level of the female white mice.

Ho2: There is no significant difference between the different concentrations of P.

pellucida (Olasiman ihalas) (250 mg/ml, 150mg/ml and 50mg/ml) in terms of the uric

acid level of female white mice

ALTERNATIVE HYPOTHESIS:

Ho1: There is an effect of Peperomia Pellucida (Olasiman Ihalas) boiled leaf in the

uric acid level of the female white mice.

Ho2: There is a significant difference between the different concentrations of P.

pellucida (Olasiman ihalas) (250 mg/ml, 150mg/ml and 50mg/ml) in terms of the uric

acid level of female white mice

1.6 SIGNIFICANCE OF THE STUDY

This study helps students and other researchers to widen their knowledge on

the effect of P. pellucida (Olasiman ihalas) boiled leaf on the uric acid of the mice.

The result of this study would benefit the following persons:

Community- this study would be a big help in society to expand their

knowledge about uric acid and P. pellucida (Olasiman ihalas), especially to those

people who suffer from hypeuricemia and also to those people who have a problem

with finances to buy a medicine for reducing uric acid.

5
 Future Researcher – this study would provide information and data to future

researchers who intend to conduct a similar study.

Health Care Workers– this study would bring new strategies to provide the

best patient care to improve the coverage of health services.

Pharmaceutical Companies- this study would bring new medicine that

improves the health and quality of life of patients around the world.

Department of Medical Technology- this study would provide knowledge

and data to the students, who intend to conduct related study.

1.7 SCOPE AND LIMITATION

This study mainly focuses on determining the effect of Olasiman ihalas (P.

pellucida) boiled leaf in reducing uric acid in female white mice. The sampling is

only focused on 33 adult female white mice that will be bought from 89-A Animas

Corner, Abanil Street Aguada, Ozamis City, Misamis Occidental 7200 Region 10.

1.8 DEFINITION OF TERMS

The following terms are defined theoretically.

Uric Acid. It determines how much uric acid is present in your blood. The test can tell
you how well your body produces and eliminates uric acid (Gabby et al. 2020).

P. pellucida (Olasiman ihalas).It refers as food, flavoring agent and as medicine. The
plant is used as medicine for treating various ailments or disorders such as asthma,
rheumatism, wound, fever, stomach problems, kidney infection, hemorrhoid pain,
joint pain, hypertension, diarrhea, snake bite and measles( Kekuda 2018).

Albino mice. It refers with a white fur and pink eyes that are commonly used in
laboratory experiment ( Ball 2016).

6
Pyrazinamide.Antituberculosis medications such as pyrazinamide have been
associated with increasing uric acid levels (Pham et al. 2014).

Hyperuricemia.It is a high uric acid level in the blood. The normal upper limit is
6.8mg/dL, and anything above 7mg/dL is considered saturated, with symptoms
possible. This elevated level is the result of increased uric acid production, decreased
uric acid excretion, or a combination of the two processes (George 2021).

Gout.It causes intense pain, swelling, and stiffness in the joints and Its main cause is
the presence of too much uric acid in the body (McIntosh 2021).

Allopurinol refers to a synthetic drug that reduces blood concentrations of uric acid
and is administered orally in the treatment of gout (Collins 2019).

Oral Gavage refers to a passage of a gavage needle into the esophagus, and this
technique often involves the animal swallowing the gavage needle as it approaches
the pharynx (Hoggatt 2010).

Diastema refers to a gap between your teeth. This can happen between any of your
teeth. Because of its position, it’s most noticeable when there’s a gap between your
upper front teeth (Brennan 2021).

7
 CHAPTER II

REVIEW OF RELATED LITERATURE

This study mainly presents the related literature and studies on the test subjects and

theory involved in this study.

Plants are free gifts of nature available at human disposal for various

pharmacological uses. They are therefore, the source of active chemical compounds

such as alkaloids tannins, flavonoids, sugars phenols, coumarins, terpenoids, saponins,

etc useful to both Man and animals. The rise in resistance of the human body system

to certain orthodox medicine has called for urgent exploration of natural products for

medicinal uses. Research carried out on medicinal plants is directed towards the

isolation of active compounds and once isolated, the active components found in these

plants are processed for the purpose of administering them in a sufficiently and more

precise dosage. The reliance of the pharmaceutical industry on organic Chemistry is

therefore immense. Although oxidation reactions are crucial for life, they can also be

damaging hence, plants and animals maintain complex systems of multiple types of

antioxidants, such as glutathione, vitamin C and vitamin E as well as enzymes such as

catalase, superoxide dismutase and various peroxidases. Oxidation reactions can

produce excess free radicals which start chain reactions that damage cells.

Antioxidants terminate these chain reactions by removing free radical intermediates

and inhibit other oxidation reactions. In recent times, antioxidant chemistry has

gained a lot of attention due to threats to biological macromolecules by oxidative

processes. Plants are the major focus of researchers for isolation of antioxidant

secondary metabolite. Peperomia pellucida also called Silver bush belongs to the

family Piperaceae. It is a herbaceous plant found in many South American and Asian

countries. The plant species has a history of ethnomedicinal uses which include

8
treatment of abdominal pain, abscesses, acne, boils, colic, fatigue, gout headache,

renal disorders, rheumatic pain and to treat breast cancer, impotence, measles, mental

disorders and small pox.However, there has not yet been validated clinical data with

regards to Peperomia pellucida dosing. The plant’s contraindications suggest that

patients with known hypersensitivity reactions to any of the components of the plant

species should avoid its use. The plant species also interferes with prostaglandin

synthesis which is contraindicating for nursing mothers. Other medicinal properties

vary depending on region. In Guyana, the plant has been used to lower cholesterol and

has been used as a cough suppressant, diuretic, emollient and treatment of cardiac

arrhythmia in the Amazon region. Numerous chemical investigations, primarily on the

essential oils of the plant are found in medical literature. One study identified 71

compounds from the essential oils of 10 piperaceae species. Flavonoids and

phytosterols such as acacetin, apigenin, isovitexin, pellucidatin campesterol and

stigmasterol, substituted styrenes and pellucidin A have also been documented. Other

compounds like the peperomins with in vitro cytotoxic or anticancer activity and

arylpropanoids such as the apiols with antifungal activity have been isolated from

Peperomia species (Chiamaka et al. 2019)

Peperomia pellucida (L.) HBK is also known as shiny bush or silver

bush belonging to family Piperaceae. In Sanskrit, it is known as Toyakandha,

Varshabhoo. Peperomia pellucida is an herbaceous plant found in many South

American and Asian countries. The species develops during rainy periods (often in

the spring) and thrives in loose, humid soils under the shade of trees. It grows in moist

habitat and is found throughout the major parts of India. In different parts of India it is

known with different names like Lochi pata in Bangali, Mashitandu chedi in

Malayalam and Pononoa in Assamese etc. Whole plant or parts of plant are used for

9
different purposes. Despite its wide range of folk medicinal uses in India sub-

continent, there is very little scientific documentation available on its pharmacological

and biological activities as well as its chemical constituents. Peperomia pellucida

leaves and stems may also be eaten as vegetable. In salads, the fresh plant has the

crispness of carrot sticks and celery. As Ethnomedicinal uses of this plant Peperomia

pellucida has been applied for treating abdominal pain, abscesses, acne, boils, colic,

fatigue, gout, headache, renal disorders, and rheumatic joint pain. In Bolivia, Altenos

Indians use the whole plant to stop hemorrhages. The roots are used to treat fevers and

the aerial parts are used as dressing for wounds. In northeastern Brazil, the plant has

been used to lower the cholesterol level. In Guyana and the Amazon region, it is a

popular cough suppressant, emollient, and diuretic. It is also used to treat proteinuria.

In the Philippines, a decoction of the plant is used to decrease uric acid levels and to

treat renal problems This plant has externally used as a facial rinse for complexion

problems. Pounded whole plant used as warm poultice for boils, pustules and pimples

and also used for headaches, rheumatic pains and impotence. Peperomia pellucida is

also used in traditional Ayurvedic medicine. It is described in Ayurveda as – Rasa –

Katu and Madhur; Guna- Lakhu, rooksha, Teekshna; and Virya- Ushna. The plant is

described to passify vitiated cough, pitta, constipation, kidney diseases, urinary

retention, dysuria, urinary tract infections, emaciation, edema and general weakness.

Infusion and decoction of leaves and stems of fresh plant are eaten as salad for the

treatment of gout and arthritis (Ganiyat et al. 2011).

