CHAPTER I

Introduction

Background of the Study

Most commonly prescribed anti-inflammatory drugs such as corticosteroid have

been shown to have potential side effects like altered response to physical stress among
[1] [19]
others and an increased risk of infection with long term use . As a result, there is a

continuous search for alternative natural anti-inflammatory agents which are safe,

relatively inexpensive and highly tolerated by many patients. In line with this and the

Republic Act 8423 (R.A. 8423), otherwise known as the Traditional and Alternative

Medicine Act (TAMA) of 1997 of the Department of Health (DOH) in promoting the use

of traditional medicine, this study aims to determine the potential anti-inflammatory

effect of Chrysophyllum cainito Linn fruit mesocarp which is traditionally used for its

anti-inflammatory effect in laryngitis and hypoglycemic properties in diabetes mellitus

[31]
.

Chrysophyllum cainito Linn, from the family Sapotaceae and commonly known

as Star Apple or Caimito, is a tree of about 25m in height and is commonly found in

tropical areas such as Southeast Asia [30]. Nine (9) polyphenolic antioxidants are present in

the fruit extract: catechin, epicatechin, gallocatechin, epigallocatechin, quercetin,

[9]
quercitrin, isoquercitrin,myricitrin, and gallic acid . Quercetin, which showed the

highest antioxidant activity in the extract, is also “known to affect immunity and

inflammation by acting mainly on leukocytes and targeting many intracellular signaling

kinases and phosphatases, enzymes and membrane proteins” [5]. Epicatechin, on the other

hand, was found to be in highest concentration in the extract and was shown to have an

1
 [34]
anti-inflammatory effect by inhibiting lipid peroxidation of arachidonic acid in vitro ,

attenuating human C - reactive protein and inhibiting NF-κB in mice models [26].

The anti-inflammatory and anti-hypersensitivity effect of crude methanolic

extracts, fractions and triterpenes of Chrysophyllum cainito L. leaves have only recently
[19]
been successfully investigated in mice but no such study has been done on the fruit.

Hence, the researchers would like to pioneer an investigation of the potential anti-

inflammatory capability of the ethyl acetate soluble fraction of Chrysophyllum cainito

Linn fruit mesocarp methanolic extract (hereon after referred to as CME) using

carageenan-induced paw edema, and histopathologic analysis of PMN infiltration and

plasma extravasation will be measured as anti-inflammatory effects.

Statement of the Problem

This study aims to determine if the ethyl acetate soluble fraction of the

Chrysophyllum cainito Linn fruit mesocarp methanolic extract (CME) is a potentially

effective anti-inflammatory agent in mice.

Specifically, it aims to answer the following question:

Is the ethyl acetate soluble fraction of Chrysophyllum cainito Linn

methanolic extract administered at doses of 30, 50 and 100 mg/kg effective as a

potential anti-inflammatory agent compared to the positive control,

Dexamethasone (2 mg/kg) measured by the following parameters:

A. Reduction in the paw thickness (mm) of carrageenan induced hind paw

edema measured grossly.

2
 B. Reduction in the degree of PMN infiltration and plasma extravasation to

the hind paw interstitium as observed microscopically in hematoxylin and

eosin stained tissue sections.

Research Objectives

The objective of this study is to provide pioneering scientific evidence of the anti-

inflammatory potential of the ethyl acetate soluble fraction of Chrysophyllum cainito

Linn fruit mesocarp methanolic extract (CME) using the mouse model of carrageenan

induced hind paw edema.

Specifically, this study aims to determine the potential efficacy of the CME (30,

50 and 100 mg/kg) in the reduction of hind paw edema compared to the control drug,

Dexamethasone (2 mg/kg), as measured by the following parameters:

a. Reduction in hind paw thickness (mm) interpreted as an increase in

percentage inhibition (%) of edema.

b. Reduction in the degree of PMN infiltration and plasma extravasation

assessed in histological sections.

Significance of the Study

Anti-inflammatory effects of certain medicinal plant extracts have long been

traditionally used to ease muscle pains, sprains, strains, and persistent inflammatory and

neuropathic pain in humans. Chrysophyllum cainito Linn., known locally as Caimito,

[31]
fruit has already been widely used as folk herbal anti-inflammatory medicine but there

is currently no scientific basis on the fruit’s anti-inflammatory and anti-hypersensitivity

properties to reduce persistent pain and inflammation. This study provides pioneering

3
scientific evidence of the anti-inflammatory properties of the ethyl acetate soluble

fraction of the Chrysophyllum cainito Linn fruit mesocarp methanolic extract with the

goal of introducing a safer yet equally effective means of managing inflammation.

Scope and Limitation

This study involved thirty-six (36) ICR mice (30-40 grams, 9-10 weeks old)

randomly selected and purchased from the Philippine Food and Drug Administration

(FDA). Only ripe caimito fruits were used and collected from three (3) different

provinces. Toxicity was evaluated using the Brine Shrimp Toxicity Assay for

determination of the LC50 and post-edema histopathologic analysis of tissue sections of

edematous paws to determine possible histopathologic effects[26].

4
Operational definition of terms

CONCEPTUAL VARIABLE DEFINTION

Anti-inflammatory Effect in the property of the substance that

reduced inflammation and swelling. Flavonoids

such as epicatechin and quercetin from

Chrysophyllum cainito Linn extract contribute

in reducing inflammation by inhibiting CRP and

NF-KB activation.

