Efficacy of Topical Binahong Leaf Ethanolic Extract Administration

on Serum TGF-β1 in Wistar Rats with Staphylococcus aureus-infected

Wounds

Rizki Andini Nawawi

, Muhammad Totong Kamaluddin

and Theodorus Theodorus

Biomedical Sciences Graduate Program, Faculty of Medicine, Universitas Sriwijaya, Palembang, Indonesia

Department of Pharmacology, Faculty of Medicine, Universitas Sriwijaya, Palembang, Indonesia

Keywords: Anredera Cordifolia, Wound Infection, Transforming Growth Factor Beta, Wound Healing, In Vivo

Abstract: Binahong (Anredera cordifolia (Ten.) Steenis) is a medicinal plant traditionally used as a topical wound

treatment. Saponin content in Binahong leaf extract has been thought to increase TGF-β secretion, which

plays an important role in accelerating wound healing. This study’s aim was to assess the efficacy of topical

Binahong leaf ethanolic extract administration on serum TGF-β1 in infected wounds. An experimental study,

in vivo, was conducted in the Biotechnology Laboratory and Animal House, Faculty of Medicine, Universitas

Sriwijaya, Palembang, from July to September 2020. Thirty male Wistar rats aged 10-12 weeks with

excisional wounds were infected with Staphylococcus aureus ATCC 25923. The rats were divided into five

groups and received three concentrations of Binahong leaf extracts (2.5%, 5%, and 10%), salve base, and

povidone iodine 10% topically twice daily for 14 days. Serum was obtained before treatment and after day 14

of treatment. Wound area was also recorded. After 14 days of topical administration of Binahong leaf extract

on rats with Staphylococcus aureus-infected wounds, a decrease in wound size was most significantly

observed in rats receiving 10% Binahong leaf extract (p = 0.02), but no significant serum TGF-β1 increase

was observed in all treatment groups.

1 INTRODUCTION

The presence of wounds poses a risk of colonization

and infection by pathogenic bacteria on the wounded

site, which might spread and cause systemic infection

if it is not adequately managed (Negut, Grumezescu,

and Grumezescu 2018). One of the most common

causative pathogens in wound and skin infections is

Staphylococcus aureus (Roy et al. 2018). S. aureus

disrupts normal wound healing process through the

release of extracellular adherence protein (Eap). Eap

release prolongs inflammation and prevents

angiogenesis, especially in the proliferative phase of

wound healing (Pereira-franchi et al. 2017; Wong,

Manikam, and Muniandy 2015). In Indonesia, a

multicenter study reported S. aureus isolation from

45.3% patients with wound infections and SSTI

(Santosaningsih et al. 2018).

https://orcid.org/0000-0002-5079-3016

https://orcid.org/0000-0002-8670-9867

https://orcid.org/0000-0003-1106-8396

The wound healing process involves a complex

interaction between the skin’s cellular components,

such as keratinocytes, fibroblasts, vascular endothelia,

immune system cells, and extracellular matrix (Martin

and Nunan 2015). These cells interact by secreting

various mediators and growth factors, one of which is

transforming growth factor-beta (TGF-β). TGF-β is a

pleiotropic growth factor secreted by platelets,

fibroblasts, and proinflammatory cells (Lichtman,

Otero-vinas, and Falanga 2016; Sutrisno et al. 2018).

TGF-β has an extensive role in wound healing, such

as stimulating collagen synthesis, angiogenesis, and

keratinocyte migration (Lichtman, Otero-vinas, and

Falanga 2016; Tejiram et al. 2016). TGF-β may also

induce epithelial-mesenchymal transition, which is an

important morphogenetic event in the formation of

scar tissue and regeneration (Martin and Nunan 2015;

Qi et al. 2018). TGF-β has three known isoforms,

Nawawi, R., Kamaluddin, M. and Theodorus, .

Efﬁcacy of Topical Binahong Leaf Ethanolic Extract Administration on Serum TGF-1 in Wistar Rats with Staphylococcus aureus-infected Wounds.

DOI: 10.5220/0010491202670272

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 267-272

ISBN: 978-989-758-499-2

267

TGF-β1 to TGF-β3, in which TGF-β1 is the most

abundant (Wang et al. 2017).

Recently, while topical therapy remains an

important modality in wound management, it has

become a concern that most available topical therapies

do not give additional benefits in speeding up the

wound healing process (Powers et al. 2019). In case of

infected wounds, rising tolerance and resistance

towards common topical antibiotics and antiseptic

agents also impose a challenge in choosing the

appropriate treatment (Hardy et al. 2018).

