The Effect of Pakoasi (Chromolaena odorata L.) Leaf Extract in

Curing Open Wound of Rabbit Skin (Oryctolagus cuniculus)

F. Ramdani

, M. Sriasih

, A. S. Dradjat

Postgraduate student at Magister Program (Manajemen Sumber Daya Peternakan) of Animal Science Faculty, University

of Mataram. Jalan Majapahit No. 62 Mataram, NTB

Faculty of Animal Science, University of Mataram, Jl. Majapahit No. 62 Mataram, NTB

Keywords:

Open Wound, Pakoasi, Rabbit

Abstract: Pakoasi (Chromolaena odorata. L) is a weed that is often used in treating wounds, but its effectiveness has

not been widely studied. The purpose of this research was to evaluate the effect of Pakoasi leaf extract on

open wound healing process in rabbit. Twenty healthy male rabbits were wounded with diameter of 2 cm

under local anaesthesia, and were divided into four groups: G0 (placebo), G1 (treated with 10% extract), G2

(treated with 20% extract) and G3 (positive control). Macroscopic and microscopic observations were

carried out, and the obtained data were analysed using analysis of variance. The macroscopic results showed

that on day 4, there was a significant difference (p <0.05) of wound diameter among groups, with G1

showing the shortest wound diameter (1.78 ± 0.08cm). Seventeen days after treatment, the wound diameter

(cm) in G1, G2, G3 and G0 were 0 ± 0; 0.08 ± 0.04; 0.17 ± 0.06 and 0.35 ± 0.08, respectively. The

complete healing process of open wound occurred as fast as 16 days after treatment in

G1 based on macroscopic and microscopic observation. This study concluded that ointment

containing 10% o f p a k o a s i extract is effective for curing open wound.

1 INTRODUCTION

Pakoasi (Chromolaena odorata L.) is a weed

belongs to Compositae family. This weed is easy to

grow, widespread and fast in tropical areas

(Madhavan, 2015) so that grassland cannot be

overgrown with grass. Some societies in West Nusa

Tenggara commonly use pakoasi as a traditional

medicine to treat open wounds, burns and other

traumas. However, the effectiveness of pakoasi as a

traditional medicine has not been widely studied yet.

Previous studies showed that the application of

pakoasi leaf extract of 2.5%, 5% and 10% in ethanol

has no effect in curing open wound in rabbits (Arif,

2016). The findings might be influenced by the

small number of samples, the number of wound

made in each rabbit and the absence of microscopic

assessment. Hence in this study, we evaluated

pakoasi (C. odorata L.) leaf extract effectiveness by

examining pakoasi ointment (10% and 20%) on the

wound healing process in male rabbits (Oryctolagus

cuniculus) skin. We made macroscopic and

microscopic evaluations.

2 MATERIALS AND METHODS

2.1 Leaf Extraction

The pakoasi leaves were removed by breaking the

stalk. They were washed thoroughly, dried and

powdered. The powder was then extracted with 96%

ethanol solution. The process was followed by

phytochemical test by gas chromatography-mass

spectrometry (GCMS) to evaluate the extract

chemical content.

2.2 Ointment Preparation

Ointment containing different concentration of

pakoasi extract was prepared as described by

Goeswin (2006). The formulation is presented in

Table 1.

Table. 1 : Component and content of pakoasi ointment.

No Component

Pakoasi extract

10 % 20%

1 Pakoasi Extract 1 2

Ramdani, F., Sriasih, M. and Drajat, A.

The Effect of Pakoasi (Chromolaena odorata L.) Leaf Extract in Curing Open Wound of Rabbit Skin (Oryctolagus cuniculus).

DOI: 10.5220/0007544904570461

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 457-461

ISBN: 978-989-758-348-3

457

2 Vaselin Albu

7.65 6.8

3 Adeps Lanae 1.35 1.2

4 Ointment 10 10

2.3 Rabbit Skin Incision

Twenty male healthy rabbits were wounded 2 cm in

diameter. Before making skin incision, the fur was

shaved around 5 cm. The skin was cleaned using

70% alcohol and was then anesthetized using

lidocaine. After 5 minutes, a circum incision of 2 cm

in diameter was performed on the top of vertebra.

The wounded rabbits were randomly divided into

four groups, with each group consisting of 5 rabbits

as follows; G1: the wounded rabbit was treated with

ointment of 10% pakoasi leaf extract, G2: treated

with 20% of pakoasi leaf extract, G3: treated with

povidone iodine as a positive control, while G0: no

treatment was given as a negative control. The

treatment was given twice a day in each group until

the wound was completely closed. The observations

were done macroscopically on day 4, 11 and 17, and

microscopically on day 7

and 21. The results were

then analysed by analysis of variance.