Inflammation Body's attempt at self-protection to remove

harmful stimuli byincrease in pro-inflammatory

cytokines: TNF-α, IL 1-Beta, white blood cells

causing redness, swelling and heat. Components

of inflammation in mice paw edema involve are

edema, leukocyte infiltration, and granuloma

formation.

Carrageenan Sulphated polygalactose from marine plants that

is a potent inducer in the inflammatory response

at localize area.

Carrageenan Induced Hind Paw A potent inducer of inflammation. Upon

Edema injection of carrageenan into the paw of a

(Lambda Carrageenan) mouse, elicits an acute inflammatory response

characterized by increase of foot thickness and

infiltration of neutrophils.

5
Chrysophyllum cainito L. Polyphenolic antioxidants extracted from the

(Star Apple or Caimito) Chrysophyllum cainito such quercetin,

epicatechin, possess anti-inflammatory

properties against inflammatory pain in mice.

Quercetin, acts mainly on leukocytes and

targeting many intracellular signaling kinases.

Epicatechin, attenuates CRP and inhibits NF-

KB activation.

Dexamethasone Cortisone-like medicine or steroid that works on

the immune system to help relieve swelling,

redness, itching and allergic reaction used as a

positive control for Carrageenan-induced hind

paw edema

Methanol Solvent for extraction purpose (methanolic

extract) to look for bioactives in medicinal

plants known to have anti-inflammatory effects

Normal Saline Solution Sterile solution of sodium chloride (0.9%) used

as a negative control for Carrageenan-induced

hind paw edema

CHAPTER II

6
 Theoretical Background

Review of Related Literature

This chapter presents all the data gathered after the thorough and in-depth study

done by the researchers. This will also present the conceptual framework and operational

definition of terms for better comprehension of the study.

Mechanism of Inflammation

Inflammation is characterized by redness, heat, pain, and swelling. The following

describes the inflammatory process: (1) increase blood supply to the infected area; (2)

increased permeability of blood vessels; and (3) migration of the WBC (neutrophils) and

macrophages to infected area [37]. Furthermore, neutrophils and macrophages, as one of

the major cells involved in inflammation, create a cytotoxic environment by releasing

noxious chemicals from cytoplasmic granules through degranulation[9].Inflammatory

cytokines such as TNF-α, IL-6 and inflammatory mediators such as NO, PGD andPGE2

are the mostly implicated chemical mediators during the process of inflammation[2].

Mechanism of Anti-inflammatory Drugs

Steroidal anti-inflammatory drugs (corticosteroids) inhibit phospholipase A 2,

during the formation of prostaglandins, thromboxane and leukotrienes [35].

Dexamethasone, an example of corticosteroid, relieves hypersensitivity reactions

[42]
such as redness, swelling and itchiness by working on the immune system . Its main

mechanism of action works through inhibition of NF-kB activation which would then

suppress the TNF-α mRNA expression and production on the targeted areas.

7
Additionally, its phospholipase A2 inhibitory proteins and lipocortins are capable of

controlling the biosynthesis of potent mediators of inflammation such as prostaglandins

and leukotrienes [40].

Chrysophyllum cainito Linn fruit

Different parts of Chrysophyllum cainito plant are known to have high therapeutic

values[37]. According to a study conducted by Shailajan & Gurjar (2014), its fruits can be

used to sooth laryngitis and pneumonia while its leaves can be used in treating diabetes

and articular rheumatism[36]. Additionally, its decoction is even used as a pectoral [31]
.

When chemically screened, results show that it also contains alkaloids, flavonoids,
[39]
phenols, sterols and triterpenes . Moreover, a study has also proven that its leaves are

capable of inhibiting myeloperoxidase activity, IL-1, IL-6 and TNF-α in mice models[19].

According to a study conducted by Dong Lou et. al.,(2002), it has been found out

that the Chrysophyllum cainito fruit contains nine polyphenolic antioxidants, the

catechin, epicatechin, gallocatechin, epigallocatechin, quercetin, quercitrin, isoquercitrin,

myricitrin and gallic acid[9].In addition to this, it has also been noted that among these

polyphenolic substances, the epicatechin is highest in concentration while quercitin

exhibits the highest antioxidant activity[9].

Quercetin reduces inflammation by acting mainly on leukocytes and targeting

many intracellular signaling kinases, phosphatases, enzymes and membrane proteins [5]. Its

mechanism of action includes targeting the cells involved in allergic inflammation such
[5]
as the basophils .Furthermore, quercetin is also capable of blocking the release of

histamine[1].

8
 [32]
Epicatechin inhibits the lipid peroxidation of arachidonic acid in vitro . In

addition to this, epicatechin is also capable of attenuating the human C - reactive protein

and inhibits the activity of NF-κB[26].

Gallic acid, another constituent of the Caimito fruit, is capable of hindering the

activity of polymorphonuclearneutrophils (PMNs) and myeloperoxidase [17].

Eleagnine, an alkaloid isolated from Chrysophyllum albidum, as described in a

study conducted in Obafemi Awolowo University, Ile-Ife Nigeria, is responsible for the

fruit’s antinociceptive, anti-inflammatory, anti-microbial and antioxidant activities.

According to their research, eleagnine effectively reduce inflammation through inhibition

of mediators involved in inflammation such as Histamine and Serotonin [28].

Animal and method

ICR mice

Johnson, in her comparative study of laboratory animals (2014), noted that the

mouse model is the most cited animal mainly due to the common biological

characteristics shared by both humans and mice. She writes, “99% of mouse genes have

human counterparts.” Aside from this, it has also been noted from this study that the use

of mice is relatively easy to experimentally manipulate, cheap and convenient to handle

[16]
.

