IN VIVO EVALUATION OF THE ANALGESIC ACTIVITY OF OKRA

(ABELMOSCHUSESCULENTUS LINN.) ON ALBINO MICE (BALB/c)

 TABLE OF CONTENTS

TITLE PAGE i
APPROVAL SHEET ii
ACKNOWLEDGEMENT iii
TABLE OF CONTENTS iv
ABSTRACT v

CHAPTER 1 INTRODUCTION
Background of the Study 1
Statement of the Problem 3
Scope and Delimitation 4
Significance of the Study 5
Definition of Terms 6

CHAPTER 2 REVIEW OF RELATED LITERATURE

Related Literature 7
Synthesis 11
Theoretical Framework 12
Conceptual Framework 13

CHAPTER 3 METHODOLOGY
Research Design 15
Research Subject 16
Research Environment 16
Research Instrument 17
Research Procedure 18
Data Analysis 19
Ethical Considerations 20
REFERENCES 22
APPENDICE

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ABSTRACT

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 CHAPTER I

INTRODUCTION

Background of the Study

Pain is a major concern in healthcare due to impairment of patients' health and

quality of life (Ali et al., 2020). Analgesics are medicines that help with pain. Ali et al.

(2020) states that pain control is important in many medical cases, from short-term

illnesses to regular treatments. Pain control makes patients more comfortable, speeds up

their healing, and makes them happier with their healthcare in general. Analgesics are a

very important part of modern medicine because delayed action to pain itself can lead to

mental problems, long-term suffering, and decreased function (Ali et al., 2020).

A lot of things could go wrong with improper use of medicines and how well they

work, even though they are very helpful. Some healthcare professionals may have lapses

in dealing with pain well may it be due to lack of training or information on the subject

(Ali et al., 2020). Doctors may also opt to not give painkillers due to its possible side

effects, such as possible teratogenicity or increase risk of needing a C-section (Ali et al.,

2020). Sometimes it's hard to control pain due to the patients lack of knowledge on

available drugs or old ideas about pain and childbirth (Ali et al., 2020). All of these issues

show how important it is to improve patient education, conversation, and access to

painkillers that would improve and individual’s overall well-being.

Okra, a plant medicine has been used as a treatment for a very long time. The

plant Okra contains ample amount of chemicals that could be used as medicine. Some of
these are found in the mucilage, seeds, and pods (Elkhalifa et al., 2021). As Sipahi et al.

(2022) stated, okra fruit has been used a lot to treat skin problems and sores under the

skin. This suggests that it may help wounds heal." The mucilage in okra is very thick and

can hold a lot of water. It has also been used in medicine to restore plasma or increase

blood volume (Elkhalifa et al., 2021). Okra is good in many ways because it has

phytochemicals in it, such as flavonoids, phenolic substances, and proanthocyanidins.

These help the plant fight off free radicals, swelling, and germs, and they also shield the

liver (Sipahi et al., 2022; Elkhalifa et al., 2021). Many years ago, okra was used in

traditional medicine. But more research is needed to find out how its chemicals work and

if they are soluble to support its potential as a supplement for a number of long-term

illnesses (Elkhalifa et al., 2021).

This study is initiated due to a possible need for new painkillers since current

methods of pain control have some complications and aren't always effective. This is why

it's important to find fresh ways to handle pain (Ali et al., 2020). That's why the idea that

okra (Abelmoschus esculentus Linn.) could be useful as a natural analgesic and it is

something that should be looked into. A lot of people are interested in okra because it is

thought to help with pain. For example, Xiong et al. (2021) and Sipahi et al. (2022) both

identified that Okra can help in wound healing and fight free radicals.

Thus, these objectives are drawn: to begin, the researchers would conduct a

controlled tests to see how well okra would resolve pain. Second, the researchers would

want to contribute to adding more knowledge about the other health benefits of okra

besides pain relief. The study looks at the chemicals in okra, how it works, and how safe

it is to get a full picture of how it could be used to treat pain and other health issues

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(Elkhalifa et al., 2021). Between the need for strong painkillers and the suggestions that

natural treatments like okra may help, this study fills in the blanks. By this study, the

healthcare community would have more knowledge and possible alternatives in treating

pain.

