Anti-inflammatory Activity Test for Ethanol Extract Moon Flower

(Tithonia diversifolia) Leaves to Male White Mice

Basyariah Lubis

, Ika Nur Saputri

, Rony Ajartha

,Sri Melda Br Bangun

, Chandra Pranata

, Novandi

Purba

, Nur Ulina M. Br. Turnip

Faculty of Midwifery, Institut Kesehatan Medistra Lubuk Pakam, Sumatera Utara, Indonesia,

Faculty of Public Health, Institut Kesehatan Medistra Lubuk Pakam, Sumatera Utara, Indonesia,

Faculty of Pharmacy Institut Kesehatan Medistra Lubuk Pakam, Sumatera Utara, Indonesia.

Keywords: Kembang Bulan Leaf (Tithonia diversifolia), Anti-inflammatory

Abstract: Introduction Hibiscus leaves (Tithonia diversifolia) are wild plants. Leaves, root bark and stems of the moon

are parts that can be used for traditional medicine. This research was conducted with the aim to determine

the effect of ethanol extracts of the moon flower (Tithonia diversifolia) as an anti-inflammatory Methods.

The method used was artificial edema of white male male foot mice with 1% carrageenan induction of 0.05

ml as an edema maker and as a positive control using Na-Diclofenac. This research was a kind of pure

experimental research. Data obtained to find the percentage of inflammation inhibition power. Data

distribution was analyzed by the Kolmogorov-Smirnov test, followed by the ANOVA test and the LSD test

with a confidence level of 95%. The research results showed that lunar leaves have an anti-inflammatory

effect expressed by the% inhibitory inflammation at the lowest dose of 50 mg / kg bw followed by 100 mg /

kg bw and 150 mg / kg bw have the highest percentage of inflammation inhibition. In the ANOVA test

showed a significant difference in each treatment group at the hours of 1 to 6 hours significantly different at

the test level (α ≤ 0.05). Discussion was expected to be able to identify the potential antioxidant activity of

the leaves of the moon flower (Tithonia diversifolia) by chromatography.

1 INTRODUCTION

The use of traditional medicine has become a

habit practiced by almost all countries in the world.

Over the past decade, the use of traditional

medicines has grown rapidly. The development of

traditional medicine continues to be done as health

care for the poor in developing countries (Karamian

et al, 2013). Traditional medicine was widely used

by the community to maintain health and was in

great demand because it is inexpensive and its

availability is affordable for the community,

especially in villages or small towns where health

centers are scarce. Compared to modern medicine,

traditional medicine has several advantages, namely

its side effects were relatively low. It must be

realized that there were dangerous traditional

medicinal ingredients if their use exceeds safe

dosages and concentrations (Katno and Pramono,

2005). The use of traditional medicines including

herbs can be beneficial for health maintenance,

prevention and treatment of diseases (Aditama,

2014).

Indonesia is a tropical country with plant

potential that has been traditionally used as

traditional medicine and has been an Indonesian

culture since centuries ago until now. Indonesia with

a tropical climate causes fertile soil so that many

types of plants can grow. Among the various types,

several types of plants have medicinal properties

(Hariana, 2013). Moon flower (Tithonia diversifolia)

is a plant species that belongs to the Asteraceae

family. The part that was used from lunar plants as a

source of chemicals, which is used for traditional

medicine is usually the leaves, but can also use the

root bark and stem. The leaves of this month's

flower plants contain alkaloids, terpenoids,

flavonoids, saponins, tannins, and polyphenols. The

benefits of lunar leaves are traditionally usually used

as a medicine for stomach aches, bloating, diarrhea

and used as a wound and anti-inflammatory drug

(anti-inflammatory) (Dalimartha, 2000).

Lubis, B., Saputri, I., Ajartha, R., Bangun, S., Pranata, C., Purba, N. and Turnip, N.

Anti-inﬂammatory Activity Test for Ethanol Extract Moon Flower (Tithonia diversifolia) Leaves to Male White Mice.

