AIP Conference Proceedings 1933, 020005 (2018); https://doi.org/10.1063/1.5023939 1933, 020005
© 2018 Author(s).
Selection of discrimination marker from various propolis for mapping and identify anti Candida albicans activity
Cite as: AIP Conference Proceedings 1933, 020005 (2018); https://doi.org/10.1063/1.5023939 Published Online: 13 February 2018
Alfiani Guntari Mahadewi, Daisy Christina, Heri Hermansyah, et al.
ARTICLES YOU MAY BE INTERESTED IN
Selection of propolis Tetragonula sp. extract solvent with flavonoids and polyphenols concentration and antioxidant activity parameters
AIP Conference Proceedings 1933, 030020 (2018); https://doi.org/10.1063/1.5023967
Indonesian honey protein isolation Apis dorsata dorsata and Tetragonula sp. as antibacterial and antioxidant agent
AIP Conference Proceedings 1933, 030007 (2018); https://doi.org/10.1063/1.5023954
Microencapsulation of ethanol extract propolis by maltodextrin and freeze-dried preparation AIP Conference Proceedings 1933, 020012 (2018); https://doi.org/10.1063/1.5023946
Selection of Discrimination Marker from Various Propolis For Mapping and Identify Anti Candida albicans Activity
Alfiani Guntari Mahadewi
1, Daisy Christina
1, Heri Hermansyah
1, Anondho Wijanarko
1, Siti Farida
2, Robiatul Adawiyah
2, Etin Rohmatin
3, and Muhamad
Sahlan
1,4,a)1Departement of Chemical Engineering, University of Indonesia, Depok, 16424, Indonesia
2 Midwifery Departement of Health Polytechnic Republic of Indonesia’s Health Ministry Tasikmalaya
3Faculty of Medicine, University of Indonesia, Depok, 16424, Indonesia
4Research Center for Biomedical Engineering (RCBE) Universitas Indonesia Kampus Baru UI Depok 16425 Indonesia
a)Corresponding author: [email protected]
Abstract. The increase in fungal resistance against antifungal drugs available in the market will reduce the effectiveness of treatment for Candidiasis. Propolis contains various compounds with antifungal properties Candida albicans, but the content of each type is very diverse. The sample used was Sulawesi propolis type smooth (taken from inside the nest), rough (taken from outside the hive) and mix (a combination of both). Anti-C. albicans molecule marker is a marker compound for selecting propolis with the ability to overcome Candidiasis. The initial step was to test the levels of flavonoids and phenolic by using UV-Vis spectrometry method. It was founded that each sample was not always superior to any substance, so propolis cannot be directly selected. In Phenolic content, mix propolis has the highest value than other 5.109%. In Flavonoid content, propolis smooth has the highest value than other, 16.38%. Furthermore, propolis selected by antifungal activity test with good diffusion method at the concentration propolis 5% either 7%, the inhibitory diameter zone propolis smooth and rough has same value 10 mm. Propolis mix has an advantage while propolis smooth and rough have the same capability range 12 mm and 13 mm. In this study, the phenolic content plays a major role in antifungal cases.
Keywords: Candida albicans, Molecular Marker, Diffuse well
INTRODUCTION
Candidiasis was caused by a common pathogenic fungus called Candida albicans. About 15% of infectious diseases were caused by fungi, and 70-90% of these fungal infections were categorized as the case of candidiasis [1].
Treatment of Candidiasis or Candida albicans fungal diseases usually use an anti-fungal therapy that requires time and high cost. Increased fungal resistance towards existing antifungal drugs can reduce the effectiveness of treatment for Candidiasis [2-4] therefore, required the new active compound to form the new drug.
Natural products are still a favorite as a source of new molecules for the latest formulation of drugs. Recently, researchers have focused on microbial extracts, plants, essential oils, secondary metabolites or new molecular syntheses that have been subjected to researchers as new antifungal and antimicrobial agents. Of the several types of natural products, propolis was known to have properties of antifungal compounds are quite effective. On the other hand, Indonesia as a tropical country has great potential for a diverse amount of propolis. Propolis from Sulawesi was available in large quantities compared to other regions. However, there were still few local researchers who examine the potential of propolis.
