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 Table of Contents  
Year : 2022  |  Volume : 6  |  Issue : 1  |  Page : 73-80

Enhanced antifungal activity of Piper betle against candidiasis infection causing Candida Albicans and In silico analysis with its virulent protein

1 Department of Microbiology, St. Peter's Institute of Higher Education and Research, Chennai, Tamil Nadu, India
2 Department of Microbiology, Ayya Nadar Janaki Ammal College, Sivakasi, Tamil Nadu, India

Date of Submission25-Jul-2021
Date of Acceptance17-Sep-2021
Date of Web Publication11-Mar-2022

Correspondence Address:
Sivakumar Thangavel
Department of Microbiology, Ayya Nadar Janaki Ammal College, Sivakasi - 626 124, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_154_21

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Background: The widespread usage of synthetic chemical drugs often contributes to the development of drug resistance in the clinical pathogens along with hazardous side effects in the human side. Among those clinical pathogens, Candida albicans is a prime consideration to explore. C. albicans is wildly causing a fungal infection of oral cavity well known as candidiasis. This study is prompted to find some novel natural compounds from a medicinal plant, Piper betle against C. albicans. Methods: Bioactive compounds were extracted from the betel leaves using different solvents. The standard drug, fluconazole was used to check anticandidal activity of P. betle against C. albicans. Plant extracts were further characterized by the antioxidant and different scavenging assays. The biocompounds were identified using gas chromatography–mass spectrometry and successfully subjected to molecular docking study. Results: Methanol and ethanol extracts were showed potential antifungal, antioxidant, and scavenging activity against C. albicans, in comparison with control drug. Twenty-seven bioactive compounds were identified in the methanol extract of P. betle. These active bioactive compounds were docked with candidapepsin-1, a proteolytic virulent enzyme of C. albicans and compared with a control drug, fluconazole (−7.8 kcal/mol), and the effective interaction was observed with specific bioactive compound, 4-hydroxy-5-imino-3,4-dimethyl-1-(4-nitrophenyl)-2-imidazolidinone (−7.5 kcal/mol). Conclusion: The present study reveals that methanol and ethanol extract of P. betle is a potential source of natural-free radical scavenging antioxidants. These findings will be great helpful in the new drug analysis for the determination of antimicrobial biocompounds against candidiasis and other clinically related infections.

Keywords: Candida albicans, Candidapepsin-1, docking, gas chromatography–mass spectrometry, Piper betle

How to cite this article:
Selvaraj GK, Wilson JJ, Kanagaraj N, Subashini E, Thangavel S. Enhanced antifungal activity of Piper betle against candidiasis infection causing Candida Albicans and In silico analysis with its virulent protein. Biomed Biotechnol Res J 2022;6:73-80

How to cite this URL:
Selvaraj GK, Wilson JJ, Kanagaraj N, Subashini E, Thangavel S. Enhanced antifungal activity of Piper betle against candidiasis infection causing Candida Albicans and In silico analysis with its virulent protein. Biomed Biotechnol Res J [serial online] 2022 [cited 2022 May 20];6:73-80. Available from: https://www.bmbtrj.org/text.asp?2022/6/1/73/339360

  Introduction Top

Candidiasis is a fungal infection caused by a group of yeast known as Candida. Some virulent species of Candida (C. albicans, C. krusei, C. tropicalis, and C. glabrata) are able to cause severe infections in humans. These species of Candida are evolved more resistant to the antifungal agents.[1],[2] Besides, these species have the ability to persist for long periods due to their formation biofilms, which leads to the development of infection in oral mucosa, dental tissues, restorative materials, and denture prostheses.[3],[4],[5] Among the above Candida spp., the most common pathogenic candidate is C. albicans.[6] C. albicans can cause severe oral infection particularly in children with weak immune systems.[7] It may lead to systemic disorders such as diabetes, respiratory and gastrointestinal tract infection, cancer, cardiovascular and neurodegenerative disorders in adult persons.[8] In general, their prevention and treatment can be done by antibacterial and antifungal chemical drugs. Due to the adverse side effects of synthetic drugs on the human, the finding of efficient drugs has moved toward the search of potential natural compounds that possess antimicrobial activity.[9] Phytochemicals present in medicinal plants which have potential antimicrobial and other various biological activities.[10] The crude extracts of plants, volatile oils, and their respective compounds are playing the better role against many pathogenic microorganisms such as Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa.[11],[12]

