The Role of Fibroblast Proliferation in Wound Healing by Different

Plants: An Experimental Study

Marisa Riliani

, Indra Kusuma

, Abdul Halim

, Aliya Muhammad

, Akbar Fitrianto

Ida Bagus Eka Narendra

Department of Anatomy, Faculty of Medicine, Universitas Yarsi, Jakarta, Indonesia

Department of Physiology, Faculty of Medicine, Universitas Yarsi, Jakarta, Indonesia

Faculty of Medicine, Universitas Yarsi, Jakarta, Indonesia

Keywords: Manihot esculenta, Ageratum conyzoides L., Fibroblast, Wound Healing

Abstract: Wound healing is a process that consisted of inflammation, proliferation, and migration of fibroblasts.

Indigenous people commonly used Manihot esculenta and Ageratum conyzoides L.as bleeding wounds

therapy. This study aimed to investigate these two plant extracts on fibroblast proliferation and migration

capabilities. Skin fibroblasts were cultured with a medium conditioned for each extract with different

concentrations (0.5%, 1%, 2%, 4%), positive and negative control groups. In evaluating proliferation,

fibroblasts were incubated by CCK-8 and measured by spectrophotometer expressed as optical density

(OD). The evaluation of migration was visualized by scratch assay. The distance between each edge of the

scratch was measured using T-scratch software and expressed as the area's closure percentage. There were

significant differences in fibroblast proliferation rate in the groups receiving 0.5% Manihot

esculenta (p=0.00) and 2% Ageratum conyzoides L. (p=0.00). The migration of fibroblasts was no different,

with 0.5% Manihot esculenta (p=0.40) and 2% Ageratum conyzoides L. (p=0.18). Manihot esculenta and

Ageratum conyzoides L. could be considered to be used as agents to accelerate wound healing by increasing

the fibroblast proliferation. Our findings suggest in vivo studies for tissue regeneration.

1 INTRODUCTION

In 2013, skin wound affected 8.2% population in

Indonesia. Metabolic diseases and infections can

influence the healing time; it could make a slow or

even non-wound healing process. Slow or non-

healing wounds affect millions of people worldwide

and result in enormous health care expenditures. The

skin is our outer part of the body, which can protect

us from many environmental stresses. Injured skin

sets into motion a series of repair mechanisms

directed to the injured tissue. Wound healing is a skin

reparative process consist of inflammation,

proliferation, and migration of fibroblasts. The role of

fibroblasts is to synthesize and integrate protein and

https://orcid.org/0000-0002-5025-793

https://orcid.org/ 0000-0001-9350-431X

https://orcid.org/0000-0003-1842-7646

https://orcid.org/ 0000-0002-9249-6044

https://orcid.org/0000-0002-8553-1550

https://orcid.org/ 0000-0001-7800-1335

elastin inside the extracellular matrix from some large

part of the mesenchyme tissue

(Simon, 2020).

Along with proper wound care products, it works

together to repair and replace devitalized tissue.

Many topical drugs are used to create and maintain a

moist environment and provide healing conditions

(Lordani et al., 2018). They are often expensive, and

drugs price increased up to 15% every year

(Blumberg, 2019). Therefore, we need to find

alternative ingredients that easy to find, at a lower

cost and have a shorter healing time. Recently, so

many plants are coming out as tools for therapeutic

application.

For decades, indigenous people have commonly

used the leaves of Manihot esculenta and Ageratum

Riliani, M., Kusuma, I., Halim, A., Muhammad, A., Fitrianto, A. and Eka Narendra, I.

The Role of Fibroblast Proliferation in Wound Healing by Different Plants: An Experimental Study.

DOI: 10.5220/0010486300050009

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 5-9

ISBN: 978-989-758-499-2

conyzoides L. as therapeutic applications for bleeding

wounds

(Oktaviani et al., 2019). In in

vivo study, Ageratum conyzoides L. increased

cellular proliferation and collagen synthesis

(Arulprakash et al., 2011). Meanwhile, Manihot

esculenta increased the gingival wound healing

process (Nisa et al., 2013). Nevertheless, these plants'

efficacy has never been found to fibroblast cells,

which is very important for the healing process.

