Effect of pH variation on chemical components and antifungal activity of ketepeng cina (Cassia alata L.) leaves extract

Ketepeng cina (Cassia alata, L.) has been widely used as a traditional medicine, one of which is as an antifungal. Previous research showed problems with the storage of ketepeng cina leaves extract, which reported changes in pH and color over time. This study explored the effect on concentration of chemical components and antifungal activity under varying pH. Antifungal activity against Candida albicans was tested using microdilution and well diffusion methods. HPTLC technique was measured the chemical components of the extract represented by aloe-emodin and kaempferol concentration. The extract with pH 4.5 had the highest aloeemodin content and antifungal activity. The result of our research were the pH variation significant influence on anti fungal activity against C.albicans, where the best results are at pH 5.5 (p<0.05). The level of active components in the extract is influenced by the pH conditions (p<0.05). Specifically, the levels of kaempferol and aloe-emodin are maximum at a pH of 5.5. It can be concluded that pH can affect the levels of chemical components and antifungal activity in ethanol extracts of ketepeng cina (Cassia alata, L.) leaves.


Introduction
One of the illnesses brought on by fungi in many tropical nations, including Indonesia, is candidiasis.Indonesia's relatively high air temperatures and humidity make it easy for people to perspire and get their skin wet, which can lead to the development of fungi on the skin.The fungus C. albicans is the primary cause of candidiasis [1].Numerous candidiasis cases in Asia reveal that, on average, 56% of the time, C. albicans is the species found in cases of candidiasis [2].Ketoconazole is one of the antifungal medications frequently used to treat candidiasis.Antifungal medication use for a long period of time, however, may result in resistance and side effects, including diarrhea, nausea, vomiting, and headache [3].
Traditional medicine made of natural ingredients is thus a treatment option for candidiasis.Traditional remedies have few negative side effects and do not lead to resistance.C. alata leaves can be used to treat pinworms and fungal infections on the skin, such as tinea versicolor, ringworm, eczema, thrush, and itching [4].Based on our initial research, the activity of ethanol extracts of C.alata leaves against C. albicans fungi shows that ethanol extracts of C.alata leaves at a concentration of 1.25% produce an inhibition zone of 18.0 ± 0 mm, where the value is greater than the 2% ketoconazole inhibition zone, which is 11.0 ± 0 mm.Empirically, C. alata leaves are widely used to treat various skin diseases, allegedly because there are active secondary metabolite compounds that can inhibit the growth of fungi and bacteria.Therefore, further research into these pH changes is necessary.C.alata leaves contain secondary metabolite compounds, such as tannins, flavonoids, alkaloids, saponins, phenols, anthraquinone compounds (rein aloe-emodin, emodin diantron), chrysophanic acid (dihydroxy methyl anthroquinone), terpenoids and steroids.Aloe-emodin and kaempferol are the main components and can be used as marker compounds in C. alata leaves [5] Numerous factors, such as the concentration of antifungal substances, the type, quantity, age, state, temperature, contact time, and chemical and physical characteristics of the growth media, such as pH, moisture content, nutrients, and the number of components in it, all have a significant impact on the ability of antifungal substances to inhibit fungal growth [6].The gel preparation of an ethanol extract of C. alata leaves experienced discoloration and a reduction in pH during storage.This is due to the ethanol extract of C. alata leaves commonly having an acidic pH.The aim of this study is to see how much of the pH change affects the level of active compounds and anti-fungi activity against Candida albicans.

Process
2.2.1.Preparation of pH Variation Solution of Ethanol Extract of C. alata Leaves.A 1,25% extract solution, then either 0,1 M HCl or 0,1% TEA was added drop-wisely onto the extracts in different sets of test tubes adjusted to various ranges of pH (pH 4,5, pH 5,5, and pH 6,5).

Agar well diffusion method.
The positive control used 2% ketoconazole, and the negative control used distilled water.Put 20 μL of sample into each well.Then, the petri dish was incubated at 30°C for 48 hours.On the surface of the media, there will be a clear area around the wells called the inhibition zone, where this inhibition zone is measured using a caliper.