Piper betle (betel) is a precious medicinal plant; vine belongs to the family, Piperaceae, mostly found in Southeast Asia and Eastern Malaysia. The betel leaves are very nutritive which contains a trace amount of vitamins and minerals.[13] It was demonstrated to have antimicrobial and wound healing properties, thereby it could act as traditional remedies for several diseases and disorders. It has been reported for the treatment of nervous pains, cough, and sore throat.[14] It is comprised of more antioxidants such as hydroxychavicol, ascorbic acid, and β-carotene. The leaves contain enzymes and amino acids such as diastase and catalase, histidine, arginine, and lysine, respectively. Therefore, it possesses many beneficial bioactivities and its extracts have been used to produce many commercial products.[15],[16] The methanol, ethanol, and chloroform extract of P. betle reported to have potential inhibition activity against pathogenic bacteria such as Plasmodium berghei[17] and Vibrio cholerae.[18]

In silico studies render the benefit of identifying novel drug candidates more quickly at a reduced cost.[19] The computational approach of drug discovery reduces the consuming time, labor, and cost of testing each and every ligand for its preliminary antimicrobial nature.[20] The Candidapepsin-1 is a proteolytic virulent enzyme from the endophytic polymorphic fungal species C. albicans and is responsible for superficial Candida infections such as oral and skin infections in immuno-compromised individuals.[21],[22] The present study aimed to focus on the various extraction methods of P. betle leaf, its antimicrobial activity and antioxidant assays. Further, it was followed by the molecular docking with proteolytic virulent enzyme of C. albicans, Candidapepsin-1.

  Methods Top

Fungal culture collection and condition

The opportunistic fungal pathogen, C. albicans was purchased from the microbial type culture collection. In the entire study, potato dextrose agar medium and nutrient broths were used as growth media, and their microbial growth was achieved at 30°C. The current study was approved by the Ayya Nadar Janaki Ammal College, Sivakasi. Ref. No. 103/2020, dated: 01.04.2020.

Sample collection extraction

The P. betle (Pann) leaves were collected from the green house garden, Sivakasi, Tamil Nadu, India. The collected betel leafs were cleaned thoroughly and dried in dark place at room temperature. The dried leaves were fine powdered and stored for further activities. 7.2, 12, and 12 g of P. betle samples (in triplicate) were extracted using the solvents, ethanol, and methanol as well as by the distilled H2O (100 ml) for 24 h. The solvents and dH2O extracts were filtered through Whatman no. 1 filter paper and centrifuged at 5000 rpm for 10 min. The supernatant was carefully collected and analyzed for antimicrobial activity. Finally, the effective solvent was tested against the C. albicans, and the antimicrobial activity was estimated by the gas chromatography–mass spectrometry (GC-MS).

Anti-candidiasis activity

The overnight culture (1.0; OD at 600 nm) of C. albicans was enriched in nutrient broth for 6–8 h at 37°C. Using a sterile cotton swab, the fungal cultures (50 μL) were aseptically swabbed on the surface of Mueller-Hinton agar plates. The different volumes (0, 10, 20, 30, and 40 μL) of betel leaves extracts (50 μg/ml) were aseptically placed over the welled the agar plates that sufficiently separated from each other to avoid overlapping of inhibition of zones. The plates were further incubated at 37°C for 24 h, and the diameter of the inhibition of zones was measured in millimeter scale.

In vitro antioxidant and free radical scavenging activity

Determination of total antioxidant capacity

The total antioxidant activity of P. betle was determined according to the method of Prieto et al.[23] Briefly, 0.3 ml of betel samples (in triplicate) was mixed with 3.0 ml reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The reaction mixture was incubated at 95°C for 90 min in the water bath. The absorbance of all the sample mixtures was measured at 695 nm within 15 min, and the ascorbic acid was used as a standard solution.

Determination of reducing power

Reducing power of the P. betle was determined using the method of Yamaguchi et al.[24] Briefly, 4 ml of reaction mixture (in triplicate) containing different concentrations of betel samples (100, 250, 500, 750, and 1000 μg) of in 0.2 M phosphate buffer (pH 6.6) was incubated with potassium ferric cyanide (1% w/v) for 20 min at 50°C. The reaction was terminated by trichloroacetic acid solution (10%, w/v). The solution was further mixed with dH2O and ferric chloride (0.1%, w/v) solution and the absorbance was measured at 700 nm.