3 RESULTS AND DISCUSSION

3.1 Phytochemical Evaluation of C.

odorata L.

Figure 1: The results of phytochemical test of C.

odorata L. leaf extract.

Figure 1 shows that there were five main

components, showing more than 5%, derived from

the extract. Those were Germacrene D (23.86%) and

seemed to be the highest content of the extract.

Germacrene D is derived from terpenoid as a

precursor of hydrocarbons (Nils et al., 2000).

Second, Trans (beta) –caryophyllene, which can be

classified as terpenoids sesquiterpen (21.07%). The

compounds have antimicrobial, antioxidant,

anesthetic and anti-inflammatory activities

(Ghelardini et al., 2001). Third, Cadinene which is a

class of terpenoids sesquiterpen (14.30%).

Sesquiterpene compounds have considerable effects

as antimicrobial, antifungal and antibiotic (Ali et al.,

2008; Guo et al., 2008). Other component was

Hexadecanoic acid (CAS) Palmitic acid (12.07%), a

derivative of saponin which has anti-inflamation and

antifungal, by damaging the structure of walls and

cell membranes of fungi. This synergistic

mechanisms is led by various compounds such as

terpenoids, which may increase the effect of

antifungal activity (Padmin et al., 2010). Last

component was Octadecatrienoic acid methyl ester

(6.30%), a class of fatty acids and has an anti-

inflammatory function (Mei Dong et al., 2000).

3.2 Macroscopic Evaluation

Figure 2 shows the measurement results of open

wound diameter in rabbits after treatment with

pakoasi leaf extract ointment

Figure 2: Healing process of each group folowing

treatment.

Data presented in Figure 2 show that healing

process began with inflammatory phase during day 1

to 4, then proliferative phase from day 4 to 11. The

process was followed by remodelling phase that

began from day 11 to 21. With 58% terpenoid and

12.07% saponin found in pakoasi extract, the wound

healing process observed from the inflammatory

phase could be accelerated by a difference of 0.35

cm within 4.5 days between G1 and G0. The

inflammatory phase causes neutrophil migration,

before progressing to the wound area that is replaced

by monocytes (Yuliani, 2012). Thus, the

inflammation process to the proliferation stage

becomes faster, and accelerates the healing process.

Terpenoids sesquiterpen on pakoasi leaf extract

has a function as an antioxidant. Antioxidant will

bind unstable free radicals that can damage cell

membranes. With this bond, free radicals are

expected to be stable, so that cell membrane damage

ICPS 2018 - 2nd International Conference Postgraduate School

458

can be reduced, and proliferation phase can be more

rapid (Ardiana et al., 2015).

Table 2. Wound diameter (mean cm ± SD) on day 4, 11

and 17 after treatments.

Note: Value with different superscript in the same

column is significantly different (p <0.05).

On day 4, there was a significant difference (p

<0.05) of wound diameter between G1 (1.75 ± 0.04)

and G2, G3 and G0. The mean and standard

deviation of wound diameter obtained by G1 on day

11 after treatment was significantly different from

mean diameter of G2 and G3 (p <0.05). The

treatment given in G2 and G3 did not cause any

significant difference (p >0.05), whereas G2 and G3

were significantly different with G1 and G0. On day

17, the average diameter and standard deviation in

G1, G2, G3 and G0 was 0 ± 0, 0.08 ± 0.04, 0.17 ±

0.06 and 0.35 ± 0.08, respectively, and all treatments

showed significant differences (p >0.05).

3.3 Microscopic Evaluation

The results of histopathologic examination on day 3

and 7 (Table 3) showed significant increases in

blood vessels and fibroblasts in G1 compared with

other groups (p <0.05).

Table 3. Scoring of epiteilization, collagenation,

fibrolast development, PMN response, and blood

vessel number of all groups on day 3, 7 and 21.

roups Epithel

isation

Colla

gen

Fibro

blast

PMN Bloo

vesse

G1 1.33±

0.57

0.67±

1.15

2.33 ±

1.15

2.00±

1.00

3.67±

0.57

G2 1.67±

0.57

0.00±

0.00

1.33±

0.57

1.67±

0.57

1.00±

0.00

G3 1.67 ±

0.57

0.67±

0.57

0.67±

0.57

1.67±

0.57

1.33±

0.57

G0 1.33 ±

0.57

0.67±

0.57

1.33±

0.57

1.33±

0.57

0.66±

0.57

Note: Value with different superscript in the same

column is significantly different (p <0.05).

