Effects of Skeletonema Costatum’s Powder on the Knee Joint’s

Diameter and the Degree of Pain of Male Rat Sparague Dawley Type

Induced by Complete Freund’s Adjuvant

Astalitha Lorel Tan

1,*

Riana Rahmawati

and Isnatin Miladiyah

Alumni, Faculty of Medicine, Universitas Islam Indonesia, Yogyakarta, 55584, Indonesia

Pharmacology Department, Faculty of Medicine, Universitas Islam Indonesia, Yogyakarta, 55584, Indonesia

Keywords: Antioxidants, Complete Freund's Adjuvant, Osteoarthritis, Skeletonema Costatum.

Abstract: Osteoarthritis is a degenerative disease caused by an abnormal inflammatory cytokine response. Complete

Freund's Adjuvant (CFA) is a chemical substance that induces osteoarthritis. Bioactive compounds in

Skeletonema costatum act as antioxidants, which could lessen inflammation. This study aims to determine

how Skeletonema costatum affects the knee joint's diameter size and the degree of pain induced by CFA in

rats. The study used thirty adult male rats (Sparague dawley), 2-3 months old, weighing 200-300 grams,

divided into five groups: negative control (K1), positive control/piroxicam (K2), and intervention groups (K1,

K2, and K3). All groups were induced with CFA, and the intervention began six weeks after induction. The

negative and positive groups received normal saline and piroxicam (10 mg/kg BW/day) for 21 days,

respectively. Skeletonema costatum was given to the intervention groups at doses of 60 mg/kg BW/day (K3),

90 mg/kg BW/day (K4), and 120 mg/kg BW/day (K5) for 21 days. The knee joint diameters and the degree

of pain were assessed using a micrometer screw and a hot water tail-flick assay on days 0, 7, 14, and 21. The

results were examined using the paired-t test, Kruskall-Wallis, and post hoc Mann-Whitney. Skeletonema

costatum lowered the diameters and pain levels of rats' knee joints before and after the trial (paired t-test,

p<0.05). All dosage groups demonstrated a beneficial effect (Kruskal Wallis, p<0.05); the higher the dose of

Skeletonema costatum, the greater the effect on reducing the knee joint's diameter and pain. A multivariate

analysis showed the reduction of knee joint diameter and pain after Skeletonema costatum's intervention was

statistically greater compared to the control/placebo group. However, this effect was lower than in the

piroxicam group (p< 0.05). Skeletonema costatum may have an anti-inflammatory effect in rats by lowering

the size of the knee joints and the degree of pain induced by CFA. Given the potential of Indonesian marine

products and the trend toward marine drug treatments, further studies are required to investigate the

inflammatory effects of Skeletonema costatum.

1 INTRODUCTION

Osteoarthritis (OA) is a chronic degenerative disease

that commonly affects older people and someone who

uses their joint too much, which causing joint

inflammation. Osteoarthritis usually affects the

interphalangeal, hip, knee, spinal, and

temporomandibular joints

(Kasper, 2015; Gs M, 2014;

Poulet, 2017; Gupta, 2017). Common symptoms of OA

include pain and limitation of daily activities due to

cartilage damage.

The incidence of OA under the age of 40 is

minimal and is commonly due to trauma. The

prevalence of OA increases between the age of 40-60

years. Globally, it is estimated that 9.6% of men and

18% of women over the age of 60 have symptomatic

osteoarthritis. According to the World Health

Organization (WHO) in 2010, OA affects more

women than men in all age groups (2.95 women per

1000 population and 1.71 men per 1000 population).

In women, the age group of 65-74 years has the

highest incidence, about 13.5 per 1000 population per

year. In men, the incidence in people over 75 years of

age is about 9 per 1000 population per year. In

Indonesia, the prevalence of OA based on

radiological findings is 15.5% in males aged 40-60

years and approximately 12.7% in females.

According to WHO, the elderly population in

Indonesia is expected to increase by 414% compared

Tania, A. L., Rahmawati, R. and Miladiyah, I.

Effects of Skeletonema Costatum’s Powder on the Knee Joint’s Diameter and the Degree of Pain of Male Rat Sparague Dawley Type Induced by Complete Freund’s Adjuvant.

