1

CHAPTER I

INTRODUCTION

1.1 Background of the Study

Inflammation is a complex biological response of vascular tissues to harmful

stimuli such as pathogens, damaged cells or irritants (physical or chemical). It is a defense

mechanism aimed to remove the injurious stimuli and initiate the tissue healing process

(Lachkar et al., 2016). Also, five cardinal clinical signs, namely redness, swelling, pain,

heat, and loss of function, characterize it. Inflammation can be acute and chronic. Many

degenerative diseases such as rheumatoid arthritis, shoulder tendonitis, gouty arthritis,

polymyalgia rheumatica, heart disease, asthma, cancer, and inflammatory bowel disease

are often associated with inflammatory processes (Lachkar et al., 2016).

Medicinal herbs have been used as a form of therapy for the relief of pain

throughout history (Almeida, 2011). The treatment of rheumatic disorder is an area in

which the practitioners of traditional medicine enjoy patronage and success.

Akapulko (Cassia alata) exhibited significant antimicrobial activity and showed

properties that support folkloric use in the treatment of some diseases as broad-spectrum

antimicrobial agents. Thus, akapulko is well anchored in its traditional uses has now found

wide-spread acceptance across the world (Bhalodia & Shukla, 2011).

Also, according to Jacinto (2012), akapulko leaves contain chrysophanic acid, a

fungicide that is used to treat fungal infections, like ringworms, scabies and eczema. These

leaves are also known to be sudorific, diuretic and purgative, used to treat intestinal

problems including intestinal parasites. It is also used as herbal medicine to treat bronchitis

and asthma. Because of akapulko’s anti-fungal and anti-inflammatory properties, it is a

 2

common ingredient in soaps, shampoos, lotions, tablet and herbal medicine. in the

Philippines. The Philippine Council for Health Research and Development (PCHRD) has

helped develop the technology for akapulko herbal medicine lotion.

This paper assesses the methanolic extract of akapulko leaf that has an anti-

inflammatory property.

1.2 Statement of the Problem

This study aims to determine the anti-inflammatory activity of methanolic extract of

akapulko leaf in albino mice.

1.3 Objectives of the Study

The researchers aims to achieve the following objectives at the end of the research:

 To determine the methanolic extract of akapulko leaf has a strong anti-

inflammatory activity that can be beneficial in medical field.

 To test the effectivity of methanolic extract of akapulko leaf.

 To prepare the capsule from akapulko as an anti-inflammatory assay.

1.4 Null Hypothesis

There is no significant decrease in the inflammation of the mice with methanolic

extract of akapulko.

1.5 Scope and Delimitation

The study will focus on the anti-inflammatory activity of the methanolic extract of

akapulko leaf using carrageenan induced paw edema method. The collection of the plant

that will be used for authentication and in conducting the research will be collected in

Orani, Bataan. This study will be conducted at Angeles University Foundation under

academic year 2018-2019. This is to determine that the akapulko leaf extract has a strong
 3

anti-inflammatory activity of methanolic extract using filtration method, and rotary

evaporation before and after the administration of methanolic extract in albino mice.

1.6 Significance of the Study

The results of this investigation could be highly significant and beneficial specifically

to the following:

Society. Researchers vision is to live in a world where people are free from the

arthritis by using methanolic extract of akapulko leaf to prevent the pain.

Medical Field. This study will provide the basis and knowledge for developing

higher quality of treatment for all the patients of inflammation. This study will also help

the doctors to treat inflammation using methanolic extract of akapulko leaf as an herbal

medicine. Also, to be the leading provider of high-quality continuing education and

learning to the medical profession.

Future Student-Researchers. The results of this study would be a good source of

information for students who will pursue further studies and experiments about the anti-

inflammatory activity of methanolic extract of akapulko leaf in treating albino mice.

1.7 Definition of Terms

 Acute. This refers to the illnesses generally develop suddenly and last a short time,

often only a few days or weeks (Gifford, 2018).

 Chronic. This refers to conditions develop slowly and may worsen over an

extended period of time—months to years (Katon, 2011).

 Degenerative Disease. This refers to a disease in which the function or structure of

the affected tissues or organs changes for the worse over time (Booker, 2007).
 4

 Pathogens. This refers to the microscopic organisms that cause or have the

potential to cause disease (Bailey, 2018).

