the role of liquid smoke coconut shell in the ...production of liquid smoke coconut shell (ls-cs)...

© 2020 Journal of Pharmacy & Pharmacognosy Research, 8 (6), 549-557, 2020 ISSN 0719-4250

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Original Article | Artículo Original

_____________________________________

The role of liquid smoke coconut shell in the proliferation phase of an oral traumatic ulcer

[El papel del humo líquido de la cáscara de coco en la fase de proliferación de una úlcera traumática oral]

Nurina Febriyanti Ayuningtyas1, Meircurius Dwi Condro Surboyo1, Diah Savitri Ernawati1*, Adiastuti Endah Parmadiati1, Hening Tuti Hendarti1, Fatma Yasmin Mahdani1, Saka Winias1, Fitriatuz Zakia2, Indira Arella Harianto2

1Department of Oral Medicine. Faculty of Dental Medicine. Universitas Airlangga, Surabaya 60132, Indonesia. 2Faculty of Dental Medicine. Universitas Airlangga, Surabaya 60132, Indonesia.

*E-mail: [email protected]

Abstract Resumen

Context: Liquid smoke coconut shell (LS-CS) contains phenolic compounds that are able to promote wound healing by interfering with the inflammation phase of wound healing. The homeostatic mechanism that decreases the inflammation releases a growth factor that enhances proliferation by increasing fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF) expression and fibroblast proliferation.

Aims: To analyze the role of LS-CS in the proliferation phase of oral traumatic ulcer healing by analyzing fibroblast numbers and FGF-2 and VEGF expressions.

Methods: Oral traumatic ulcers were induced in diabetes mellitus Wistar rats via an alloxan injection. A traumatic ulcer as long as 10 mm was made in the inferior fornix incisive labial area. The oral traumatic ulcer was then topically treated with LS-CS using a dose of 20 μL / 20 g body weight once daily for three, five and seven days. The fibroblast number of the oral traumatic ulcers’ tissue was then analyzed using hematoxylin-eosin, and the FGF-2 and VEGF expression were analyzed via immunohistochemistry staining. The difference in fibroblast numbers and FGF-2 and VEGF expressions were analyzed by one-way ANOVA and post hoc tests with a significance of p<0.05.

Results: The LS-CS affected fibroblast numbers (p=0.001) and FGF-2 expression (p=0.000) after topical treatment for seven days. There was a significant difference in the expression of VEGF after topical treatment with LS-CS for five days compared to the control group (p=0.000) and benzydamine hydrochloride (p=0.005). The topical treatment of LS-CS for seven days affected the expression of VEGF compared to the control group (p=0.000) and benzydamine hydrochloride (p=0.019).

Conclusions: LS-CS could improve the healing of oral traumatic ulcers by increasing fibroblast numbers and FGF-2 and VEGF expression after seven days of treatment.

Contexto: El humo líquido de la cáscara de coco (LS-CS) contiene compuestos fenólicos que pueden promover la curación de heridas al interferir con la fase de inflamación de la curación de heridas. El mecanismo homeostático que disminuye la inflamación libera un factor de crecimiento que mejora la proliferación al aumentar el factor de crecimiento de fibroblastos-2 (FGF-2), la expresión del factor de crecimiento endotelial vascular (VEGF) y la proliferación de fibroblastos.

Objetivos: Analizar el papel de LS-CS en la fase de proliferación de la cicatrización de la úlcera traumática oral mediante el análisis de los números de fibroblastos y las expresiones FGF-2 y VEGF.

Métodos: Se indujeron úlceras traumáticas orales en ratas Wistar con diabetes mellitus mediante una inyección de aloxano. Se realizó una úlcera traumática de hasta 10 mm en el área labial incisiva del fórnix inferior. La úlcera traumática oral se trató por vía tópica con LS-CS (dosis de 20 μL/20 g de peso corporal) una vez al día durante tres, cinco y siete días. El número de fibroblastos del tejido de las úlceras traumáticas orales se analizó luego utilizando hematoxilina-eosina, y las expresiones de FGF-2 y VEGF se analizaron mediante tinción inmunohistoquímica. La diferencia en los números de fibroblastos y las expresiones FGF-2 y VEGF se analizaron mediante ANOVA unidireccional y pruebas post hoc con una significación de p<0,05.