Natural products and their active compounds are

starting to be considered in the development of novel

products for wound management (Istyastono and

Yuliani 2016). Anredera cordifolia (Ten.) Steenis

(Binahong) is a plant from the Basellaceae family,

which has long been used in traditional medicine

around the world (Astuti et al. 2011; Leliqia,

Sukandar, and Fidrianny 2017). In Indonesia,

Binahong has been traditionally used for various

diseases, including in the treatment of wounds and

bacterial infections (Astuti et al. 2011; Sukandar and

Kurniati 2014).

A number of studies have been conducted on the

efficacy of A. cordifolia extract in wounds, and so far

there have been no data yet on the in vivo efficacy of

A. cordifolia extract on TGF-β1 concentrations in

infected wounds. This study aimed to determine the

efficacy of A. cordifolia ethanolic extract on

increasing serum TGF-β1 in rats with Staphylococcus

aureus-infected wounds.

2 MATERIALS AND METHODS

An experimental study, in vivo, was conducted at the

Biotechnology Laboratory and Animal House,

Faculty of Medicine, Universitas Sriwijaya,

Palembang, in July to September 2020. The study

population was male Wistar rats. There were 30 male

Wistar rats aged 10-12 weeks, weighing 150-200

grams which fulfilled the inclusion criteria.

Rats showing signs of infection in the first 24

hours after S. aureus inoculation were included in

this study. Ethical clearance had been approved by

Health Research Ethics Committee, Faculty of

Medicine, Universitas Sriwijaya, prior to the

commencement of the study (Certificate No.

024/kepkrsmhfkunsri/2020).

2.1

Extract Preparation

Four hundred grams of dried Binahong leaves

obtained from Karangpandan, Tawangmangu,

Central Java (elevation of 800 m above sea level),

was extracted by maceration with 96% ethanol which

is in accordance with the Indonesian Herbal

Pharmacopoeia and concentrated through rotary

evaporation (Kementerian Kesehatan Republik

Indonesia 2009). The obtained concentrated extract

was then formulated into salve with vaseline album

and adeps lanae base. Three concentrations of salves

were formulated, each containing 2.5%, 5%, and

10% Binahong leaf ethanolic extract, respectively.

Salve base was used as negative control and

povidone iodine 10% (Betadine®, PT Mahakam

Beta Farma, Jakarta, Indonesia, Batch No. GB20045)

was used as positive control.

2.2

In Vivo Efficacy Test

Rats were anesthetized by using ketamine, and their

dorsal skin was depilated with scissors and depilatory

cream before wounding. A 2 cm

circular excision

wound was made with scalpels and surgical scissors,

followed by inoculation with Staphylococcus aureus

ATCC 25923 suspension containing 2 x 10

cfu and

a 24-hour incubation period. The rats were then

divided into 5 treatment groups, each receiving salve

base (negative control), three concentrations of

Binahong leaf ethanolic extract salves, and povidone

iodine 10% (positive control). All groups received

treatments twice daily for 14 days.

Wounds were photographed on days 4, 7, 10, and

14 of treatment, and wound area was measured with

image processing software. Serum samples were

obtained during the 24-hour incubation period before

treatment started and on the 14

day of treatment.

TGF-β1 assay was performed by ELISA

(MyBioSource, San Diego, CA, USA), following

protocols specified by the manufacturer.

2.3

Statistical Analysis

Homogenity and normality of data was assessed prior

to further analysis. Efficacy of each treatment was

assessed by using paired T test. Efficacy comparison

between treatment groups and controls was

performed by using unpaired T test, and significance

test was performed with Post Hoc test. Significance

is assumed at p < 0.05.

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Table 1: Efficacy of topical Binahong leaf ethanolic extract on decreasing wound area.

Treatment Group Wound area (cm

) p

Pre-

treatment

Mean

Pos

treatment

Mean

Salve base

Binahong leaf ethanolic

extract

2.5%

10%

Povidone iodine 10%

2.403

2.368

1.427

2.069

2.208

0.356

0.225

0.259

0.198

0.302

0.001

Paired T test, p = 0.05

3 RESULTS

Wound area was significantly decreased in all

treatment groups (Table 1).

Figure 1: Wound area contraction in all treatment groups

during the course of the experiment.