Statement of the Problem

The goal of this study is to evaluate the in vivo activity of the vegetable Okra

(Abelmoschus esculentus Linn.) if it contains analgesic properties which would be done

on laboratory mice and to see if Okra could be used as a natural alternative to painkillers.

Specifically, it aims to:

1. Determine the effectiveness of okra in relieving pain compared to conventional

analgesic medications in animal subjects, assessed through measures such as paw

licking and withdrawal latency from a heat stimulus as indicators of pain

response, using the hot plate method.

2. Elucidate the underlying mechanisms of action of okra in alleviating pain,

including its impact on inflammatory pathways and neurotransmitter modulation,

with a focus on investigating the presence of specific phytochemicals present in

okra using relevant analytical techniques.

3. Investigate the safety profile of okra as an analgesic agent, including potential

adverse effects and long-term implications on physiological parameters in animal

subjects, as young fruit is utilized in the study.

Scope and Delimitation

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 The study includes, Okra (Abelmoschus esculentus Linn.) as a possible natural

option for pain relief using white rats as test subjects in live experiments. This study

focuses on three main areas: finding out how well okra works at relieving pain compared

to other common painkillers in animals; studying how okra works to relieve pain,

including how it affects inflammatory pathways and neurotransmitter regulation; and

looking into how safe okra is as an analgesic, including any possible side effects and

long-term effects on different physiological parameters. However, the study is limited by

a number of things. It only looks at models for animals and not at clinical studies on

people. The study only looks at models of acute and chronic pain in albino rats. It doesn't

look at other animal models or pain types. Mechanistic studies mostly look at how

inflammation works and how neurotransmitters are changed, but they don't look into

many other possible processes. Also, safety evaluations mostly look at short-term side

effects and biochemical factors in animals, with not much research into long-term effects

or chronic toxins. The study doesn't look at the pharmacokinetics or pharmacodynamics

of okra chemicals, how to make the best mixture, or how to give the right amount. Even

with these limits, this study aims to have more knowledge about Okra's pain-relieving

properties and whether it could be used as a natural replacement to painkillers in

experimental animal studies.

Significance of the Study

The significance of this study may have far-reaching implications for various

stakeholders:

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 Health Care Professionals: This study shows healthcare professionals how okra

might work as a natural pain reliever, giving patients more choices for managing pain

besides traditional drugs. Using medicines based on okra might make treatments safer

and easier to handle, which would improve patient results and overall well-being.

Patients: For patients, the fact that okra might be able to help with pain naturally

gives them hope for better and more tolerable treatment options. Patients may feel more

in control of their pain management and general health if they have access to treatments

based on okra.

Pharmaceutical Industry: The study's results could encourage drug companies

to make painkillers from okra, which would expand treatment choices and meet the desire

for natural solutions in the market. This new idea could help the development of drugs

based on natural products.

Research Community: This study encourages more research into natural

medicines and their healing power by showing that okra can help with pain. It encourages

people to work together and share their information, which will lead to future

improvements in healthcare.

Public Health: Finding out that okra might be able to relieve pain encourages

people to use better and longer-lasting pain relief methods, giving them the power to look

into natural treatments. This helps to lower the cost of health care and improve people's

health in neighborhoods.

Definition of Terms

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 The following terms are operationally defined for a clearer and better

understanding of the study:

Abelmoschus esculentus Linn., or Okra: Okra is an ornamental plant in the

mallow family (Malvaceae). Its formal name is Abelmoschus esculentus. It makes green

seed pods that can be eaten and are often used in cooking, especially in dishes from

Africa, the Middle East, and South Asia. Within this research, okra is being looked into

to see if it could be used to relieve pain.

Analgesic Activity: An analgesic is a medicine or drug that eases pain without

making you lose awareness. Analgesic activity is a measure of how well a drug or

treatment reduces pain. It is the main goal of this study to find out if okra has any

qualities that can ease or reduce pain.