ICR mice, an albino outbred strain of mice originated in Switzerland as described

by Johnson, et. al., (2014), is a general purpose animal model used in the research of

different disciplines particularly in Toxicology, Neurobiology, Oncology, Infection,

Pharmacology, and in Product Safety Testing. According to their works, this breed is

9
characterized by its docile nature, high productivity, rapid growth rate and low incidence

of spontaneous tumor [16].

Brine Shrimp

Artemia salina or brine shrimp is a suitable organism in ecotoxicology studies due

to its intrinsic features. In fact, it was highlighted on a review entitled Use of the genus

Artemia in Ecotoxicity testing by Nunes, et al., (2015) that it can adapt to a wide range of
[25]
salinity (5-250 g/L) and temperature (6-35ºC) . Also, it was described in this review

that this non-selective breeder has a short lifespan, high fecundity and small body size

which make it a convenient model organism [28].

Carrageenan

Carrageenan, a highly sulfated group of polysaccharides made up of repeating

galactose-related monomers, has the ability to cause inflammation in different

experimentations such as animal studies that dwell on mediators of inflammation and

[4]
anti-inflammatory therapeutics .Specifically, it induces a local inflammation

characterized by increased arachidonic acid metabolism, increased vascular permeability,

edema, and neutrophil extravasation according to Hassimoto et al (2013) [13]. Carrageenan-

induced hind-paw inflammation is inhibited by prostaglandin synthesis inhibitors and this

assay predicts the clinical success of anti-inflammatory agents in reducing peripheral

inflammation.

Carrageenan induced hind paw edema

According to a study entitled Carrageenan-Induced Edema in Hind Paw of the

Rat as an Assay for Anti-inflammatory Drugs by Winter et al. (1962), a carrageenan

induced inflammation is acute, non-immune, well-researched, and highly reproducible

10
[43]
. Additionally, it has also been emphasized in this study that the edema induced, is

brought about by the production of pro-inflammatory agents such as bradykinin,

histamine, tachykinins, complement, reactive oxygen and nitrogen species in the early

phase and migration of neutrophils in the late phase after the introduction of carrageenan

into the rats [43]. Paw edema can then be measured in terms of increase in paw size, which

is observable within five (5) hours subsequent to carrageenan induction [41].

The histopathologic analysis of paw edema has significantly contributed in the

[22]
demonstration of neutrophil infiltration during acute inflammation . It’s well

characterized cellular and molecular mechanism as described by Cuzzocrea et al., (2000),

[7]
is divided into two phases, early phase and the late phase . The former is characterized

by the production of the chemical mediators of inflammation while the latter can be

distinguished by the appearance of neutrophil infiltration, production of neutrophil-

derived free radicals and oxidants and the release of other neutrophil-derived mediators

that causes tissue necrosis [7].

11
Conceptual framework

Confounding Variable:

Body Mass, Age, and Sex of Mice

Anti-inflammatory effect:

Reduction in:
Chrysophyllum cainito L. fruit Hind paw edema in terms of:
mesocarp Methanolic a. Reduction of paw thickness
ExtractEthyl Acetate Soluble (mm)
b. Reduction of inflammatory
Fraction
infiltration
30 mg/kg
50 mg/kg
100 mg/kg

Figure 1.Conceptual framework of the study.

Figure 1 illustrates that CME, at doses of 30, 50 and 100 mg/kg, was evaluated for

its potential anti-inflammatory activity demonstrated as a reduction in hind paw edema

measured in terms of reduction of paw thickness and inflammatory infiltration to the site

of inflammation. Confounding variables were identified as the mouse’s body mass, age

and sex at the time of experimentation. Variations caused by these variables were

controlled by the inclusion of only 30-40 kg mice that were 9-10 weeks old. Variations in

the responses according to sex were beyond the scope of this study

12
 CHAPTER III

Methodology

A. STUDY DESIGN

The study followed an experimental design with the introduction of CME given at

three (3) different doses, 30 mg/kg, 50 mg/kg and 100 mg/kg, as the intervening variable.

The study was controlled by a standard drug (2 mg/kg Dexamethasone), a negative

control (sterile saline) and a non-treatment group.

B. STUDY SETTING

The study was conducted in the Veterinary Hospital of De La Salle Araneta

University, Malabon City, Metro Manila.

C. STUDY POPULATION AND SAMPLING

Thirty-six (36) ICR mice, eighteen (18) males and eighteen (18) females (30-40 g,

9-10 weeks old) purchased from the Philippine Food and Drug Administration (FDA),

were used in the study. Six (6) groups of six (6) mice were used in the carrageenan-

induced hind paw edema assay, three (3) males and three (3) females each group.

D. DATA COLLECTION AND PROCEDURE

Plant Material

The C. cainito L. fruits were collected from three (3) different provinces, namely,

Bulacan, Albay and Zambales. The taxonomic identification of representative samples

was confirmed by the National Museum of the Philippines.

13
Extraction

The extraction was performed by the Institute of Pharmaceutical Sciences,

University of the Philippines Manila. Fresh Chrysophyllum cainito Linn fruit mesocarp

was collected (8 kg) discarding the seed and the peel and homogenized prior to extraction

with methanol. The crude methanolic extract was then partitioned with hexane and ethyl

acetate sequentially, discarding the hexane soluble fraction.