 Stimulus. This refers to anything, literally anything that induces a response in your

nervous system (Karosia, 2017).

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CHAPTER 2

REVIEW OF RELATED LITERATURES AND STUDIES

This chapter deals with the related studies and literatures after the through and in-

depth search by the researchers that will help the researchers to fully understand what to

be done and pursue their experimentation that will guide them through. The medicinal

capabilities of akapulko are certainly undeniable with more than a few studies supporting

the claim.

2.1 Review of Related Literature

2.1.1 An introduction to Akapulko (Cassia alata) and its medicinal benefits

For its medicinal purposes, Akapulko (Cassia alata) plays a vital role in

Philippines’ herbal medicine plant. Its seeds are used as anthelmintic. The leaves are

vermifuge and can be used as medicinal drug to cure skin diseases. When the flowers and

leaves have been extracted, it can be served as expectorator for bronchitis, asthma, and as

a mouthwash in inflammation of the mouth.

It is an erect tropical, annual herb with leathery compounded leaves. It belongs to

the Fabaceae family. It grows up to about 8m tall and can be found in diverse habitats. This

perennial shrub has erect waxy yellow spikes that resemble fat candles before the

individual blossoms open. The large leaves are bilateral-symmetrical opposed and fold

together at night. The fruit is a pod, while the seeds are small and square. Extracts from the

leaves of this species has shown several pharmacological properties such as antimicrobial

and antifungal activities, antiseptic, anti-inflammatory and analgesic and anti-

hyperglycemic. It has also shown therapeutic and anti-ageing activities. The plant is a

source of chrysoeriol, kaempferol, quercetin, 5,7,4'-trihydroflavanone, kaempferol-3-O-β-

 6

D-glucopyranoside, kaempferol-3-O- β -D-glucopyranosyl-(1->6)-βD-

glucopyranoside,17-hydrotetratriacontane, n-dotriacontanol, n-triacontanol, palmitic acid

ceryl ester,stearic acid, palmitic acid. Several other flavonoids and anthraquinones have

been isolated from the plant. There is only a report on the constituents of its volatile oil

(Victor et al., 2008).

On the different aspect, since Akapulko can be used as herbal medicine plant, it

possesses different various activities in antibacterial, antimicrobial and anti-inflammatory.

With its properties, the different parts of the plant can be used as an extract that can cure

different diseases such as skin problems, stomach problems, lung problems, and mouth

problems (Jacinto, 2012).

In addition, medicinal plants with anti-inflammatory activity are considerably

employed in the traditional treatment of disorders of inflammation. The inflammatory

responses involve a complex array of enzyme activation, mediator released fluid

extravasations, cell migration, tissue breakdown and repair, which are aimed at host

defence and usually activated in most disease conditions. These different reactions in the

inflammatory response cascade are therapeutic targets; which anti-inflammatory agents,

including medicinal plants interfere to suppress exacerbated inflammatory responses,

usually evoked in such disorders as rheumatoid arthritis, in infection or injury. Inhibition

of the synthesis of pro-inflammatory prostaglandins is one of such therapeutic targets to

which some of the potent anti-inflammatory agents of clinical relevance. Several anti-

inflammatory agents have also demonstrated the ability to inhibit the synthesis of synthesis

of prostaglandins (Sani et al., 2010).

 7

2.1.2 Akapulko (Cassia alata) in response with various activities

In the study of Otto and Onegi (2015), Ether and methanol extracts were prepared

from the plant parts. 12-375mg/ml of serially diluted ether extracts in DMSO and methanol

extracts in water were tested using agar-well diffusion method against Neisseria gonorrhea

clinical isolate cultured on MTM agar. MICs were determined from corresponding

concentration-response curves. Ceftriaxone was used as positive control, whereas DMSO

and water as negative controls. All the crude extracts showed concentration-dependent

Neisseria gonorrhea inhibition. Ether extracts for both leaves and roots gave lower MICs

compared to those of methanol. Ether root extract showed the highest potency. Both the

leaf and the root of Akapulko plant have activity against clinically resistant Neisseria

gonorrhea; the root having the higher activity. Lipophilic solvent, ether, gives more potent

antigonorrhoeal extracts.