Macrophages and neutrophils play roles during

inflammatory phase. Neutrophil immediately exits

blood vessels, and its number increases from 24 to

48 hours (Regan and Barbul, 1994). The number

decreases on day 3 as it is replaced by macrophages.

Macrophages and neutrophils have functions in

preventing infection by phagocytosis of

microorganisms that enter the wounded area. In the

absence of infection in the wounded area,

polymorphonuclear leukocytes are relatively short in

time, and the number decreases rapidly after day 3

(Regan and Barbul, 1994).

The results of histopathology examination on day

3 and 7 in Table 3 show a significant increase in the

number of blood vessels and fibroblasts in G1

compared to other groups. On day 3, the average

number of blood vessels that are at that time blood

vessel buds or endothelial progenitor cells go to the

blood circulation to the granulation tissue become

mature endothelium that will initiate angiogenesis

(Icha et al., 2016).

The next phase is proliferation or migration. The

cells that play roles in this phase are macrophages,

lymphocytes, fibroblasts and endothelial cells.

Macrophages have a longer life span than

polymorphonuclear leukocytes, and they remain

until healing process has complete (Yuliani and

Viktor 2015).

Observation on day 7 showed the

highest number of blood vessels due to endothelial

cells experiencing the peak of the proliferation

phase. Saponin content in pakoasi leaf extract

increases monocyte proliferation. This leads to

increasing number of macrophages. Macrophages

produce and secrete growth factors to attract more

fibroblasts to the wounded area, to synthesize

collagen and to increase proliferation of capillary

blood vessels (Ardiana et al., 2015). Therefore, G1

had a high proportion of fibroblasts and blood

Groups Treatments

The day

10%

Pakoasi

extract

1.72±

0.04

0.65±

0.05

0±0

20%

Pakoasi

extract

1.81±

0.02

0.91±

0.07

0.08±

0.04

Povidone

Iodine

1.79±

0.05

0.93±

0.08

0.17±

0.06

G0 Placebo

1.87±

0.04

1.28±

0.08

0.35±

0.08

The Effect of Pakoasi (Chromolaena odorata L.) Leaf Extract in Curing Open Wound of Rabbit Skin (Oryctolagus cuniculus)

459

vessels in microscopic examination compared to

other groups (Table 3).

The presence of growth factors may increase cell

propagation or proliferation, and this will become a

frame of fibroblast receptorization and proliferation.

When fibrin clumped, fibroblasts will proliferate,

and the number of fibroblasts will increase

(Diegelmann, 2004). According to Thang et al.

(1998), the active components present in C.odorata

can stimulate fibroblast proliferation .

In the remodeling phase, the wound surface

restores epithelial integrity and epithelialization

from the basement membrane. Wound epithelial

cells begin to show increasing mitotic activity and

migrate the living connective tissue. Marginal basal

cells on the edges of the wound become loosely

bonded from the dermis nearby, enlarge and migrate

to the surface of the wound that has filled the

previous matrix (Singer and Clark, 1999).

The

microscopic results showed that the treatment given

in G1 influenced re-epithelization, collagen

formation, fibroblast formation, PMN cell

inflammation, and blood vessel formation faster than

G2, G3 and G0. This is consistent with a study

carried out by Henshaw et al. (2017) that showed

that the ethanol extract of C. odorata L. significantly

increased the number of red blood cells,

haemoglobin concentration and cell volume in white

mouse animal experiments.

4 CONCLUSIONS

Pakoasi leaf extract ointment with concentration of

10% cure open wound faster than 20% concentration

ointment, positive control (povidon iodine) and

negative control (without treatment).

ACKNOWLEDGEMENTS

Acknowledgments are given to Mr. Suparman for

technical assistance, Kholik DVM, M.Vet and

Chandra Dwiatma DVM, M.Si for assisting in

macroscopic and microscopic evaluations.

REFERENCES

Ardiana T, Andina R. P. dan Muhammad D F. 2015.

Efektivitas Pemberian Gel Binahong (Anredera

Cordifolia) 5% Terhadap Jumlah Sel Fibroblast Pada

Soket Pasca Pencabutan Gigi Marmut (Cavia

Cobaya). ODONTO Dental Journal. Volume 2. Nomer

1. Juli 2015.

Arif M. Z. 2016. Formulasi Sediaan Salep Ekstrak Etanol

Daun Kirinyuh (Euphatorium Odoratum L.) Sebagai

Penyembuh Luka Terbuka Pada Kelinci. Fakultas

Farmasi Universitas Muhammadiyah Surakarta

Ali N.A, Martina W, Arnold N, Lindequist U, Wessjohan

L. 2008. Essential Oil Composition from Oleogum

Resin of Soqotraen Commiphora kua, Rec. Nat. Prod.