Animals

All animals were housed by three’s in plastic cages with adequate perforations

kept in a room with a controlled temperature of 25 ± 2°C, 40-60% humidity maintained

under a 12-hour light/dark cycle with adequate lighting and ventilation. Ethical clearance

for this experimental protocol was obtained from the Philippine Bureau of Animal

Industry. The animals were allowed to acclimatize to the laboratory setting for fourteen

(14) days and were fed with standard pellet diet and water ad libitum and were deprived

of food overnight (12 hours) prior to the experiment.

Brine Shrimp Lethality Assay

To determine the LC50 on which the CME dosages will be based, the Brine Shrimp

Lethality Assay was performed according to the method described by Rahman et. al.,

(2005). Brine shrimp (Artemia salina) eggs hatched in artificial sea water prepared from

commercial sea salt 38 g/L. A lamp was placed above the open side of the tank to attract

the hatched shrimps close to the tank wall. After 48 hours, the shrimps matured as nauplii

and were ready for the assay. The brine shrimp lethality bioassay was carried out on the

CME using the standard procedure:

14
 Prepare vials for testing each fraction initially at 1000, 100, and 10 μg/ml. Prepare

three (3) replicates for each concentration making a total of nine (9) vials. Weigh 20 mg

of sample and add 2 ml of organic solvent (20 mg/2 ml). From this solution, transfer 500,

50, or 5 μl to vials corresponding to 1000, 100, or 10 μg/ml, respectively. Evaporate

solvent under ambient air and then place under high vacuum for about 30 minutes for the

volatile organic solvents to evaporate overnight. After two (2) days (when the brine

shrimp larvae have matured), add 5 ml “sea water” to each vial and add 10 shrimps per

vial with the help of Pasteur pipette (30 shrimps per dilution). The vials are maintained

under illumination. After 24 hours have elapsed, count and record the number of

surviving shrimps, with the aid of a 3× magnifying glass. The data was analyzed with a

Finney computer program (Probit analysis) to determine LC 50 values at 95% confidence

intervals.

Carrageenan-induced hind paw edema

The assay was performed according to the method published by Rahman et. al.

(2005) with minor modifications. Thirty (36) ICR mice, 25-35 g and 9-10 weeks old,

were divided into six (6) groups of six (6): non-treatment, negative, positive and the three

experimental groups. A paw edema was induced by subplantar injection of 50uL of

carrageenan (10% w/v) into the left hind paw and an equal volume of vehicle (0.9%

normal saline solution) was injected into the contralateral paw. The CME (30, 50 or 100

mg/kg), the reference drug, dexamethasone (2 mg/kg), or the vehicle (10 ml/kg of 0.9%

NSS) was given intraperitoneally to the corresponding groups thirty (30) minutes prior to

subplantar injection of carrageenan. The thickness of both hind-paws was measured with

a vernier caliper immediately before (0 hour) and 1, 2, 3, 4 and 5 hours after the

15
carrageenan was injected. The difference in the thickness between hind paws was the

measure of the edema (mm). Percentage of inhibition of inflammation was calculated

using the formula, % inhibition = 100 [(Tc-Tt)/Tc], where ‘Tc’ represents edema

thickness in the control and ‘Tt’ the edema thickness in the group treated with CME or

Dexamethasone.

Histopathologic Examination

Histological analysis of edematous paws was performed to evaluate the anti-

inflammatory effects and/or toxic effects of the CME compared to positive control drug

(dexamethasone). Five (5) hours after carrageenan injection, three (3) mice of each group

were sacrificed, and the edematous paws were fixed in 10% Neutral Buffered Formalin

for one (1) week and decalcified in 5% Nitric Acid for 24-48 hours. After decalcification,

the tissues were washed with Phosphate Buffered Saline and underwent routine

histological tissue processing. Four slices of five (5) μm sagittal sections were placed on

adhesive glass and stained with H&E for histopathologic evaluation conducted by Dr.

Cynthia M. Nalo-Ochona, DVM, MSc, VCPSP, Veterinary Pathologist. To eliminate

subjective bias the researchers used single blinded study in determining the rate of PMN

infiltration and edema fluid formation.

The semi quantitative analysis of tissues were graded according to the degree of

PMN infiltration and edema fluid formation using the scale provided by a certified

veterinary pathologist: 1- PMN infiltration and edema fluid formation absent, 1.5- very

few, 2- mild, 2.5- mild to moderate, 3- moderate, 3.5- moderate to severe, and 4- severe.

16
E. DATA PROCESSING AND ANALYSIS

The data from the study was expressed as the mean ± standard error of the mean

(SEM). Statistical analysis and significance in carrageenan induced paw edema were

determined by Repeated Measures ANOVA followed by Tukey’s test with p ≤ 0.05.

Statistical analysis for PMN infiltration and plasma extravasation was computed using

mean. Lethality (%) in brine shrimp lethality assay was calculated from the mean survival

larvae for both extract-treated and control group. LC 50 values were obtained from the

best-fit line by regression analysis (STATA, Version 13.0). The significance was

accepted when p ≤ 0.05.