Therefore, in this study, we aimed at investigating

fibroblast proliferation and migration capabilities in

vitro in the presence of ethanol extracts of Manihot

esculenta and Ageratum conyzoides L.

2 MATERIALS AND METHODS

2.1 Research Design

The cells were cultured with a medium conditioned

for each extract with different concentrations (0.5%,

1%, 2%, 4%), positive and negative control groups.

For migration assay, cell culture was divided into

four groups; two groups received the most

significantly induced fibroblast proliferation for each

extract; 0.5% Manihot esculenta and 2% Ageratum

conyzoides L., two other groups as positive and

negative control groups.

2.1.1 Time and Place

The experiment was conducted from October to

December 2019 at Laboratorium Penelitian Terpadu

Universitas Yarsi.

2.1.2 Populations and Samples

The preputium skin fibroblast from Biorepository of

Universitas YARSI was routinely cultured in

Dulbecco's modified Eagle's medium (DMEM), low

glucose supplemented with 2 mM L-glutamine and

10% fetal bovine serum (FBS). All cells were grown

in 100 units/mL penicillin, 100 ug/mL streptomycin,

and 0,25 ug/mL amphotericin B. Cells for the

experiments were used at passage 3.

Plant materials of Manihot esculenta and

Ageratum conyzoides L. were collected in October

2019 in the Caringin village, west java. The leaves

were dried at room temperature for five days and then

were cut into fragments of approximately 5 cm. The

fragments were soaked in 70% ethanol and filtered. A

rotatory evaporation machine evaporated the filtrate.

2.1.3 Viability and Proliferation Assay

Fibroblasts were seeded at the concentration of 3000

cells/well in a 96 well plate and allowed to incubate

overnight. After the cells attached to the well surface,

cells were washed in phosphate-buffered saline

(PBS). The cells were cultured with a medium

conditioned for each extract with different

concentrations (0.5%, 1%, 2%, 4%), positive and

negative control groups for 48hr. The culture medium

was discarded every indicated time points and added

10 µl CCK-8 and 90 µL PBS. After 90 minutes of

incubation with CCK-8, absorbance detected at 450

nm using a Tecan microplate reader. The results

expressed in optical density (OD) units as compared

to untreated cells.

2.1.4 Migration Assay

Fibroblasts were seeded at a concentration of 40.000

cells/well in a 12 well plate and incubated until

confluence. A scratch was made using a 10 µl pipette

tip for each well. After removed the media, the cells

were washed in PBS and then added medium

conditioned with 0.5% Manihot esculenta and 2%

Ageratum conyzoides L. The distance between scratch

areas was measured by optical microscopy every two

hours for 48 hours using Nikon advanced research

elements 3.21.00 software and expressed as a

percentage of the area's closure compared to untreated

cells.

2.1.5 Data Analysis

Three technical replicates performed the experiments

for each treatment. All statistical analyses were

performed by comparing 0.5% Manihot esculenta and

2% Ageratum conyzoides L using unpaired Student's

t-test.

3 RESULTS

3.1 Cell Viability and Proliferation

At 48 hours, the extracts of 2% Ageratum conyzoides

L. was the most significantly induced fibroblast

proliferation at 0.22 ± 0.02 (Figure 1) and showed

intact fibroblast morphology (Figure 3, panel D). The

extracts of 0.5% Manihot esculenta was the most

significantly induced fibroblast proliferation at 0.16 ±

0.01 (Figure 2) and showed intact fibroblast

morphology (Figure 4, panel B). DMSO significantly

decreased fibroblast proliferation at 0.01 ± 0.00 and

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

showed damage to fibroblast morphology. The

control group indicated fibroblast proliferation at 0.07

± 0.01 and showed intact fibroblast morphology.

There were significant differences in fibroblast

proliferation rate in 2% Ageratum conyzoides L.

(p=0.00) and 0.5% Manihot esculenta (p=0.00)

compared to the control group.

Figure 1: Fibroblast viability and proliferation in different

concentrations of Ageratum conyzoides L. at 48 hours.

Table 1: T-test for fibroblast proliferation in different

concentrations of Ageratum conyzoides L. at 48 hours.

