The effects of fresh leaf-to-water ratio and heating time on the antifungal and antioxidant activities of betel leaf (Piper betle L.) extract

More than half of the Indonesian people use herbal medicines to maintain their health, including ones that are served immediately after preparation or also known as jamu gendong. This plant-based medicine is prepared traditionally by boiling using different ratios of leaves to water and heating time. The study was designed to determine the heating time and the fresh leaf-to-water ratio that exhibited the highest antioxidant and antifungal activities against Candida albicans. Fresh betel leaves were extracted by boiling and infusion methods, with the ratio of 1:5 and 1:10 and the heating time of 5 and 10 minutes for boiling and 15 minutes for infusion. The excess water in the resulting extract was removed using a freeze dryer, then the total phenolic content was determined by FolinCiocalteu spectrophotometry. Meanwhile, the antioxidant and antifungal activities were tested by the DPPH method and microdilution, respectively. The results showed that betel leaf extract with the highest yield and polyphenol content was obtained by infusion with the sample-to-water ratio of 1:10, and this was positively correlated with antioxidant activity against DPPH (IC50= 17.4 ppm) and antifungal effect against Candida albicans (MIC= 0.5%).


INTRODUCTION
Among the Indonesian population, the leaves of betel (Piper betle L.) are known as candidiasis preventive agents. Whether for personal consumption or sale, fresh betel leaves are still processed traditionally by boiling with varying ratios of fresh leaves to water and heating time, producing a beverage called jamu gendong. This herbal remedy can be consumed on its own or combined with other traditional medicines.
According to several studies, betel leaves show antioxidant activity (Ali et al., 2018;Chauhan et al., 2016;Sarma et al., 2018), antibacterial activity against Streptococcus mutans (Thangavelu, 2013;Nalina and Rahim, 2007;Rahim and Thurairajah, 2011), and antifungal activity against Candida albicans (Ali et al., 2010). They contain bioactive components, such as hydroxychavicol, eugenol, isoeugenol, and allyl pyrocatechol 3,4-diacetate (Ali et al., 2018). Temperature and extraction time reportedly affect the resulting extract (Muruganandam et al., 2017). For instance, Soxhlet extraction typically produces ethanol extracts containing hydroxy-chavicol (69.46%), 4-Chromanol (24%), and eugenol (4.86%). Meanwhile, drying at 80 o C can cause the main components of the betel leaf to undergo decomposition, and the yield of extraction using water multiplies as temperature and leaf-towater ratio increase (Pin et al., 2006). This study has examined the effects of heating time and the ratio of fresh betel leaves to water on the total phenolic content, antioxidant activity, and anti-candidiasis activity of betel leaf-based remedies. The result of this study can be applied to the traditional processing of betel leaves in society or by jamu gendong vendors.

Extraction of betel leaves
Fresh betel leaves that had been initially sorted were cut into small sizes of 1-2 cm 2 . These leaves were then extracted using the leaf-to-water ratio of 1:5 and 1:10, and each sample was boiled for 5 and 10 minutes and infused at 90 o C for 15 minutes. The extracts obtained were lyophilized, and the yields were calculated.

Determination of total phenolic content
The total phenolic content was analyzed according to the method proposed by Farmakope Herbal Indonesia (Kemenkes RI, 2011), with gallic acid as the standard. The extract weighing 10 mg was dissolved in 25 mL of methanol. One ml of the solution was pipetted, added with 5 mL of 7.5% Folin-Ciocalteu, allowed to stand for 8 minutes, and added with 1% NaOH. After one hour of incubation, the mixture was measured at  = 755 m.

Determination of antioxidant activity
The antioxidant activity was determined by the DPPH method using a microplate ELISA reader. The extract was dissolved in analytical-grade methanol to obtain 100 ppm concentration. Samples with varying volumes, namely, 10, 20, 30, 40, 50 µl, were placed into the well. Then, each well was added with 60 µl of 40 mM DPPH solution, and the volume was filled up to 200 µl with DPPH solution. After 30-minute incubation at room temperature, the absorbance of each sample was analyzed at  of 515 nm. The percentage of DPPH scavenging was calculated using the formula below: % DPPH Radical Scavenging = (Ablank -Asample)/ Ablank x 100% Meanwhile, the IC50 was computed based on the regression equation y = ax +b.

Determination of antifungal activity against Candida albicans
The antifungal activity against C. albicans was evaluated based on the minimum inhibitory concentration (MIC) by microdilution, in which a 48-well plate containing potato dextrose broth with turbidity equivalent to 1% of the McFarland standards was incubated for 2 x 24 hours.

Data Analysis
The average yield of the extract and the total phenolic content (TPC) of the lyophilized aqueous betel leaf extract were first expressed in mean ± SD. These data were then statistically analyzed by the Student's t-test, and the results were considered significant when P<0.05.