Hydrogen peroxide scavenging assay

The free radical scavenging activity of the P. betle was determined by hydrogen peroxide assay.[25] Hydrogen peroxide (10 mM) solution was prepared using phosphate-buffered saline (0.1 M, pH 7.4). The 1 ml of different concentration of betel extracts (100, 250, 500, 750, and 1000 μg) were rapidly mixed with 2 ml of hydrogen peroxide solution. The absorbance (OD at 230 nm) was measured using the ultraviolet spectrophotometer after 10-min incubation at 37°C against a blank (without hydrogen peroxide). The percentage of scavenging of hydrogen peroxide was calculated using the formula: percentage scavenging (H2O2) = (absorbance of control [A0] − absorbance of sample [A1])/absorbance of control [A0] ×100.

1-1-diphenyl-2-picrylhydrazyl radical scavenging assay

The free radical scavenging activity of P. betle was also measured by the 1-1-diphenyl-2-picrylhydrazyl (DPPH) method.[26] DPPH was used as a reagent which offers a convenient and accurate method for titrating the oxidizable groups of natural (or) synthetic antioxidants. The 0.1 mM solution of DPPH in methanol was prepared and 1 ml of this solution was added to 3 ml of P. betle of different concentration (100, 250, 500, 750, and 1000 μg). Ascorbic acid was used as standard solution and for the blank, samples were substituted by methanol. After 10 min, absorbance was measured at 517 nm. The percentage scavenging activity values were calculated according to the above-mentioned formula ([A0 − A1]/A0) ×100).

Gas chromatography–mass spectrometry analysis

The powdered P. betle (20 g) was soaked and dissolved in 75 ml of methanol for 24 h. Then, the filtrate was collected as evaporated form under the liquid nitrogen. The GC-MS analysis was carried out using Clarus 500 PerkinElmer (AutoSystem XL) gas chromatograph equipped and coupled with a mass detector TurboMass Gold– Perking Elmer TurboMass 5.2 spectrometer with an Elite-1 (100% dimethylpolysiloxane), 300 m × 0.25 mm × 1 × m df capillary column. Test methodology followed by protocol. The instrument was set to an initial temperature of 110°C and maintained for 2 min after that, the oven temperature was raised to 280°C, at the rate of an increase of 5°C/min maintained for 9 min. Injection port temperature was ensured at 250°C and the helium flow rate was maintained at 1 ml/min and their ionization voltage was 70 eV. The triplicate samples were injected in split mode as 10:1. Mass spectral scan range was set at 45–450 (MHz). The fragmentation patterns of GC-mass spectra were compared with those stored in the spectrometer database using the National Institute of Standards and Technology Mass Spectral Database. The percentage of each component was calculated from their respective relative peak area of each component in the chromatogram.

Molecular docking

The effective C. albicans enzymes of Candidapepsin-1 (2QZW) were docked to the bioactive compound of P. betle and fluconazole (control) drugs through molecular modeling simulation software, AutoDock. AutoDock tool is dependent on two techniques, namely, the rapid grid-based estimation and systematic search of torsional freedom for the ligand-protein molecular docking.[28]

  Results Top

Antimicrobial activity against Candida albicans

The P. betle leaf extract was determined for antimicrobial activity against opportunistic fungal pathogen C. albicans. It shows an excellent inhibitory effect with a methanol extract (with maximum zone with 17 mm against 40 μL) [Table 1]. Among the experimental solvents used in this study, methanol extract possess enhanced antimicrobial activity against C. albicans. The extracts from young betel leaf have showed higher anticandidal activity (>12) than the mature betel leaves.
Table 1: Antimicrobial activity of Piper betle extracts against Candida albicans

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Total antioxidant capacity and reducing power of Piper betle extract