17
 CHAPTER IV

Results and Discussion

CME was tested for anti-inflammatory effects using a carrageenan-induced hind-

paw edema model as a classic in vivo activity screening model. Carrageenan, into the

hind paw of the mouse induces a local inflammation characterized by increased

arachidonic acid metabolism, increased vascular permeability, edema, and neutrophil

extravasation. Carrageenan-induced hind-paw inflammation is inhibited by prostaglandin

synthesis inhibitors, and this assay predicts the clinical success of such anti-inflammatory

agents in reducing peripheral inflammation [13]. Variations in responses according to sex

explained that hormone testosterone of male ICR mice aggravates inflammation

demonstrating increase in TNF-α which regulates the extravasation and migration of

neutrophils in histopathological basis [20] while hormone estrogen of female ICR mice

attenuates inflammation by limiting neutrophil, macrophage infiltration and suppressing

[31]
the production of numerous cytokines such as TNF-α. Age, as one of the confounding

variables of the mice also plays an important issue since older mice respond well with a

consistent inflammatory pattern to carrageenan that displays a biphasic edema. [32]

The results of the Brine Shrimp Lethality Assay may be found in Figure 4. The

concentration with 50% probability of mortality (LC50) of CME was calculated as 113.30

ppm or mg/kg, which was 12% higher than the CME’s highest administered dose (100

mg/kg).

18
Figure 4.Best Fit Curve of LC50 of Chrysophyllum cainito methanolic extract. LC50 =

The mean of paw edema (mm) and the percentage inhibition (%) of each

treatment group are presented in Table 4.1 Dexamethasone was used as the positive

control and showed maximum percentage inhibition of 75% in male and 72.73% in

female at the fifth hour. The CME at a dose of 100 mg/kg, on the other hand, showed a

maximum percentage inhibition of 100% and 95% in male and female respectively

beginning at the fourth hour while the CME at 30 mg/kg showed a maximum percentage

inhibition of 87.5% in males and 63.64% in females and 50 mg/kg showed 55% and

81.82% percent inhibition in male and female respectively at the fifth hour.

19
 Table 4.1.Effect of Chrysophyllum cainito Extract (CME) on a Carrageenan-induced
Paw Edema
Treatment Dose Gender Edema Measurement in mm (percent inhibition)
Groups (mg/k
N=5 g) 0hr 1hr 2hr 3hr 4hr 5hr

Male 0 1 1 1.3 1.3 1.3

NSS 10ml/
kg Female 0 0.83 1.17 1.83 1.83 1.83

0 0.5 0.5 0.5 0.5 0.3

2 Male (0) (50) (50) (62.5) (62.5) (75)
DEX 0 1 0.67 0.5 0.5 0.5
Female (0) (-20) (42.86) (72.73) (72.73) (72.73)
0 0.7 0.7 0.7 0.3 0.2
Male (0) (33.33) (33.33) (50) (75) (87.5)
CME 30 0 1 1.67 1 1 0.67
Female (0) (-20) (0) (45.45) (45.45) (63.64)
0 0.8 0.5 0.7 0.5 0.6
50 Male (0) (16.67) (50) (50) (62.5) (55)
CME 0 0.67 0.5 0.83 0.33 0.33
Female (0) (20) (57.14) (54.55) (81.82) (81.82)
0 0.7 0.3 0.2 0 0
100 Male (0) (33.33) (66.67) (87.5) (100) (100)
CME 0 0.33 0.67 0.5 0.1 0.1
Female (0) (60) (42.86) (72.73) (94.55) (94.55)

CME=Chrysophyllum cainito extract;DEX=Dexamethasone; The values represent the

mean of hind paw thickness and percent inhibition measured at 1, 2, 3, 4 and 5 hr. N=30
mice. Repeated measures ANOVA followed by Tukey’s test at p ≤ 0.05. Statistically
significant when compared to control.

There was no significant difference between the percent inhibition of CME at

doses of 100, 30 and 50 mg/kg and the positive control, Dexamethasone, when p<0.05, in

both male and female but dose of 100 mg/kg showed significant difference from doses 30

mg/kg in females and 50 mg/kg in males.

20
 A B

C D

Figure 4.2.The interaction plot of mean edema (mm) in male (A) and female (B) and
percent inhibition in male (C) and female (D) of carrageenan-induced hind paw edema in
mice measured at hourly intervals for 5 hours. CME at 100 mg/kg showed a consistently
lower mean paw edema and a higher percent inhibition compared to positive control (p ≤
0.05). The same pattern is observed at each time interval. CME=Chrysophyllum cainito
methanolic extract.

21
 Fig A B

C D
Figure 4.3.Distribution of mean edema in male (A) and female (B) and percent
inhibition in male (C) and female (D) in carrageenan-induced hind paw edema in
mice. This represents the upper, lower and median value of each group with CME
100 mg/kg showing the lowest mean paw edema volume and highest percentage
inhibition. CME=Chrysophyllum cainito extract.

Examination of histological sections of the mouse paws showed comparable

results with that of the positive control except for the CME at dose 100 mg/kg, which

showed moderate to severe PMN infiltration with only mild to moderate edema (Figure

4.4).

GRADING DESCRIPTION

1.0- ABSENT 0 cells /lpf

1.5- VERY FEW 1-10 cells /lpf

2.0- MILD 10-35 cells /lpf

areas with mild infiltration and areas with

2.5- MILD TO MODERATE moderate infiltration

3.0- MODERATE 35-65 cells /lpf

22
 areas with moderate infiltration and areas with
3.5- MODERATE TO SEVERE severe infiltration