According to Ethno-botanical studies the whole plant has been in medicinal

use for since long. It is crushed and mixed with water to form a mixture, heated, and

administered orally to cure hemorrhage. It has also been applied against coughing,

fever, common cold, headache, sore throat, diarrhea, kidney and prostate problems,

10
and against high blood pressure. The analgesic properties of the plant seem to be

related to its effect on prostaglandin synthesis. It may have potential as a broad-

spectrum antibiotic, as demonstrated in tests against Staphylococcus aureus, Bacillus

subtilis, Pseudomonas aeruginosa, and Escherichia coli. Chloroform extracts from

dried leaves of P. pellucida have been shown to exhibit antifungal activity against

Trichophyton menta- grophytes in vitro. Although the plant can cause asthma-like

symptoms in patients with known hypersensitivity reactions to the species, no clinical

data have yet been reported on human toxicity (Sheikh et al. 2021).

The plant Peperomia pellucida was tested for its anti-hyperuricemic effects.

Thirty white male rats were distributed into three groups given placebo, allopurinol,

and Peperomia extract after induction of hyperuricema with the use of potassium

oxonate and uric acid. Fasting blood uric acid levels were taken at 12, 24, 36, and 48

hours after test drug administration. In the allopurinol group, the mean blood uric acid

level significantly decreased by 32.28% by the 36th hour and 55.26% by the 48th

hour. In the Peperomia group, the mean blood uric acid level significantly decreased

by 30.29% by the 36th hour and 61.14% by the 48th hour. This study confirmed the

anti-hyperuricemic potential of the plant Peperomia pellucida.(Alvero et al. 1992)

LOCAL STUDY OF P. PELLUCIDA

The Piperaceae is a large family of angiosperms composed of around 3700

species (Christenhusz and Byng, 2016). It is among the oldest of the pan-tropical

plants and its species are mainly distributed in two genera: Piper with around 2000

species (Quijano-Abril et al., 2006) and Peperomia with approximately 1600 (Frenzke

et al., 2015). The group Peperomia is one of the largest genera of basal angiosperms

(Wanke et al., 2006) and is composed of herbs usually perennial (Shu, 1999).

11
Several Peperomia species are cultivated as ornamentals because of the beauty of their

foliage (Guimarães and Carvalho-Silva, 2012). Its species are dispersed pantropically,

having the greatest biodiversity in the Neotropics followed by Southern Asia (about

100 species), Madagascar (about 40 species), Africa (about 20 species), Australia and

New Zealand (less than 20 species) (Wanke et al., 2006).

Peperomia pellucida (L.) Kunth is known for its variety of pharmacological

properties. This plant is an herbaceous herb with succulent, alternate oval leaves and

inflorescences in terminal spikes, axillary and opposite to the leaves. The species

grows well in humid and loose soils in places with reduced solar radiation with

preference to rainy periods (Arrigoni-Blank et al., 2004). Stems are succulent,

translucent green, erect or ascending, and internodes are usually 3–8 cm long,

glabrous and hairless (Majumder, 2011). It is an annual, fasciculate short-rooted herb

usually having a height of 15–45 cm (Majumder and Kumar, 2011). The plant is a

weed with heart-shaped leaves and tiny seeds attaching to the cord-like spikes

(Mosango, 2008, Ooi et al., 2012). Its scientific name is usually written in books and

publications in South America as P. pellucida (L.) H.B.K. However, P. pellucida (L.)

Kunth should be applied since Kunth was responsible for the present classification of

this species (previously named by Linnaeus – 1753 – as Piper pellucidum L.) in the

genus Peperomia (Mathieu and Posada, 2006). The abbreviation H.B.K. is possibly

used because of its taxonomic information published in the book “Nova Genera et

Species Plantarum” which was written by Humboldt et al. (1815) – H.B.K.

Traditionally, Peperomia pellucida has been utilized in the folk medicine of

several pantropical countries to treat a wide spectrum of ailments and diseases such as

skin sores (Arrigoni-Blank et al., 2002), gastrointestinal disorders (Mollik et al.,

2010), dysentery, diarrhea, indigestion (Mollik et al., 2010), abscesses and injuries

12
(Bojo et al., 1994). In the literature, numerous studies on chemical and

pharmacological properties have been reported for its EO and extracts, which include

cytotoxic (Xu et al., 2006), analgesic (Aziba et al., 2001), antibacterial (Khan and

Omoloso, 2002), and anti-inflammatory potential (Arrigoni-Blank et al.,

2004). Peperomia pellucida essential oils (EOs) and extracts are typically composed

of phenylpropanoids followed by sesquiterpenes (De Diaz et al., 1988). However, the

chemical composition can vary significantly depending on its place of origin.The aim

of this review is to evaluate the present ethnomedicinal

applications, phytochemical and pharmacological studies of P. pellucida EOs and

extracts from different locations around the world under a critical view. A similar

review on P. pellucida was published by Raghavendra and Prashith Kekuda (2018).

However, a more detailed survey with a critical analysis of the available data is still

needed to understand the state of the research on this species. In the present work,

there is a more extensive coverage of this plant's ethnopharmacological and

phytochemical characteristics. The biological activities and ethnopharmacological

aspects reported also include new references. Furthermore, a critical selection of the

material included was also done since several studies have described pharmacological

properties with high concentrations of extracts and/or essential oils of P. pellucida. In

general, the literature previously published failed to report several scientific details,

which emphasizes the importance of conducting more complete studies focused on P.

pellucida ethnomedicinal, pharmacological potential and phytochemical composition

(Alves et al. 2019)

URIC ACID

Gout and hyperuricemia are metabolic disorders associated with abnormal

amounts of uric acid in the body, resulting in the deposition of urate crystals in the

13
joints and kidneys, causing inflammation as well as gouty arthritis and uric acid

nephrolithiasis (Kramer and Curhan, 2002, Tomita et al., 2000). Xanthine

dehydrogenase (XDH) and xanthine oxidase (XO), which are present in significant

concentration only in liver and intestine, oxidizes hypoxanthine and xanthine to uric

acid in the purine catabolic pathway. Treatment and prevention of gout and

hyperuricemia entail to reduce serum uric acid concentration in the body. XDH and

XO inhibitors are available to block the final step in uric acid synthesis, reducing the

production of uric acid, as well as anti-inflammatory agents to relieve the symptoms

of the disease (Kelley, 1991, Star and Hochberg, 1993, Wortmann, 1993). Allopurinol

is the only inhibitor of the enzyme in clinical use, but its use can result in a number of

adverse side effects, such as allergic and hypersensitivity reactions, nephropathy, and

enhancement of 6-mercaptopurine toxicity (Hande et al., 1984, Urban et al., 1995,

Sumi and Wada, 1996). Thus, the development of new hypouricemic agents of greater

effectiveness and safety is highly warranted.A Chinese herbal medicine Ermiao wan,

which is composed of the phellodendri cortex and atractylodis rhizome, has long been

used in traditional medicine. According to Dan-Xi-Xin-Fa (Zhu Zhenhen, Yuan

dynasty: a Chinese medicine book named Danxi’s Experiences in Medicine), Ermiao

wan was descried to treat acute gout through eliminating heat and excreting dampness

in terms of traditional Chinese medicine. Ermiao wan is recorded in State

Pharmacopoeia of People’s Republic of China at all times for the successful treatment

of gout and hyperuricemia. Despite the facts that phellodendri cortex and atractylodis

rhizome have shown antioxidant and anti-inflammatory activities (Endo et al., 1979,

Yamahara et al., 1990, Resch et al., 1998, Lee et al., 2000), it is not clear whether

Ermiao wan can reduce serum uric acid level in a hyperuricemia model and inhibit

XDH and XO activities. The aim of the present study was to evaluate the effects of

14
Ermiao wan in vivo on reduction of serum uric acid level in hyperuricemic mice and

the activities of mouse liver XDH and XO.In normal mice, serum uric acid level was

3.24 ± 0.41 mg/dl. In hyperuricemic animals, serum uric acid level was elevated to

5.73 ± 0.15 mg/dl. ( Kong LD et al. 2004).