In our study, the total antioxidant activity of P. betle was analyzed and compared with the standard ascorbic acid. The activities of P. betle were calculated based on inhibition percentage and it accounts for 84.41% ± 0.12% [Figure 1]a. The selected plant had an efficient activity against the selected fungal pathogen. The reducing power of P. betle (absorbance, 0.12–1.01) was compared with the standard ascorbic acid (0.363–1.543) [Figure 1]b.
Figure 1: The bar diagrams illustrating the total antioxidant activity of P. betle with standard ascorbic acid (a), reducing power of P. betle with standard ascorbic acid (b), H2O2 scavenging assay of P. betle with standard Gallic acid (c), DPPH scavenging assay of Piper betle with standard gallic acid (d), P. betle: Piper betle, DPPH: 1-1-diphenyl-2-picrylhydrazyl

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Scavenging activity of Piper betle extract

The activity observed in hydrogen peroxide scavenging assay was directly proportional to change and as found to be concentration gradients also directly proportional to change. The activities of hydrogen peroxide inhibition for the P. betle in this study were 80.12% ± 0.29% [Figure 1]c. The activity observed in the DPPH radical scavenging assay for the P. betle was 69.27% ± 0.92% [Figure 1]d. The ethanol extract of P. betle showed 11.81%, 20.78%, 26.69%, 31.50%, 37.41%, 56.67%, 71.99%, and 87.52% scavenging activity at the different concentrations (20–800 μg/ml), where the highest scavenging activity of P. betle extracted from ethanol was 87.52% at concentration 800 μg/ml.

Gas chromatography–mass spectrometry analysis

In the present study, the spectrum of GC-MS analysis was used to identify a number of phytocompounds from the methanol fractions of the P. betle leaves. GC-MS analysis was revealed the presence of key active compounds of the P. betle leaf extracts such as 6.729 (3,5-dimethylbenzoic acid, 2,5-dimethylbenzoic acid, methionine), 12.024 (2-propanone, 1-[2-benzoxazolylthio]-1-fluoro-, cyclopropyl [3,4-dimethoxyphenyl] methanone, 4-hydroxy-5-imino-3,4-dimethyl-1-[4-nitrophenyl]-2-imidazolidinone), and 13.594 (ethyl 3, 4, 5-trimethyl-1H-pyrrole-2-carboxylate, 2-[1-adamantyl]-N-methylacetamide, benzenamine, N-[phenylmethylene]-) [[Figure 2] and the detailed list of bioactive compounds was given in [Table 2]]. In overall, GC-MS analysis of P. betle leaf extract revealed the presence of eleven compounds peak with representation 33 active compounds. There were merely 27 compounds elucidated by this instrumentation study.
Figure 2: The spectrum of GC-MS showing the active compounds derived from the methanol fractions of Piper betle leaves. GC-MS: Gas chromatography–mass spectrometr

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Table 2: Characterization of bioactive compounds identified from the methanol extracts of Piper betle leaf using gas chromatography-mass spectrometry

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Molecular docking

The structure of Candidapepsin-1 (2QZW) was retrieved from protein data bank, and the structures of bioactive compounds were retrieved from drug bank. All the ligand molecules were made to dock against the active sites of the target Candidapepsin-1 using AutoDock Vina. The docking scores were given in detail in [Table S1]. Candidapepsin-1 (SAPs) of C. albicans was successfully molecular docking with bioactive compounds and control drug, fluconazole that represented in [Figure 3]. The effective interaction and scores of bioactive compound compare to control drugs such as 4-hydroxy-5-imino-3,4-dimethyl-1-(4-nitrophenyl)-2-imidazolidinone (−7.5 kcal/mol) interact with Gly34, Gly220, Tyr225, Ser301, Ala303, Ile305 (3H-bond); ethyl 6,8-difluoro-4-hydroxyquinoline-3-carboxylate interact with Gly34, Asp32, Asp86, Asp218, Gly220 (1H-bond); and fluconazole interact with Pro162, Ala281, Leu297, respectively. From these results, fluconazole drugs have no hydrogen bond with any amino acids, but the 4-hydroxy-5-imino-3,4-dimethyl-1-(4-nitrophenyl)-2-imidazolidinone (P. betle) effective has 3 hydrogens and van der Waals force with more amino acids.
Figure 3: Docking image of Candidapepsin-1(SAPs) compound of Candida albicans. Control drug - Fluconozole (a) and C. albicans - bioactive compound (b)