In Vivo Evaluation: When an experiment or study is done on a live thing,

usually an animal example, to see how a drug, treatment, or action works, this is called

"in vivo evaluation." There is evidence in this study that the analgesic (pain-relieving)

effects of okra are tested on live things, most likely animals.

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 CHAPTER II

REVIEW OF RELATED LITERATURE

This chapter presents the comprehensive review of the acquired relevant

literatures and studies that support the present research. It also discusses the synthesis of

the gathered studies, gaps to be bridged by the study and the theoretical and conceptual

foundations of the study.

Pain Management

Pain treatment that works well is very important for making sure that people are

healthy and comfortable. An investigation by Alkhatib et al. (2020) looked at how much

healthcare workers in basic medical centres in Jordan knew and felt about managing pain.

The results showed that both nurses and doctors didn't know much about pain control and

had bad feelings about it. El-Tallawy et al.'s (2020) study was also about the problems

that came up when trying to treat chronic pain during the COVID-19 outbreak. It showed

how healthcare delivery systems need improvement to make sure that pain is managed

best while still following safety and social distance rules. Also, the study by Mankelow et

al. (2022) looked at how primary care doctors understood pain and how to treat it after

learning about pain science. It focuses on how hard it is to use a biopsychosocial method

to pain treatment in everyday clinical practice, even though people are generally positive

about it. Manchikanti et al. (2020) also stated at how lessons for better pain management

have changed over time, stressing how important it is to understand past methods in order

to shape future approaches. Lastly, Gido et al. (2021) looked at how often doctors in
Ethiopian state hospitals used maternal drugs to help women who were labouring. There

was no doubt that women who are in labor should be able to get the right drugs and

training to make sure they feel better. One issue that still needs to be fixed is the lack of

knowledge among healthcare workers and problems with the way things are done. There

are also scenarios for full pain management standards to connect what is known to what

is done.

Okra (Abelmoschus esculentus Linn.)

Okra has been used as medicine for a long time, mostly to treat pain and other

health issues. The okra plant is called Abelmoschus esculentus Linn. More research has

been done on how it works and how it might be used as a drug. Zhu et al. (2020)

conducted a study on okra polysaccharides (OPs) and how they can help lower blood

sugar, fight inflammation, and boost the immune system. The study conducted looked

into not only how Okra polysaccharides worked but also what chemicals are in them.

Esmaeilzadeh et al. wrote another study in 2020 that talked about how okra can help

people with metabolic syndrome, diabetes, or high cholesterol. Researcher Nikpayam et

al. (2021) conducted a study that focuses on how okra products affects cholesterol and

blood sugar levels. Based on these tests, okra-based foods might be good for people with

diabetes. Wu et al. (2020) indentified that okra have different amounts of vitamins and

organic chemicals. For some reason, these foods naturally stops enzymes in the gut from

doing their job and produces vitamins. In 2021, Sipahi et al. conducted a study on how

well the vegetable okra could heal cuts. They identified that Okra has antibacterial

properties, good for inflammation, and is an antioxidant. A study by Xiong et al. (2021)

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looked at how the acid-soluble pectin in okra can help fight inflammation and free

radicals. Okra may be a healthy way aquire vitamins and feel better. Romdhane et al.

(2020) looked into the phytochemicals that are beneficial for individuals in Tunisian okra

for a long time. These chemicals were studied to find out how they kill cells, keep cells

from getting damage, and halts enzymes from its normal activity. The studies

abovementioned indicates that okra is beneficial vegetable and is a possible alternative

drug to reduce pain.

Analgesic Properties of Okra

Okra has been used in traditional medicine for a long time. In fact, several studies

have looked at its healthy components and how they might benefit health. In 2022,

Agregán et al. looked into the phytochemicals that okra contains. Specifically, they

looked at flavonoids and catechins. Flavonoids and catechins are good components that

can aid in immune defense, diabetes, cancer, and high blood pressure. Mohammed et al.