Concentration

Ageratum

conyzoides L

T-test

0.5% 0.15 0.00

1% 0.19 0.00

2% 0.22 0.00

4% 0.18 0.01

dmso 0.01 0.00

control 0.07

Data were expressed as optical density (OD)

Figure 2: Fibroblast Viability and Proliferation in different

concentrations of Manihot esculenta at 48 hours.

Table 2: T-test for fibroblast proliferation in different

concentrations of Manihot esculenta at 48 hours.

Concentration

Manihot

esculenta

T-test

0.5% 0.16 0.00

1% 0.15 0.00

2% 0.14 0.00

4% 0.12 0.00

dmso 0.01 0.00

control 0.06

Data were expressed as optical density (OD)

Figure 3: Fibroblast Morphology in different concentration

of Ageratum conyzoides L. at 48 hour. (A) control group,

(B) 0.5%, (C) 1%, (D) 2% and (E) 4% extract in complete

medium, (F) 10% DMSO.

Figure 4: Fibroblast Morphology in different concentration

of Manihot esculenta at 48 hour. (A) control group, (B)

0.5%, (C) 1%, (D) 2% and (E) 4% extract in complete

medium, (F) 10% DMSO.

3.2 Cell Migration

At 48 hours, the wound was almost closed for each

group. However, there were no significant differences

in fibroblast migration rate in 2% Ageratum

conyzoides L. (p=0.18) and 0.5% Manihot esculenta

(p=0.40) compared to the control group.

Table 3: Migration of fibroblast.

Control

2% Ageratum

con

zoides L

0.5% Manihot

esculenta

61.42 ± 7.88 64.35 ± 6.25 66.65 ± 4.17

31.08 ± 12.29 38.28 ± 8.89 35.36 ± 11.21

0.60 ± 0.15 2.07 ± 2.86 0.43 ± 0.2

Data were expressed as wound closure percentage (%). At

48 hours, 2% Ageratum conyzoides L. at 2.07 ± 2.86, and

0.5% Manihot esculenta at 0.43 ± 0.2 .

(OD)

The Role of Fibroblast Proliferation in Wound Healing by Different Plants: An Experimental Study

Figure 5: Analysis of fibroblast migration during wound

closure. Control group at (a): 0hr, (b): 24hr, (c): 48hr. Cells

treated by 2% Ageratum conyzoides L. at (d): 0hr , (e): 24hr ,

(f): 48hr. Cells treated by 0.5% Manihot esculenta at (g):

0hr , (h): 24hr , (i): 48hr.

4 DISCUSSION

Wound healing time can take a year or more to finish.

Some wounds do not heal in a timely and orderly

manner. It is disturbed by various factors such as

infection, tissue hypoxia, necrosis, exudate, and

excess inflammatory cytokines. The wound healing

process has four overlapping stages: homeostasis,

inflammation, proliferation, and migration

(Addis et

al., 2020). While platelets have a role in clot

formation during homeostasis, inflammation cells

debride injured tissue during the inflammation phase.

At the proliferative phase, occur epithelialization,

fibroplasia, and angiogenesis (DesJardins et al.,

2018).

Meanwhile, granulation tissue forms and the

wound begins to contract. In the maturation phase,

collagen forms tight cross-links to other collagen and

with protein molecules. Fibroblasts have a crucial

role in all of these phases, including the deposition of

extracellular matrix (ECM) components, wound

contraction, and new ECM remodeling

(Sumbayak,

2016).

Plants and herbs are active medicinal, are used to

stimulate stem cell proliferation, regeneration, and

rehabilitation in damaged tissue

(Cragg and Newman,

2017). Several studies found that the leaves' active

ingredients mainly activate stem cell regeneration

potential (Maioli et al., 2010). Plant extracts or plant-

derived compounds are preferred because of fewer

side effects and widespread availability

(Agyare et al.,

2014). Moreover, wound healing management can be

elicited by the antioxidant activity of some plant

extracts

(Süntar et al., 2012).