Many reviews about potential propolis as antifungal agent and report with a chemical compound it has, the content
However, on the other hand, the composition of the propolis content varies depending on the vegetation of the resin consumed by bees, in fact, the difference location of propolis in one nest has different characteristics. There were two kinds of propolis from one nest that we called the fine and rough propolis. Fine propolis originating from within the nest has distinct characteristics with the propolis of the carri on located outside the nest called rough propolis. The absence of clear standards regarding the function of propolis due to differences in the content.
The research on standardization of propolis function was required as the standard, in this case, to determine the role of propolis as an anti-Candida albicans. The purpose of this research was to know the relation between the quantitative amount of phenol and flavonoid in Sulawesi propolis wax and its function to anti- C. albicans. By observing the quantitative amount and antifungal of these molecules (phenol/flavonoid) in other samples, it was expected to illustrate the ability of these samples for candidiasis drugs. The process of classifying propolis based on the that will be the basis of selection of quality propolis to be an active ingredient for candidiasis drug. Then its potential to screening propolis which has strong anti-fungal for making best and safe drug to fight c. albicans.
METHOD Material
The main materials used in this study were Sulawesi propolis wax from RIN Biotech. Propolis wax consists of smooth propolis from inside hive with a soft texture or commonly called by the name of regular propolis; rough propolis originating from outside the hive with a rough texture and commonly referred to as coral; and a combined propolis mix of both with a 6: 4 ratios. Propolis wax is the residue of the propolis extraction process, so there was still a resin from the propolis. It was a mixture of different elements of propolis and has a more complex element of beeswax [8]. The ingredients in the wax propolis include resins from propolis, beeswax, plant wax, and essential oils from plants. The material used for testing the levels of flavonoids and phenols consist of the standard quercetin and gallic acid obtained from Sigma-Aldrich. Antifungal activity test used Mueller Hinton agar and a commercial disk containing Nystatin antifungal compounds.
Method
This research was divided into two main procedures. The first procedure was to analyze the quantitative content of Phytochemistry properties like flavonoid and phenol from propolis wax; these properties were the main properties of an antifungal agent. This test uses the colorimetric method with UV-Vis spectrometer. The second was to analyze the ability of the antifungal activity by the diffusion method so that it has been by CLSI (Clinical and Laboratory Standards Institute) standards to obtain the inhibitory diameter.
Quantitative Phenolic Content
Total phenol content in each extract was determined using the Foline Ciocalteu reagent method described [9] The gallic acid compound was used as a standard curve with concentrations 10,20,40,60 and 80 ppm. The diluted extract (for propolis sample) or gallic acid (for standard curve) (0,5 mL) was added to 5 mL Foline Ciocalteu reagent (5-fold diluted with distilled water) and mixed thoroughly for three minutes. Sodium carbonate (4 mL, 10% w/v) was added to the mixture, and the mixture could stand for 15 min at room temperature. The absorbance of the mixture was measured at 765 nm using a UV-VIS. For a sample, make mother stock for 10,000 ppm (μg/ml) with MtOH 100%, then dilute with aqua dest for make 2000 ppm, then each of it mix with 5 mL Foline Ciocalteu reagent and Sodium carbonate (4 mL, 10% w/v). The absorbance of the sample was repeated for 3 times.
Quantitative Flavonoid Content
Total flavonoid content was measured UV-Vis assay used aluminum chloride as reagent to react with flavonoid compound [10]. In this method, quercetin was used to make the calibration curve. Quercetin is one type of flavonoid
class of flavonoids. Some studies say ideal quercetin was used as a reference curve standard in determining total flavonoids [11] . 10 mg of quercetin was dissolved in methanol and then diluted to 25, 50, 75, 100, 125, 150, 175, 200, 225 μg/ml. A calibration curve was made by measuring the absorbance of the solution at 415 nm. The solution was contained 0,5mL quercetin solution; 1,5 mL MtOH; 0,1 mL sodium acetate 1M and 2,8 mL aqua dest. The addition of Aluminum chloride will form a stable acid complex with ring A and B or carbonyl and hydroxyl groups on the flavonoid structure, so the color turns yellow [12]. For a sample, make mother stock for 10,000 ppm (μg/ml) with MtOH 100%, then dilute with aqua dest for make 2000 ppm. 0,5ml of propolis wax solution, mixed with 1,5 mL MtOH; 0,1 mL sodium acetate 1M and 2,8 mL aqua dest, the absorbance was read after 30 min of incubation. The absorbance of the sample was repeated for 3 times.