(2024) did a study in western Ethiopia that okra is often eaten to feel better after being

sick, it heals cuts faster, and keeps people from getting malaria. Phasate chemistry studies

showed that the amount of nutrients in different types of okra varied. Kushi et al. (2023)

also looked into how pregnant women in western Ethiopia traditionally thought about and

used okra. They concluded that pregnant women would eat okra leaves and then grind

them up to keep them fresh for a long time.

People with diabetic nephropathy were given different amounts of dried okra to

eat in a 2022 study by Nikpayam et al. It was found that people who took okra pills ate

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less carbs and sugar. To put it another way, the vitamins might help people with diabetes

watch what they eat. Sipahi et al. (2022) also looked at how well okra fruit could heal

wounds on living things and in the laboratory; the researchers concluded that okra

products can kill germs, lower inflammation, and keep cells from damage. This backs up

the idea that they can be used to treat skin problems in traditional medicine.

It took Romdhane et al. (2020) longer to learn more about the nutrients and

vitamins that can be found in Tunisian okra pods. They found that they have a lot of

protein, carbs, and vitamins. Another thing the researchers stated that okra products

might kill cells, protect cells from damage, or stop enzymes from working. When

Adekanmi et al. (2020) looked into the phytochemical properties of okra, they found

flavonoids, saponins, alkaloids, and other good things. Okra has been used for a long time

to help with many health issues. It works as a medicine because it has chemicals in it.

Tests show that okra has a lot of chemicals that may be beneficial for an

individual’s health. Hormones may help people with long-term illnesses like diabetes and

to keep track of what they eat and speed up the healing of cuts. The researchers would

like to conduct further investigation to understand how okra can be used as a medicine.

Ibuprofen

A nonsteroidal anti-inflammatory drug (NSAID) called ibuprofen is widely used

and has been studied a lot to see how well it works for controlling different kinds of pain.

Studies like those by Bailey et al. (2013) and Moore et al. (2014) have shown that it can

help with pain after surgery, like having your wisdom teeth taken out or having surgery

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on your hand. Ilyas et al. (2018, 2019) and Joshi et al. (2004) also found that ibuprofen

was helpful at controlling pain after surgery for carpal tunnel release and third tooth

extraction. Additionally, ibuprofen's safety profile has been well-established, as shown by

Rainsford (2012) and Vladiskovic and Masciocchi (2015). This makes it a popular choice

for pain treatment in many professional settings. It has been compared to painkillers like

ibuprofen and opioids, and studies like those by Derry et al. (2015a, 2015b) show that it

works just as well or better at easing pain. Ibuprofen can be used as a positive control in

clinical studies that test new painkillers because it is known to work and be safe. Using

ibuprofen as a standard, researchers can compare how well and safely new painkillers

work, giving us important information about how they might be used in the real world.

Behavioral and Biochemical Parameters

Many studies have helped us learn more about the different ways that pain can be

changed and how to create models of neuropathic pain in rodents, mostly rats and mice.

Lehner et al. (2006) looked into how rats react to painful stimuli and found that different

ways of dealing with pain were linked to different types of behavior. Rats that were more

sensitive froze more often, while rats that were less sensitive made louder noises. These

differences were linked to changes in the amount of serotonin and brain activity in

different parts of the brain. Guida et al. (2020) studied changes in behavior, biochemistry,

and electrophysiology in the spared nerve injury (SNI) model of neuropathic pain. They

found that the mice showed signs of intolerance to pain, behavior similar to depression,

and problems with their ability to think and learn. Changes in gene expression linked to

immune reaction and inflammation were seen in the hippocampus, as shown by

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electrophysiological studies of changes in brain firing activity. Medeiros et al. (2019)

studied changes caused by a modified chronic constriction injury (CCI) model. They

found that the model caused mechanical allodynia, movement delays, anxiety and

depression-like behaviors, and cognitive problems in mice. Neurotransmitter levels in the

prefrontal cortex changed when biochemical tests were done. Alsalem et al. (2020)

looked at the pain-relieving effects of cannabis and opioid mixtures in rat models of

chronic pain. They found that these combinations caused pain-relieving effects without

increasing the risk of making it harder to move.