DOI: 10.5220/0013084800003873

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 1st International Conference on Medical Science and Health (ICOMESH 2023), pages 5-10

ISBN: 978-989-758-740-5

to 1990 by 2025 due to the increase in life expectancy

in Indonesia.

The development of OA is influenced by many

risk factors such as: continuous mechanical stress

occurs in multiple parts of joints, inducing the release

of pro-inflammatory mediators and degradation

enzymes such as IL-1, IL-6, IL- 1 β, NO, and TNF-α,

resulting in joint inflammation (

McCance, 2014).

Inflammation results from an imbalance between

pro-inflammatory and anti-inflammatory cytokines.

Pro-inflammatory mediators induce the formation of

reactive oxidative stress (ROS), leading to

exacerbated inflammation. Another consequence of

the release of pro-inflammatory mediators is that the

chondrocyte components in the turnover process

become imbalance and the joint undergoes excessive

destructive processes leading to chondrocyte

apoptosis (

McCance, 2014). Increased chondrocyte

apoptosis reduces the number of proteoglycans in

cartilage. Bonds between collagens weaken due to

decreased synthesis of type II collagen and increased

collagen degradation.

Molecules resulting from the breakdown of

collagen and proteoglycans are destroyed by synovial

macrophages causing an increase in the number of

pro-inflammatory cytokines. These cytokines bind to

chondrocyte receptors and trigger the release of

metalloproteinase (MMP), inhibiting type II collagen

production and continuing cartilage degradation. In

response to mechanical and biochemical stimuli,

chondrocytes overproduce its MMP, collagenase,

stromelysin, and gelatinase, causing greater joint

damage. Metalloproteinase also trigger the release of

ROS, namely hydrogen peroxide, hypochlorite ion,

hydroxyl radical, or superoxide anion

. Chondrocytes

normally produce small amounts of nitric oxide (NO)

and superoxide anion. These two ions will form

peroxynitrite and hydrogen peroxide. Hydrogen

peroxide can convert its form to hydroxyl radical,

forming lipid peroxide in chondrocytes and causing

further degradation (Gupta, 2017).

Our cells can naturally reduce excess ROS by

using antioxidants. Antioxidants are divided into two

groups: enzymatic and non-enzymatic. Antioxidants

use superoxide dismutase (SOD), catalase (CAT),

glutathione peroxidase (GPX) and some fat- and

water soluble small molecules to prevent the

formation of ROS. It acts as a ROS scavenger by

repairing damage that has occurred (Gupta, 2017; Sun

AR, 2017). However, when the antioxidant capacity

is reduced, ROS damage the extracellular matrix,

nucleus, and cell membrane, leading to cell death,

causing dead cells to release oxidative molecules,

triggering the release of synovial macrophages, pro-

inflammatory cytokines, ROS and MMP, which lead

to chronic inflammation (Sun AR, 2017).

Initial treatment of OA uses an oral non-steroidal

anti-inflammatory drug (NSAID) such as piroxicam.

It aims to reduce the inflammatory response that

occurs in the joints. Additionally, corticosteroid

injections, oral administration of glucosamine and

chondroitin sulfate can reduce joint inflammation. It

acts as an anti-inflammatory agent, causes a

scavenging effect, and plays a role in lipid peroxidase

mechanism by exploiting the mechanism of reducing

ROS production (Gunawan SG, 2016). Because

NSAID therapy adversely affects the gastrointestinal

system when taken long term (Gunawan SG, 2016).

Another alternative from a marine alga, Skeletonema

costatum, may help reduce the inflammatory effects

of OA.

Antioxidants are protective agents which inhibit

disease progression and the formation of ROS from

chondrocytes, thus reducing the progression of OA.

Skeletonema costatum is a microalga belonging to the

class Coscinodiscophyceae and the Skeletonemacea

family (Miyashita K, 2009). This microalgae contains

antioxidants such as carotenes and unsaturated fatty

acids. The types of carotene they contain include

fucoxanthin and astaxanthin (Miyashita K, 2009; Foo

SC, 2017). The total fucoxanthin contained in this

microalgae is 0.36 ± 0.00 with a total carotene of 0.97

± 0.24 (Foo SC, 2017). While levels of omega 3:

0.911 – 3.738%; omega 6: 15.591 – 38.002% and

omega 9: 0.292 – 15.112% (Erlina A, 2004). The

antioxidants in Skeletonema costatum act as anti-

inflammatory agents by reducing intracellular ROS,

SOD, and NO levels, increasing type II collagen

synthesis and enabling joint repair.