RESULTS AND DISCUSSION Extraction yield and total phenolic content
This study analyzed the fresh betel leaves used commonly by the community, including jamu gendong vendors. A hundred leaves with a length of 10-15 cm and width in the range of 7.5 and 11 cm were sorted and placed on a scale. These leaves, which were averagely 232.12 g in weight, were then washed, drained, and cut into small sizes of 1-2 cm 2 . Afterward, the leaves were grouped based on treatments and the independent variable, i.e., the ratio of leaf to water (1:5 and 1:10). The extraction yield and total phenolic content of the aqueous betel leaf extract obtained by boiling (5 and 10 minutes) were compared with those produced by 15 minutes of infusion. The results of both techniques were then filtered and lyophilized, and the characteristics of the extract obtained by boiling and infusion were evaluated (Table I). Note: A= Lyophilizate extracted by 5-minute boiling, with leaf-to-water ratio 1:5 B = Lyophilizate extracted by 10-minute boiling, with leaf-to-water ratio 1:5 C = Lyophilizate extracted by 15-minute infusion, with leaf-to-water ratio 1:5 D = Lyophilizate extracted by 5-minute boiling, with leaf-to-water ratio 1:10 E = Lyophilizate extracted by 10-minute boiling, with leaf-to-water ratio 1:10 F = Lyophilizate extracted by 15-minute infusion, with leaf-to-water ratio 1:10 Compared to 1:5, the 1:10 ratio created a higher yield of extract potentially due to the low or insoluble chemical components in water, indicating that a higher water volume is needed. Also, with this 1:10 ratio, 15 minutes of heating at 90 o C in the infusion method resulted in higher yields. The highest phenolic content (27.28%) was found in Sample F, which was extracted by infusion with the 1:10 ratio. However, this result does not differ significantly from that of Sample B (extraction by 10 minutes of boiling with 1:5 ratio) and Sample D (extraction by 5 minutes of boiling, with 1:10 ratio). Among the above treatments, infusion with the leaf-to-water ratio of 1:10 provided the highest yield. Multilevel extraction of betel leaves using hexane, ethyl acetate, methanol, and water has proven that the highest total phenolic content is found in the ethyl acetate extract, followed by the hexane, methanol, and lastly water extracts (Abrahim et al., 2012). A study of the nutraceuticals properties of Piper betel (Paan) (Chauhan et al., 2016) also confirms that, in the order of highest to lowest, phenols can be found in methanol, ethanol, ethanol-water, and water extracts. Another study reports that the water extract of dried betel leaves that have been macerated for 4 hours with hot water has a total phenolic content of 128.89 mg/g extract or approximately 12.9% w/w (Taukoorah et al., 2016). Furthermore, boiling fresh betel leaves with the leaf-to-water ratio of 1:50 for 30 seconds produces extracts containing a total phenolic compound of 676 mg per 100 g of fresh leaves (0.68%) (Tan et al., 2014). In other words, the total phenolic content of betel leaf extract varies depending on the extraction method, the solvent used, and the leaf-to-water ratio. Despite the use of water as a solvent, this study obtained relatively high levels of phenolics. This finding is potentially caused by the manner of heating and the use of fresh leaves. The content of the hydroxy catechol in betel leaf can be increased by raising the extraction temperature to 60 o C with an equilibrium point of 30 minutes; drying at 50-60°C is found to be optimum, whereas rehydration at 40°C is claimed to produce the best or most acceptable product (Pin et al., 2006;Balasubramanian et al., 2011).

Antioxidant activity
The in vitro test by the DPPH method revealed that the highest antioxidant activity was exhibited by Sample F (extracted by infusion with the 1:10 ratio). A smaller IC50 value can lead to a stronger antioxidant property, which is associated with total phenolic compounds. Sample F showed a relatively high total phenolic level, and the lowest IC50 value against DPPH was compared to the other samples.  indicates antioxidant effects against DPPH (Sharma et al., 2010). Furthermore, betel leaf extract has a proton-donating ability and can serve as a free radical inhibitor or scavenger (Umar et al., 2018).

Minimum inhibitory concentration (MIC)
Based on the results of the antifungal activity test against C. albicans (microdilution), the lyophilized aqueous betel leaf extract has the MIC of 0.5%-1%, as shown in Table III  Boiling and infusion using the leaf-to-water ratio of 1:10 exhibited higher antifungal activities against Candida albicans, with the MIC of 0.5% (Figure 2 and Table III). Although, Sample F (infusion with a 1:10 ratio) has the same MIC as Samples D and E, at a concentration of 0.25%, the growth of C. albicans in Samples D and E was higher than in Sample F ( Figure 2); therefore, this study recommends infusion for optimal fresh betel leaf extraction. On the other hand, boiling dried betel leaves using distilled water with the leaf-to-water ratio of 1:10 for 5-6 hours produces extracts with a higher MIC, that is, 12 mg/mL (1.2%) (Himratul-Aznita et al., 2011). Ali et al. (2010 suggest that hydroxychavicol isolated from aqueous betel leaf extracts inhibit the biofilm formation by several Candida albicans isolates, with an MIC of around 125-500 ppm.

CONCLUSION
With 1:10 leaf-to-water ratio and 15 minutes of heating, extraction by infusion produces betel leaf extracts that have the highest antioxidant and antifungal activities against Candida albicans.