DOI: 10.5220/0009974705510557

In Proceedings of the International Conference on Health Informatics and Medical Application Technology (ICHIMAT 2019), pages 551-557

ISBN: 978-989-758-460-2

551

Inflammation is a process that involves a series of

events that can be caused by various stimuli such as

infectious substances, ischemia, antigen-antibody

interactions, and injuries due to heat or other physical

injuries (Goodman and Gilman, 2014). Each type of

stimulus has common signs of inflammation such as

swelling, pain, redness, heat and loss of cell function

that causes discomfort for sufferers so treatment

therapy was needed to overcome them, by using

modern drugs or medicines derived from plants

(Supriyatna et al., 2015).

Therapy that can be given to patients with

inflammatory complaints can be given with Non-

Steroid Anti-Inflammatory Drugs (NSAIDs) Non-

Steroidal Anti-Inflammatory Drugs (NSAIDs) that

can relieve symptoms, maintain cell or tissue

function and slow down or stop processes that

damage tissue (Katzung et al., 2017). Non-steroidal

Anti-Inflammatory Drugs (NSAIDs) which are

widely circulating are modern drugs that can inhibit

various inflammatory mediators (Pinzon, 2007). The

use of NSAIDs orally generally cause various side

effects problems such as central nervous system

(headache), cardiovascular (fluid retention,

hypertension), gastrointestinal tract (abdominal pain,

dysplasia), hemotology, liver, lung, skin and kidney

(Katzung, 2017) . Therefore the use of anti-

inflammatory drugs from plants can be used as an

alternative treatment with relatively smaller side

effects than modern medicine (Kinanti, 2016).

Based on the research of Widia, et al. (2016)

showed that the formulation of ethanol extract of

moon leaves leaves has the potential to inhibit

Staphylococcus aureus using well diffusion method.

Hanifa, (2015) showed that the antioxidant activity

of ethanol extracts from lunar leaves contained total

flavonoid levels of 4.209 mg QE / gram extract.

Meanwhile, Suherman (2013) reported that testing

the antioxidant activity of Tithonia diversifolia leaf

extract produced IC 50 against free radical DPPH of

27.88 μg / ml. The results of the separation of lunar

leaf extract by thin layer chromatography obtained

flavonoid compounds namely 5,7,8,3',4'-

pentahidroksiflavonol or 5,6,7,3',4'-

pentahidroksiflavonol (Aisha, et al., 2015).

Flavonoid compounds have anti-inflammatory

activity by inhibiting the release of serotonin and

histamine to the site of inflammation and inhibiting

the synthesis of prostaglandin from arachidonic acid

by inhibiting the action of cyclooxygenase (COX)

(Hasanah, 2011). Based on Ramadhani and Sri's

research (2017) flavonoid compounds have anti-

inflammatory activity. The strength of the anti-

inflammatory effect was indicated by the percentage

of edema inhibition. Based on the description above,

the moon flower leaves contain flavonoids which

were expected to be used as new drugs in anti-

inflammatory treatment. The author conducted

research to determine the anti-inflammatory effects

of ethanol extracts of lunar leaves in male white

mice carrageenan-induced.

2 RESEARCH METHODS

This type of research used in this study was

purely experimental methods. The research phase

includes sample preparation, sampling, preparation

of experimental animals, simplicia characteristics,

phytochemical screening, extraction methods and

testing of anti-inflammatory effects on white male

mice. The basis of this method was to make an

edema on the sole of the back foot of the mouse

using 1% karegenan. The research data were

analyzed with Analysis of Variance (ANOVA) with

95% confidence so that it can be known whether the

differences obtained are significant or not, if there

are significant differences followed by the Least

Square Difference test (LSD). The research site was

carried out at the Lubuk Pakam Institute of Health

Chemistry Laboratory in the manufacture of reagent

solutions, characteristics of phytochemicals and

phytochemical screening, simplicia extraction

processes and anti-inflammatory testing. The time of

the study was carried out in March - May 2019.

