The Spray of Pegagan Leaf Extract as an Antifungal of Vulvovaginal Candidiasis: A Narrative Review

Vulvovaginal Candidiasis (VVC) is a type of infection caused by the fungus Candida spp. The treatment of candidiasis usually uses antifungal drugs against Candida albicans. Pegagan (Centella asiatica (L.) Urban) is one of wild plants that have been used by the community as a drug. The secondary metabolite compounds found in pegagan, such as triterpenoids, alkaloids, flavonoids, and saponins can act as antifungal agents. A literature review of national, international journals and digital books originated from various sites was carried out online. The result of the narrative analysis showed that the ethanol extract of pegagan leaves with a concentration of 75 x 10 3 ppm can inhibit the growth of C. albicans. The results of the toxicity prediction with three parameters showed that the active compounds of pegagan leaves are weak inhibitors, non-carcinogenic and in the toxicity test, it at most belongs to category III. In addition, the spray formulation with a concentration of 1% (w/w) of pegagan leaf extract was found to be safe and non-irritant to skin.


INTRODUCTION
The feminine area is one of the sensitive parts of women which is prone to health problems, such as infections caused by bacteria, fungi, or parasites.Vulvovaginal candidiasis (VVC) is a type of infection caused by fungus Candida spp.VVC is characterized by infection of the genital mucosa, particularly the vulva and vagina.Common clinical symptoms found are itching, burning, pain, and redness which is often accompanied by whitish vaginal discharges (Willems et al. 2020).
Vulvovaginal candidiasis is a type of infection in feminine area that often occurs, especially to women of reproductive age.It is estimated around 70-75% of reproductive age women have experienced VVC once in their lifetime and 40-50% of them tend to experience recurrence (Brandolt et al. 2017).According to Willems et al. (2020), repeated VVC which occurs three times a year is experienced by almost 8% of women globally.
Almost 80-90% of vulvovaginal candidiasis is caused by Candida albicans which is a normal microbiota of the vagina.The infection occurs usually caused by various predisposing factors that support the growth of fungi (Willems et al. 2020).According to Brandolt et al. (2017), several exogenous factors that influence are the increase of climate, heat, and humidity and poor hygiene.
The treatment of candidiasis usually uses antifungal drugs on Candida albicans.There are four categories of commonly used antifungal drugs, namely echinocandins, polyenes, azoles, and fluoro-pyrimidines.Excessive use of these drugs can cause side effects, such as nausea, vomiting, diarrhea, and even resistance.Amphotericin B and nystatin in long-term use can also cause kidney damage (Ksiezopolska and Galbadon 2020).
Pegagan (Centella asiatica (L.) Urban) is one of wild plants that have been used by the community as a medicinal plant either in the form of fresh, dry, or concoction.The secondary metabolite compounds found in pegagan, includes triterpenoids, alkaloids, flavonoids, and saponins.The saponin compounds in pegagan extract are known to have antifungal activities and hinder the growth of microbes by destroying the cell membrane of the organism's tissue.The terpenoid can interfere with the fungal cell wall by inhibiting the synthesis of 1,3-β-Dglucan for the fungal cell to become lysis (Gintjee et al. 2020).Triterpenes are a heterogeneous group of bioactive compounds with a structure consisting of triterpene agiterones (sapogenins) and one or more sugars that bind to acetal glycosidic (ester).Triterpenoid compounds are bioactive which can inhibit the growth of microbes including fungi (Yusuf et al. 2017).Based on the content of secondary metabolites, pegagan leaf extract has the potential as an antifungal particularly on Candida albicans.Therefore, the spray of pegagan leaf extract is expected to be a practical and effective preparation in overcoming vulvovaginal candidiasis.
Studies on pegagan leaves extract and their bioactive potential have been conducted.However, there are no reviews that specifically analyze the role of focused on analyzing the antifungal potential of pegagan leaf extract against the fungus Candida albicans which causes vulvovaginal candidiasis and observing a spray dosage formulation for its application.

METHODOLOGY
The methods used are review of literature in the form of national, international journals and digital books originated from various sites, such as ResearchGate, PubMed, Science Direct, NCBI, Elsevier, and Gramedia Digital.Other than that, predictive analysis of active compounds is carried out using the PubChem and admetSAR1 sites to support the data obtained.The keywords used in the literature searching process, namely the Candida cell membrane, Centella asiatica active compound, inhibition of Candida albicans, toxicity, pegagan spray and skin irritation.