Uric acid, an antioxidant has been discovered to protect against declining lung

function due to aging or lung disease, particularly in women. High uric acid levels can

cause health problems such as gout and renal damage, but this study found that it

protects females from lung function decline. The function of uric acid and other

antioxidants in the lungs, as well as gender differences, will most likely be considered

for future lung disease management. They began by developing a murine lung disease

model with elevated uric acid levels in the blood. Because mouse uric acid levels are

low in comparison to humans, the researchers used gene disruption and inhibitor

treatments to suppress uricase (UOX), a murine urate-metabolizing enzyme, in order

to raise uric acid levels. They then developed a lung disease mouse model with

emphysema or chronic obstructive pulmonary disease (COPD) and high uric acid

blood levels. They discovered that female mice with UOX gene disruption or UOX

inhibitor treatments had elevated uric acid levels, and that their respiratory functions

had improved. When uric acid levels in male mice were raised, however, symptoms of

lung disease did not improve or worsen. This unexpected discovery suggests that uric

acid protects female mice's lungs. Uric acid also reduces oxidative stress in human

lung epithelial cells, according to further research. In the presence of female

hormones, the antioxidant effect of uric acid was lost in female lung epithelial cells.

In other words, women with lower levels of female hormones are more likely to

benefit from uric acid's antioxidant effect in lung tissue. This suggests that uric acid

15
may have a strong protective effect in elderly women with low levels of female

hormones (Shuto 2020).

Age-related cognitive dysfunction is a neurodegenerative disorder that mainly

affects intellectual abilities used for perceptions, acquiring, understanding, and

responding to information presented to a person. At present, there are more than 15%

of Chinese middle-aged and older people suffering from cognitive impairment

reducing their quality of life. This condition brings a heavy burden on the workforce,

economy, and society at the same time, which highlights the critical importance of

delaying cognitive decline.High plasma uric acid levels have been proved to be

associated with increased risk of gout, cardiovascular diseases, impaired fasting

glucose or diabetes, and chronic kidney diseases. However, lack of sufficient uric acid

may also harm health, with evidence from several studies reporting an association

between lower uric acid levels and higher all-cause and cardiovascular mortality. As a

primary natural antioxidant, uric acid acts as a free radical scavenger that may protect

nerves from oxidative injury and decrease the risk of neurodegenerative conditions.

Nevertheless, epidemiological studies on the association between uric acid and

diverse cognitive outcomes have yielded controversial conclusions. Some studies

found that participants with higher baseline uric acid levels had better subsequent

cognitive performance or decreased risk of dementia, while others reported the

opposite association. Furthermore, most of these studies were limited due to the cross-

sectional design, lack of representativeness, or small sample sizes. In particular, most

previous studies included participants with hyperuricemia, which might make the

association between plasma uric acid levels and cognitive function complex, due to

the impact of the potential use of urate-lowering medications and adverse health

outcomes associated with hyperuricemia. Additionally, few studies considered the

16
association of lower uric acid on cognitive function, especially in the Chinese

population.This study thus aimed to examine the longitudinal association of baseline

plasma uric acid levels with subsequent cognitive function in Chinese adults without

hyperuricemia ( Huang et al. 2022).

BLOOD COLLECTION

Twenty years ago, tail incision for collecting serial blood samples from mice

was introduced as a new technique. Despite a number of advantages over established

methods, it has not become a frequently used technique. This report describes

modifications of blood collection from mice by tail incision that allow obtaining rapid

(1–1.5 min) serial blood samples (40–150 μl) from unanaesthetized laboratory mice.

Evaluation of corticosterone concentrations in the blood plasma from repeated

samples indicated that subsequent samples were unaffected by the procedure.

Furthermore, histopathological examination demonstrated that repeated bleeding did

not cause any lasting harm to the animals. Blood collection by tail incision may,

therefore, be of particular interest for studies that attempt to relate physiological

measures to behavioral responses in laboratory mice, and may contribute to the

refinement of animal experimentation according to the principles of the Three Rs

(Durschlag et al. 1996).

The saphenous venipuncture method was compared with a modified tail-clip

technique that requires minimal restraint. Mice were evaluated through behavioral

observation and plasma corticosterone levels. The results showed that the 2 methods

produced similar corticosterone responses and that the tail-clip method produced

fewer behavioral reactions. In addition, the effects of saphenous venipuncture method

appeared to be dependent on the handler's technical expertise. When a series of 4

17
blood collections were performed over 1 week, the 2 methods yielded similar

corticosterone levels that did not increase over time. Some of the behavioral signs

appeared to increase over the series of blood collections obtained by the saphenous

venipuncture method. Serial complete blood counts showed that the tail vessels

yielded higher total white blood cell, neutrophil, and lymphocyte counts than did the

saphenous vein. Neither method appeared to cause stress-associated changes in the

leukogram after serial blood collection. Overall, the effects of modified tail-clip

method were similar to those of the saphenous venipuncture method in unanesthetized

mice (Omorodola et al. 2008).

PLANT BOILED

Pansit-pansitan (family: Piperaceae) is an herbal medicine also known as

Ulasiman-bato, olasiman-ihalas & tangon-tangon in the Philippines. English name:

peperomia. It is a small herb that grows from 1 to 1 1/2 feet. Pansit-pansitan can be

found wild on lightly shaded and damp areas such as nooks, walls, yards and even

roofs. Pansit-pansitan has heart shaped leaves, succulent stems with tiny flowers on a

spike. When matured, the small fruits bear one seed which fall of the ground and

propagate.The leaves and stalk of pansit-pansitan are edible. It can be harvested,

washed and eaten as fresh salad. Taken as a salad, pansit-pansitan helps relive

rheumatic pains and gout. An infusion or decoction (boil 1 cup of leaves/stem in 2

cups of water) can also be made and taken orally - 1 cup in the morning and another

cup in the evening.For the herbal treatment of skin disorders like abscesses, pimples

and boils, pound the leaves and/or the stalks and make a poultice (boil in water for a

minute or two then pounded) then applied directly to the afflicted area. Likewise a

decoction can be used as a rinse to treat skin disorders.( Philippine Herbal Medicine

2005-2020)

18
 Fresh samples of three culinary herbs, viz. P. crispum (Acc. no. NK-134), T.

foenum-graecum (Acc. no. 03660), and C. citratus (Acc. No. 129848), were collected

from Hamza Nursery Farm, Rawalpindi and their accession numbers were obtained

from Herbarium of Quaid-eAzam University and National Agricultural Research

Center, Islamabad. Similarly, processed samples of these herbs were taken for

analyses from Central Store Department (CSD)Rawalpindi.Three heat treatments,

namely roasting (150–180°C), baking (200°C), and boiling (100°C), were given to

each herb for 10, 20 and 30 min, whereas untreated samples were used as a

control.After heat treatments, samples (1.25 g each) were placed in reagent bottles

and remained water-logged in 25 mL of distilled water for four days at 25 ± 2°C.

Thereafter, the mixture was filtered using a muslin cloth and a Whatman filter paper

and then concentrated using a water bath at 50°C to yield crude extracts. Besides

lipophilic compounds, culinary herbs are also rich in hydrophilic compounds such as

phenols and flavonoids.(Nadeem et al. 2022).

Making a Peperomia pellucida steeping Fresh P.pellucida plants (leaves and

stems) were washed under running water. Following withering processfor 1 day, the

washed plantswere then dried directly under the sunlight for 7 daysto obtain 1 kg dry

plants (0% water). Dried plants were storedin aluminum foilto keep it

dry.Theplantwas brewed with boiling water making thedosageof 150, 300, and 600

mg/kg, respectively.These dosages weredissolved to 3.6 ml/200gramsof ratbody

weight. Brewed P. pellucidawas dissolved with 200 ml of warm waterto easen the

brewing process. Because of the conversion factor for 70 kg of human to 200 g of rat

is 0.018, the dosages 150, 300 and 600 mg/kg of human body weight were converted

to 1.665, 3.333, and 6.666 g/kg rat body weight, respectively. Afterwards, these

19
steepingwere stirred and taken as many as 3.6 ml/200g of ratbody weight ( Hernayanti

et al. 2020).

The plant extracts (broth solution) were prepared to prepare zinc oxide

nanoparticles on the basis of ecofriendly measures, time and cost effectiveness. The

plant materials were thoroughly cleaned with running tap water to remove debris and

other contaminations, followed by double distilled water and air dried at room

temperature. All the parts were separated and finely chopped into small pieces (Figs.