Comparatively to the study of Elysha Nur, Somchit and Abdul Rahim (2012),

Crude ethanol and water extract of leaves from akapulko were tested in vitro against

bacteria (Staphylococcus aureus and Escherichia coli) and in vivo to evaluate the effect of

both extracts in liver cells of mice. Antibacterial activity of akapulko (Cassia alata)

extracts on Staphylococcus aureus was detected. The water extract exhibited higher

antibacterial activity than the ethanol extract from leaves (inhibition zone of 11 to 14 mm

and 9 to 11 mm respectively). Escherichia coli showed resistant to both extract. Results

were compared to commercial antibiotics, chloramphenicol, penicillin and aerofloxaxine,

which had 19 mm, 29 mm and 22 mm respectively. Histological examination on the liver

cells suggests that both extract groups contribute to mild hepatocyte degeneration.

Concentrations at a higher dosage suggest the lesion was dose-dependent. Based on the
 8

current findings, it can be concluded that this plant has antibacterial activity, which is as

potent as standard antibacterial drugs against certain microorganisms. The effect of

akapulko extract has on the liver of mice is dose- dependent.

A study conducted by Owoyale et al (2005) present that the phytochemical

screening of the leaves and roots of C. alata revealed the presence of alkaloids,

carbohydrates, tannins, saponins, phenols, flavonoids, anthraquinones and cardiac

glycosides.Drugs present in plants are known as active principles and these serves to

protect the plants themselves against microbial (bacteria, fungi, viruses) attacks as well as

predation by pests and animal. The inhibitory activities exhibited by the extracts tends to

agree with the reports, all of whom linked antimicrobial properties of plants to the presence

of bioactive secondary metabolites like alkaloids, tannins, saponins, flavonoids, phenols,

glycosides and diterpenes.

2.2 Review of Related Studies

2.2.1 Akapulko (Cassia alata) in response with various activities

In the study of Sy et. al. (2012) the akapulko has been found to exhibit a great

antifungal activity against some human pathogenic fungi that had justified the traditional

use of this plant in managing fungal diseases. The extracts were used for evaluating

antifungal activity against five clinical isolates of pathogenic fungi namely Candida

albicans, Microsporum canis, Trichophyton mentagrophyte, Penicillium notatum, and

Aspergillus niger. The extracts inhibited the growth of Candida albicans, Microsporum

canis and Trichophyton mentagrophyte better than the ketoconazole 200 mg used as

positive control. The minimum inhibitory concentration of the water leaf extract of C. alata

for Candida albicans, Aspergillus niger, Penicillium notatum, Microsporium canis and
 9

Trichophyton metagrophytes were 26.90 mg, 32.40 mg, 29.50 mg, 30.30 mg, and 27.80

mg respectively. While that of ethanol leaf extract of C. alata for Candida albicans,

Aspergillus niger, Penicillium notatum, Microsporium canis and Trichophyton

mentagrophytes were 5.60 mg, 3.50 mg, 4.90 mg, 12.60 mg, and 9.80 respectively.

Another study by Sule et al. (2010) entitled, In vitro antifungal activity of akapulko

crude leaf extract found that extracts of akapulko leaf crude extract have high potential as

antimicrobial agent. It showed varying degrees of activities against all the tested

dermatophytes with better antifungal activity against Microsporum canis, Trichophyton

verrucosum, Tricophyton metagrophytes and Epidermophyton jloccosum. The

phytochemical analysis revealed the presence of important secondary metabolite

(alkaloids, saponins, tannis, steroid, and anthraquinones), thus indicating the therapeutic

potentils of akapulko leaf. The formalin test in mice is a valid and reliable model of

nociception and is sensitive for various classes of analgesic drugs. The noxious stimulus is

an injection of dilute formalin (1% in saline) under the skin of the dorsal surface of the

right hindpaw. The response is the amount of time the animals spend licking the injected

paw. Two distinct periods of high licking activity can be identified, an early phase lasting

the first 5 min and a late phase lasting from 20 to 30 min after the injection of formalin. In

order to elucidate the involvement of inflammatory processes in the two phases, we tested

different classes of drugs in the two phases independently. Morphine, codeine, nefopam

and orphenadrine, as examples of centrally acting analgesics, were antinociceptive in both

phases. In contrast, the non-steroid anti-inflammatory drugs indomethacin and naproxen

and the steroids dexamethasone and hydrocortisone inhibited only the late phase, while

acetylsalicylic acid (ASA) and paracetamol were antinociceptive in both phases. The
 10

results demonstrate that the two phases in the formalin test may have different nociceptive

mechanisms. It is suggested that the early phase is due to a direct effect on nociceptors and

that prostaglandins do not play an important role during this phase. The late phase seems

to be an inflammatory response with inflammatory pain that can be inhibited by anti-

inflammatory drugs. ASA and paracetamol seem to have actions independent of their

inhibition of prostaglandin synthesis and they also have effects on non-inflammatory pain.