Pathogenesis of Vulvovaginal Candidiasis
Vulvovaginal candidiasis (VVC) is a superficial infection mostly caused by Candida albicans.Broadly speaking, the process of VVC starts from the presence of predisposing factors that make the C. albicans easier to attach to mucosal epithelial cells to form colonization.Furthermore, the fungus will release keratolytics which hydrolyze the phospholipids of the epithelial cell membrane, thereby facilitating invasion of the tissue.In the tissue, C. albicans will secrete neutrophil chemotactic factors which cause acute inflammatory reactions and manifest as areas of hyperemia or erythema in the vulva and vaginal mucosa.The keratolytic substances that are released by Candida will continue to damage the mucosal epithelium, causing shallow ulcers which become heavier by scratching, resulting in erosion.The rest of the necrotic tissue, epithelial cells, and fungi will form white lumps called whitish vaginal discharge (Willems et al. 2020;Brandolt et al. 2017).Pathogenesis of C. albicans is strongly influenced by changes in the commensal form of fungi into hyphae during the colonization process (Mba and Nwaze, 2020).

Antifungal Mechanism of Action
The cell wall of a fungus is composed of mannoproteins, β-glucans matrix, and a phospholipid bilayer whose main component is ergosterol (Freiesleben and Jager 2014).According to Ksiezopolska and Gabaldon (2018), C. albicans cell walls and membranes are common targets for commercial antifungal drugs.

The Active Compounds of Pegagan Leaf
According to the results of a review by Gray et al. (2018), there are 57 active compounds of pegagan leaves which can be seen in (Table 2).A literature study of pegagan leaves extracted with different solvents (Table 3) shows different active compound contents due to the influence of solvent polarity.A compound will dissolve in a solvent that has the same polarity (Leksono et al. 2018).Among the six solvents used, ethanol produced the maximum bioactive compound, while hexane produced the minimum bioactive compound.The content of active compounds of pegagan plant is very high and has an important role in medicinal applications, namely triterpenes (Senthilkumar 2018).Triterpene in pegagan contains many compounds including a siatic acid, madecassic acid, asiaticoside, madecassoside, brahmoside, brahmic acid, brahminoside, thankiniside, isothankunisode, centelloside, madasiatic acid, centic acid, and cenelli acid (Seevaratnam et al. 2012).Apart from triterpenes, pegagan also contains high total phenolics derived from flavonoid derivatives, such as quercetin, kaempferol, patuletin, rutin, apigenin, castilliferol, castillicetin, and myricetin (Orhan 2012).
Terpenoids can interfere with the fungal cell wall formation by inhibiting the synthesis of 1,3-β-D-glucan for the fungal cells to become lysis (Gintjee et al. 2020).Derivative compounds, such as saponins, exhibit antifungal activity by damaging the cell membrane of the fungus (Freiesleben and Jager 2014).Flavonoid is a substance that is known to have antibacterial and antifungal properties (Mickymaray 2019).In general, the way flavonoids work in inhibiting fungal growth includes disrupting the integrity of cell membranes or mitochondrial function and inhibiting cell wall formation, cell division, and RNA and protein synthesis (Al Aboody and Mickymaray 2019).
Flavonoid derivatives, such as quercetin, have been reported as strong inhibitors of the growth of Candida albicans (Li et al. 2012).Research by Bitencourt et al. (2013), also stated that quercetin has antifungal properties and works synergistically with fluconazole in inhibiting the action of the fatty acid synthase enzyme which plays an important role in the synthesis of endogenous fatty acids in fungal cell membranes.Apigenin is known to have antifungal activity by inhibiting biofilm formation and stimulating disruption of the fungal cell membrane resulting in a decrease in cell size and leakage of intracellular components (Lee et al. 2018).

The Inhibition of Candida albicans
The inhibition of Candida albicans by pegagan leaf extract (Table 3) generated with various solvents, such as hexane, chloroform, ethyl acetate, ethanol, petroleum ether, and distilled water.A comparison to the inhibition of Candida albicans is nystatin, one of the commercial antifungal drugs commonly used to inhibit Candida albicans.Most of the extracts of pegagan show an inhibitory power against Candida albicans, except at concentrations of 62.5 ppm and 25 x 10 3 ppm from various extracts and hexane extracts with a concentration of 50 x 10 3 diameter of the antifungal inhibition zone are zero.
The comparison for the inhibition of Candida albicans called nystatin shows the inhibition zone diameter of 17.35 mm (Balafif et al. 2017).Pegagan leaf extract which has an inhibition zone diameter approaching the nystatin inhibition zone, or even higher, are the ethanol extract of pegagan leaves with a concentration of 75 x 10 3 ppm resulted in 17.5 mm inhibition, the ethanol extract of pegagan leaves with a concentration of 100 x 10 3 ppm produced 21.5 mm inhibition, and the ethyl acetate extract of pegagan leaves with a concentration of 100 x 10 3 ppm generated in 18.4 ppm inhibition.
According to Senthilkumar (2018), the ethanol extract of pegagan shows maximum inhibition on Candida albicans, causing at a lower concentration of 75 x 10 3 ppm, the ethanol extract of pegagan has an inhibition zone diameter that is scarcely different from the commercial drug, nystatin.Meanwhile, the ethanol and ethyl acetate extract of pegagan leaves have a concentration of 100 x 10 3 ppm.Even though it has a higher inhibition zone diameter, the required concentration is high.

