4.10 (b) Repeat first for MIC test of fraction 9-15 against 66 Candida albicans and fraction 9 against Candida parapsilosis. 4.11 (b) Second repetition for MIC test of fraction 9-15 against 67 Candida albicans and fraction 9 against Candida parapsilosis.
INTRODUCTION
Plant Metabolites
One of the reasons is due to the widespread use of the antifungal drugs (Campoy and L. Adrio, 2017). Thus, the studies and research on antifungal properties of plants are important to discover the potential of the plant species to become an alternative to other currently known antifungal agents.
Minimum Inhibitory Concentration (MIC)
Moreover, plants are relatively inexpensive biological material consisting of various bioactive compounds with certain biological activities (Tiwari and Mishra, 2011).
Minimum Fungicidal Concentration (MFC)
Mangifera species
- Origin
- Taxonomy
- Species of Mangifera and their pharmacological activities
- Botanical description
- Present-day cultivation and usage
The fruit peel of Mangifera foetida, which is rich in gallic acid, protocatechuic acid and vanillic acid, showed antioxidant activity, while the leaf extract of M. Mangifera sylvatica pulp contains mangiferin, quercetin, kaemferol, p-coumaric acid, gallic acid and ellagic acid. pickles.
Candida species
- History of Candida Infections
- Candida albicans
- Candida parapsilosis
- Current anti-Candida drug
An estimated 75% of the population worldwide has Candida albicans in their oral cavity. In the United States, Candida parapsilosis is one of the common pathogens that come after Candida albicans.
Objectives of the Project
13 Polyenes such as nystatin and amphotericin-B will act on the cellular membrane of the fungus containing ergosterol by developing ion channels and affecting the proton gradient (Martins et al., 2015). Azoles such as fluconazole and ketoconazole will affect the biosynthesis of the ergosterol, while allylamines and thiocarbamates will cause ergosterol depletion and increase the levels of squalene which is toxic and will cause the cell membrane to become permeable and affect the cellular organization (Martins et al., 2015). . ).
Natural compounds in Mangifera pajang
- Phytochemical compounds in the kernel of Mangifera pajang
The methanol extract of the kernel of M. pajang will be tested for its antifungal properties against C. 2009) stated that the methanol extract of the kernel contained a very high amount of total phenolic content which is about 10% of its total weight. 18 In the kernel, ferulic acid and diosmin are the two most important chemical components with a concentration of 5.334 mg/g and 2.386 mg/g, respectively (Preedy, Watson and Patel, 2011).
Antifungal activity of Mangifera pajang
They were incubated at 30-37oC for 24-48 hours and the diameters of the zones of complete inhibition were measured (Ahmad et al., 2015). Kong Chee Kei, my co-supervisor's master's student, had tested the antifungal activity of crude methanol extract of Mangifera pajang kernel and found that it had significant antifungal property against Candida albicans (C.K. Kong 2018, personal communication, January 24). The differences in the result may be because the method used to determine the antifungal properties was different.
Microbroth dilution method was used by the student to determine the MIC of the crude extract instead of the agar diffusion method. On the other hand, the master's student also extracted the kernel successively, twice each time with hexane, chloroform, ethyl acetate, ethanol, methanol and water for two days. 20 to extract the kernel would affect the compounds extracted into the solvent, which would in turn affect the antifungal properties of those particular extracts.
Antifungal activity of other Mangifera species
- Tannins
Derivatives of phenolic acids isolated from some plant sources show antifungal activity, which is shown in Table 2.3. Phenolic acid derivatives extracted from different sources may have variable MIC values against Candida species due to different amounts of phenolic acid derivatives present in different plants, different solvents used for extraction, and other compounds in the extracts may act synergistically with phenolic acids on they increase the overall antifungal activity (Teodoro et al., 2015). The antifungal activity of flavonoids may be due to their ability to form a complex with extracellular and soluble proteins and complex with fungal cell walls (Sachikonye and Mukanganyama, 2016).
27 supported by a study that reported that the antifungal activity of flavonoids increases as the length of the alkyl chain increases (Sachikonye and Mukanganyama, 2016). This class of compounds has been reported to activate the macrophages, which could treat fungal infection (Tiwari and Mishra had reported that the antifungal activity of the monosubstituted coumarins studied was independent of the substitution pattern in the coumarin core and also the properties of the substituents. a planar molecule and contains various substituent groups attached to it (Tiwari and Mishra had reported that the existence of a hydroxyl group on the structure was essential for its antifungal activity and the nature of the substituent group will affect its antifungal activity.
Thin-Layer Chromatography (TLC)
- Preparation of Extract for TLC
- Preparation of Mobile Phases for TLC
- Preparation of Developing Chamber
- Preparation of TLC Plates
- TLC Test
A Camag's® developer chamber, also known as a baby chamber, was used to develop TLC. A length of 1 cm was measured from the bottom of the TLC and a line was drawn with a pencil. Then 0.5 cm from the top of the TLC plate was measured and a line was drawn with a pencil.
The TLC plate was then carefully inserted into the development chamber and sealed with a lid. After the mobile phase had traveled to the solvent front on the TLC plate, the TLC plate was removed from the development chamber and set aside in the fume hood until all the solvent had evaporated. After that, the TLC plate was placed in the chamber for a few seconds with the lid on.