The most significant decrease in wound area was

found in treatment groups receiving 10% Binahong

leaf extract, where a significant difference in efficacy

was found in comparison to negative control group (p

= 0.02), but there was no significant efficacy

difference in comparison to povidone iodine 10%

(Table 2).

After 14 days of topical Binahong leaf ethanolic

extract administration, there was no significant

increase in serum TGF-β1 concentration in all

treatment groups (Table 3).

4 DISCUSSION

This study aimed to assess the efficacy of topical

Binahong leaf ethanolic extract administration on

serum TGF-β1 in infected wounds, while its efficacy

in decreasing wound area was also examined for

baseline data. Topical administration of Binahong

leaf ethanolic extract did not increase TGF-β1 levels,

but effectively decreased wound area. Treatment

with 10% Binahong leaf ethanolic extract was shown

to be most efficacious in decreasing wound area.

Table 2: Efficacy comparison between different doses of topical Binahong leaf ethanolic extract and controls on decreasing

wound area after 14 days of administration.

Comparison Group Treatment Group p

Salve base

(negative control)

2.5% Binahong leaf extrac

5% Binahong leaf extract

10% Binahong leaf extract

Povidone iodine 10%

0.129

0.109

0.022

0.427

Povidone iodine 10%

(positive control)

Salve base

2.5% Binahong leaf extract

5% Binahong leaf extract

10% Binahon

leaf extrac

0.427

0.323

0.360

0.063

Unpaired T test, p = 0.05

Efﬁcacy of Topical Binahong Leaf Ethanolic Extract Administration on Serum TGF-1 in Wistar Rats with Staphylococcus aureus-infected

Wounds

269

Table 3: Efficacy of topical Binahong leaf ethanolic extract on decreasing wound area.

Treatment Group Serum TGF-β1 (p

/ml) p

Pre-treatment

Mean

Pos

-treatment

Mean

Salve base

Binahong leaf ethanolic extract

2.5%

10%

Povidone iodine 10%

1,036.736

1,034.923

1,018.898

1,065.949

1,072.551

1,076.681

952.615

998.705

969.090

1,006.461

0.128

0.225

0.753

0.463

0.249

Paired T test, p = 0.05

Presently, studies conducted on the efficacy of

Binahong leaf extract in wound healing have

reported various findings. A study by Paju, Yamlean

and Kojong (2013) in rabbits with incisional wounds

reported the efficacy of 10% Binahong leaf ethanolic

extract in wound healing, which is similar to our

findings (Paju, Yamlean, and Kojong 2013).

Meanwhile, Sukrama et al. (2017) reported that

concentrated Binahong leaf ethanolic extract

effectively decreased the area of burn wounds in

murine models (Sukrama, Wihandani, and Manuaba

2017). A study on excisional wounds in guinea pigs

by Miladiyah and Prabowo (2012) showed that 40%

Binahong leaf ethanolic extract in distilled water was

efficacious in decreasing wound area (Miladiyah and

Prabowo 2012). Histopathological studies on rats

receiving 5% Binahong leaf extract showed a greater

decrease in polymorphonuclear (PMN) infiltration

and increase in collagen deposition, angiogenesis,

and fibrosis in comparison to silver sulfadiazine

(Yuniarti and Lukiswanto 2017).

Three main bioactive compound classes in

Binahong leaves are known to play important roles in

the wound healing process, namely saponin, tannin,

and flavonoid (Leliqia, Sukandar, and Fidrianny

2017; Yuniarti and Lukiswanto 2017). Saponin

enhances wound healing process through stimulation

of procollagen synthesis. Saponin also enhances the

proliferation of monocytes, which will differentiate

into macrophages and secrete various growth factors.

In the reepithelialization process, saponin stimulates

fibroblast proliferation and keratinocyte migration

(Yuniarti and Lukiswanto 2017). Fibroblasts will also

secrete growth factors, such as VEGF, interleukins,

and TGF-β (Sukrama, Wihandani, and Manuaba

2017; Sutrisno et al. 2018). Flavonoid and tannin are

aromatic compound classes known for their

astringent properties. The astringent properties of

flavonoid and tannin compounds cause the skin pores

to contract, hence stopping capillary bleeding and

exudation and stimulates wound contraction (Ibrahim

et al. 2018). In addition, Flavonoid enhances wound

healing through stimulation of collagen matrix

rearrangement, while tannin stimulates wound

contraction through its role in fibroblast migration

and proliferation (Budovsky, Yarmolinsky, and Ben-

shabat 2015; Ibrahim et al. 2018; Yuniarti and

Lukiswanto 2017).