3AB-8AB). About 5 gm of finely cut leaves, stems and roots were kept in a 250 ml

Erlenmeyer flask with 50 ml of sterile double distilled water and then the mixture was

boiled for 30 min. The aqueous plant extracts were cooled down and filtered by

standard filtration method with Whatman filter paper no.1 and stored at 4 oC in

refrigerator for further experimentation. 2.3. Preparation of precursor Zinc Nitrate

hexahydrate [Zn(NO3)26H2O] (Merck, Mumbai) was used as a precursor to

synthesize ZnO nanoparticles in the present study. 1mM Zinc nitrate solution was

prepared using sterile double distilled water and stored in brown bottle at 4 oC for

further use to synthesize ZnO nanoparticles from leaf, stems and roots of P. pellucida

and C. argentea (Shekhawat et al. 2016)

Ten medicinal plants including Peperomia pellucida were obtained from

Lampang Herb Conservation, Thailand during March- June 2012. Plant materials

were dried in oven at 60°C for 72 h and the dried plants were ground. After that, dried

plants (250 g) were soaked with two solvents: distilled water and 95% ethanol in the

ratio of 1:10 (w/v). The dried plants were marcerated with distilled water at 45°C for

3 h, while the procedure with 95% ethanol was performed in a shaker for 72 h at room

temperature. The extract was filtered using Whatman No.1 filter paper.(kaewkod et al.

2015)

20
URIC ACID TEST

Marquardt et al. (1983) reported the development of a high-performance

liquid chromatographic (HPLC) procedure for the assay of uric acid in poultry excreta.

They demonstrated that nearly all of the ultraviolet absorbing material in a perchloric

acid (PCA) extract of excreta was attributable to uric acid. The results suggested that

the uric acid content of excreta could be quantitated directly using a

spectrophotometric procedure. The objective of the current study was to develop a

simple and rapid spectrophotometric method for the direct quantitation of the content

of uric acid in poultry excreta.The source of chemicals and excreta, the type of

equipment utilized, and some of the procedures have been described (Marquardt et al.,

1983). Analysis of variance was calculated using general linear models as outlined by

Helwig and Council (1979). Student-NewmanKuels multiple range test, regression

analysis, and standard error determinations were performed as outlined by Snedecor

and Cochran (1967). Ultraviolet Scans and Extinction Coefficients of Uric Acid. A 1

m/W solution of sodium ureate (MW, 190.1) was prepared in distilled water and

diluted 10-fold with 9 volumes of either .1 N NaOH, .1 N HC1, or 5% PCA. The

solutions were scanned against the appropriate blanks using a DU8 spectrophotometer

(Beckman, Irvine, CA). The absorbance at individual wavelengths was also

determined for a 1-cm light path. Preparation of Standards and Unknown Samples.

Most of these procedures have been outlined before (Marquardt et al., 1983). A uric

acid standard was prepared by dissolving sodium ureate (MW, 190.1) in water or uric

acid (MW, 168.1) in .1 M glycine buffer, pH 9.3, to form a .3 mM stock standard.

This standard, which was stable for 7 days at 2 C, was diluted with different amounts

of water and, prior to use, with an equal volume of 10% PCA. Absorbance was

21
determined against the appropriate blank at the same wavelength as the unknowns

(285 nm). Finely ground excreta (50 mg) was added to 100 ml of .1 M glycine buffer,

pH 9.3, and extracted at 40 C with constant mixing for 1 hr. The suspension was

allowed to settle, and an aliquot was rediluted 15-fold with 14 volumes of 5.35% PCA.

The sample was centrifuged at 20,000 x g for 15 min or filtered through a 13 mm x 5

jum cellulose acetate filter prior to reading the absorbance values. Calculation of Uric

Acid Content of Excreta. The amount of uric acid in the excreta was determined from

the following equation: uric acid in sample (mg) = (A X 100 x 168.1 X 15)/Z where A

= absorbance of sample at 285 nm for a 1-cm light path, 100 is milliliters of original

extraction solution, 168.1 is the molecular weight of uric acid, 15 is the dilution factor,

and 2 is the molar extinction coefficient at a given wavelength (i.e., 11,500 at 285 nm).

The percent uric acid equals the amount of uric acid in the sample (mg) X 100/sample

weight 2106 SPECTROPHOTOMETRIC METHOD: URIC ACID 2107 (50 mg).

Under certain conditions it may be desirable to establish extinction coefficients from a

standard uric acid solution. The determined molar extinction coefficient (£) would

equal A (absorbance of the stock solution when diluted with equal volumes of 10%

PCA) X 103 /C (concentration of diluted stock solution: .15 m/W). The

concentrations of unknowns can also be determined from a standard curve. Treatment

of Samples with Uricase, Percent Recovery of Uric Acid, and Comparison of

Spectrophotometry and HPLC Results. Excreta samples from barley-fed (n = 2) and

starved birds (n = 2) were incubated separately in the presence and absence of uricase

(E.C. 1.7.3.3) for 12 hr at 25 C in pH 9.3 glycine buffer followed by the addition of

PCA as described previously (Marquardt et al., 1983). Uric acid recoveries were

estimated in duplicate for two excreta samples, each of which contained 0, 10, 20, 40,

and 60% added uric acid. The samples, obtained from the previous study, were

22
analyzed for uric acid as described in this study. Excreta samples were also analyzed

in duplicate for uric acid content using the current and an HPLC (Marquardt etal.,

1983) procedure. Excreta from corn-fed (n = 5), barley-fed (n = 5), and starved birds

(n = 9) were obtained as described previousl. (Marquardt et al.,1983).

PLANT CONCENTRATION

Ficus Exasperata, popularly referred to as “sandpaper leaf tree,” finds wide

application in ethnomedicine. In African traditional medicine, all the plant parts are

considered medicinally important, but the leaves are much valued in the treatment of

diseases. The leaf extract has been shown to cause a significant decrease in

hyperglycemia, polyurea, and hyperlipidemia and enhanced serum insulin levels in

streptozotocin-induced diabetic rats. In south-eastern Nigeria, the leaves are mainly

used for the treatment of topical wounds and for the management of diabetes and

hypertension. Since no studies have been done to validate its wound-healing activity

and its safety in managing chronic ailments, we evaluated the wound-healing activity

of the aqueous extract and fractions, as well as its safety after subchronic

administration in albino rats. The shade-dried leaves of Ficus Exasperata were

pulverized, cold macerated for 24 h, and filtered, and the filtrate was concentrated

using freeze-drier. Part of the freeze-dried extract (100 g) was adsorbed on silica gel

and eluted in succession with n-hexane, chloroform, ethyl acetate, and methanol. The

filtrate so obtained was evaporated to a constant weight using rotary evaporator at

40°C. Agar well diffusion technique, as described by Adeniyi et al. was used. The

bacterial isolates of Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi,

Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, and Proteus vulgaris

were used. The extract concentrations used were 10, 50, 100, 150, 200, and 250

mg/ml at an equal volume of 1000 μl. Each concentration was tested in triplicate and

23
the zones of inhibition were measured in millimeters. The fusion method was adopted

in the formulation of the herbal ointment. The required quantity of the ointment base

was weighed and melted at a temperature of about 70°C in a water bath. The

designated quantity of the extract (or fraction) was added to the melted base at 40°C

in a water bath, stirred gently and continuously until a homogenous dispersion was

obtained. The above exercise was repeated using different weights of the crude extract

(or fractions) in order to obtain the above-mentioned concentrations. (Umeh et al.

2014)

TOXICITY

Peperomia pellucida is widely used in traditional medicine for the treatment

of abscesses, acne, boils, arthritis, wound healing, inflammation and gout. This study

was to evaluate the phytochemical composition, antimicrobial activities and

toxicological profiles of the chloroform and methanol extracts of P. pellucida.

Chemical and chromatographic tests were employed in phytochemical investigations.

Inhibitory activities of chloroform and methanol extracts against clinical strains

of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella

pneumonia, Bacillus subtilis and Candida albicans were compared with those of

gentamycin. Acute toxicological evaluation was carried out in mice while 14-day

assessment was done in rats. Phytochemical studies revealed the presence of alkaloids,

tannins and flavonoids. The extracts inhibited the growth of Pseudomonas aeruginosa,

Klebsiella pneumonia, and Bacillus subtilis to varying extents, but only the methanol

extract and the positive control, gentamycin inhibited growth of Staphylococcus

aureus, Escherichia coli and Candida albicans. Oral doses as high as 5 g/kg did not

cause death or toxicological symptoms in mice. Histopathological effects on the liver,

spleen, heart and kidney of rats administrated the aqueous methanol extract showed

24
mild to moderate congestions and infiltrations of chronic inflammatory

cells. Peperomia pellucida showed mild toxicity in certain organs and it will be

necessary to fully establish its safety profile, as this may likely unlock its mechanism

of action as an anti-inflammatory agent. It also demonstrated antimicrobial activity

against clinical strains of selected microorganisms, thereby justifying its usage in

traditional medicine. (Abere et al. 2012).