The tools used in this study are laboratory

glassware, UgoBasille®- Plethysmometer, mortar

and stamper, injection syringe, mouse cage, digital

camera, analytical balance, vacuum rotary rotary

evaporator, oral sonde, tissue rolls, labels, blenders,

animal scales. The materials used in this study were

lunar leaves, chemicals used sodium diclofenac

(PT.IndoFarma factory), Carboxy Methyl Cellulose

(CMC), λ-carrageenan, sodium chloride solution

0.9%, Pb (II) acetate, iron (III) chloride P, mercury

(II) chloride, potassium iodide, iodine, α-naphthol,

nitric acid, bismuth nitrate, ether, clofrom,

isopropanolol, ethanol, methanol, sodium sulfate

anhydrous, ethyl acetate, magnesium powder,

bismuth nitrate, ether, chloroform , isopropanolol,

ethanol, methanol, sodium sulfate anhydrous, ethyl

acetate, magnesium powder, zinc powder,

hydrochloric acid P, ether, sulfuric acid P and

distilled water.

The experimental animals used were white male

mice with a body weight of 20-25 g and 8 weeks of

age of 25 animals. Sample was done purposively,

without comparing with the same material from

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552

other regions. Samples taken are old and flowering.

Single leaf, carved until half the length of the leaf

bone, jagged, alternating, pointed tip and base,

pinnate, and green. The sample used was the moon

flower leaves (Tithonia diversifolia) obtained from

Tigaras, Kec. Dolok Pardamean, Kab. Simalungun,

North Sumatra Province.

Leaves that have been taken were washed using

running water drained, then weighed wet weight.

Then dried for three days, then sorting was dry and

chopped. Furthermore, it was dried in a drying

cabinet until it was brittle after it was blended into

powder, weighed then put into a tightly closed

plastic bottle container and stored at room

temperature. To see the characteristics of simplicia,

macroscopic and microscopic examination was

performed. The phytochemical screening of samples

as follows:

Alkaloid: 0.5 gram of simplicia powder was

weighed, added 1 ml of 2 N hydrochloric acid and 9

ml of distilled water, heated over a water bath for 2

minutes, cooled and filtered, the filtrate was used for

the alkaloids test. 3 test tubes were taken, then 0.5

ml of filtrate were put into each test tube. In tube I: 2

drops of Mayer's reagent are added, forming white

or yellow lumpy deposits, in tube II: 2 drops of

Dragendrof reagent are added, a brown or orange-

brown precipitate is formed, in tube III: added 2

drops of Bouchardat reagent, will form a brown

sediment to black. The alkaloid is positive if there is

sedimentation or turbidity in two or three of the

above experiments (MOH RI, 1995). Saponin: 0.5

gram of simplicia powder was weighed, put in a test

tube, added 10 ml of hot distilled water, cooled, then

shaken vigorously for 10 seconds. Saponin is

positive if a stable foam is formed not less than 10

minutes as high as 1 to 10 cm and with the addition

of 1 drop of 2 N hydrochloric acid the foam does not

disappear (MOH RI, 1995). Tannin: As much as 0.5

gram of simplicia powder was weighed, then mixed

with 10 ml distilled water, the filtrate was diluted

with water until it was colorless. The solution is

taken as much as 2 ml and added 1-2 drops of

reagent iron (III) chloride 1%. If there is a blue or

blackish green color indicates the presence of

tannins (Farnsworth, 1966).

Preparation of Ethanol Extract Simplicia powder

was extracted by maceration using 70% ethanol

solvent. Procedure: Samples were weighed as much

as 500 grams, then put into maceration containers.

Soaked with solvent until completely submerged and

then covered and stored at room temperature. Stirring

once a day for five days. After that the solvent is

separated from the pulp by pouring the solvent in

another container, and the remaining solvent in the

pulp is mixed and filtered. To ensure the extraction

process takes place perfectly, the pulp that has been

kneaded was soaked again using a new ethanol

solvent. Left for two days while stirring every day,

then kneaded and filtered. Do the same treatment

until the solvent is colorless. All maserates are

combined and evaporated using a rotary evaporator at

± 400C until a thick extract is obtained.