Cost Effectiveness of Plants Compared To Commercialized Analgesic

Numerous research have shown that using plants as analgesics is more affordable

than using commercially available analgesic drugs. An evaluation of the anti-

inflammatory, analgesic, and antipyretic properties of a number of plant extracts, such as

those from Afrormosia laxiflora, Cyathula prostrata, Ficus glomerata, Lantana camara,

Lippia geminata, Lippia nodiflora, and Synedrella nodiflora, was carried out by Forestieri

et al. in 1996. According to their research, these plant extracts have notable

pharmacological effects, which suggests that using plants to treat pain might be

reasonably priced.

The analgesic qualities of extracts from Artemisia vulgaris and Achillea

millefolium, which are often used as analgesics, were examined by Pires et al. (2008).

Their findings supported the plant-based therapies' cost-effectiveness by demonstrating

considerable analgesic benefits.

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 The antiulcerogenic and analgesic effects of traditional medicinal plants utilized

for their analgesic properties—Maytenus aquifolium, Sorocea bomplandii, and Zolernia

ilicifolia—were assessed by Gonzalez et al. (2001). According to their research, these

plants may have analgesic properties, which adds credence to the idea that plant-based

analgesics might be reasonably priced.

Tamarindus indica's potential as an anti-inflammatory and analgesic was reviewed

by Komakech et al. (2019). Strong pharmacological evidence supporting its analgesic

properties was discovered by them, suggesting that it may be an affordable natural

painkiller.

Aloe ferox's analgesic activity was assessed by Mwale and Masika (2010), who

discovered notable analgesic effects. These findings confirm the analgesic agent's cost-

effectiveness. These studies demonstrate the promise of natural remedies in pain

treatment by showing that utilizing plant-based analgesics may be more affordable than

using commercially available analgesic drugs.

Synthesis

A study on Pain Management and the Analgesic Properties of Okra shed light on

the issues people have and what possible solutions they can use when they want to treat

pain and use okra for medical reasons. Some health care workers don't preffer pain

control and don't know much about it, according to Alkhatib et al. (2020). Both

Mankelow et al. (2022) and El-Tallawy et al. (2020) said that it is hard to follow through

with pain treatment plans that work. This was especially true during the COVID-19

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outbreak and in real life when biopsychosocial methods were used. Manchikanti et al.

(2020) said that we need to know what people thought about pain management in the past

in order to change what they think about it, going forward.

Studies by Agregán et al. (2022) and Mohammed et al. (2024) show that okra has

been used to treat pain for a long time. The phytochemicals in okra were looked at, as

well as how it has been used to treat pain. Some of the good chemicals found in okra are

flavonoids and catechins. These chemicals might be able to help with pain and other

health issues. Nikpayam et al. (2022) and Sipahi et al. (2022) also did studies that showed

okra vitamins and foods that can help with diabetic neuropathy and wound healing.

Both sets of studies look at important parts of how to treat pain and how okra can

help the body heal, but there is a big gap in how traditional knowledge and tried-and-true

methods are put together. Studies on how to deal with pain show how hard it is to use

biopsychosocial methods. Studies that look at okra's pain-relieving properties, on the

other hand, mostly look at its chemical make-up and therapeutic effects. To fill this gap,

researchers could look into how old practices can be used to teach new ways to deal with

pain, especially when it comes to natural remedies like okra. It's possible that this will

help us find better ways to treat pain.

Theoretical Framework

Researcher Varro E. Tyler was the first person to write about the Pharmacognosy

Theory in 1997. Researchers can learn about natural things, like medicinal plants, in

order to innovate newer drug alternatives and medicines. People learned how to use

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plants as medicine and got better because of Tyler’s work. There are lots of natural

things, mostly plants, that can be used as medicine. The Pharmacognosy Theory is

important because of this. A planned study of these natural sources can help scientists

find drugs that are useful and learn how they work. This lets new medicines be made.