PYRAZINAMIDE

Hyperuricemia results in an increased level of blood uric acid, a prerequisite

of gout. Commonly prescribed agents for the treatment of hyperuricemia include

allopurinol, febuxostat, and probenecid. Multiple adverse effects like hypersensitivity,

gastrointestinal upsets and hepatotoxicity limit their use. Sixty-eight male mice (Swiss

Albino) were separated into four groups. Group A mice were labelled as negative

control and mice in this group were given chow & glucose water. Group B mice

received 500mg/kg Pyrazinamide (PZA) added in glucose water once daily. Group C

mice were given low dose Nigella sativa seeds powder in a dose of 500 mg/kg

suspended in the glucose water accompanied by PZA in a dose of 500mg/kg. The

mice in group D received high dose of Nigella Sativa seeds powder 1000 mg/kg

suspended in glucose water along with 500mg/kg of PZA. All the doses of

Pyrazinamide and Nigella sativa seeds suspension were given orally for six weeks.

Blood sample was collected three times from each group. On day 0, sample from two

mice from each group was taken for baseline uric acid levels and of five mice from all

groups in mid of study to check uric acid levels. On 42nd day, the blood from

remaining 10 mice in each group was taken to check the serum uric acid levels.

Analysis of data was done using Graph Pad Prism Version 8, p value <0.05 was

considered significant. (Hafeez et al.2022)

25
ALLOPURINOL

Hyperuricemia has recently been recognized to be a risk factor for

nephropathy in the diabetic subject. We tested the hypothesis that lowering uric acid

with a xanthine oxidase inhibitor might reduce renal injury in the diabetic mouse.

Diabetic (db/db) mice were treated with allopurinol or no treatment for 8 wk. Serum

uric acid, renal function, and histology were assessed at death. The direct effect of

uric acid in human proximal tubular epithelial cells was also evaluated under normal

or high glucose condition. We found that db/db mice developed hyperuricemia,

albuminuria, mesangial matrix expansion, and mild tubulointerstitial disease.

Allopurinol treatment significantly lowered uric acid levels, reduced albuminuria, and

ameliorated tubulointerstitial injury, but it did not prevent mesangial expansion. The

mechanism for protection was shown to be due to a reduction in inflammatory cells

mediated by a reduction in ICAM-1 expression by tubular epithelial cells.

Interestingly, allopurinol did not reduce oxidative stress in the kidney. An

inflammatory role of uric acid on tubular cells was also confirmed by our in vitro

evidence that uric acid directly induced ICAM-1 expression in the human proximal

tubular cell. In conclusion, hyperuricemia has a pathogenic role in the mild

tubulointerstitial injury associated with diabetic nephropathy but not glomerular

damage in db/db mice. Lowering uric acid may reduce tubulointerstitial injury in

diabetes.(Kosugi et al. 2009)

Gout and diabetes are common chronic conditions causally associated with

the metabolic syndrome and obesity. Individuals with gout have a higher prevalence

of diabetes than individuals without gout, and the number of individuals with co-

morbid diabetes and gout is likely to rise given the increasing incidence of both

conditions. Poorly controlled gout may lead to greater use of systemic steroids, which

26
exacerbates hyperglycemia and the risk of diabetes complications. Gout and diabetes

are each independently associated with cardiovascular disease, meaning many

individuals with co-morbid diabetes and gout may be at substantially elevated

cardiovascular risk.The most commonly used chronic therapy for gout is the xanthine

oxidase inhibitor allopurinol, which is effective at preventing acute gout attacks and

complications. Recently, there has been growing interest in a potentially novel role for

allopurinol in people with diabetes (with or without hyperuricemia) to reduce the risk

of major adverse cardiovascular events, with clinical trials reported or ongoing.

However, one potential concern is that poor adherence to allopurinol could be a major

barrier to broader implementation for cardiovascular risk reduction.Less than half of

individuals with gout prescribed allopurinol have high adherence by one year

following the initial prescription (defined as having medication supply available for

more than 80% of all days in a given time frame), and 54 to 87% of individuals

discontinue allopurinol within the first year. This is perhaps due to adverse effects,

poor understanding of the use of this medication as a chronic preventative therapy

rather than for treatment of acute gout flares, or other reasons. However, there is

limited understanding on predictors of allopurinol adherence, patterns of allopurinol

use over longer periods of time, or the influence of other comorbid conditions such as

diabetes on allopurinol adherence.Allopurinol adherence and persistence in

individuals with diabetes might be better than in individuals without diabetes due to

enhanced frequency or intensity of care. Conversely, multimorbidity, both in

individuals without diabetes and specifically within diabetes populations, is associated

with worse adherence to medications in general. In individuals with gout and diabetes,

those with diabetes complications such as cardiovascular or kidney disease may have

less focus on gout management because of the prioritization of these other important

27
conditions, though this has not been studied. One half to two-thirds of people with

diabetes are adherent to antihyperglycemic, antihypertensive and lipid-lowering

medications, with older age and greater comorbidity being associated with higher

adherence. We are aware of only one study that has evaluated medication adherence

to non-diabetes medications in people with diabetes (i.e. for a competing priority

condition), which demonstrated that adherence and persistence to medications

for overactive bladder were higher among those with diabetes than those without

diabetes.We previously demonstrated in a large retrospective cohort study that among

older adults with diabetes, allopurinol exposure was associated with a reduction in

mortality and cardiovascular events. In the current study, we conducted a secondary

analysis of this cohort to evaluate long-term allopurinol adherence, persistence and

patterns of use, as well as their predictors, in individuals with diabetes and gout

(Weisman et al. 2021)

WHITE MICE

Mice and rats have long served as the preferred species for biomedical

research animal models due to their anatomical, physiological, and genetic similarity

to humans. Advantages of rodents include their small size, ease of maintenance, short

life cycle, and abundant genetic resources. The Rat Resource and Research Center

(RRRC) and the MU Mutant Mouse Regional Resource Center (MMRRC) serve as

centralized repositories for the preservation and distribution of the ever-increasing

number of rodent models. Mention mice and rats to most people and images of

unsanitary conditions and urban decay come to mind. Rats have been vilified as the

carriers of the infected fleas that led to the dreaded Black Plague that decimated

Europe, North Africa and Central Asia in the fourteenth century. It has been

suggested recently that an apology is in order and that other influences, not rodents,

28
were to blame. More recently, infected mice have resulted in Hantavirus outbreaks

including the recent scare in Yosemite National Park where many campers contracted

the deadly virus from mice living in the cabins. Laboratory rats and mice provide

ideal animal models for biomedical research and comparative medicine studies

because they have many similarities to humans in terms of anatomy and physiology.

Likewise, rats, mice, and humans each have approximately 30,000 genes of which

approximately 95% are shared by all three species use of rodents for research

purposes has economic advantages: mice and rats are relatively small and require little

space or resources to maintain, have short gestation times but relatively large numbers

of offspring, and have fairly rapid development to adulthood and relatively short life

spans. For example, mice have a gestation period of approximately 19–21 days; can

be weaned at three to four weeks of age, and reach sexual maturity by five to six

weeks of age, allowing large numbers of mice to be generated for studies fairly

quickly. The use of rodents also provides advantages related to the wealth of genetic

information available to scientists. The human genome was sequenced in 2001, with

those of the mouse and rat following in 2002 and 2004 respectively The availability of

the complete nucleotide sequences for all three species has enabled genome-wide

comparisons across species which have been critical for the identification and

characterization of genes. The ability to use sophisticated molecular genetic

techniques to manipulate the genes in mice and more recently rats, allows genes to be

“knocked out” (no expression) or expressed at designated times of development or in

select tissues in order to better understand their normal function and/or role in disease.

Since mice are small in size and generally cost less to maintain, and because the tools

to genetically manipulate their genomes have been available since the 1980s, mice are

often the first choice as a rodent model. However, there are many areas of

29
investigation where rats are preferred, including cardiovascular research, behavioral

studies and toxicology (Bryda 2013).