Resultados: El LS-CS afectó los números de fibroblastos (p=0,001) y la expresión de FGF-2 (p=0,000) después del tratamiento tópico durante siete días. Hubo una diferencia significativa en la expresión de VEGF después del tratamiento tópico con LS-CS durante cinco días en comparación con el grupo control (p=0,000) y clorhidrato de bencidamina (p=0,005). El tratamiento tópico de LS-CS durante siete días afectó la expresión de VEGF en comparación con el grupo control (p=0,000) y el clorhidrato de bencidamina (p=0,019).

Conclusiones: LS-CS podría mejorar la curación de las úlceras traumáticas orales al aumentar el número de fibroblastos y la expresión de FGF-2 y VEGF después de siete días de tratamiento.

Keywords: FGF-2; fibroblast; liquid smoke coconut shell; oral traumatic ulcer; VEGF.

Palabras Clave: cáscara coco; FGF-2; fibroblastos; humo líquido; úlcera traumática oral; VEGF.

ARTICLE INFO Received: June 15, 2020. Received in revised form: July 21, 2020. Accepted: July 26, 2020. Available Online: August 17, 2020. Declaration of interests: The authors declare no conflict of interest. Funding: This research was funded by the Ministry Higher of Education, Republic of Indonesia 2020 (Grant No. 596/UN3.14/PT/2020).


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Ayuningtyas et al. Liquid smoke role in oral traumatic ulcer

http://jppres.com/jppres J Pharm Pharmacogn Res (2020) 8(6): 550

INTRODUCTION

Diabetes mellitus is one of the most systemic diseases in the world, and the prevalence rate is getting higher every year (Guariguata et al., 2014). Diabetes mellitus patients experience oral mucosal disorders more easily than people who do not suf-fer from the disease. It can manifest in the oral cavity, which is in the delayed healing of oral ul-cers (González-Serrano et al., 2016; Mauri-Obradors et al., 2017).

An oral traumatic ulcer is a damaged mucous membrane with loss of surface tissue, disintegra-tion and epithelial tissue necrosis, which causes pain and a burning sensation and is uncomfortable for patients due to inflammation and thus needs proper handling (Nandakumar, 2015; Silva et al., 2015; Lim et al., 2016). In general, cases of traumat-ic ulcers are treated with anti-inflammatory and antiseptic drugs to accelerate healing, but it has been found that in patients with diabetes mellitus, these ulcers are difficult to cure despite the provi-sion of anti-inflammatory and antiseptic drugs (Tripathi and Tripathi, 2015). Therefore, an alter-native medicine containing other ingredients such as herbs is needed.

The coconut tree (Cocos nucifera L., Arecaceae) is a plant that originates in Southeast Asian countries and is widely grown and used in tropical countries such as Indonesia, but the use of coconut in Indo-nesia is of less interest to the national community (Lima et al., 2015). Coconut shell is known to con-tain cellulose, hemicellulose and lignin, which al-low coconut shell to be used as liquid smoke by pyrolysis (Budijanto et al., 2008; Ayudiarti and Sari, 2010). Liquid smoke coconut contains phenol-ic compounds that are known to have the ability of anti-inflammatory in wound healing, such as 2-methoxyphenols (guaiacol), phenol, 4-ethyl-2- methoxyphenol (EMP), ascorbic acid and flavo-noid (Surboyo et al., 2019a). Phenol and guaiacol can decrease the expression of pro-inflammatory

cytokines such as tumor necrosis factor- (TNF-),

interleukin 6 (IL-6) and interleukin 1 (IL-1) by

binding to reactive oxygen species (ROS) and in-hibiting nitric oxide (NO), and they reduce prosta-glandin E2 synthesis by inhibiting cyclooxygenase (Surboyo et al., 2019b). Liquid smoke coconut shell also promotes wound healing by increasing fibro-blast proliferation and collagen (Tarawan et al., 2017).

Considering liquid smoke coconut shell’s abili-ties in the inflammation phase of wound healing, the purpose of this study is to analyze the wound healing activities of liquid smoke coconut shell during the proliferation phase by analyzing fibro-blast numbers and fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) expressions.

MATERIAL AND METHODS

Vegetable material

The coconut shell came from an eight- to 10-month-old coconut fruit that was collected from a coconut seller at the local market in Surabaya, In-donesia, during November-December 2016. The sample of coconut fruit and the coconut shell were examined and identified as Cocos nucifera L and as being from genus Cocos by the Indonesian Institute of Science, Plant Conservation Centre Purwodadi, Indonesia with registration number 1694/IPH.6/HM/X/2016.