Toxicity Prediction Analysis
The toxicity analysis of 57 active compounds of pegagan leaves showed that there were 40 active compounds detected by PubChem which were then analyzed by the admetSAR site.The toxicity analysis focuses on three parameters, namely carcinogenicity, Human ether-a-go-go-related gene (hERG), and acute oral toxicity.Carcinogenicity is the ability of a substance or compound to form cancer (Astutiningsih et al. 2010).The results of the toxicity test analysis (Table 4) showed that 40 active compounds of pegagan leaves are non-carcinogenic indicating that they do not have the potential to form cancer.
Human ether-a-go-go-related gene (hERG) is a gene related to encoding the pore formation subunit of the K + channel which plays an important role in the repolarization of the heart muscle.The inhibition or reduction of hERG activities causes loss consciousness and sudden death that occurs in patients with cardiac ischemia (Lamothe et al. 2016).The results of the toxicity prediction in (Table 4) showed that the active compounds of pegagan leaves are weak inhibitors of hERG.
Acute oral toxicity is an essential test to observe the toxicity of a drug or compound when it enters the digestive system within a certain time after giving a single dose (Zulfiana, 2014).The level of toxicity is divided into 5 categories based on the LD 50 score, including category 1 (≤5 mg/kg), category 2 (5 mg/kg < LD50 ≤ 50 mg/kg), category 3 (50 mg/kg < LD50 ≤ 300 mg/kg), category 4 (300 mg/kg < LD50 ≤ 2000 mg/kg) and category 5 (2000 mg/kg < LD50 ≤ 5000 mg/kg) (Son and Yen, 2014).The results of the toxicity prediction show that all ligands belong to category III, except for campesterol, sitosterol, and stigmasterol which are classified as category I, quercetin, myricetin, kaempferol, and chavicol are classified as category II and Epicatechin belongs to category IV.

The Spray Formulation of Pegagan Leaf Extract
The spray formulation used refers to the research of Sawatdee et al. (2016) and the results of a literature review on the inhibition of Candida albicans.The formulation uses pegagan leaves ethanol extract with a concentration of 75 x 10 3 ppm.The additional ingredients are HP-β-CD, eudragit E100 and copovidone as a polymer coating, glycerol and PEG 400 as a humectant, and ethanol and distilled water as solvents.
The process of making spray begins by mixing 1% (w/w) the extract and 2% HP-β-CD into 7% distilled water.Furthermore, 2% eudragit E 100 and 6% copovidone are dissolved in 70% absolute ethanol.Both solutions are mixed and stirred at a speed of 300 rpm until it is clear.After that, add 10% PEG 400 and 5% glycerol and stir until the mixture is homogeneous.This formulation is used as a reference as it has the best physical properties (appearance, pH value and viscosity) and spreadability (Sawatdee et al., 2016).

Skin Irritation Analysis
The analysis of the impact of spray from pegagan leaf extract on the skin carried out by Sawatdee et al. (2016) showed that no edema was detected in primary skin irritation studies on selected formulations which also have the potential to be used as formulations for spraying pegagan extract for Candida albicans antifungal.Therefore, pegagan leaf extract was found to be safe and non-irritant to skin.
In conclusion, The ethanol extract of pegagan leaves with a concentration of 75 x 10 3 ppm had the best inhibition against Candida albicans at 17.55 mm.The content of secondary metabolites of ethanol extract of pegagan, such as flavonoids, terpenoids, alkaloids, and saponins, have antifungal properties that work in different ways.The spray of pegagan leaf extract with an extract concentration of 1% (w/w) and several additional ingredients, such as HP-β-CD, eudragit E100, copovidone, glycerol, PEG 400, ethanol, and distilled water have the physical properties (appearance, pH value and viscosity) and the best dispersibility and are safe for the skin.

Figure 1
Figure1The structure of the cell walls and membranes of fungi (Freiesleben and Jager 2014).

Table 2
The Active compounds of Pegagan Leaves and inhibition of Candida albicans

Table 3
Toxicity prediction results