Minimum Inhibition Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
test.The test was performed by using the round bottom 96-microtiter plate.Extracts were diluted to 1,25%; 0,62%; 0,31%; 0,16%; 0,08%; 0,04%; 0,02%; 0,01%; and 0,005%, respectively in saboraud dextrose agar and incubated at 30℃ for 48 h.Solutions of ketoconazole 2% were included in each experiment as the positive control.The solutions of dimethyl sulphoxide 1,25% were used as negative controls.The solutions of hydrochloric acid 0.1 M and triethanolamine 0.1% as a pH control serve to determine that the pH control solution has no antifungal activity.Wells containing media and C.albicans suspension as a control of fungal growth.The MIC was defined as the minimum concentration of extracts at which no observable growth occurred.To ascertain the MBC, 3 loops of culture were extracted from each broth tube in which no growth was observed at the MIC concentration.These culture samples were then inoculated onto fresh Saboraud Dextrose Agar (SDA) plates.Following a 48-hour incubation period, the plates were examined for any signs of growth.The concentration of the extracts that resulted in no growth was documented as the MBC.Based on testing the antifungal activity of ethanol extracts of C.alata leaves using diffusion method showed on Figure 1, the diameter of the inhibition zone of the extract with pH 4.5 ± 0.2 is greater than the diameter of the inhibition zone of the initial pH (pH 5.32), extract with pH 5.5 ± 0.2, extract with pH 6.5 ± 0.2.The activity of phytochemical compounds in plant extracts can increase in acidic conditions [7].The inhibition zone formed can be associated with one of the active compounds contained in antifungal substances [8].The antifungal effect of kaempferol against C. albicans occurs by inhibiting nucleic acid synthesis in fungal cells [9].The antifungal activity of kaempferol compounds produces MIC values of 256 μg/mL -512 μg/mL against C. albicans [10].Testing the ethanol extract of C.alata leaves using the liquid microdilution method is used to determine the MIC and MBC values in a sample quantitatively.In this test, the highest concentration will be used at 1.25% as seen on Figure 2. The microdilution test results in Table 2, ethanol extract of C. alata leaves with pH 4.5 ± 0.2 has the highest antifungal activity with MIC of 0.08% and MBC of 0.16%.Microorganisms have an optimum pH to grow best and a minimum pH (acid) that causes their growth to be inhibited.[11].Other research results, states that the antimicrobial activity of Cosmos caudatus extract increases at an acidic pH (pH 3) [12].The reduction in Salmonella sp. is faster at room temperature at acidic pH (pH 3.3-3.4)[13].The antimicrobial activity of Allium sativum extract and Zingiber officinale extract increased in an acidic atmosphere [14].Data analyzed using the Normality test resulted in a normal distribution (p > 0.05), while the results of the Homogeneity test showed that the data had an inhomogeneous variety (p < 0.05).Because of this normal and inhomogeneous data, the requirements for conducting the ANOVA test are not met.Data can be analyzed using the Kruskal-Wallis test, which shows a p = 0.003 (p < 0.05).Then, continued with the Mann-Whitney test, which stated significant differences between treatment groups except in the negative control and solvent groups and the pH control and pH 5.5 extract groups.