Production of liquid smoke coconut shell (LS-CS)

The LS-CS used in this research was a product of Surboyo et al. (2019a). The LS-CS was obtained for 5 kg of dried coconut shells. The coconut shell was heated at a temperature of 400°C for 4.5 hours, producing a smoke that flowed and was cooled using an evaporation pipe to form a liquid. The liquid was deposited then filtered using filter pa-per and distilled at 120 to 150°C. The number of components identified was 32. The most common components in LS-CS are phenol (36.6%), furfural (17.8%) and 2-methoxyphenol (14.4%) (Surboyo et al., 2019a).




Table 1. Treatment groups.

Treatment Dose Duration Group

Distilled water

20 μL/20 g topically in the ulcer

Once a day for 3 days Control - 3



Benzydamine hydrochloride

Once a day for 3 days Benzydamine - 3



LS-CS Once a day for 3 days LS-CS - 3

Once a day for 5 days LS-CS - 5

Once a day for 7 days LS-CS - 7

LS-CS: liquid smoke coconut shell.

Animal

Ethical approval was obtained from the Health Experiment Committee, Faculty of Dental Medi-cine, Universitas Airlangga in Surabaya with regis-tered number 046/HRECC.FODM/II/2020. This research involved 36 male, two-month-old Wistar rats weighing 120 to 160 g divided into nine groups (Table 1).

They were induced with alloxan (alloxan mon-ohydrate A7413, Sigma Aldrich., St). Alloxan in-traperitoneal was given to the Wistar rats to reach a fasting blood glucose of >200 mg/dL as a diabe-tes mellitus condition (Etuk, 2010; Bako et al., 2014). Oral traumatic ulcers 10 mm in length were made in the inferior fornix labial region using a round stainless steel blade (Kiliç et al., 2013) to attain a white or yellowish ulcer base and reddish edge to match the clinical appearance of an oral traumatic ulcer (Muñoz-Corcuera et al., 2009). During the process of making the traumatic ulcers, all of the animals were under anesthesia, which was produced using a ketamine/xylazine cocktail.

The oral traumatic ulcers were then treated with LS-CS topically at a dose of 20 μL/20 g body weight for three, five and seven days (Hitomi et al., 2015; Surboyo et al., 2019a). The other groups’ ulcers were treated using the same dose of benzy-damine hydrochloride or distilled water (control).

Oral traumatic ulcer tissue preparation

After all of the groups had been treated for three, five and seven days, the oral traumatic ul-cers in the inferior fornix labial region were biop-sied. The oral traumatic ulcer tissue was fixed us-ing a 10% formalin buffer solution and then pro-cessed in a paraffin block.

Fibroblast analysis

The oral traumatic ulcer tissue from the paraffin block was sliced to be 4 μm using a rotary micro-tome. The tissue was then stained with hematoxy-lin-eosin (HE) in order to analyze the fibroblast numbers, which was performed via a histological view at 400 times magnification with a light micro-scope in a single-blinded manner (Chan, 2014).

FGF-2 and VEGF expression

The oral traumatic ulcer tissue from the paraffin block was sliced to be 4 μm using a rotary micro-tome. The tissue was then stained using the indi-rect immunohistochemistry method in order to analyze the FGF-2 and VEGF expressions. The anti-FGF-2 antibody used was made in Germany by Antibodies-online.com, while the anti-VEGF antibody used was made by Bioenzy. During the preparations, it was stained with diaminobenzi-dine tetrahydrochloride (DAB). Fibroblast cells that expressed FGF-2 and the endothelial cells that




expressed VEGF at 400 times magnification with a light microscope were counted in a single-blinded manner (Brizeno et al., 2016).

Statistical analysis

The data obtained from the results of the study were analyzed using Statistical Package for the Social Science Software (SPSS) version 23.00. The data is presented in the form of mean ± standard deviation. The One-Sample Kolmogorov Smirnov Test was the homogeneity test used, and the Levene Test was the normality test used. The dif-ferences in fibroblast numbers and FGF-2 and VEGF expressions were analyzed via a one-way ANOVA and post hoc test. If the results showed p<0.05, then the results were considered statistical-ly significant.

RESULTS

Fibroblast numbers

The presence of a fibroblast was confirmed by the presence of the red-stained cytoplasm of a fi-broblast. There was no difference in the number of fibroblasts when topically treating with LS-CS compared to benzydamine hydrochloride and the control in the three-day treatment group (Fig. 1A).