FEMALE WHITE MICE

Type 2 diabetes mellitus is a complex disease characterized by a state of

insulin resistance of peripheral tissues such as skeletal muscle, white adipose tissue

and liver. In order to manage diabetes mellitus, blood glucose must be controlled,

especially in postprandial condition since its regulation implies, in this case, the

actions of insulin in suppressing hepatic glucose production and triggering glucose

uptake in skeletal muscle and adipose tissue. Inositol, formerly referred to as vitamin

B7, stands for 1,2,3,4,5,6-cyclohexanehexol and includes nine distinct stereoisomers

(named allo-, d-chiro-, l-chiro-, cis-, epi-, muco-, myo-, neo- and scyllo-inositol)

through epimerization in hydroxyl group configuration. In addition to various

biological functions for the treatment of diseases such as depression, panic disorder or

polycystic ovary syndrome.Some inositol derivatives exhibit insulin-mimetic activity

and are efficient in lowering blood glucose level. For instance, administration of d-

chiro-inositol to diabetic rats, rhesus monkeys, mice and humans increases glucose

disposal and improves insulin sensitivity. D-Pinitol, the 3-O-methylated form of d-

chiro-inositol, is also one of the naturally occurring inositol derivatives. d-Pinitol

from soybeans or Bougainvillea spectabilis leaves has also been reported to reduce

blood glucose level in streptozotocin-induced diabetic rats and in patients with

diabetes mellitus. D-Chiro-inositol and d-pinitol are rare inositol derivatives, whereas

among all the nine inositol isomers, myo-inositol is the only one to be widely

distributed in tissues and foodstuffs. Since myo-inositol can be converted to d-chiro-

inositol in insulin sensitive tissues, namely, skeletal muscle, adipose tissue and

liver, myo-inositol has been mainly regarded as a nutritional source of d-chiro-inositol.

30
Ortmeyer demonstrated that administration of a high dose of myo-inositol (1.5 g kg−1)

to diabetic monkeys was efficient in lowering blood glucose level. Recent studies

demonstrated that oral administration of myo-inositol is able to improve glucose

tolerance and trigger skeletal muscle GLUT-4 translocation in C57/BL6 mice,

suggesting an improvement of insulin sensitivity. However, in vitro studies of glucose

uptake on rat L6 myotubes as well as some experiments on rodents yielded conflicting

results. To date, the effect of a chronic treatment with myo-inositol, which would be

more compatible with a therapeutic-targeted strategy for diabetes mellitus prevention

or care, remains to be documented. Hence, we tested in the present study the effect of

15 days of treatment with myo-inositol on insulin sensitivity in female CD-1 Swiss

mouse (Croze et al. 2013).

31
 CHAPTER III

RESEARCH METHODOLOGY

This chapter deals with the methods and procedure that is used in the study.

This includes the research design, research locale, sampling procedure, research

instrument, and their validity, data gathering procedure.

3.1 RESEARCH DESIGN

The study used a Quantitative research design, experimental research, we’re

an analysis of the effect of P. pellucida (Olasiman ihalas) leave on the uric acid of

female white mice. The data collected using scientific experiment procedures. An

experimental research design that attempts to maintain control factors that may affect

the result of an experiment.

3.2 STUDY AREA

The Plant used in this study were collected in officer’s line, Barangay Panggao

Saduc, Marawi city, Lanao del Sur, BARMM. The Animal species were collected at

89-A Animas Corner, Abanil Street Aguada, Ozamis City, Misamis Occidental 7200

Region 10. The study conducted at the Laboratory of Adventist Medical Center

College. The P. pellucida (Olasiman ihalas) extract and Blood collection test for uric

acid were done at Medical Technology Laboratory Department from Adventist

Medical Center College.

32
 Source: Map of the Iligan City,Lanao del Norte (Bing Maps App)
Figure 2. Location of the Adventist Medical Center College, Iligan City

Source: Map of the Marawi City, Lanao del sur (Bing Maps App)
Figure 3. Location of Officers Line, Panggao Saduc, Marawi City

Source: Map of Ozamis City, Misamis Occidental (Google Map App)

Figure 4. Location of A-Animas Corner, Abanil Street Aguada, Ozamis City.

33
3.3 STUDY SUBJECT

In this research, the study subjects used female white mice which are collected

from 89-A Animas Corner, Abanil Street Aguada, Ozamis City, Misamis Occidental

7200 Region 10. The researchers selected 24 adult female white mice for the control

and induced factors of this study. The researcher chose adult female white mice for

this study because it is intrinsically more variable than males.

3.4 SAMPLING PROCEDURES

The researcher used 24 female white mice for replicates and final

concentration and control factors. The researcher would like to conduct a trial-and-

error for the confirmation of the study, 15 for final induced mice and 9 for replicates

induced mice. Pyrazinamide used for hyperuricemia of mice by mixing the drug to the

food of mice. Allopurinol will be the standard drug to the positive control (decreasing

uric acid). The drug used to decrease high uric acid levels of the control positive mice

and P. pellucida (Olasiman ihalas) served as a treatment for lowering the uric acid

level of the induced mice. The blood collection procedure for the uric acid test and for

the preparation of plant were performed at the Adventist Medical Center College,

Laboratory in Iligan City.

34
 33 mice

9 Replicate mice 15 Final Induced mice

6 mice for Positive Control 6 mice Negative Control

(Allopurinol) (Water)

Pyrazinamide

6 mice for 250 mg/ml

6 mice for 150 mg/ml

6 mice for 50 mg/ml

Figure 5. Sampling Procedure

35
3.5 INSTRUMENTATION

In carrying out this study, aside from the personal protective equipment such

as gloves and mask for safety purposes and avoid contamination, a total of 24 adult

female white mice were used for the experiment. The materials for an experiment

such as the P. pellucida (Olasiman ihalas) boiled leaf were used for the female white

mice. Other instruments that were also needed during the course of the study such as

spectrophotometry, Capillary tube, Glass tube, Reagents for uric acid, 50ul Pippete,

1000ul pippete 33 Female White mice, Foods (Pallet chips), 1ml Syringe, Innovation

Gavage crop tube (3cc), fabric clothes (for extraction of the plant), Cottons, Gloves,

Transpore tape, Gauze pad, Mask, Tissue paper, Isoprophyl Alcohol, sterile Scissor,

Distilled Water and other necessity for mice.

Purchased sample and Ingredient

In this study, female white mice sample were obtained from 89-A Animas

Corner, Abanil Street Aguada, Ozamis City, Misamis Occidental 7200 Region 10.

Peperomia pellucida (Olasiman ihalas) sample were obtained from the homegrown at

Officer’s line Panggao Saduc,Marawi city Lanao del sur (LDS).

Other ingredients used to boiled P. pellucida (Olasiman ihalas) sample is

Distilled water obtained from the Blue Water (Water station) at Andres Bonifacio

Avenue, Barangay San Miguel, Iligan City.

36
Authentication of Plant (Peperomia Pellucida)

The Plant was authenticated by a botanist from Mindanao State University-

Iligan Institute of Technology (MSU-IIT) in Iligan City. The plant was identified as

Peperomia Pellucida (L.) under the family of piperaceae using the reference of Co’s

Digital Flora of the Philippines this January 19, 2022.

3.6 DATA GATHERING PROCEDURES

A. Preparation of plant samples. The sample plant were collected at officer’s line,

Barangay Panggao Saduc, Marawi city, Lanao del Sur, BARMM. The sample washed

thoroughly to remove any excess dirty.

B. The preparation of boiled plant. The 250g dried plant leaf were extracted with

1L distilled water within 30 minutes and then filtered through fabric clothes, the

boiled leaf were placed at 1L empty bottle, each group of female white mice drink a

specific amount of (250 mg/ml, 150mg/ml and 50mg/ml) replicates and for final

concentration,( 250 mg/ml, 150mg/ml and 50mg/ml )induce mice of Peperomia

pellucida (Olasiman ihalas) extract every day once a day.

Washing

Rinse herbs under cool running water after harvesting, turning constantly, until

thoroughly clean. Allow herbs to drip-dry for a few moments over the sink. Examine

and dispose of any remaining damaged leaves.

37
 Drying

After washing method, the P. pellucida (olasiman ihals) was sundried for 3

consecutive days at the rooftop. Sundrying is the traditional method for reducing the

moisture content.

Cutting

The researcher used scissor to cut the leafs of P. pellucida (Olasiman ihalas)

about 6 inches from the top and just below a leaf joint (No bruise, unwanted cuts).

Strip the leaves off the cutting with the exception of a few sets of leaves on the top

and wash with tap water to remove excess dirt into 5 minutes.

Weighing

The P. pellucida (Olasiman ihalas) leaf weighing the plant dried leaf into

250g.

Boiled

The 250g dried plant were put to 1 liter distilled water and boiled for 30

minutes and then filtered through a clean cloth fabric as filtration. The boiled leaf

placed at 1L empty bottle and stored to refrigerator.