These medicines come from plants and have been used for a long time in

traditional medicine. This idea takes that into account. Modern study methods are also

used to show that these treatments are safe and effective. The Pharmacognosy Theory

helps us understand how okra (Abelmoschus esculentus) can ease pain for this study.

They can use pharmacognostic rules to find, sort, and describe the chemicals in okra that

may be good for one’s health now that they know they come from living things. Tyler

wants to make new drugs and medicines by using the healing qualities of plants. This

way works, which is good. The Pharmacognosy Theory is a general way of thinking

about how to study the healing qualities of natural things like okra. It helps us understand

how these substances might be used in therapy and makes pharmaceutical research and

drug development better.

Conceptual Framework

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 Figure 1. The Conceptual Framework of the Study

For the problem statement about figuring out how soothing okra (Abelmoschus

esculentus Linn.) is, the IPO (Input, Process, Output) framework gives us an organised

way to look into its possibilities as a natural option for pain management. Inputs for the

study include okra juice that has been found and made for testing, as well as animals that

can be used for in vivo studies. There must also be regular painkillers available for

comparison, so that a full assessment of okra's effectiveness can be made. Having lab

supplies and equipment on hand is very important for doing studies in a controlled

environment.

There are several important steps in the process. The first step in testing for

analgesic action is giving okra extract to one group of animals and regular painkillers to

another. Next, known pain models are used to test for pain relief. The second part of the

study looks into the processes of action to find out how okra eases pain. This means

looking into how it changes chemicals that control how an individual feel pain and how it

affects inflammatory pathways. One way to do this is to check the levels of factors that

cause inflammation. A safety check is also done to see if using okra as a medicine could

have any bad effects. Keeping an eye on body factors and any long-term effects on organ

function and tissue changes is an important part of this process.

A lot of important things will be learned from the study. As a first step, a study

will compare how well okra and popular drugs work to ease animal pain. This will help

the researchers understand how okra deals with pain by showing them how it changes

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hormones and routes for inflammation. It is also helpful to know how safe okra is as a

medicine because it can reveal a lot about its possible side effects and long-term effects

on animal bodies.On top of that, the IPO framework's feedback system makes sure that

the study is always open to new information and issues. When experts get feedback at

every step, they can make their methods better over time. This makes their results more

reliable and helps them learn more about how okra eases pain.

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 CHAPTER III
METHODOLOGY

This chapter presents the research design, locale, respondents, instruments, and

data gathering procedures, data analysis, and ethical considerations. This chapter laid out

the study's parameters and procedure upon its conduct. It helps the study to be

materialized.

Research Design

The research design for the study assessing the analgesic activity of okra

(Abelmoschus esculentus Linn.) entails a controlled experimental approach within a

laboratory setting. Animal test subjects, particularly mice, are chosen due to their

physiological similarity to humans and are meticulously assigned to different treatment

groups. Some receive okra extract, while others are administered regular painkillers for

comparison. To conduct the interventions, specific concentrations of okra extract, such as

5mg, 15mg, and 25mg, are utilized, with positive and negative controls incorporated into

the study design. A phytochemical analysis would be used to analyze both qualitative and

quantitative component of the plant okra. For the positive control, ibuprofen, a commonly

used painkiller, is employed. The treatment plans are based on previous study and test

studies. During the study, various types of measures are employed to assess the efficacy

of okra extract in alleviating pain. These measures encompass specific behavioral tests,
biochemical assays, and bodily assessments. In terms of behavioral tests, commonly

utilized methods such as the hot plate test and the tail flick test are employed to evaluate

nociceptive responses and pain sensitivity in animal subjects. Biochemical analyses

involve assessing inflammatory markers such as prostaglandin E2 (PGE2) levels or

cytokine concentrations in tissue samples to determine the anti-inflammatory effects of

okra extract. Additionally, bodily measurements may include quantifying changes in paw

edema or joint swelling using calipers or plethysmometers. Pain induction in animal

subjects may be achieved through methods such as thermal or chemical stimuli, with pain

intensity assessed using standardized pain scales or behavioral scoring systems.