After five days, the fibroblast number of those being topically treated with LS-CS (20.50 ± 1.29) was higher than that of the control group (17.75 ± 1.70; p=0.028) and those being treated with benzy-damine hydrochloride (15.00 ± 1.41; p=0.001). There was also a higher number of fibroblasts after five days of topical treatment with benzydamine hydrochloride compared to the control group (p=0.028). However, after seven days of treatment with LS-CS (22.50 ± 1.29), a higher number com-pared to the control group (18.50 ± 2.38; p=0.016) was demonstrated (Fig. 1B).

FGF-2 expression

FGF-2 expression was confirmed by the pres-ence of brown-stained cytoplasm in the fibroblast cells (Fig. 2A). Topically treating with LS-CS for seven days provided a significant difference in

FGF-2 expression (15.25 ± 1.30) compared to topi-cally treating with benzydamine hydrochloride (8.50 ± 0.96; p=0.000) and the control group (3.25 ± 0.96; p=0.000) (Fig. 2B).

VEGF expression

VEGF expression was confirmed by the pres-ence of brown-stained cytoplasm (Fig. 3A). The VEGF expression of the LC-CS group (7.50 ± 0.85) was no different to the benzydamine hydrochlo-ride (6.75 ± 0.5; p=0.302) and control groups (8.00 ± 0.82; p=0.135) after three days of treatment. After five days, the VEGF expression in oral traumatic ulcers being topically treated with LS-CS (14.00 ± 1.41) was higher than in those treated with benzy-damine hydrochloride (10.75 ± 1.5; p = 0.019) and the control (7.5 ± 0.85; p=0.000). The benzydamine hydrochloride group’s VEGF expression also showed an increase after five days compared to the control group (p=0.001) (Fig. 3B).

After topically treated with LS-CS for seven days, a higher expression of VEGF (12.75 ± 1.70) was exhibited compared to topically treating with benzydamine hydrochloride (10.00 ± 1.41; p=0.019) and the control group (5.00 ± 0.82; p=0.000). After treating with benzydamine hydrochloride for sev-en days, the VEGF expression also showed statisti-cally significant differences compared to the con-trol group (p=0.001) (Fig. 3B).

DISCUSSION

The healing of traumatic ulcers goes through several phases, with healing starting with homeo-stasis and inflammation, then moving on to prolif-eration and ending with the remodeling phase. The homeostasis phase occurs after bleeding, and then coagulation occurs in response to inflamma-tion (Brizeno et al., 2016). The inflammatory phase transpires from when the wound is formed until the fifth day and is characterized by the formation of pro-inflammatory cytokines, such as IL-1β, TNF-α and gamma interferon (IFN-γ), by macro-phages. Macrophages also produce PDGF, TGFβ, FGF and VEGF, which can stimulate the formation of granulation tissue (Brizeno et al., 2016). These




Figure 1. The histological view of fibroblasts in oral traumatic ulcer tissue with hematoxylin-eosin staining after treatment for three, five and seven days in the control (A, D, G), benzydamine hydrochloride (B, E, H) and LS-CS (C, F, I) groups (magnification 400×) (A); The number of fibroblasts (B).

The same character at the top of each bar indicates statistically significant differences in the ANOVA and post hoc LSD test (p<0.05).

cytokines play a role in the proliferation phase, which occurs from day three to 14 after the ulcer is formed. The remodeling phase involves a balance between synthesis and degradation and the for-mation of collagen (Ornitz and Itoh, 2015).

The way the control group was used was based on a study by Surboyo et al. (2019b). A control group was used for this study to prevent the for-mation of pseudomembranous. Benzydamine hy-drochloride was used due to its analgesic effects being similar to those of LS-CS. Benzydamine hy-drochloride does not inhibit cyclooxygenase (COX), but it maintains cell membrane stabiliza-

tion so that arachidonic acid does not come out of the cells and prostaglandins do not form (Gos-wami et al., 2018; Surboyo et al., 2019b). LS-CS contains phenol, which has an antioxidant func-tion that can inhibit the formation of lipid peroxi-dation, which promotes fibroblast activity, that is, fibronectin synthesis, which can promote wound healing (Goswami er al., 2018).