In the present study, CFA induction can induce

inflammation due to OA after 6 weeks after intra-

articular injection. No research explains the anti-

inflammatory activity by Skeletonema costatum,

based on data sources, so it is necessary to research to

examine the effect of Skeletonema costatum on the

diameter of the knee joint and the value of pain in rats

induced by Complete Freund's Adjuvant

(CFA).

2 METHOD

This type of research was conducted using a

laboratory experimental method with a pre-post-

study design and a post-test-only control group

design. The research was conducted in January-

February 2020 at the Nutrition Laboratory of the

Center for Food and Nutrition Studies, Gadjah Mada

University, and obtained research ethics permit from

ICOMESH 2023 - INTERNATIONAL CONFERENCE ON MEDICAL SCIENCE AND HEALTH

the Ethics Committee of the Faculty of Medicine,

Public Health and Nursing, Gadjah Mada University

with protocol number KE/0851/07/2019.

The inclusion criteria used in this study were adult

male rats of the Sprague Dawley strain, aged 3-4

months and weighing 200-300 grams, the rats were

obtained from the same breeding grounds with the

same food and drink, the rats were healthy and had no

marked defects with the animal trying to move

actively, the fur looks clean and doesn't fall out.

Meanwhile, the exclusion criteria were sick and dead

rats during the study.

The grouping of the sample was carried out using

the simple random sampling method. The size of the

research sample is determined by the Federer

formula.

Thirty adult male rats (Sparague dawley), 2-3

months old, weighing 200-300 grams, were utilized

in the study and were divided into five groups:

negative control (K1), positive control (K2), and

intervention groups (K1, K2, and K3). CFA was used

to induce all the groups, and the intervention started

six weeks after induction. Normal saline and

piroxicam 10 mg/kg BW/day for 21 days were

administered to the negative and positive groups.

Skeletonema costatum was administered to the

intervention groups for 21 days at various dosages of

60 mg/kg BW/day (K3), 90 mg/kg BW/day (K4), and

120 mg/kg BW/day (K5). On days 0, 7, 14, and 21,

the groups joint diameter and pain levels were

measured using a micrometer screw and a hot water

tail-flick assay. A paired-t test, Kruskall-Wallis, and

post hoc Mann-Whitney were used to examine the

results. On the 21st day, the groups were terminated.

SPSS software version 20.0 for Windows (SPSS

Inc. Chicago, USA). Paired-t test (data are normally

distributed) and Wilcoxon test (data are not normally

distributed) were performed to confirm the difference

in mean size of right knee joint diameters in rats

before and after treatment (Sastroasmoro, 2011)

To confirm the decrease in joint diameter and

comparison of pain response between groups, the

non-parametric Kruskal-Wallis tes was performed

because the requirements of the Anova test were not

met. To identify significant differences in each group,

we performed Mann-Whitney as a follow-up test to

Kruskall Wallis.

3 RESULTS

3.1 Measurement of Rat Right Knee

Joint Diameter

Right knee joint diameters in rats were measured

using a screw micrometer calibrated based on the

Clinical Assessment of Experimentally Induced

Osteoarthritis Rat Model In Relation To Time to see

OA before and after treatment. Table 1 observed

diameter of the right knee joints of rats in the five

groups.

Based on the test results, the right knee joint

diameter of rat in the negative control group (K1)

tended to increase. On the other hand, the positive

control group (K2) (piroxicam 10 mg/kg BW) and all

dose treatment groups (K3, K4, K5) decreased the

diameter of the right knee joint of rats. A decrease in

the diameter of the rat's right knee joint began to be

seen on day 7.