2.2.2 The use of albino mice in medicine, specifically as anti-inflammatory

All animals procedure are conducted by (Hammel et al., 2016) guidelines for the

ethical treatment of experimental animals. All experiments were conducted using 260–280

g male Sprague–Dawley rats housed in individual cages on a 12 h/12 h dark/light reversed

cycle and allowed access to food and water ad libitum. A total of 54 rats were used in the

experiments described here of which 21 were used as naive controls and 23 were subjected

to adjuvant-induced arthritis. To induce monoarthritis, animals were anesthetized (2–4%

isoflurane) and one knee joint injected with 100 μL complete Freud’s adjuvant (CFA)

(DIFCO Laboratories, Detroit, MI, USA) emulsion (2 mg/mL diluted in 1:1 normal

saline:peanut oil). Rats were returned to their home cages and monitored daily. Joint

circumference and pain-related behaviours were assessed prior to CFA injection and daily

beginning on day 3 after CFA (days 3–7).

In the present study, the researchers used a model of carrageenan-induced arthritis

to further evaluate the effects of joint inflammation on NGF level. These studies showed

that experimentally-induced arthritis in rats caused a significant increase in NGF in the

perivascular area of the synovium. It also showed that injection into the synovium of

purified NGF did not cause inflammation per se and that the destruction of peripheral
 11

sympathetic innervation significantly reduced both the inflammation and the level of NGF

following carrageenan injection.

The study of Lewis and Levy (2011), it investigates the anti-inflammatory effects

of a hexane extract of akapulko leaves in complete Freund's adjuvant (CFA) arthritis in

rats. A hexane extract of akapulko leaves was administered by oral gavage to CFA arthritic

rats (500 mg/kg, n = 6). Controls received corn oil (2 ml, n = 6). The CFA arthritic model

was induced by the injection of 0.5 ml (CFA) into the synovial cavity of the right knee joint

of the hind leg of rats. The ability of the plant extract to reduce swelling as a sign of arthritic

inflammation was assessed by obtaining the circumference of the knee joint before and for

twenty eight days post arthritis induction. Reduction of leukocyte infiltration into the blood

and synovial cavity of the arthritic rats were assessed using automated counting and

Wrights method. Protection against cartilage erosion was also assessed histologically.

Akapulko extract significantly (p = 0.0032) reduced knee circumference (swelling) in the

CFA arthritic rats. Total and differential leukocyte counts in both blood and synovial fluid

from akapulko treated animals were significantly (p < or = 0.05) lower than in control

animals. Protective effects against cartilage degradation on the femoral head of the knee

joint were observed in akapulko treated animals, as normal cartilage structure and

chondrocyte arrangement were maintained. The results suggest that akapulko exhibits anti-

inflammatory activities that should be further examined and potentially exploited for anti-

arthritic therapies.
 12

CHAPTER III

RESEARCH METHODOLOGY

3.1 Introduction

This chapter discusses in detail all the research methods adopted to conduct the

study. Research design, materials/equipment, sample subjects, locale, and procedures that

the researchers will follow to carry out the experiment. The researchers will conduct an

experimental study on the anti-inflammatory activity of methanolic extract of akapulko

(Cassia alata) leaf in treating albino mice.

3.2 Research Design

The researchers used the experimental type of research in this study. Experimental

research design is the method that will allow the researchers to test their hypothesis to have

valid conclusions regarding independent and dependent variables. This will also

manipulate and control the tests to understand causal processes. Experimental type of

research is utilized to prove the anti-inflammatory activity of methanolic extract of

akapulko (Cassia alata) leaf using the carrageenan induced paw method.