4.0- SEVERE 65-90 cells/lpf

Table 4.2.Histopathologic grading of Polymorphonuclear cell infiltration

Table 4.3.Histopathologic grading of plasma extravasation/edema

GRADING DESCRIPTION

1.0- ABSENT No edema

1.5- VERY FEW 1-10% edema of affected area

2.0- MILD 10-35% edema of affected area

2.5- MILD TO MODERATE areas with mild edema and areas with moderate edema

3- MODERATE 35-65% edema of affected area

3.5- MODERATE TO areas with moderate edema and areas with severe
SEVERE edema

4.0- SEVERE 65-90% edema of affected area

A B C

D E 23
Figure 4.4.Histological changes in edema paws 5 h after injection of carrageenan. Paws
were harvested 5 h after injection of carrageenan and subjected to histochemical staining.
(A) Non-treatment control group shows no proliferation of inflammatory cells, no edema;
(B) Dexamethasone (2mg/kg)-treated group shows mild to moderate proliferation of
inflammatory cells and mild to moderate edema; (C) 30 mg/kg of CME-treated group
shows mild to moderate proliferation of inflammatory cells, mild to moderate edema (D)
50 mg/kg of CME-treated group shows mild proliferation of inflammatory cells, mild
edema; (E) 100 mg/kg of CME-treated group shows moderate to severe proliferation of
inflammatory cells, mild to moderate edema.; magnification ×100.

The mean grading of the tissue slides of the CME and Dexamethasone treated

groups may be seen in Table 4.2 and 4.3. The tables 4.4 and 4.5 showed that the effect of

CME in PMN infiltration and plasma extravasation was comparable to positive control.

The dose of 100 mg/kg gave the highest mean in PMN infiltration and plasma

extravasation in both male and female and dose of 50 mg/kg gave the lowest mean of

PMN infiltration in both gender. Dose of 50 mg/kg also showed the same mean of plasma

extravasation with the positive control in males while it has the same result with 30

mg/kg in females.

Treatment (MEAN)

Gender DEX 30 mg/kg 50 mg/kg 100 mg/kg CME

24
 CME CME

Male 1.5 2 2.5 3.5

3.5 2.5 2.5

Female 2.5 3 2 2

2.5 2.5 3.5

Male 1.5 2.75 2.5 3

AVERAG Femal 2.5 2.75 2.25 2.75

E e

Table 4.4.Histopathologicalmean grading of Dexamethasone and CME in terms of

Polymorphonuclear cell Infiltration in male and female ICR mice.
The values represent the mean of the histologic grading of inflammation in the paw
tissues of the different groups in terms of polymorphomuclear infiltration. CME =
Chrysophyllum cainito methanolic extract; DEX = Dexamethasone.

Table 4.5.Histopathological mean grading of Dexamethasone and CME in terms of

Plasma Extravasation/Edema in male and female ICR mice.

Treatment (MEAN)

Gender DEX 30 mg/kg CME 50 mg/kg CME 100 mg/kg CME

25
 Male 2 2.5 2 2.5

2.5 2 2.5

Female 2.5 2 2 3.5

2.5 2.5 2.5

Male 2 2.5 2 2.5

AVERAGE Female 2.5 2.25 2.25 3

The values represent the mean of the histologic grading of inflammation in the paw
tissues of the different groups in terms of plasma extravasation;CME=Chrysophyllum
cainito methanolic extract; DEX=Dexamethasone

CHAPTER V

Conclusion and recommendation

According to our findings, the CME at 30, 50 and 100 mg/kg is an

effective anti-inflammatory agent compared to control (p < 0.05) with all three (3)

concentrations showing no significant difference from positive control.

26
 In terms of gross reduction of local paw swelling, 100 mg/kg of CME

showed significantly higher results than the CME at 30 and 50 mg/kg exhibiting 100%

inhibition of edema as early as the fourth hour.

In terms of reduction in PMN infiltration and plasma extravasation graded

microscopically (p ≤ 0.05), all three concentrations of CME showed no significant

difference from the positive control with 50 mg/kg of CME showing almost the same

effect as Dexamethasone followed by 30 mg/kg CME.

Taking into consideration both the gross and microscopic assays, the

researchers conclude that 50 mg/kg and 30 mg/kg of CME are the most recommended

doses showing statistically similar results with the control drug, Dexamethasone, with no

increase in degree of PMN infiltration as seen in the 100 mg/kg CME.

Recommendations

For further investigation, the researchers recommend the following:

1. Investigate the acute oral toxicity study on mice (LD50)

2. Perform trials on other doses of Chrysophyllum cainito Linn methanolic extract

3. Use more specific assays like Myeloperoxidase Assay and TNF alpha

measurement

4. Study the correlation between Polymorphonuclear cell infiltration and edema in

100 mg/kg dose

5. Compare the effectivity of the CME doses to different doses of Dexamethasone

27
 6. Perform the separation of the three major components of CME (quercitin,

epicatechin, and gallic acid) via HPLC to identify which among them is the most

effective anti-inflammatory agent

Proposed Applications of CME

1. Development of plant-based anti-inflammatory drug without the side effects

manifested in steroidal drugs

2. Possible alternative to dexamethasone suppression test used for the diagnosis of

Cushing’s syndrome

REFERENCES

[1] Agnihorti, S., Wakode, S., &Agnihorti, A. (2010). “An overview on an anti-
inflammatory properties and chemo profiles of plants used in
traditional medicine.” Indian Journal of Natural Products and
Resources, Vol. 1(2), 150-167.