Table 1: Rat’s right knee joint diameter on days 0 to 21

Treatment Group

Mean diameter of right knee joint ± SD (mm)

Day-0 Day-7 Day-14 Day-21

K1 9.07±0.45 9.13±0.35 9.39±0.26 9.49±0.23

K2 8.62±0.33 6.48±0.35 3.97±0.44 2.50±0.23

K3 8.64±0.28 8.23±0.07 7.37±0.29 5.40±0.20

K4 8.77±0.50 8.00±0.07 6.51±0.29 4.58±0.21

K5 8.70±0.25 7.53±0.41 5.11±0.20 3.11±0.22

The paired-t test in table 2 was performed to

confirm size group mean difference in the diameter of

the right knee joint (data for all groups were normally

distributed). A Kruskall-Walis test was then

performed to confirm significant comparisons

between groups (data not normally distributed until

day 7). Then proceed to the Mann-Whitney test to

check for significant differences between groups

(table 4).

Effects of Skeletonema Costatum’s Powder on the Knee Joint’s Diameter and the Degree of Pain of Male Rat Sparague Dawley Type

Induced by Complete Freund’s Adjuvant

Table 2: Test results paired -t-test on groups rat’s right knee

joint diameter.

Group Pair P Value,

Confidence Interval Value

(CI) 95%

Days 0

and 7

Days 0

and 14

Days 0

and 21

K1 before

–

K1 afte

0.527

0 .029 * 0 .029 *

K2 before

–

K2 afte

0,000 * 0.000 *

K3 before

–

K3 afte

0 .021 *

K4 before

–

K4 afte

0.013 *

K5 before

–

K5 afte

0.004 *

Table 2 shows that joint diameter remained at day

7 and increased significantly at day 14 and 21 in

group K1 (p<0.05). For K2 and all test groups (K3,

K4, and K5) the mean joint diameter was significantly

decreased on day 7, 14, and 21 (p<0.05). From the

data in tables 1 and 2, we concluded, Skeletonema

costatum (60 mg / kgBB; 90 mg / kgBB; 120 mg /

kgBB) significantly reduced the size of diameter of

right knee joint as seen in the piroxicam-treated

group.

Table 3: Mann -Whitney post hoc test in groups of rat’s right

knee joint diameter.

Intergroup

Relations

P value

Decline

(0-7 days)

Decline

(0-14

)

Decline

(0-21

)

K1 K2 0.004* 0.004* 0.004*

K3 0.004* 0.004* 0.004*

K4 0.004* 0.004* 0.004*

K5 0.004* 0.004* 0.004*

K2 K3 0.004* 0.004* 0.004*

K4 0.004* 0.004* 0.004*

K5 0.005* 0.004* 0.008*

K3 K4 0.004* 0.004* 0.004*

K5 0.004* 0.004* 0.004*

K4 K5 0.004* 0.004* 0.004*

The results of the Kruskall-Walis test revealed

that there was a significant difference (p<0.05) in the

mean reduction in diameter of the right knee joints of

rats between groups in all groups. Table 3 shows a

significant (p<0.05) mean reduction in diameter of

the right knee joint of rats in each group. The negative

control (K1) is significantly different from the

positive control (K2) or test group (K3, K4, K5).

Although the right knee joint diameters of rats are

decreased, Table 3 shows that the mean decrease in

diameter in the various test groups is significant in the

positive control group (K2). In the study groups,

significant differences in joint diameter reduction

were observed between the 60 mg/kg BW (K3), 90

mg/kg BW (K4), and 120 mg/kg BW (K5).

3.2 Measuring Pain Response of Rats

Assessment of the degree of pain in rats using the hot

water tail-flick assay. The longer the rat lifts its tail

from the water bath, the better the pain response.

Below is a table of the observed pain scores of the rats

in the five groups.

Table 4: Pain response rats on days 0 to 21.

Treatment

Group

Rat

ain score ± SD (seconds)

Day-0 Day-7 Day-14 Day-21

K1 0.42±0.07 0.32±0.04 0.25±0.40 0.23±0.03

K2 0.45±0.92 0.92±0.06 1.08±0.06 1.98±0.05

K3 0.45±0.05 0.51±0.02 0.66±0.03 0.78±0.05

K4 0.48±0.07 0.62±0.03 0.86±0.04 0.96±0.02

K5 0.45±0.03 0.80±0.04 0.95±0.04 1.47±0.22

Based on table 4, the pain scores of rats in the

negative control group (K1) tended to decrease or

showed no improvement in pain response with tail

raising. In the positive control group (piroxicam 10

mg/kg BW) and all dose treatment groups (60 mg/kg

BW; 90 mg/kg BW and 120 mg/kg BW), there was

an increase in the tail-lifthing time which began to

appear on the day 7.