Content of Main Chemical Components in C. alata Extract
A standard curve was made to measure the levels of aloe-emodin and kaempferol.Compound levels can be calculated by entering the area obtained into the line equation on the standard calibration curve as seen on Figure 3.
Figure 3 shows the standard calibration curve of aloe-emodin and kaempferol.In the aloe-emodin standard calibration curve, the linear regression of the relationship between concentration and the standard area is y = 2.10-5-05x + 0.0006, where x is the concentration and y is the area.The regression coefficient (R 2 ) obtained is 0.9942, where this value is good, in the range of 0.9˂R 2 ˂1.As for the kaempferol standard calibration curve, the linear regression relationship between the concentration and area of the standard solution is y = 6.10-5-05x -0.0033.The regression coefficient (R 2 ) obtained was 0.9961.The response given by the device to the analyte concentration has met the requirements set, namely R 2 >0.99.Thus, it can be said that the device is in good condition, and the straight-line equation obtained can be used to calculate the sample concentration.Based on the results obtained in Table 3 and Figure 4, ethanol extract of C.alata leaves with pH 4.5 ± 0.2 contains the highest aloe emodin content with an average of 49.5 μg/mL.This is possible because hydrolysis occurs in extracts with an acidic atmosphere (pH 4.5), making its activity more active at pH 4.5 ± 0.2 than at other higher pH [15].This is in line with the research of [16], which shows that aloin compounds undergo oxidative degradation into derivatives, namely aloe-emodin and 10-hydroxyaloin A and B, where aloe-emodin is formed at pH 5 and below, while 10-hydroxyaloin A and B are formed at alkaline or neutral pH.Thus, more aloe-emodin compounds are formed in extracts with acidic pH, and the levels are higher.This is reinforced by research conducted by [17], which shows that aloin A and B decompose faster in alkaline pH solutions than solutions with acidic pH.In addition, this study also tested the kaempferol content of ethanol extract of C. alata leaves as seen on Figure 5. Based on the results obtained in Table 4, ethanol extract of C.alata leaves with pH 4.5 ± 0.2 contains the highest kaempferol content with an average of 199.7778 μg/mL.Kaempferol undergoes autooxidation in alkaline solutions and is oxidized by oxygen (O2) at pH seven, which causes the fragmentation process to run fast.These high levels of aloe-emodin and kaempferol can affect antifungal activity, where the extract with pH 4.5 ± 0.2 has the largest inhibition zone.The hydroxyl group in kaempferol will bind to the phospholipid membrane of fungal cells, increasing membrane permeability and eventually damaging the cells.
Research on the effect of pH on kaempferol levels has not been widely studied.Other research that supports the research conducted by [18] showed that karamunting (Rhodomyrtus tomentosa) fruit extract is used as a pH indicator because it contains anthocyanin flavonoid compounds, which are natural dyes in plants that can produce red to purplish pigments according to pH conditions.The occurrence of color changes is due to changes in the anthocyanin structure due to the influence of H+ and OH-ions.The extraction process of flavonoid compounds should be done in an acidic atmosphere [19].This is because acid plays a role in denaturing plant cell membranes, causing anthocyanin pigments to leave the cell and preventing flavonoid oxidation.Higher acidity levels will cause an increase in the amount of anthocyanin pigments in the form of colored flavilium or oxinium cations.In addition, research by [20], which discusses the effect of pH on delphinidin anthocyanin compounds in rosella petals, states that the compound can dissolve in acids and is unstable in neutral or alkaline solutions.Note.The concentration of all extracts was 1.25% The results of this study are also supported by statistical analysis using the One Way ANOVA test followed by the Tukey test to determine the real differences between treatments.The One-way ANOVA test shows that pH influences aloe-emodin and kaempferol levels, which significantly differ between treatments with a significance value of p = 0.000 (p < 0.05).The results of this study could respond to an obstacle our initial research related to the storage of preparations of ethanol gel extract leaves C. alata which also turned out to cause changes in anti-fungal activity against C. albicans.

Conclusion
The pH of the extract can influence the levels of chemical components and antifungal activity of an extract.Ethanol extract of C. alata leaves with pH 4.5 ± 0.2 in the well diffusion and microdilution methods showed the highest antifungal activity results.Testing the effect of pH on the levels of chemical components, namely aloe-emodin and kaempferol, has the highest levels in ethanol extracts of C.alata leaves with pH 4.5 ± 0.2.

Table 1 .
Well diffusion test result of ethanol extract of C.alata leaves

Table 2 .
Microdilution test result of ethanol extract of C.alata leaves

Table 3 .
Aloe-emodin content in ethanol extract of C. alata leaves

Table 4 .
Kaempferol content in ethanol extract of C. alata leaves