3.3 Research Materials and Instruments

The materials and instruments used to conduct the experiment are 1 alcohol lamp, 1

beaker, 1 erlenmeyer flask, 1 glass funnel, 1 graduated cylinder, 2 filter paper, 6 albino

rats, 150 mL of methanol extract, 500 mL of distilled water, 500g of akapulko leaves, male

or female rats weighing >100 g upon arrival, Formalin solution, 0.9% (w/v) saline, sterile,

Scales, accurate to 0.1 and 0.0001 g, ½ -in needle, 3-ml glass syringe, 27-G, ½ -in needle,1-

ml syringes and 6mm biopsy punch

 13

3.4 Samples/Subjects of the Study

The primary objective of this research study is to test the inflammation using the

Akapulko (Cassia alata) leaf. Akapulko (Cassia alata) commonly known as the candle

bush. The plant sample will be authenticated at University of the Philippines Diliman

Institute of Biology.

3.5 Research Locale

The plant sample, Akapulko (Cassia alata) was collected from the forest located in

Orani Bataan, Philippines. The experiment including the extraction and assay will be

conducted in Biology Laboratory, Angeles University Foundation –Sta. Barbara Campus,

Angeles City under the academic year 2018 - 2019.

3.6 Procedures of the Study

Collection of plant

The researchers searched for Akapulko (Cassia alata) leaves in their chosen area.

Akapulko (Cassia alata) plants were rooted to the ground: thus, the use of scissors to

separate them from the ground was necessary. The collected plants were stored inside a

container for future usage

Extraction of plant

The dried leaves of Akapulko (Cassia alata) were pulverized using a blender. 500g

each of the powdered leaves of Akapulko (Cassia alata) was extracted with 800ml of 80

% MeOH. The extract solution was evaporated to dryness under reduced pressure (below

40 °C) to yield crude methanolic extract.

 14

Experimental animals

Albino mice was kept under standard environmental conditions at 25 °C with 12:12

h light–dark cycle in ventilated plastic cages. Animals were fed with a standard rodent diet

and water supplied ad libitium.

Anti-inflammatory activity

The mice was randomly divided into three groups of 3 animals each. Before

treatment, the right hind paw of each animal was measured. To determine the anti-

inflammatory effect of the extract on mice, a formalin induced inflammation test was

carried out (Safari et al., 2016). The formalin is injected in mice paws at a dose of 2.5%.

Thirty minutes after the injection of fomalin, all the groups are injected in the laboratory

mice to check all their anti-inflammatory activity. The response of mice is the amount of

time the animals spend licking the injected paw. Hourly changes in paw sizes was

determined using a vernier caliper. The administered drugs was injected to the mice (0.9%

saline) or standard drug (Diclofenac at 50mg/kg), methanolic extract at 10 and 100 mg/kg

respectively derived from Akapulko (Cassia alata) were given intramuscular to the

animals. After injecting all of this in the right hind paw of mice, the researcher measured

the reduction of edema around the paw that was determined using a vernier caliper.
 15

CHAPTER IV

PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

This chapter provides the results gathered in tabular and graphical form of the

experimental study. It presents the results concisely and discusses the relation to the

research objectives by comparing and contrasting relative studies to answer the problems

presented with the following data.

4.1 Presentation of Data

The Formalin-induced Paw Edema test was used to determine the ability of

akapulko (Cassia alata) leaf extract to reduce inflammation. In this test, the reduction of

diameter of edema around the paw of the mice was measured in millimeter and recorded

before and every hour for three hours after the administration of test substances. Then, the

percentage measurement of edema was determined. The presence of the anti-inflammatory

activity in the akapulko (Cassia alata) extract depends on the reduction of the diameter or

percentage of the edema in the paw of the mice.

Table 1

Percent and diameter change in paw diameter after the administration of

Methanolic extract of akapulko (Cassia alata) leaf

Group Treatment 0 hr 1 hr 2 hr 3 hr

100% 97.56% 94.31% 88.62%

Normal
Control 1.23 1.20 1.16 1.09
Saline
mm mm mm mm

Standard Diclofenac 100% 83.74% 75.61% 61.79%

 16

1.23 1.03 0.93 0.76

mm mm mm mm

Methanolic 100% 95.93% 91.06% 86.18%

and

Butanolic

Test-1 extract of 1.23 1.18 1.12 1.06

akapulko mm mm mm mm

(Cassia

alata) leaf

Table 2: The anti-inflammatory activity interpreted from the reduction of paw diameter of

Normal saline, Diclofenac and Methanolic and Butanolic extract of akapulko (Cassia alata)

leaf represented by percentage.