38
 Plant Boiled

Cut the leaf of P. pellucida (Olasiman ihalas) about 6 inches from the
top and just below a leaf joint (No bruise, unwanted cuts)

Strip the leaves off the cutting with the exception of a few sets of leaves on the top and
wash with tap water to remove excess dirt into 5 minutes and sun-dried it for 3 days

The P. pellucida (Olasiman ihalas) leaf will weighing the plant leaf into
250g.

The 250g plant will be kept with 1 liter distilled water and boiled for
30-40 minutes and then filtered through a clean cloth fabric as filtration and
placed it at 1L empty bottle . And stored at refrigerator.

Figure 6. Step for Plant Extraction

C. Oral Gavaging Procedure. The researcher performed force-feeding (Gavaging) to

the female white mice through placing the gavage tube in the diastema of the mouth.

The tube is gently advanced along the upper palate until the esophagus is reach. The

tube should Pass easily into the esophagus.

39
 Oral Gavaging Step

Gently but firmly grasp the mouse by

the base of the tail

Then use the thumb and index finger to gently grasp the nape
of the neck. You may use your middle finger to stabilize the
head

Once you have a good grasp, lift the mice and hold in an upright
position freeing the legs from the bench surface.

The mouse should appear comfortable, judged my monitoring respiratory

rate that is within normal limits.

Figure 7. Oral Gavaging Step

D. Peparation of the animal sample. The female white mice were kept at the

Ammar Apartment, Andres Bonifacio Avenue, Brgy San Miguel, Iligan City for about

3 days before extracting blood sample. After 3 days of keeping the animal sample, the

female white mice were immediately brought to the Adventist Medical Center

College Laboratory at Medical Technology Department to get the initial/baseline of

uric acid level of the female white mice.

40
E. Induction of Hyperuricemia

Pyrazinamide Drug

Is an anti-therapeutic drug associated with increased of uric acid levels and

were obtained from Pharmacy. The metabolite pyrazinoic acid is oxidized by xanthine

oxidase and is likely responsible for the hyperuricemia effect. Pyrazinamide inhibits

the renal excretion of uric acid, which may frequently result in precipitation or

exacerbation of gout. Therapy with pyrazinamide should be administered cautiously

in patients with hyperuricemia. This will be served as a drug for hyperuricemia for the

female white mice. Once the drug crush this drug will mix with the food and feed it to

the mice. The everyday routine of the drug is 1ml in twice a day.

Allopurinol Drug

It is a synthetic drug used to lower the level of uric acid and were obtained

from pharmacy. The drug used as a standard drug for the positive control mice. Once

the drug crush into powder it will dissolve in the distilled water. The everyday routine

of the drug is 0.5ml once a day.

Uric Acid Test

Uric Acid is a normal waste product that’s made when the body breaks down

chemicals called purine. Most of the uric acid dissolves in blood and then goes to the

kidney, it leaves the body through the urine if the body makes too much uric acid, or

it doesn’t release enough into the urine it can make crystals that form in joints, and it

is known as Gout. The Uric acid test measures the amount of uric acid in the blood.

This test is to determine the origin/normal uric acid of mice. And once we get

the uric acid results from the mice, we're going to raise the uric acid of the mice using

41
drugs (Pyrazinamide) by mixing the drug to the foods of the mice. Every 3 days, we'll

run a uric acid test to see if the mice's uric acid has been successfully raised for about

3 days, at this point we began using the P. pellucida (Olasiman ihalas) leaf as a

medication to reduce the uric acid of the mice. The researcher were having a two

control for the validity of the study, the positive control and negative control. The

researchers were used another drug (Allopurinol) as a standard drug for positive

control for lowering the uric acid level of the female white mice. The negative control,

there is no medication or drug will be involved, only water will be served as a

treatment. The data that will record were used as support for the results and discussion.

Uric Acid Test

Blood samples were obtained from tail of female white mice. The blood
samples were collected at capillary tube and centrifuged for 15 min in
3400 rpm.

Get a 25uL serum sample in capillary tube and place in

test tube

Add a 1000uL uric acid reagents and 1000uL for control reagent
in separate test tube and put in water bath for 5 minutes.

Aspirate Reagent blank in spectrophotometry and follow

by the control reagent and then the sample test tube

Wait for the result

Figure 8. Step for Uric acid test

42
F. Introduction of the Plant to the Mice. The researcher gave a P. pellucida

(Olasiman ihalas) leaf extract through performing oral gavaging technique with a

different concentration ( 250 mg/ml, 150mg/ml and 50mg/ml) for replicates to induce

the uric acid level of the mice and be served as a treatment for their uric acid.

G. The preparation of blood extract. The researchers used a cutting technique and

capillary tube to collect blood samples from the tail of the mice.

Blood Collection

Place the mice in restraint device

exposing the entire tail

Massage in 1 min and wipe a cotton with a alcohol the tail of the mice

Cut tail with sanitized sharp scissors

Place tip tail into the capillary tube to coolect

blood ( avoid hemolyzed blood)

Apply manual pressure to stop bleeding

Figure 9. Step for Blood Collection

H. Monitoring of Uric Acid Level. The researcher monitored the uric acid level of

the 24 mice in 12 days and each of the mice undergo in uric acid test every 3 days.

Pyrazinamide were used for hyperuricemia (Increasing uric acid in the blood) of mice

by mixing the drug to the food of mice and P. pellucida (Olasiman ihalas) served as a

treatment for lowering the uric acid level of the mice.

43
I. Terminated animal after experiment. The researcher terminate the 24 mice after

testing because Once an experiment is complete, mice are typically rendered

extraneous since they can no longer be used as a control, and are humanely

euthanized.

3.7 STATISTICAL TREATMENT

The researchers used analysis of variance, or ANOVA, is a statistical method

that separates observed variance data into different components to use for additional

tests. A one-way ANOVA is used for three or more groups of data, to gain

information about the relationship between the dependent and independent variables.

44
 Preparation of materials

Collection of Sample and

Extracting

Uric Acid Test TREATMENT

Pyrazinamide

Uric acid test-prepare P. pellucida (Olasiman

material ihalas) 1ml liquid

Blood Collection Using 250g plant leaf

cutting method (tail)
Mixed with their food
and Capillary tube
Wash with tap water
and sun-dried

Uric acid test using

Put in 1L distilled Feed to the mice twice
serum
water. a day

Monitoring of Uric Boiled within 30-40 mins

Acid Level
filtered through a clean
cloth fabric

Concentration

(250mg/ml, 150mg/ml and 50mg/ml)

Figure 10. Research Methodology Flow

45
 Control Induced Mice

Positive Negative
(250mg/ml, 150mg/ml and
100mg/ml)

Allopurinol (Aloprim,
Zyloprim)

Figure 11. Research Methodology Flow

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 CHAPTER 4

RESULTS AND DISCUSSION

This chapter presents analysis and interprets the data gathered by the

researchers. It also includes the materials used, procedures undertaken during the

study, the results obtained from the testing of uric acid of the mice and the results of

effectiveness of the Peperomia pellucida leaf.

1. What is the baseline of Uric Acid level of female white mice before

administration of P. pellucida boiled leaf.

Table 1

Group of mice P-
N Mean SD F
(Concentration) value

Baseline 50 mg/mL 3 1.87 0.6469

150 mg/mL 3 2.24 0.0173 1.95 0.266

250 mg/mL 3 1.42 0.5225

Positive Control 3 1.63 0.6363

Negative Control 3 1.98 0.6181

Note: ns-not significant at 0.05 level

47
 The table 1. shows the result of the initial uric acid level of the female white

mice, they were divided in 3 every group of concetrations.The n is the number of

samples . Out of 3 in 250mg/ml group the mean average is 1.42. Out 3 in 150mg/dl

group the mean average is 2.24. Out of 3 in50mg/dl group the mean average is 1.87.

Out of 3 in positive control group the mean average is 1.63 and Out of 3 in Negative

group the mean average is 1.98. According to (kong LD et al. 2004) normal mice,

serum uric acid level was 3.24 ± 0.41 mg/dl. Using One-way ANOVA, the result gave

a very small F-value = 1.95 and p-value = 0.266 which is above the chosen alpha of

0.05, this means that before the administration of the treatment there is no significant

difference between the groups.

2. What is the uric acid level of female white mice after administration of

Pyrazinamide.