Preparation of Control Test Materials and

Comparative Medicines Hibiscus leaf extract with a

dose of 50 mg / kg bw, 100 mg / kg bw, 150 mg / kg

bw (test material), Diclofenac sodium suspension

dose 6.5 mg / kg bw (positive control), CMC

suspension 0.5 % (negative control), carrageenan

1% (induction). The preparations consist of: Making

a 0.5% CMC suspension: A total of 0.5 g CMC was

sprinkled evenly into a mortar containing 35 ml of

hot distilled water, allowed to stand for 15 minutes

until obtained a transparent mass, crushed to form a

gel and then diluted with a little water, put in a 100

ml flask, then added distilled water to the mark line.

Preparation of diclofenac sodium suspension: 6,5 mg

of diclofenac sodium was added and then crushed

with the addition of 0,5% CMC suspension until

homogeneous, put into 10 ml flask, then added 0,5%

cmc suspension to the mark line. Preparation of

ethanol extract suspension: Weighed 50 mg, 1000

mg, 150 mg extract of moon flower leaves. Each

crushed by adding 0,5% CMC suspension until

homogeneous, put into a 10 ml flask, sufficient to

the mark with 0,5% CMC suspension. Praparation of

inflammation indicator: Weighed 100 mg of

carrageenan lambda, then homogenized with 0,9%

sodium chloride solution, then put in a 10 ml flask,

then supplemented with 0,9% sodium chloride

solution until the mark line was than allowed to

stand and incubated at 37

C for 24 hours.

Figure 1: Experiment Sample: (a) Tithonia diversifolia (b)

Simplisia Powder (Tithonia diversifolia), (c) Animal

Research.

Anti-inﬂammatory Activity Test for Ethanol Extract Moon Flower (Tithonia diversifolia) Leaves to Male White Mice

553

2.1 The Anti-inflammatory Procedure

Were Tested with the following

Scheme

Before testing, mice were fasted for 18 hours while

still being given a drink. Mice were grouped into 5

groups, namely the negative control group (0.5%

CMC suspension), the test material group (three

doses of lunar extract extract suspension) and the

positive control (diclofenac sodium). On the day of

the test, each animal was weighed and marked on its

left leg and tail, then the left leg of the mice was put

in a cell containing a reservoir solution that had been

prepared beforehand until the liquid rose at the

upper boundary line, the pedal was then held,

recorded figures on the monitor as the initial volume

(Vo), which is the foot volume before treatment is

given. The preparation is given orally with a volume

of giving to mice as much as 1 ml in accordance

with the treatment group as follows: Group I: 5 mice

were given a 0.5% w / v Na-CMC suspension orally

as a negative control; Group II: 5 mice were given

orally diclofenac sodium solution (positive control);

Group III: 5 mice were given orally extracts of

hibiscus leaves at a dose of 50 mg / KgBB; Group

IV: 5 mice were given orally extracts of hibiscus

leaves at a dose of 100 mg / kg; dan Group V: 5

mice were given lime leaves extract at a dose of 150

mg / KgBW orally. One hour later, each mouse was

induced with 0.05 ml of carrageenan 1% intraplantar

then measured the initial volume of the mice's feet.

After that measured the volume of mice edema feet

after treatment every interval of 1 hour for 6 hours.

The edema volume is determined based on the

increase in mercury in the plathysmometer.