2 (3): 70- 75

Diegelmann 2004. Wound Healing: An Overview of Acute,

Fibrotic, and Delayed Healing. Frontiers in

Bioscience, hal 283-289.

Ghelardini C, Galeotti N, Di Cesare Mannelli L, Mazzanti

G, and Bartolini A. 2001. Local anaesthetic activity of

β-caryophyllene/ Farmaco, vol. 56, no. 5-7, pp. 387–

389.

Goeswin A. 2006. Pengembangan Sediaan Farmasi.

Institut Teknolgi Bandung. Bandung.

Guo L, Jin-zong W, Tin H, Tong C and Khalid R. 2008.

Chemical Composition, Antifungal and Antitumor

Properties of Ether Extracts of Scapania verrucosa

Heeg. and its Endophytic Fungus Chaetomium

fusiforme, Molecules, 13: 2114-2125, DOI:

10.3390/molecules 13092114

Henshaw U O, Ifeyinwa M.O, Emmanuel K.U, Item J.A.

2017. Some hematological parameters of Wistar rats

treated with Chromolaena odorata leave extracts.

JoJournal of Biological Research; volume 90:6210

Icha Nofikasari, Afifah Rufaida, Chynintia Dewi

Aqmarina, Failaso, Annisa Rahmi Fauzia, Juni

Handajani. 2016. Efek aplikasi topikal gel ekstrak

pandan wangi terhadap penyembuhan luka gingiva.

Majalah Kedokteran Gigi Indonesia Vol 2 No 2 –

Agustus 2016 ISSN 2460-0164 (print), ISSN 2442-

2576

Madhavan M. 2015. Quantitative Estimation of total

phenols and antibacterial studies of leaves extracts of

Chromolaena odorata (L.) King & H.E. Robins

International Journal of Herbal Medicine 2015; 3(2):

20-23

Mei Dong, Yukiko Oda, Mitsuru Hirota. 2000. 10E,12Z-

90Hdroxy-10,12,15-octadecatrinic acid Methyl Ester

as an Anti-Inflammatory CompoundFrom Ehretia

dicksonii. The United Graduate Sholl of Agricultural

Science, Gifu University, Japan. Biosci. Biotecnol,

Biovhem., 64 (4), 882-886.

Nils Bulow, Wilfried A Konig. 2000. The role of

germacrene D as a precursor in sesquiterpene

biosynthesis: investigations of acid catalyzed,

photochemically and thermally induced

rearrangements. Phytochemistry Volume 55, Issue 2,

September 2000, Pages 141-168

Regan, M.C., and Barbul, A., 1994. The Cellular Biology

of Wound Healing in Fibrin Sealing in Surgical and

Nonsurgical Fields.

Singer, J.A. and Clark, R.A.F., 1999. Cutaneous Wound

Healing. Departements of Emergency and

Dermatology. State University of New York. Stony

ICPS 2018 - 2nd International Conference Postgraduate School

460

Brook. New York. The New England Journal of

Medicine; Vol 341.

Thang, T.P., Hughes, M.A., and Cherry, G.W.,1998.

Enhanced Proliferation of Fibroblast and Endothelial

Cells Treated with an Extract of the Leaves of

Chromolaena odorata (Eupolin), an Herbal Remedy

for Treating Wound. Plasttic Recontr. Surgery. 101.

Padmin E.A, Valarmathi A, and Rani M.U. 2010.

Comparative Analysis of Chemical Composition and

Antibacterial Activities of Mentha spicata and

Camellia sinennsis. Asian J. Exp. Biol. Sci, 1 (4) : 772

– 781

Yuliani S H. 2012. Formulasi Sediaan Hidrogel

Penyembuhan Luka Ekstrak Etanol Daun Binahong

(Anredera Cordifolia (Ten) Steenis) Program Pasca

Sarjana. Program Study Ilmu Farmasi Fakultas

Farmasi UGM. Yoyakarta.

Yuliani N.S dan Viktor L. (2015). Pengaruh Ekstrak Daun

C.Odorata Terhadap Proses Kesembuhan Luka Insisi

Pada Tikus Sprague-Dawley. Jurnal Kajian Veteriner

Desember 2015 Vol. 3 No. 2 : 93-99.

The Effect of Pakoasi (Chromolaena odorata L.) Leaf Extract in Curing Open Wound of Rabbit Skin (Oryctolagus cuniculus)

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