Table 2

Concentration N Mean SD

Induced UA level 50 mg/mL 2 6.29 0.04243

150 mg/mL 2 6.95 0.00000

250 mg/mL 2 6.33 0.00707

Positive Control 3 6.65 0.33501

Negative Control 3 6.25 0.01155

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 The table 2. shows the uric acid level of mice after the administration of the

pyrazinamide. .The n is the number of samples . Out of 2 in 50mg/ml group the mean

average is 6.29. Out 2 in 150mg/dl group the mean average is 6.95. Out of 2 in

250mg/dl group the mean average is 6.33. Out of 3 in positive control group the

mean average is 6.65 and Out of 3 in Negative group the mean average is 6.25.

According to (kong LD et al. 2004.) In hyperuricemic animals, serum uric acid level

was elevated to 5.73 ± 0.15 mg/dl .Therefore the researcher conclude that all female

white mice uric acid is higher.

3. What is the uric acid level of female white mice after administration of P.

pellucida (Olasiman ihalas) boiled leaf?

Table 3.

Concentration N Mean SD

Treated UA 50 mg/mL 2 2.46 0.0495

150 mg/mL 2 3.13 0.1768

250 mg/mL 2 2.71 0.4172

Positive Contro( Allopurinol) 3 1.22 0.0153

Negative Control( Water) 3 2.45 0.0416

The table 3. shows the uric acid level of mice after the administration of the P.

pellucida (Olasiman ihalas) boiled leaf. This table shows the treated uric acid level of

49
female white mice.The n is the number of samples . Out of 2 in 50mg/ml group the

mean average is 2.46. Out 2 in 150mg/dl group the mean average is 3.13. Out of 2 in

250mg/dl group the mean average is 2.71. Out of 3 in positive control group which is

induce by allopurinol has the mean average of 1.22 and Out of 3 in Negative group

which is induce by water only and has the mean average of 2.45 . According to (kong

LD 2004) normal mice, serum uric acid level was 3.24 ± 0.41 mg/dl.Therefore the

researcher conclude that all female white mice uric acid is normal. According to

(Ahmad et al. 2017) Peperomia pellucida is a traditionally used as an analgesic, anti-

hyperuricemic, anti-diabetes mellitus, and antihypertension.

50
 4. Is there a significant decrease in the uric acid after the administration of P.

pellucida (Olasiman ihalas) boiled leaf?

Paired Samples T-Test

Paired Samples T-Test

Statistic p

Induced UA Treated UA Wilcoxon W 78.0 < .001

N Mean Median SD SE

Induced UA 12 6.49 6.32 0.305 0.0881

Treated UA 12 2.30 2.44 0.710 0.2050

51
 The table shows the difference result of the treated and induced

uric acid level of the female white mice. The researcher used Paired

sample T-test to see the changes of before and after administration of P.

pellucida. On the other hand, using Paired sample t-test specifically

Wilcoxon W = 78.0 and has a p-value < 0.001 which obviously very low

comapared to the chosen alpha = 0.05 (95% CI). Therefore P.pellucida

significantly decreases uric acid level of female white mice. According to

study of Alvero et al. 1992 that they confirmed the anti-hyperuricemic

potential of the plant Peperomia pellucida.

5. Is there a significant difference of uric acid decrease across different

concentration of P. pellucida.

One-Way ANOVA (Non-parametric)

Kruskal-Wallis

χ² df p

Decrease 0.286 2 0.867

52
 Concentration N Mean SD

Decrease 50 mg/mL 2 3.83 0.0919

150 mg/mL 2 3.83 0.1768

250 mg/mL 2 3.62 0.4243

The table below shows the differences of uric acid decrease across

the different concetration of P. pellucida. Out of 2 in concetration of

50mg/dl it has mean average of 3.83, Out of 2 in 150mg/dl it has mean

average of 3.83, Out of 2 in 250mg/dl it has mean average of 3.62. and

there p-value is 0.867.According to study of Alvero et al. 1992 that they

confirmed the anti-hyperuricemic potential of the plant Peperomia

pellucida. Using non-parametric test Kruskall-Wallis, p-value = 0.867

(since p-value > 0.05, significant at p-value < 0.05) which means that there

is no significant difference of decrease across difference concentration of

P.pellucida with control groups. Therefore it does not matter what

concentration of the treatment the effect of decreasing uric acid still

relatively same.

53
 CHAPTER 5

SUMMARY, CONCLUSION & RECOMMENDATION

This chapter presents the summary of the study, major findings, conclusion,

implication and recommendations.

Summary

This study aim to determine how the effect of Peperomia pellucida (Olasiman

ihalas)boiled Leaf on the Uric Acid of the Female White Mice. The Peperomia

Pellucida is known as shiny bush or silver bush belonging to family piperaceae.

Peperomia Pellucida is an herbaceous plant found in man south American and Asia

countries. Peperomia Pellucida showed mild toxicity in certain organs and it will be

necessary to fully establish it safety profile as this may likely unlock its mechanism of

action as an anti-inflammatory agent. It also demonstrates anti-microbial activity

against clinical strains of selected microorganisms, thereby justifying its usage in

tradition medicine and not cause death or toxicological symptoms in mice, extract leaf

showed mild to moderate congestions and infiltration of chronic inflammatory cells.

Therefore As we made our trial and error we came across that the Peperomia

Pellucida (Olasiman ihalas) boiled leaf is effective on the white Mice base on the

reaction of the mice. We first get the blood sample from the tail of the mice through a

laboratory experiment done in Medical Technology laboratory of Adventist Medical

Center College. The 33 White Mice sample used for replicates and final concentration

and control factors that were randomly collected. Each group of female white mice

drink a specific amount of ( 250 mg/ml, 150mg/ml and 50mg/ml) replicates and for

54
final concentration( 250 mg/ml, 150mg/ml and 50mg/ml) for inducing of Peperomia

pellucida (Olasiman ihalas) boiled leaf every 3 days.

The researchers conducted a trial-and-error for the replication of the study, 9

replicates of female mice. The results of the study clearly shows In trial 1 and 2 the

uric acid level of a female white mice increased due to inducing them by a

pyrazinamide drug while in trial 3 shows the uric acid level of female white mice

decreased when the researcher started induced Peperomia Pellucida boiled leaf is

effective on the white Mice from the blood sample collection in the tail through a

laboratory experiment done in Medical Technology laboratory of Adventist Medical

Center College. The 33 White Mice sample used for replicates and final concentration

and control factors that were randomly collected. Each group of female white mice

drink a specific amount of (250 mg/ml, 150mg/ml and 50mg/ml) replicates and for

final concentration( 250 mg/ml, 150mg/ml and 50mg/ml) for inducing of Peperomia

pellucida (Olasiman ihalas) extract every 3 days.

The researchers conducted a trial-and-error for the replication of the study,9

replicates of female mice. The results of the study clearly shows that in trial 1 and 2

the uric acid level of a female white mice increased due to inducing them by a

pyrazinamide drug while in trial 3 shows the uric acid level of female white mice

decreased when the researcher started induced Peperomia pellucida boiled leaf.

Conclusions

Plants have played a significant role in maintaining human health (Craig, 199)

and improving the quality of human life for thousands of years and have served

humans well as valuable components of food and medicines (Winston, 1999). The

plant P. pellucida (Olasiman ihalas) will be tested for its anti-hyperuricemic effect

55
 Based on the findings of this study and from the result of the laboratory

experiment, this study hereby concludes that;

1) The first hypothesis is accepted that there’s an effectiveness of Peperomia

pellucida ( Olasiman Ihalas) boiled leaf in the uric acid level of the female

white mice.

2) The second hypothesis is accepted that there’s no significant difference

between the different concetrations of P.pellucida (Olasiman Ihalas)

( 250mg/ml, 150mg/ml and 50mg/ml) in terms of the uric acid level of the

female white mice.

Therefore, the researchers conclude that the Peperomia pellucida ( Olasiman Ihalas)

boiled leaf is effective in different concetration ( 50mg/dl, 150mg/dl, 250mg/dl) on

treating the uric acid level of the female white mice

Recommendations

Based on the findings and conclusions stated from the study, the following

recommendations and future directions were given below.

1) Future researchers may use this as a reference or basis for consideration to

their study relating to this research.

2) Community of society may make use of these outcomes to look into other well

studied plants with potent treatment activity for other source of alternative safe

home-remedy for treating typical illnesses such as goat caused by high uric

acid and other particular illness.

3) Further study on determination of their safeness and deep understating of their

treatment is highly recommended by the researchers.

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