3 RESULTS AND DISCUSSION

Macroscopic examination results of fresh moon

flowers are leaves single, etched to half the length of

the leaf bone, jagged, alternating, leaf length 26-32

cm, width 15-25 cm, tip and base of pointed leaves,

pinnate, green leaves. Microscopic examination

results showed fresh moon flowers the presence of

multicellular single hair closures, cuticles, upper

epidermis, palisades, spiral trachea, spongy tissue,

lower epidermis, and leaf mouth Diitic type. This

Phytochemical screening of the simplicia leaves of

the moon flower to show the class of secondary

metabolite compounds contained therein. The

examination carried out on the simplified powder of

the moon flower is the examination of the group of

alkaloid compounds, flavonoids, saponins, and

tannins. The results of phytochemical screening of

the simplicia leaves of the moon flower are alkaloids

(-), flavonoids (+), saponins (-), and tannins (+).

Description (+) positive means it contains a class of

compounds and (-) negative means that it does not

contain compounds. Extraction result of moon

flower leaves are 500 gram sample weight, 64 gram

extract weight, solvent volume ( ethanol 70 %) 3

liters, and soaking time 5 x 24 hours.

Inflammation is a disorder that is often

experienced by humans and animals that cause pain

in the surrounding area. So the need for prevention

or treatment to reduce pain, fight or control pain due

to swelling. In this anti-inflammatory study the

method used was the formation of artificial edema

on the soles of mice's feet using carrageenan as an

induction of edema. This method was chosen

because it is one of the methods of testing

anbtiinflamasi activity that is simple, easy to do and

often used. The use of carrageenan as an inducer of

edema has several advantages including not leaving

a scar, not causing tissue damage and giving a more

sensitive response to anti-inflammatory drugs

(Fitriyani, 2008).

Carrageenan as an irritant compound induces cell

injury through the release of mediators that initiate

the inflammatory process. In ssat release of

inflammatory mediators occurs maximal edema and

lasts several hours. Inflammation induced by

carrageenan is characterized by increased pain,

swelling, and prostaglandin synthesis up to 4-5

times. Udem caused by carrageenan induction lasts

for 6 hours and gradually decreases within 24 hours

(Taufiq, 2008). EEDKB anti-inflammatory activity

testing used 25 test animals, with 5 treatment

groups. The group consisted of positive control

given Na diclofenac at a dose of 6.5 mg / kgBW

orally, negative control given CMC Na treatment

0.5% orally, the extract treatment group dose 50 mg

/ kgBW, the extract treatment group dose 100 mg /

kg kgBW, and the 150 mg / KgBW dose extract

group. The mice were fasted for ± 18 hours, then the

mice were weighed marked on the tail and left ankle

of the rat. Before each group was given ethanol

extract of lunar leaves, the volume of mice's feet was

measured first as the initial volume (Vo). After that,

each group was given ethanol extracts of lunar

leaves ie group I was given a 0.5% Na-CMC

suspension, group II was given a diclofenac sodium

suspension of 6.5 mg / KgBW, groups III and IV

and V were each given an EEDKB suspension dose

50, 100, 150 mg / kgBB orally. One hour later, each

foot of the mice's foot was injected intraplantar with

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554

0.05 mL of 1% λ-carrageenan solution.

Measurements were made using a pletismometer

with measurement principles based on Archhimedes'

law. After 30 minutes, the measurement is carried

out by dipping the feet of the mice into the

pletismometer cells that contain special fluid until

the solution reaches the upper limit, and the pedal is

held. Numbers are recorded on the monitor. The

change in liquid volume that occurs is recorded as

the volume of the feet of mice (Vt). Measurements

were made every 30 minutes for 360 minutes.

Changes in mice foot volume, can be calculated

percent inflammation in mice feet. Next, a graph of

changes in the average inflammation of the feet of

mice was made. The percent inflammation group in

the feet of mice smaller than the control group

showed that the test material was able to suppress

inflammation caused by carrageenan. The results of

the percent inflammation measurement can be seen

in Figure 2:

Figure 2: Percent graph inflammation of the average foot

mice

In Figure 2 it can be seen that the sodium

diclofenac suspension of 6.5 mg / kgBW has a

smaller inflammation percentage than EEDKB doses

of 50, 100, and 150 mg / kgBW, and the EEDKB

dose of 150 mg / kgBW has a smaller percent

inflammation than EEDKB doses of 100 and 50 mg /

kg body weight. The formation of inflammation by

carrageenan produces acute inflammation, and does

not cause tissue damage, although inflammation can

last for 360 minutes and gradually diminish for one

day.