Systematic observation and measurement of relevant factors are used to collect data. Both

numeric and qualitative data are collected to get a full picture of the anaesthetic benefits

and any possible side effects. Ethical concerns are very important, and the study followed

institutional and legal rules to make sure the animals were treated humanely and in the

best way possible. Overall, the study plan is strict and organised. The goal is to find

scientifically sound proof of the pain-relieving effects of okra extract and its promise as a

natural option for managing pain.

Research Subjects

In the study investigating the efficacy of okra (Abelmoschus esculentus Linn.) in

pain relief, albino mice (BALB/c strain) are pivotal subjects. These mice are selected due

to their biological resemblance to humans, facilitating the examination of pain responses

and potential therapeutic effects. Employing albino mice allows for controlled

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experiments and facilitates the translation of findings to human subjects. The animal test

subjects are randomly allocated to different treatment groups.

For inclusion criteria, albino mice of both genders, aged between 6 to 8 weeks,

and weighing between 20 to 25 grams are included in the study. Exclusion criteria

involve mice with pre-existing health conditions, pregnant or lactating mice, and those

showing signs of distress or malnourishment.

A total of 100 albino mice will be included in the study, with five treatment

groups: one control group receiving no treatment, and four experimental groups receiving

different concentrations of okra extract (5mg, 10mg, 15mg, and 20mg). Each treatment

group will consist of 20 albino mice. Random sampling will be conducted using a

random number generator to assign albino mice to each treatment group, ensuring an

unbiased selection process.

Research Environment

The Matias H. Aznar Memorial College of Medicine laboratory will serve as the

primary facility for the study due to its controlled environment conducive to laboratory

research. While the laboratory provides essential facilities, tools, and data required for

tests and studies, researchers will ensure the availability of specific instruments needed

for the study. In the event of any lacking instruments, the researchers will make

arrangements to acquire or provide them as necessary.

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Research Instrument

Various methods will be employed to evaluate the efficacy of Okra as an

analgesic. Initially, photographs will be taken to document and monitor changes in the

animals' responses to pain following the administration of okra extract. The hot plate

method will be utilized to assess the analgesic effect of okra by observing the reaction of

the mice's tails to thermal stimuli. This test will provide insights into the intensity and

duration of pain relief. To ensure fairness in comparing treatment groups, meticulous

observations of in vivo pain reactions in laboratory mice will be conducted. The study

will investigate the levels of cytokines responsible for inflammation and prostaglandins,

which are neurochemicals involved in pain signaling. Vital signs of the mice, including

heart rate, respiratory rate, and body temperature, will be monitored before and after the

administration of okra extract, particularly okra juice, to assess any overall positive or

negative effects. By employing these methods, researchers can comprehensively examine

the analgesic properties of okra and determine its safety and effectiveness.

Table 1. Observations of Pain Response in Mice

Mouse No. Vital Signs Vital Signs Pain Reaction Pain Reaction
(Before) (After) (Before) (After)

Note: Vital signs include heart rate, respiratory rate, and body temperature. Pain reaction

assessments will be based on observed behaviors and responses during the hot plate test.

Research Procedure

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 A systematic procedure will be implemented to ensure the accurate execution of

the study and data collection aimed at investigating the potential pain-relieving properties

of okra. Initially, mice approved by the Institutional Animal Care and Use Committee

(IACUC) and compliant with animal welfare regulations will be selected, ensuring their

suitability for the study. After acclimatization, okra juice will be extracted at

concentrations of 5 mg/mL, 10 mg/mL, and 20 mg/mL, prepared using standard methods

such as maceration or extraction with a solvent like ethanol. These concentrations will be

chosen based on preliminary studies and existing literature on okra's analgesic effects.

For the control group, ibuprofen will be administered at a dosage of 10 mg/kg

body weight, based on previous research indicating its efficacy as an analgesic agent in

rodents. The dosage of ibuprofen will be calculated based on the weight of the mice and

administered orally using appropriate methods to ensure precise delivery.