The antioxidant flavonoid of Ageratum

conyzoides L. has anti-inflammatory effects,

especially quercetin, that can inhibit β-glucuronidase,

decrease leukotriene, inhibit histamine, inhibit some

enzymes such as ATPase, phosphodiesterase, and

protein kinases

(Galati, 2008). Meanwhile, the extract

of Manihot esculenta has an anti-inflammatory effect

since having gallic acid, vitamin C, flavonoid,

saponin, tannin, and triterpenoid as antioxidant

compounds

(Oktaviani et al., 2019).

Reactive oxygen species are involved in many

infections, degenerative diseases, cancer, and even

wound healing. Antioxidants enhance the healing of

wounds by reducing the damage caused by oxygen

radicals. Plant-derived antioxidants benefit from their

redox properties, which allow them to act as hydrogen

donors, reducing agents, hydroxyl radicals (OH), or

superoxide radicals (O2) scavengers

(Geethalakshmi

et al., 2013).

These two extracts have toxic components that

may interfere with the healing process since the data

showed no significant difference in fibroblast

migration. Ageratum conyzoides L. contains

pyrrolizidine alkaloids (PAs), which have been

reported to be hepatotoxic, mutagenic, and

carcinogenic

(Bosi et al., 2013). Manihot esculenta

contains toxic agents cyanogenic glycosides, made up

of 95% linamarin and 5% lotaustralin

(Faezah et al.,

2016).

Pyrrolizidine alkaloids are widely distributed in

plants throughout the world. Alkaloids present a

lipophilic character, soluble in apolar organic

solvents and alcohol. Pyrrolizidine alkaloids can

penetrate the nucleus and react with DNA, causing

DNA cross-link and DNA-protein cross-link to elicit

an abnormal function, which will cause damage

(Moreira et al., 2018).

Cyanogenic glycosides are bioactive plant

products derived from amino acids—the primary

biological function is as a plant defense system

against the effects of distinct animals. Acute

poisoning of animals and humans from cyanogenic

consumption can induce rapid and drastic inhibition

of the respiration system in mitochondria

(Vetter,

2017).

Our data showed that the extracts could enhance

wound healing by stimulating the proliferation of

fibroblast. Within this context, the extracts of

Ageratum conyzoides L. and Manihot esculenta could

be used in the future as a topical therapeutic

application to stimulate the wound healing process

and antioxidant responses in damaged skin.

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

5 CONCLUSION

The study of Ageratum conyzoides L and Manihot

esculenta extract's leaves proved it could accelerate

wound healing. The extracts increase fibroblast

proliferation in vitro. Further in vivo study is needed

for tissue regeneration applications.

ACKNOWLEDGEMENTS

We want to thank Herbal Laboratory-Universitas

Yarsi and Yarsi University Foundation for the

support.

REFERENCES

Addis, R., Cruciani, S., Santaniello, S., Bellu, E., Sarais, G.,

Ventura, C., Maioli, M. and Pintore, G., 2020.

Fibroblast Proliferation and Migration in Wound

Healing by Phytochemicals: Evidence for a Novel

Synergic Outcome. International Journal of Medical

Sciences, 17(8), p.1030.

Agyare, C., Adarkwa-Yiadom, M. and Osei-Asante, S.,

2014. Medicinal plants used for treatment of wounds

and skin infections: Assessment of wound healing and

antimicrobial properties of Mallotus oppositifolius and

Momordica charantia.

Arulprakash, K., Murugan, R., Ponrasu, T., Iyappan, K.,

Gayathri, V.S. and Suguna, L., 2012. Efficacy of

Ageratum conyzoides on tissue repair and collagen

formation in rats. Clinical and Experimental

Dermatology: Experimental dermatology, 37(4),

pp.418-424.

Blumberg, Y., 2019. Here's why many prescription drugs in

the US cost so much—and it's not innovation or

improvement. CNBC. Retrieved from:

https://www.cnbc.com/2019/01/10/why-prescription-

drugs-in-the-us-cost-so-much.html

Bosi, C.F., Rosa, D.W., Grougnet, R., Lemonakis, N.,

Halabalaki, M., Skaltsounis, A.L. and Biavatti, M.W.,

2013. Pyrrolizidine alkaloids in medicinal tea of

Ageratum conyzoides. Revista Brasileira de

Farmacognosia, 23(3), pp.425-432.