Carrageenan as the cause of inflammation can be

influenced by anti-inflammatory drugs. Its response

to anti-inflammatory drugs is more sensitive than

other irritants (Juheini, et al., 1990). The percentage

of mice foot inflammation smaller than the control

showed that the diclofenac sodium suspension and

EEDKB suspension were able to inhibit

inflammation in the mouse feet caused by

carrageenan.

The ability to inhibit this inflammation, called

inflammation inhibition, can be seen in Figure 3.

Figure 3: Percent graph of the average inflammation of the

feet of mice

In Figure 3 it can be seen that EEDKB 50 mg /

kgBW has a smaller percentage of inflammation

inhibition than EEDKB 100, 150 mg / kgBW and

with diclofenac sodium suspension at a dose of 6.5

mg / kgBW, EEDKB 100 mg / kgBW has percent

inflammation inhibition smaller than EEDS 150 mg /

kgBW and with diclofenac sodium suspension 6.5

mg / kgBW, and EEDKB 150 mg / kgBW have a

smaller percentage of inflammation inhibition than

diclofenac sodium suspension at a dose of 6.5 mg /

kgBW.

Data obtained in the normality test by the

Kalmigorov-Smirnov method to see the distribution

of percent data of mice foot inflammation inhibition

to the treatment group showed that all treatment

groups were normally distributed and not

significantly different. Then homogeneity test was

performed using the Levene method to see data on

the percentage of inflammation inhibition of

homogeneous mouse mice or not, the results showed

that all treatment groups were homogeneously

distributed (α≥0.05). Because the data meet the

homogeneity requirements, ANOVA was continued

to look at the average percentage of mice foot

inflammation inhibition in the treatment group to see

significantly different or insignificant values with a

95% confidence level. Least Square Difference

(LSD) test was performed. The results showed that

the percentage of mice foot inflammation inhibition

throughout the initial volume group in each

treatment did not differ significantly, in the

induction volume group EEDKB 50 mg / kgBW and

100 mg / kgBW were significantly different, at hour

to 1-6 for each treatment is significantly different at

the 0.05 test level.

Based on these test results, it can be concluded

that the administration of ethanol extracts of lunar

leaves at a dose of 50 mg / kg bw, 100 mg / kg bw,

150 mg / kg bw can reduce inflammation in the soles

Anti-inﬂammatory Activity Test for Ethanol Extract Moon Flower (Tithonia diversifolia) Leaves to Male White Mice

555

of male white mice induced by 1% carrageenan.

This research has also been carried out by Verawati,

et al (2011), it has been reported that the ethanol

extract of the moon flower leaves has anti-

inflammatory activity. This is seen from the

decrease in exudate volume in the back

inflammation of female white mice that are given

topically. In the test studies the anti-inflammatory

effects of ethanol extracts of lunar leaves showed

that the effect was dose dependent on increasing

certain doses. Anti inflammatory effects can be seen

from the content of lunar leaves in which flavonoid

compounds have anti-inflammatory activity by

inhibiting the release of serotonin and histamine to

the site of inflammation and inhibiting the synthesis

of prostaglandin from arachidonic acid by inhibiting

the action of cyclooxygenase (COX) (Hasanah,

2011).

4 CONCLUSIONS

Ethanol extract of the moon flower (Thitonia

diversifolia) can provide anti-inflammatory effects.

The obtained extracted ethanol of the moon flower

leaves (Thitonia diversifolia) are 50, 100, and 150

mg / KgBW. The dose of 150mg/kgBW has an anti-

inflammatory effect in inhibiting mouse foot edema

and the dose of 150 mg/kgBW has smallest

percentage of inflammation among the doses used.

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