Glass Column Chromatography
- Preparation of the Column
- Separation of Compounds in Plant Extract
- Preparation of Reagents and Fungus
- Culture medium, RPMI-1640
- Amphotericin B (Antibiotics)
- Preparation of Agar Plates
- Cultures of Fresh Batch of Candida albicans and Candida parapsilosis The master plates of Candida albicans and Candida parapsilosis were used to
- Preparation of filtered and unfiltered methanol: water solution (2:1) In order to prepare filtered methanol: water mixture (2:1), 30 mL of methanol was
- Preparation of Fractions for bioassay
- Process of Bioassay
- Orders of Addition of Reagents and Fractions
- Serial dilution for fractions and Amphotericin B
- Addition of p-iodonitrophenyltetrazolium violet (INT) indicator During the next day of incubation, the plates were took out from the incubator
- Determination of Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC)
A portion of the silica gel was transferred to a 250 ml beaker and mixed with an amount of 100% chloroform sufficient to make a slurry mixture. A dropper was used to flush the column wall with chloroform to wash down any silica gel along with the extract. It was ensured that the level of the mobile phase in the column was not below the silica gel level in the column.
The column was therefore continuously refilled with the mobile phase needed before the mobile phase near the surface of the silica gel. Then distilled water was added until the total volume of the solution became 1 liter. 43 µL of the medium to all the wells in the fraction columns, the column for amphotericin B and the positive control.
High Performance Liquid Chromatography (HPLC)
Thin-Layer Chromatography
The tail formation is due to the presence of acids, bases or highly polar compounds in the crude extract, which adhere strongly to the absorbent. This will cause the compounds to move upwards, disrupting the migration of the solutes across the stationary phase. Adding a trace amount of acids or bases can minimize the strong interaction between the compound and the adsorbent as well as increase the polarity of the mobile phase.
After testing, formic acid reduced the stain tail effect much better than acetic acid and ammonia. When the polarity of the mobile phase increased with the increase in the percentage of ethyl acetate, the spots traveled a longer distance on the TLC plate mean. 51 that compounds in situ have a higher polarity because it is more soluble in the mobile phase of higher polarity and migrate further.
Glass Column Chromatography
These fractions were further pooled into 15 fractions by performing TLC on each fraction to verify their purity. Those that had a nearly similar or exactly identical TLC separation pattern were mixed to become a single fraction. For fractions 4, 5, 6, and 7, each of them was considered as a single fraction because the TLC result showed no significant similarities.
From fraction 8, the polarity of the compounds increased and the preceding mobile phase cannot elute the compounds from baseline. Although the tail effect on fractions 13 to 27 makes it impossible to clearly distinguish one fraction from another, it is not a significant problem as the main goal of this project is to determine the antifungal activity of the Mangifera pajang extract. The recovery yield of the fractions was not 100% because some of the compounds adhered strongly to the silica gel of the column which could not be removed even though the silica gel was eluted with 100% ethanol and 100%.
MIC (Minimum Inhibitory Concentration) Test for Bioassay
- MIC against Candida albicans
First, 10 mg/mL solution was prepared from each fraction and used for the MIC test. However, only fractions 1, 2 and 14 showed MIC values while the rest did not form any precipitate. If the concentration of the fraction was 10 mg/mL, then the concentration of the first well will be 2.5 mg/mL due to the dilution with medium and fungi.
The precipitates formed in fraction 9 for both replicates against Candida parapsilosis had quite a large difference as shown in Figures 4.8 (a) and 4.9 (a). Since almost all the fractions had quite high antifungal activity against Candida parapsilosis, another concentration of fractions was prepared at 0.63 mg/ml except for fraction 1 because the amount of it was very small and the whole of fraction 1 had been used up to determination of MIC against Candida parapsilosis. Fractions 7, 8 and 9 showed a very trace amount of precipitates in the last wells and their MIC values cannot be determined because the first concentration at which the precipitates formed are less than 50% of the precipitates observed in the growth control well was unknown.
MFC (Minimum Fungicidal Concentration) Test for Bioassay
71 The table showed that fraction 8 and 9 had the lowest MFC values against Candida parapsilosis, while fraction 7, 8 and 9 showed the lowest MFC values against Candida albicans. Almost all of the fractions have similar MIC and MFC values, meaning that the compounds in the fractions were strong enough to kill the fungal population rather than just inhibit their growth. Among the MIC and MFC values of all fractions, fraction 8 and 9 had the lowest MIC and MFC against Candida parapsilosis, while fraction 7, 8 and 9 had the lowest MIC and MFC values against Candida albicans.
However, only fraction 8 and 9 became the fraction of interest because they both exhibited strong antifungal properties against both fungi and their amount was large enough to continue further work. Thus, fraction 8 and 9 were analyzed using HPLC to determine the number of compounds present within each fraction.
High Performance Liquid Chromatography (HPLC)
The blank chromatogram shows that the peaks beyond 30 minutes belong to the solvent. The peaks in the chromatogram of fraction 8 and 9 beyond 30 minutes belong to the solvent rather than the compounds. By biological assay, the methanol extract of Mangifera pajang kernel contains strong antifungal properties against Candida albicans and Candida parapsilosis.
This was proven by the data in which almost each of the fractions has similar MIC and MFC values, meaning that the compounds in the fractions kill the fungal population rather than just inhibit their growth. Inhibitors of the glyoxylate cycle enzyme ICL1 in Candida albicans for potential use as antifungal agents. Antifungal Activity of Coumarin Mammeisin Isolated from Species of the Kielmeyera Genre (Family: Clusiaceae or Guttiferae).