28
[2] Ashley, N., Weil, Z., & Nelson, R. (2012). Inflammation: Mechanisms, Costs, and
Natural Variation. doi: 10.1146/annurev-ecolsys-040212-092530

[3] Berg, R., Mogensen, T. &Paludan, S. (2007). Mechanisms of Dexamethasone-

Mediated Inhibition of Toll-Like Receptor Signaling Induced by Neisseria
meningitidis and Streptococcus pneumonia. doi:10.1128/IAI.00856-07

[4] Borthakur, A., Bhattacharyya, S.,Natarajan, A. A.,Kumar A., Dudeja, P. K., &
Tobacman, J. K. (2012). Prolongation of Carrageenan-induced
Inflammation in Human Colonic Epithelial Cells by Activation of an NFκB
– BCL10 Loop.doi: 10.1016/j.bbadis.2012.05.001
[5] Chen, M.L., Hsieh, C.C., Chiang B.L., & Lin, B.F. (2014). Triterpenoids and
Polysaccharide Fractions of GanodermatsugaeExert Different
Effects on Antiallergic Activities.doi.org/10.1155/2015/754836

[6] Chirumbolo S., (2010). The role of quercetin, flavonols and flavones in modulating
inflammatory cell function. National Center for Biotechnology
Information, U.S. National Library of Medicine: Bethesda, MD, USA.

[7] Cuzzocrea S., Santagati S., Sautebin L., Mazzon E., Calabro G., Serraino I., Caputi
A.P., & Maggi, A. 17b-Estradiol Anti-inflammatory Activity in
Carrageenan Induced Pleurisy. Endocrinology. Vol 141(4). 1455-1463.

[8] Denny C., Melo, P., Franchin, M. ,Massarioli, A., Bergamaschi, K., de Alencar, S.
& Rosalen, P. (2013). Guava pomace: a new source of anti-
inflammatory and analgesic bioactives. doi:10.1186/1472-6882
13- 235

[9] Dong Luo, X., Basile, M., & Kennelly, E. (2002). Polyphenolic Antioxidants from
the Fruits of Chrysophyllum cainito L. (Star Apple).doi:
10.1021/jf011178n.

[10] DrugBank: Dexamethasone (DB01234). (n.d.). Retrieved March 15, 2015, from
http://www.drugbank.ca/

[11] Fields, T.R. (2009). Steroid Side Effects: How to reduce corticosteroid side effects
Retrieved on August 13, 2015 from http://www.hss.edu/

[12] Ferreira, R.T., Coutinho, M.A. S., Malvar, D., Costa, E.A., Florentino, I.F.,Costa
S.S., & Vanderlinde, F.A., (2014). Mechanisms Underlying the
Antinociceptive, Antiedematogenic, and Anti-Inflammatory Activity of the
Main Flavonoid from Kalanchoepinnata.
doi.org/10.1155/2014/429256\

[13] Hassimotto, N., Moreira, V. et al.(2013) Inhibition of Carrageenan-Induced Acute

Inflammation in Mice by Oral Administration of Anthocyanin Mixture

29
 from Wild Mulberry and Cyanidin-3-Glucoside
http://dx.doi.org/10.1155/2013/146716

[14] Iroko Pharmaceuticals, LLC. (2014). Briefing Documents for FDA Joint Meeting of
the Arthritis Advisory Committee (AAC) and Drug Safety and Risk
Management Advisory Committee (DSARM). Silver Spring, MD:USA.

[15] Jang, M., Jeong, S., Cho, S., Ahn, K., Lee, J.H., Yang, D.C., & Kim, J.C.,
(2014).Anti-Inflammatory Effects of an Ethanolic Extract of Guava
(Psidiumguajava L.) Leaves In Vitro and In Vivo.doi:
10.1089/jmf.2013.293.

[16] Johnson, M. (2014). “Laboratory Mice and Rats.” Mater Methods. Vol. 2. 113

[17] Kroes B.H., van den Berg A.J., Quarles van Ufford H.C., Van Dijk H. &Labad,
R.P. (1992). “Anti-inflammatory activity of gallic acid.” Planta
Medica. Vol. 58(6). 499-504.

[18] Kacew S. & Festing M.F. (1999). “Role of rat strain in the differential sensitivity to
pharmaceutical agents and naturally occurring substances.” Journal of
Toxicology and Environmental Health. Vol 47(1). 1-30.

[19] Ma, Y., Li, Yue & Li, Xiufeng.(2013). Anti-Inflammatory Effects of 4-
Methylcyclopentadecanone on Edema Models in Mice.
doi:10.3390/ijms141223980

[20] Malkin, C., Pugh, P. et al., (2013). The Effect of Testosterone Replacement on
Endogenous Inflammatory Cytokines and Lipid Profiles in Hypogonadal
Men. DOI: http://dx.doi.org/10.1210/jc.2003-031069. Retrieved
from:http://press.endocrine.org/doi/full/10.1210/jc.2003-031069

[21] Matalka, K., Tuntunji, M., Abu-Baker, M., & Abu Baker, Y. (2005). “Measurement
of protein cytokines in tissue extracts by enzyme-linked immunosorbent
assays: Application to lipopolysaccharide-induced differential milieu of
cytokines.” Neuroendocrinology Letters, Vol.26(No.3), 231–236.

[22] Mazue, G., Richez, P. &Berthe, J. (1983). Pharmacology and comparative

toxicology of non-steroidal anti-inflammatory agents. doi:10.1007/978-94-
0096604-8_32

[23] Meira, N., Klein, L., Rocha, L., Quintal, Z., Monache, F., Filho, C., &Quintão, N.
(2014). Anti-inflammatory and anti-hypersensitive effects of the crude
extract, fractions and triterpenes obtained from Chrysophyllum cainito
leaves in mice.doi:10.1016/j.jep.2013.12.014.