Antifungal Test
The stock of culture has been available at the Parasitology Laboratory of Faculty of Medicine Universitas Indonesia for later inoculum prepared by standard turbidity. The standard turbidity equivalent to the value of its microorganism content has been established by the standard term Mc Farland. The Mc Farland type used in this study is Mc Farland 0.5 which is equivalent to 1.5 x 108 / ml cell density. For positive control, we used Nystatin was proven as antibiotic for Candida albicans. Before being used, the disks were soaked in the sample for three minutes and then placed above the surface, so that has been applied with microbes. Incubation was performed at room temperature for 24 hours and measuring the diameter of inhibition using a caliper.
RESULT AND DISCUSSION
Quantitative Phenol and Flavonoid Content
By using the standard gallic acid for phenol measurement and quercetin for flavonoid measurement, the standard curve will be known. The standard curve consists of the x axis of standard concentration whereas the y-axis is the result of UV-Vis absorbance. Both data are plotted to produce a line equation to determine the amount of phenol and flavonoid compounds in the sample. The standard phenol curve with standard gallic acid has a line equation y = 0.0076x + 0.0307 with R² = 0.9908, while the standard curve of flavonoid with quercetin standard has an equation y = 0.0048x + 0.0116 with R² = 0.9986.
The absorbance or sample concentration was measured by three times, the sample absorbance data was then made on average and vulnerable to error and visualized as graph 2. The value of phenol content in propolis taken from inside the hive (smooth), outside the hive (rough) and mixed have differences; it was proven that the characteristics of propolis in one hive have differences. Through graphs two phenolic compounds contained in propolis mix reaches 5.109%, at roughly 4.943% propolis and propolis smooth reach 4.256% when compared to the other propolis, propolis Sulawesi kind of smooth, rough and mix had a phenol content of 4,25- 5,11%. This value is lower than the value of phenol content in Argentine propolis with the same method and reagent having a content up to 36.23% [13]. In the flavonoid content, the value of% b/b of smooth propolis 16.38%; Rough 8.08%; And the mix of 12.38%. Based on the reference value using the same method, Sulawesi propolis has a high flavonoid content. Taiwanese propolis contains flavds ranged from 2.82 to 3.07%; Brazil ranged from 3.26%; China ranges from 5.37-7.73% [14]; Romania ranges from 4.24 to 19.07% [15].
FIGURE 1. Flavonoid and Phenol Content in Smooth, Rough, and Mix Propolis. The black color indicates the smooth propolis;
dark grey indicates the rough propolis and light grey indicates the mix propolis
Through graph two, the diversity of the results of the number of phytochemical compounds in each sample. The results show that each sample does not always have the greatest compound content in any compound. The value of the content of phytochemical compounds in propolis mix cannot be predicted by the value of propolis smooth or rough, because propolis mix was not a propolis wax with the smooth and rough mixture, but propolis mix was obtained from the result of the extraction process smooth propolis combined with rough. By comparing with other propolis as having been mentioned in the previous section, Sulawesi propolis flavonoids were superior to propolis Brazil, China and Romania. It can be concluded that the smooth, rough and mixed propolis of Sulawesi has flavonoid value that is quite competitive with other propolis in the world.
Antifungal Test
After incubation, for 24 hours the results will be known as shown in Figure 3. On the control of nystatin and smooth propolis, there are some blank zones that there was no colony due to the uneven distribution of counter strike application when applying inoculum to the medium surface. Nevertheless, the positive value of nystatin control is still valid because it is the value corresponding to the literature that is more than 20 mm.
Positive control in the form of nystatin substances has available commercial disk so ready for use without any immersion first. While for the propolis sample, using a blank disk that does not contain any substance, then done soaking blank disk with sample for 3 minutes. Before determining the immersion time of 3 minutes, the optimization of the immersion time is 3 and 10 minutes, where at 10 minutes the disc is destroyed so that the time is 3 minutes.