Subsequently, the mice will undergo various pain assessment tests to evaluate the

efficacy of okra in alleviating pain. These tests may include the tail flick test and the hot

plate test, among others. Observers will monitor the animals for responses indicative of

pain relief, such as changes in licking or flinching behaviors. The dosage of okra juice

and ibuprofen will be accurately calculated using standardized dosing methods, ensuring

consistency and accuracy in the administration of treatments. Additionally, the amounts

of juice administered will be adjusted based on the specific concentrations prepared and

the desired dosage regimen.

Then, common tests like the tail flick test and hot plate test are used to assess the

effectiveness of okra in reducing pain. During these tests, observers monitor the animal's

responses, such as licking or flinching its paw, to gauge its reaction to pain stimuli.

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Standardized pain rate scales, such as the Von Frey filament test, Facial Grimace Scale,

are employed to quantify the intensity and duration of pain experienced by the animals.

It's important to note that these pain rate scales have been validated in previous studies.

Additionally, biological studies are conducted concurrently to investigate the mechanisms

underlying okra's analgesic effects. These studies involve analyzing the levels of

neurotransmitters (e.g., serotonin, dopamine), inflammatory cytokines (e.g., interleukin-

1β, tumor necrosis factor-alpha), and prostaglandins associated with pain. Blood or tissue

samples are collected from the animals for this purpose. Regarding tissue samples,

specific histologic changes, such as cellular inflammation, nerve damage, or tissue

degeneration, would be examined to assess the impact of okra on pain-related pathways

and physiological responses.

During the whole study, strict rules are followed for collecting and entering data

to make sure that the results are correct and can be repeated. The safety of okra as a pain

healer is carefully studied by keeping careful records of any bad effects or changes in the

animals' bodies that were seen. This all-around method helps experts figure out whether

okra is safe and effective for treating pain.

Data Analysis

That study about how okra (Abelmoschus esculentus Linn.) can help with pain

will take some time to make sense of. As part of these steps, scientists will look at proof

from both biology and psychology. The first thing that will be looked at is how the mice

would behave during pain tests like the hot plate test. During this study, appropriate

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statistical tests such as t-tests or analysis of variance (ANOVA) may be used to compare

between treatment groups the time it took to remove the arm or the number of times and

lengths of actions caused by pain.

Assays for enzyme-linked immunosorbent assays (ELISA) or high-performance

liquid chromatography (HPLC) could be used to find out how much of a neurotransmitter

is present in this study. Statistical tests will be used to find biological factors that are

significantly different between the treatment groups.

The ideas and study goals will be used to figure out what the results mean once all

the data has been gathered and looked at. To find out how well okra eases pain,

researchers will compare the bodies and behaviors of animals that were given okra to

those that were not. By looking at the genetic data and how much pain has gone down,

researchers will be able to figure out how the drugs really work. They might be able to

tell, for instance, that they change the way cells work or the way inflammation happens.

Ethical Considerations

To ensure everyone involved in the study is treated fairly and ethically,

researchers got to consider some important factors. First and foremost, the researchers

would comply the needed permission from the organizations required for the conduction

of research study to ensure that the research complies with all applicable laws and

regulations on the humane treatment of animals.

The Three Rs: Reduction, Replacement, and Refinement—come next. This

indicates that wherever feasible, the researchers try to use non-animal approaches.

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Researchers must use as few mice as possible while still getting good results. They must

also be careful that the animals do not suffer more than is necessary for the study.

The animals' well-being is the first thing that the investigators think about. To

keep the animals healthy, the experts will make sure they have enough air flow, light, and

a nice place to live. They have to make sure that if they do get sick, they get the right care

and treatment to get better.

When an animal is in terrible pain and the experts can't help, they will arrange for

a licenced professional to euthanize the animal in a way that is both ethical and caring,

following strict guidelines.

In addition, it is very important that the scholars be open and honest about any

personal biases or interests that might affect our study. When they talk about their

studies, they stress fairness and teaching people the right way to deal with pain.

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