Cragg, G.M. and Newman, D.J., 2013. Natural products: a

continuing source of novel drug leads. Biochimica et

Biophysica Acta (BBA)-General Subjects, 1830(6),

pp.3670-3695.

desJardins-Park, H.E., Foster, D.S. and Longaker, M.T.,

2018. Fibroblasts and wound healing: an update.

Regenerative medicine, 13(5). Retrieved from

https://www.futuremedicine.com/doi/full/10.2217/rme

-2018-0073

Nur, F.O., Siti, A.H. and Umi, K.Y., 2013. Comparative

evaluation of organic and inorganic fertilizers on total

phenolic, total flavonoid, antioxidant activity and

cyanogenic glycosides in cassava (Manihot

esculenta). African Journal of Biotechnology, 12(18).

Galati, E.M., Miceli, N., Taviano, M.F., Sanogo, R. and

Raneri, E., 2001. Anti-inflammatory and antioxidant

activity of Ageratum conyzoides. Pharmaceutical

biology, 39(5), pp.336-339.

Geethalakshmi, R., Sakravarthi, C., Kritika, T., Arul

Kirubakaran, M. and Sarada, D.V.L., 2013. Evaluation

of antioxidant and wound healing potentials of

Sphaeranthus amaranthoides Burm. f. BioMed research

international, 2013.

Gonzalez, A.C.D.O., Costa, T.F., Andrade, Z.D.A. and

Medrado, A.R.A.P., 2016. Wound healing-A literature

review. Anais brasileiros de dermatologia, 91(5),

pp.614-620.

Lordani, T.V.A., de Lara, C.E., Ferreira, F.B.P., de Souza

Terron Monich, M., Mesquita da Silva, C., Felicetti

Lordani, C.R., Giacomini Bueno, F., Vieira Teixeira,

J.J. and Lonardoni, M.V.C., 2018. Therapeutic effects

of medicinal plants on cutaneous wound healing in

humans: a systematic review. Mediators of

inflammation, 2018.

Maioli, M., Santaniello, S., Montella, A., Bandiera, P.,

Cantoni, S., Cavallini, C., Bianchi, F., Lionetti, V.,

Rizzolio, F., Marchesi, I. and Bagella, L., 2010.

Hyaluronan esters drive Smad gene expression and

signaling enhancing cardiogenesis in mouse embryonic

and human mesenchymal stem cells. PLoS One, 5(11),

p.e15151.

Moreira, R., Pereira, D.M., Valentão, P. and Andrade, P.B.,

2018. Pyrrolizidine alkaloids: chemistry,

pharmacology, toxicology and food

safety. International journal of molecular

sciences, 19(6), p.1668.

Nisa, V.M., 2013. Efek Pemberian Ekstrak Daun Singkong

(Manihot esculenta) Terhadap Proses Penyembuhan

Luka Gingiva Tikus (Rattus norvegicus). Repository

unej. Retrieved from

http://repository.unej.ac.id/bitstream/handle/123456789

/59375/Vina%20M.Nisa.pdf?sequence=1

Oktaviani, D.J., Widiyastuti, S., Maharani, D.A., Amalia,

A.N., Ishak, A.M. and Zuhrotun, A., 2019. Bahan

Alami Penyembuh Luka. Majalah Farmasetika, 4(3),

pp.45-56.

Simon, P.E. 2020Skin wound healing. Medscape. Retrieved

from https://emedicine.medscape.com/article/884594

Sumbayak, E.M., 2015. Fibroblas: Struktur dan Peranannya

dalam Penyembuhan Luka. Jurnal Kedokteran Meditek,

21(57). Retrieved from

http://ejournal.ukrida.ac.id/ojs/index.php/Meditek/articl

e/view/1169

Süntar, I., Akkol, E.K., Nahar, L. and Sarker, S.D., 2012.

Wound healing and antioxidant properties: do they

coexist in plants?. Free Radicals and Antioxidants, 2(2),

pp.1-7.

Vetter, J. 2017. Plant Toxins : Plant Cyanogenic

Glycosides. Dordrecht, The Netherlands: Springer.

The Role of Fibroblast Proliferation in Wound Healing by Different Plants: An Experimental Study