0 2 4 6 8 10 12 14 16 18 20
Flavonoid Fenol
%w/w
FIGURE 2. Inhibitory diameter zone (a) nystatin as a control (b) smooth propolis (c) rough propolis (d) mix propolis The resulting inhibitory diameter is described as shown in the figure. 4 demonstrating that the propolis type with the most superior diameter is propolis mix. At 5% and 7% propolis concentrations, smooth and rough propolis produces a 10-mm inhibitory diameter, while propolis mix is 12 and 13mm. In the anti-fungal test, it appears the effect of the concentration of 5% and 7% not much different; it was because the concentration on propolis does not differ much. When viewed between propolis, the value of propolis mix is greater than the value of smooth and rough propolis inhibitory diameter. It was due to the synergy between the antifungal content of the little and rough propolis that was interpreted as a mixed sample.
FIGURE 3. Inhibitory Diameter Zone in Sulawesi Propolis Result. The light grey indicate the smooth propolis, grey indicate the rough propolis, light black indicates the mix propolis and black indicate the nystatin as positive control.
Meanwhile, the value of propolis wax of the smooth, rough and third mixed sample was smaller than nystatin control. Based on the reference [16, 17] it has been mentioned that at concentrations of propolis 10% inhibitory diameter ranges from 10 mm. The diameter of 10mm has an inhibitory moderate. Based on the SIR (Sensitive, Intermediate and Resistance) categories, the anti-fungal abilities of the three samples fall into the middle category because they have more than 2 mm and have a difference of more than 3 mm from the control. Thus, to compare the effective concentration for feed control needs further research.
CONCLUSION
The amount of flavonoid and phenol compounds is not always superior in every sample. Based on the anti-fungal activity test, propolis mix has the advantage of smooth and rough type, where propolis mix itself has a low number of flavonoids and high phenols. In this case, the determination of anti-C. albicans marker by testing phytochemical compounds leads to phenol content of propolis. Synergies effect can be seen from the type of anti-fungal compounds contained in the smooth, rough and mix propolis. Maybe there are compounds which only appeared in rough propolis or smooth propolis, so in propolis mix, there is synergistic in the presence of compounds contained in rough and smooth propolis, so to define the actual marker we need further study
ACKNOWLEDGEMENT
The author would like to thank the financial support from DRPM-UI Grant of Indexed International Publication of Student Final Project (Publikasi Internasional Terindeks untuk Tugas Akhir Mahasiswa/PITTA) 2017.
0 5 10 15 20 25 30
5 7
Inhibitory Diamter (mm)
Concentration of Propolis (%)
REFERENCE
1. Delaloye, Calandra and Thierry, Virulance 5(1), 161-169 (2014).
2. J. A. Lemos, Braz J Microbiol 40(1), 163–169 (2009).
3. R. S. Traboulsi, Int J Antimicrob Agents 31(3), 272–276 (2008).
4. V. P. L. Agarwal, J Microbiol Immunol Infect 43(5), 447–451 (2010).
5. A. Farnesi, Genet Mol Res 8(2), 635-640 (2009).
6. J. C. Montero, Braz Oral Res 26(4), 318–322 (2012).
7. K. F. D. Dota, M. E. L. Consolaro, T. I. E. Svidzinski and M. L. Bruschi, Evidence-Based Complementary and Alternative Medicine 2011, 1-8 (2011).
8. M. Hudnall, Composition Containing Fractionated Bee Propolis, U.S. Patent No. US 7294351 B2 (13 November 2007).
9. A. Pourali, M. Afrouziyeh and Moghaddaszadeh-ahra, European Journal of Experimental Biology 4(1), 74-176 (2014).
10. L. A. da Silva, B. R. Pezzin and L. Soares, Pharmacogn Mag 11(41), 96-101 (2015).
11. G. C. Bag , P. G. Devi and T. Bhigyabati, Int. J. Pharm. Sci. Rev. Res. 30(1), 154-159 (2015).
12. M. Popova, V. Bankova, V. Petkov and Damayantova, Phytochemical Analysis 15(4), 230-240, (2004).
13. M. Gonzalez, B. Guzman, R. Rudyk and E. Romano, J. Pharm 22(3), 243-248, (2003).
14. C. C. Chang, M. H. Yang, H. M. Wen and J. Chern, Journal of Food and Drug Analysis 10(3), 178-182 (2002).
15. C. M. Mihai , L. Marghitas, D. Dezmirean and Tăma, Animal Science and Biotechnology 43(1), 407-410 (2010).
16. N. K. Hendi, H. S. Naher and A. H. Al-Charrakh, Journal of Medicinal Plants Research 5(20), (2011).
17. S. Gavanji and B. Larki , Chin J Integr Med 23(3), 201-207 (2017).