LS-CS’s effect on an oral traumatic ulcer in a di-abetes mellitus rat was exhibited after topical treatment for seven days. LS-CS contains 2-methoxyphenol (guaiacol) and phenols, which have strong antioxidant properties. Phenol, as an




Figure 2. The histological view of FGF-2 expression in oral traumatic ulcer tissue with immunohistochemistry staining after treatment for three, five and seven days in the control (A, D, G), benzydamine hydrochloride (B, E, H) and LS-CS (C, F, I) groups (magnification 400×) (A); The FGF-2 expression (B).


antioxidant, plays a role in inhibiting the for-mation of prostaglandins through the inhibition of cyclooxygenase (Surboyo et al., 2019a). Phenols can also inhibit the formation of lipid peroxidation in fibroblast cell membranes and increase fibron-ectin, causing the proliferation of fibroblasts. Guaiacol can accelerate the inflammation phase by inhibiting pro-inflammatory cytokines and trigger a transfer to the proliferation phase, which is char-acterized by high FGF-2 expression. It is thought that the increase in FGF-2 is able to activate the AKT pathway, which could increase cell survival and stimulates the growth and proliferation of

fibroblasts (Ornitz and Itoh, 2015; Tarawan et al., 2017; Surboyo et al., 2019b).

This study shows an improvement in the VEGF expression in the group treated with LS-CS. LS-CS promotes endothelial proliferation by increasing the VEGF expression via the induction of hypoxia-

inducible factor 1 (HIF-1) to stimulate the pro-duction of VEGF in endothelial cells. Phenol and guaiacol, as antioxidants, play a role in wound healing through the inhibition of lipid oxidation by binding to free radicals produced by the lipid oxidation process (Surboyo et al., 2019a). The inhi-bition of lipid peroxidation can reduce the amount




Figure 3. The histological view of VEGF expression in oral traumatic ulcer tissue with immunohistochemistry staining after treatment for three, five and seven days in the control (A, D, G), benzydamine hydrochloride (B, E, H) and LS-CS (C, F, I) groups (magnification 400×) (A); The FGF-2 expression (B).


of ROS to normal amounts and can also induce

HIF-1, which affects the increase of VEGF expres-sion (Kim and Byzova, 2014). The flavonoid, as it is also known, is one of the components of LS-CS that can increase the VEGF expression through the

inhibition of HIF-1 (Park et al., 2008; Tarawan et al., 2017).

Topically treating with LS-CS for seven days al-so showed a higher expression of VEGF compared to topically treating with benzydamine hydrochlo-ride and the control group. This indicates that LS-

CS has the potential to increase VEGF expression better than distilled water and benzydamine hy-drochloride. This is because LS-CS’s ability to in-

hibit nuclear factor kappa B (NF-B) is superior to that of benzydamine hydrochloride’s (Surboyo et al., 2020). LS-CS is also able to bind to free radicals and inhibit superoxide radicals, which affect the production of pro-inflammatory and pro-angiogenic cytokines, and can stabilize the amount

of HIF-1, which plays a role in increasing VEGF production (Scozzafava et al., 2014; Tan et al., 2019).




A limitation of this study is that it only ana-lyzed the proliferation phase by evaluating fibro-blast numbers and FGF-2 and VEGF expressions. More markers need to be analyzed for it to prove that topical treatment can affect the proliferation phase.

CONCLUSIONS

This research shows that there is an increase in fibroblast numbers and the expression of FGF-2 and VEGF after treatment with liquid smoke coco-nut shell (Cocos nucifera L.). The results show that liquid smoke coconut shell could play a significant role in healing oral traumatic ulcers in people with diabetes mellitus.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

This research was funded by the Ministry Higher of Edu-cation, Republic of Indonesia 2020 (Grant No. 596/UN3.14/PT/2020).

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AUTHOR CONTRIBUTION:

Contribution Ayuningtyas NF Surboyo MDC Ernawati DS Parmadiati AE Hendarti HT Mahdani FY Winias S Zakia F Harianto IA

Concepts or ideas x x

Design x x x

Definition of intellectual content x x

Literature search x x

Experimental studies x x x

Data acquisition x

Data analysis x x

Statistical analysis x

Manuscript preparation x x x

Manuscript editing x x x

Manuscript review x x x x x x x x x

Citation Format: Ayuningtyas NF, Surboyo MDC, Ernawati DS, Parmadiati AE, Hendarti HT, Mahdani FY, Winias S, Zakia F, Harianto IA (2020) The role of liquid smoke coconut shell in the proliferation phase of an oral traumatic ulcer. J Pharm Pharmacogn Res 8(6): 549–557.


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