Paired-t test (normally distributed data on days 7

and 21) and Wilcoxon (non-normally distributed data

on day 14) were used to count difference in mean pain

response before and after treatmnet. Tables 5 and 6

show the averages of various test results.

Table 5: Test results paired -t-test on groups pain response

rat for days 0 and 7 and days 0 and 21

Group Pair

P value

Days 0 and 7 Days 0 and 21

K1 before

–

K1 afte

0,000 *

0.003 *

K2 before

–

K2 afte

0.000 *

K3 before

–

K3 afte

K4 before

–

K4 afte

K5 before

–

K5 afte

Table 6: Wilcoxon test results on groups pain response rat

for days 0 and 14.

Group Pair

P value

s 0 and 14

K1 before

–

K1 afte

0.027 * _

K2 before

–

K2 afte

0 .024 *

K3 before

–

K3 afte

0.027 *

K4 before

–

K4 afte

0.027 *

K5 before

–

K5 afte

0.028 *

Tables 5 and 6 for the negative control group (K1)

show a reduction in time pain response on days 7, 14,

or 21. This is linear with the data of measurement of

the right knee joint diameter of rats (table 1) which

illustrates enhanced inflammation in the K1 group.

ICOMESH 2023 - INTERNATIONAL CONFERENCE ON MEDICAL SCIENCE AND HEALTH

Otherwise, the means pain scores before and after

treatment, the positive control group (K2) or all test

groups (K3, K4, and K5) were significantly different

(p<0.05) in improving tail lift time in rats that

indicates the pain response is improved.

The other test is to compare the average of

decreased score pain on days 7, 14, and 21 using the

Kruskall-Wallis test. The data for day 14 are not

normally distributed and therefore did not meet the

requirements for the Anova test. According to the

results of the Kruskall-Walis test, there was a

significant difference between groups in all post

treatment observation periods (p<0.05). To see which

groups differed, Mann-Whitney performed a post hoc

test.

Table 7: Further test results post hoc Mann-Whitney on the

pain response group.

Intergroup Relations

P value

Day-7 Day-14 Day-21

K2 0.004* 0.004* 0.004*

K3 0.003* 0.004* 0.004*

K4 0.004* 0.004* 0.004*

K5 0.004* 0.004* 0.004*

K3 0.003* 0.004* 0.004*

K4 0.004* 0.004* 0.004*

K5 0.012* 0.005* 0.004*

K4 0.003* 0.004* 0.003*

K5 0.003* 0.004* 0.004*

K4 K5 0.004* 0.004* 0.004*

Table 7 shows this significant difference in pain

score in all groups. Regarding the effective reduction

of right knee joint diameter in rats, this study shows

that the groups (K3, K4, and K5) are significantly

different from the negative control group (K1) or the

positive control group (K2). There are also significant

differences between the Skeletonema costatum

groups (60 mg/kg BW (K3); 90 mg/kg BW (K4), and

120 mg/kg BW (K5)).

4 DISCUSSION

This study showed that the negative control group has

a decrease in pain response and an increase in the

diameter of right knee joint rat, that is, there was

inflammation caused by OA by Complete Freund's

Adjuvants (CFA). While in the positive control group

(piroxicam 10 mg/ kg BW) and in all treatment

groups (Skeletonema costatum 60 mg/ kg BW; 90

mg/kg BW; 120 mg/ kg BW) there was an increase in

pain response along with a decrease in the size of the

right knee joint diameter of the rats(p<0.05).

This study, also demonstrated that Skeletonema

costatum has the effect of reducting joint diameter

and improving pain response before and after

treatment (paired t-test, p<0.05). Although efficacy

was seen in all groups of Skeletonema costatum, it has

been reported that the higher the Skeletonema

costatum dose, the greater the reduction in joint

diameter and the greater the improvement in pain

response. Reduction in right knee joint diameter of

rats and improvement of pain response after

administration of Skeletonema costatum was better

than negative control, but anot as good as using

piroxicam compared with positive control.