The percentage represents the measurement of the paw edema on the mice. If the

percentage is low, it means that the edema has subsided over time and the lowest percentage

would correspond to the fastest group. If the percentage is high, it means that the edema

has not subsided yet and the highest percentage would be the slowest group. In comparison

to the positive control or Diclofenac, results show that the percentage diameter of edema

is lower than the extracted Methanolic extract of akapulko (Cassia alata) leaf. These results

show that the said extracted Methanolic extract of akapulko (Cassia alata) leaf does not

exhibit anti-inflammatory activity compare to the commercialized product.

 17

4.2 Intepretation of Data

Table 2

Data summary

Samples

1 2 3 Total

N 6 6 6 18

∑𝑿 3.45 2.72 3.36 9.53

Mean 1.15 0.9067 1.12 3.1676

∑ 𝑿𝟐 11.9025 7.3984 11.2896 30.5905

Variance 0.00625 0.018633335 0.0036 0.0284833335

Std. Dev. 0.0790569415 0.1365039743 0.06 0.2755609158

Std. Err. 0.04564354646 0.07881060631 0.0346101615 0.1590951689

The table above reveals that there is a significant difference in the anti-

inflammatory activity of the test extract, Diclofenac and the negative control. Since the

computed probability value was higher than 0.05, the researchers accept the null

hypothesis. This means that there is no significant difference between the groups.

To further elaborate the findings of the study using the data acquired from the one

way ANOVA test indicating the difference of each group, the researchers used Post Hoc

Analysis specifically Tukey’s HSD Post Hoc test.

 18

Table 4

Interpretation of the results from one-way ANOVA by the means of Tukey’s HSD

Post Hoc test

Groups Hypothesis

Group 1 (Normal Saline) vs. Group 2 (Diclofenac) Significant difference

Group 1 (Normal saline) vs. Group 3 (Methanolic extract No Significant difference

of akapulko (Cassia alata) leaf)

Group 2 (Diclofenac) vs. Group 3 (Methanolic extract of Significant difference

akapulko (Cassia alata) leaf)

The table shows that there is a significant difference in Normal Saline and

Diclofenac since they are the positive and negative control group. However, when Normal

Saline is compared to the Methanolic extract of akapulko (Cassia alata) leaf there is no

significant difference. This simply means that the Methanolic extract of akapulko (Cassia

alata) leaf is negative in anti-inflammatory activity.

4.3 Results

Using the Formalin-induced Paw Edema Test, the researchers gathered data that

proved that the Methanolic extract of akapulko (Cassia alata) leaf does not exhibit anti-

inflammatory activity. This statement is supported by Table 2 shown above.

 19

CHAPTER V

SUMMARY, RECOMMENDATION AND CONCLUSION

This chapter provides the summary of the findings, conclusion of the study and the

recommendations that needs to be improved.

5.1 Summary of Findings

Three groups were tested on mice using a Formalin-induced Paw Edema Test.

These three include Normal Saline as the control or negative control group, Diclofenac or

the commercially produced anti-inflammatory medicine as the standard or positive control

group, and the Methanolic extract of akapulko (Cassia alata) leaf as Test-1. To show that

the Methanolic extract of akapulko (Cassia alata) leaf has an anti-inflammatory activity,

the diameter of paw edema on the mice needs to be on the same with or a lower percentage

of paw edema than the positive control.

With the use of the Formalin-induced Paw Edema Test, the researchers collected

the following data: 88.62% diameter of paw edema in Normal saline, 61.79% diameter of

paw edema in Diclofenac, and 86.18% diameter of paw edema in Scoparia extract. The

findings show that the negative control and Methanolic extract of akapulko (Cassia alata)

has a higher percentage than the positive control. This result equate to the percentage of

edema in Methanolic extract of akapulko (Cassia alata) being higher than that of

Diclofenac. From this test, the researchers found out that Methanolic extract of akapulko

(Cassia alata) does not have an anti-inflammatory activity.

5.2 Conclusion

The results of the data gathered shows that the extract from the Methanolic extract

of akapulko (Cassia alata) leaf does not exhibit anti-inflammatory activity.

 20

5.3 Recommendations

1. Find an alternative solution that has a high anti-inflammatory activity.

2. Use more mice for accurately and precise result.

3. Improve procedures.
 21

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