No significant increase in serum TGF-β1 was

observed in all treatment groups. A previous study

suggested that low concentration povidone iodine

(0.5%) administered topically on clean wounds could

stimulate TGF-β secretion (Wang et al. 2017).

Therefore, higher concentration of povidone iodine

administered and wound infection might have

significantly impaired povidone iodine’s effects on

TGF-β. Saponin content in Binahong leaves has been

thought to indirectly increase TGF-β secretion

through stimulating fibroblast and monocyte

proliferation (Sukrama, Wihandani, and Manuaba

2017; Sutrisno et al. 2018). Harvesting conditions

might have influenced the saponin content of

Binahong leaves prior to extraction. A previous study

quantified more saponin content in older Binahong

leaves than younger ones (2.36 µg/mg and 1.37

µg/mg, respectively) (Hasbullah 2016).

Most previous studies on the role of saponin

compounds in increasing TGF-β release had not

specified the exact TGF-β isoform studied. Among

all TGF-β isoforms, TGF-β1 has so far been

considered more important as it is also the most

abundant. TGF-β1 has been known to induce integrin

expression from keratinocytes in the skin epidermis,

which facilitates the migratory components of

reepithelialization (Ibrahim et al. 2018). Recently,

TGF-β3, another isoform of TGF-β, has also been

reported to play an important role in the later stages

of wound healing. A study in murine models reported

that TGF-β1 and TGF-β3 affected cell cycle

progression and cell migration differently, which

showed clinically in the formation of scar tissues.

Higher TGF-β1 concentration tended to cause scar

tissue formation, while higher TGF-β3 concentration

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

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270

tended to promote scarless wound healing

(Lichtman, Otero-vinas, and Falanga 2016).

Binahong leaf extract’s effect on fibroblast and

macrophage proliferation might have enhanced

secretions of other growth factors and mediators as

well. Fibroblasts and macrophages secrete a plethora

of growth factors and cytokines, such as interleukins

(IL-1, IL-6, IL-11, IL-17, IL-18), TNF-α, IFN-γ,

VEGF, PDGF, and granulocyte-macrophage colony-

stimulating factor (GM-CSF), which have known

roles in the wound healing process (Gonzalez et al.

2016; Zeinali, Rezaee, and Hosseinzadeh 2017).

Previously, a study on rats with Pseudomonas

aeruginosa-infected burn wounds showed an

increase of IL-6 and VEGF concentrations after

administration of Binahong leaf concentrated extract

(Sukrama, Wihandani, and Manuaba 2017).

Our findings have reinforced existing evidence

on the efficacy of Binahong leaf ethanolic extract

administration in enhancing wound healing, hence

further suggesting its possible clinical application

both as a single wound management product and in

combination with other agents. However, our current

study’s findings were limited in that serum TGF-β1

was assayed only before treatment and after day 14

of treatment, which corresponded with later phases

of wound healing. Considering the vast influence of

TGF-β in all phases of wound healing, possible

increases in TGF-β1 during the earlier phases of

wound healing still needs to be determined in order

to better comprehend the effects of Binahong leaf

ethanolic extract administration on TGF-β1

secretion. Another limitation was that this study

focused on a single isoform of TGF-β, and the

possible effects of Binahong leaf ethanolic extract on

other TGF-β isoforms remains to be elucidated.

5 CONCLUSION

In rats with Staphylococcus aureus-infected wounds,

topical administration of Binahong (Anredera

cordifolia (Ten.) Steenis) leaf extract for 14 days was

efficacious in decreasing wound area but did not

increase serum TGF-β1 levels. Further studies need

to assess the effects of Binahong leaf ethanolic

extract administration on TGF-β1 levels in

accordance with each phase of wound healing and

investigate the effects of Binahong leaf ethanolic

extract on other isoforms of TGF-β.

ACKNOWLEDGEMENTS

The authors wish to thank Dr. dr. M. Irsan Saleh,

M.Biomed, dr. Iqmal Perlianta, SpBP-RE, and dr.

Ella Amalia, M.Kes for their valuable inputs in this

study.

The authors also thank Maisa Pusrita and Laila

Wardani Hasanah for their assistance with

interpreting the ELISA results.

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