30
[24] Mendelson, J. (2014). Anti-inflammatory Drugs for Aspirin Allergic People.
Retrieved from http://www.livestrong.com/ on February 21, 2015.

[25] Moore A. R., Ayoub S. S., &Seed M. P. (2008). Cyclooxygenase Enzymes and
Their Products in the Carrageenan-Induced Pleurisy in Rats.
doi:10.1007/978-159745-364-6_17

[26] Morrison M, van der Heijden R, Heeringa P, Kaijzel E, Verschuren L, Blomhoff

R, &Kooistra T, Kleemann R. (2014). Epicatechin attenuates
atherosclerosis and exerts anti-inflammatory effects on diet-induced
human-CRP and NFκB in vivo.doi: 10.1016/j.atherosclerosis.2013.12.027

[27] Murillo, E., Britton, G., & . Durant, A. (2012). Antioxidant activity and polyphenol
content in cultivated and wild edible fruits grown in Panama.
doi:10.4103/0975-7406.103261

[28] Nunes, B. S., Carvalho F. D., Guilhermino, L. M., Van Stappen G. (2006). “Use of
the genus Artemia in ecotoxicity testing.” Environmental Pollution.Vol.
144(2). 453-62

[29] Oloyede F.M. &Oloyede F.A. (2014). “The Antioxidant and Food value of
Chrysophyllum albidium G. Don”. Scholars Journal of Agriculture and
Veterinary Sciences 2014; 1(1):1-5.

[30] Orwa et al. (2009). Chrysophyllum cainito L. sapotacea. Retrieved on January 11,
2015, from http://www.worldagroforestry.org/

[31] Oskouei, T. et al,(2009). The Impact of Gender on the Inflammatory

Parameters and Angiogenesis in the Rat Air Pouch Model of Inflammation.
Retrieved from:
http://www.mums.ac.ir/shares/basic_medical/basicmedjou/88/summer/Eteraf.pdf

[32] Posadas, I., Bucci, M., et al. (2004).Carrageenan-induced mouse paw edema is
biphasic, age-weight dependent and displays differential nitric oxide
cyclooxygenase-2 expression. doi: 10.1038/sj.bjp.0705650

[33] Rahman, A. U., Choudhary M. & Thomsen, W. (2005). Bioassay Techniques for
Drug Development. Harwood Academic Publishers, Amsterdam,
Netherlands. pp.8-9.

[34] Research models.(n.d.). Retrieved March 15, 2015, from

http://www.janvierlabs.com/

31
[35] Ricciotti, E, & FitzGerald, G.A. (2011). Prostaglandins and
Inflammation.doi: 10.1161/ATVBAHA.110.207449

[36] Stevens, C. (2010). Clinical immunology & serology: A laboratory perspective. 3rd
ed. F.A. Davis, Philadelphia, PA: USA.

[37] Strzelecka M, Bzowska M, Kozieł J, Szuba B, Dubiel O, Riviera Núńez D, Heinrich

M, Bereta J. (2005). “Anti-inflammatory Effects of Extracts From Some
Traditional Mediterranean Diet.” Journal of Physiology and
Pharmacology. 139-156.

[38] Shailajan, S. &Gurjar, D., (2014). “Pharmacognostic and Phytochemical Evaluation

of Chrysophyllum cainito Linn. Leaves.” International
Journal of Pharmaceutical Sciences Review &
Research, Vol. 26(1), Article 17, 106- 111.

[39] Vane, J.R. & Botting, R.M. (1987). “Inflammation and the mechanism of action of
anti-inflammatory drugs.” The FASEB Journal. Vol. 1(2). 89-96.

[40] Vane, J.R.& Botting, R.M. (1998). “Anti-inflammatory drugs and their mechanism
of action.” Inflammation Research. Vol. 47 Suppl 2. 78-87.

[41] Vasconcelos, P.C.D., Seito, L.N., Di Stasi, L.C., Hiruma-Lima, C.A., & Pellizzon,
C.H. (2012). “Epicatechin Used in the Treatment of Intestinal
Inflammatory Disease: An Analysis by Experimental Models.” Evidence-
Based Complementary and Alternative Medicine, Vol. 2012

[42] Vergara, M. M., (2003) Acute Oral Toxicity Study in Rats with Argentyn 23
(EPA/OECD Guidelines). Natural Immunogenics Corporation: Miami, FL.
Covance Study Number 7417-100

[43] Vinegar, R., Truax, J.F., &Selph, J.L., (1973). “Some Quantitative Temporal
Characteristics of Carrageenin-induced pleurisy in the rat.”
ProcSocExpBiol Med 143:711-714.

[44] Wang, H. & Ran Cao, Z. (2014). “Anti-inflammatory Effects of (-)-Epicatechin in

Lipopolysaccharide-Stimulated Raw 264.7 Macrophages.” Tropical
Journal of Pharmaceutical Research. September 2014; 13 (9): 1415-1419.
[45] Winter, R. D., Risley, E. A., Nuss, G. J. E., Komaitis, M. (1962). “Carrageenan
Induced Edema in the Hind Paw of the Rat as an Assay for
Antiinflammatory Drugs.” Proceedings of the Society for Experimental
Biology and Medicine,111:544-547.

[46] Zhang, Y., Nicholatos, J., Dreier, J.R., Ricoult, S.J., Widenmaier,
S.B., Hotamisligil,G.S., Kwiatkowsk, D.J.& Manning, B.D. (2014).
32
Coordinated regulation of protein synthesis and degradation by mTORC1.
doi: 10.1038/nature13492

33