It reduces inflammatory markers since

Skeletonema costatum acts as an anti-inflammatory

agent. This is achived by inhibiting the release of

cytokine by macrophages and neutrophils because

Skeletonema costatum contains fatty acids, trace

elements, and antioxidants. Antioxidants act as

inhibitors of inflammation by reducing the formation

of pro-inflammatory cytokine (Health L, 2008). The

inhibition of inflammation was supported by

researchers who staid that the methanol extract of

Skeletonema costatum produced the highest phenolic

content compared with the hexane extract which was

0.644 mg / gallic acid equivalent (GAE)/g, where the

phenolic content of the hexane extract wass 0.392 mg

GAE/g (Health L, 2008). In addition, it said that the

free radical scavenging activity of the Skeletonema

costatum methanol extract is 59% at a concentration

of 3.2 mg/ml (Lenin T, 2015).

The anti-inflammatory and antioxidant effects

may be related to vitamins contained in Skeletonema

costatum. Vitamins are trace minerals that play a role

in metabolism, cell repair, immunity, and more.

Vitamin content of Skeletonema costatum, among

other things; vitamin A 141 µg, vitamin D 11 µg,

vitamin E 108 µg, vitamin K 5.5 µg, vitamin B1 710

µg, B2 37 µg, B6 134 µg, B12 117 µg, vitamin C 59

µg and vitamin PP 511 µg (Roeck-holtzhauer, 1991).

Vitamins A, C, and E have antioxidant roles in

reducing reactive oxidative stress (ROS). According

to the 2012 Framingham Cohort Study, vitamin E can

reduce the progression of OA in men, but has no

significant effect on the incidence of OA (Roeck-

holtzhauer, 1991). At the same time, high doses of

vitamin C can slow the progression of OA induced by

surgery in guinea pigs because vitamin C has a

protective effect on cartilage by stimulating collagen

and proteoglycans (Roeck-holtzhauer, 1991).

In this study, the best anti-inflammatory effect of

Skeletonema costatum was achieved after day 21 and

using the highest dose of 120 mg/kg BW. Consistent

with study Andari (2016) it was also found that

lemuru fish oil which is rich in omega-3 and omega-

6 had the best time to reduce TNF-α in joint cartilage

Effects of Skeletonema Costatum’s Powder on the Knee Joint’s Diameter and the Degree of Pain of Male Rat Sparague Dawley Type

Induced by Complete Freund’s Adjuvant

induced by CFA on day 21. In agreement with the

study of Bahtiar, it was shown that the longer the

administration of Skeletonema costatum, the higher

the antioxidant level in the tissue and the better the

ability of Skeletonema costatum to inhibit the

inflammatory process (Bahtiar A, 2011).

To the researchers’ knowledge, there are currently

no publications on the benefits of Skeletonema

costatum as an anti-inflammatory agent in OA. The

results of this study could lead to a pilot study on the

efficacy of Skeletonema costatum as an anti-

inflammatory agent. In this study, the anti-

inflammatory effects of Skeletonema costatum were

not yet comparable to the administration of piroxicam

as the standard, but using a higher dose of

Skeletonema costatum, the effect was improved with

a decrease in the diameter of the right knee joint

diameter in rats. It can be observed in pain response.

This could inform the next study to determine the

dose of Skeletonema costatum to be tested.

5 CONCLUSIONS

Based on the study and results, it can be concluded

that the administration of Skeletonema costatum

reduced the diameter of the right knee joint in rats and

improved the pain response before and after treatment

(paired t-test, p<0.05). Although effects were

observed in all groups of Skeletonema costatum

groups, higher dose of Skeletonema costatum

reported improved reduction in joint diameter and

improvement in pain response (closer to the positive

control). The mean reduction in right knee joint

diameter and improvement in pain response in rats

following administration of Skeletonema costatum is

superior to the negative control (no treatment), but not

as good as piroxicam (10 mg/kg BW) as a positive

control (p<0.05).

Further investigation into the inflammatory

effects of Skeletonema costatum needs to be carried

out especially to determine the therapeutic dose of

Skeletonema costatum, the time needed to reach

maximum therapeutic levels, and the toxicity level of

Skeletonema costatum. Apart from that, given the

potential of Indonesia, which is rich in marine

products, and the trend toward marine drug

treatments, further studies are essential to study the

effect of Skeletonema costatum on other types of

diseases and other organs, for example, metabolic

syndrome and cancer.

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