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  Discussion Top

In the present study, C. albicans presumptive identification is tested based on a germ tube method. Similarly, Abiroo et al.[29] were also used the positive modified germ tube test for the presumptive identification of C. albicans. The present study is mainly focused on the phytochemical analysis found to be positive for alkaloids, glycosides, terpenoids, flavonoids, tannins, and saponins. Chen et al.[30] and Nakanishi[31] revealed that many compounds have been used in the form of whole plants or plant extracts for food or medical applications due to the plants are the natural reservoir of many antimicrobial, anticancer agents, analgesics, antidiarrheal, antifungal as well as various therapeutic activities. Natural products are in the great stipulate for their extensive biological properties and bioactive components which had been proved to be useful against varieties of causative agents of diseases.[32]

Balaji et al.[33] reported that the aqueous and ethanol extract of the leaves of P. betle leaf was reported to have antibacterial activity against three Gram-positives (Micrococcus luteus, Bacillus subtilis, and S. aureus) and two Gram-negative bacteria (E. coli and P. aeruginosa). These tested bacteria are mainly known for the multidrug resistance and food-related diseases. Most of the phytochemical studies have used different solvents such as acetone, ethanol, ethyl acetate, water, and dichloromethane. Among these solvents, ethanol has reported to have more antibacterial properties than other solvents. However, in the present study, we have found that methanol extract possess enhanced antimicrobial activity against C. albicans. Sivareddy et al.[34] reported that ethyl acetate extract of P. betle is more effective as an anticandidal agent against C. albicans which compared with tulsi and fluconazole (control). Mani et al.[35] described that Piper nigrum and P. betle have high antimicrobial activity against opportunistic pathogen C. albicans causing oral candidiasis. Moreover, the extracts from young betel leaf showed higher anticandidal activity than the mature betel leaves. It is mainly due to the release of more secondary metabolites from young leaves because of the fragility of their physical defenses such as lack of a thick waxy cuticle and low cell wall rigidity.[36]

In general, antioxidants can scavenge human disease-associated free radicals and reduce their impact. In addition, natural photochemicals have ability to protecting against the reactive oxygen species-mediated damages in the oxidative stress.[37] The P. betle exhibited with an efficient activity against C. albicans. Likewise, Rathee et al.[36] have extracted the total phenolic content of P. betle using the enzymatic treatment. They revealed that this phenolic content might also contribute to higher antioxidant activities as well as effective free radical scavengers and antioxidants.

Dwivedi et al.[38] revealed the reducing power of P. betle with the increased absorbance (Fe+ 3 to Fe+ 2 transformations) of the reaction mix as well as their commercial antioxidants was estimated spectrophotometrically at 700 nm. Higher absorbance of the reaction mixture indicated the greater reducing power. The higher antioxidant activity with the methanol extracts showing the highest reducing power as same like our findings. Bursal and Gulcin[27] have demonstrated that reducing power and antioxidant potential of compounds. As seen in the case of other analyses, the reducing power of the aqueous extract was seen to increase with enzymatic treatment. Proteolytic as well as carbohydrate hydrolyzing enzymes might be degrading the complex plant material thereby leading to the formation and exposure of other materials, which may influence the antioxidant activity.

Many human diseases are mainly assorted with the accretion of free radicals. In this study, free radical scavenging activities were evaluated by several standard methods using spectrophotometer. Fazal et al.[25] reported that better ability of antioxidant activity of P. betle leaf extract and H2O2 might account for the results. The range of DPPH radical scavenging assay for the P. betle was 69.27% ± 0.92% [Figure 1]d. In the same way, Mayer et al. (2013)[39] reported that the DPPH free radical scavenging activity of the P. betle leaf extract and ascorbic acid. The highest scavenging activity of P. betle extracted from ethanol was 87.52% at concentration 800 μg/ml. Furthermore, Jaiswal et al.[26] observed the enhanced scavenging activity (80%) of P. betle using ethyl acetate extract (79 mg CE/g dw) and the lowest scavenging activity using the water extract of (6.45 mg CE/g dw). In overall, ethanol extract of P. betle is prospective source for the natural bioactive compounds which provide as an efficient free radical scavenger.

P. betle consists of wide range of bioactive compounds and those specific concentrations usually depend on its plant diversity, soil wealth, harvesting period, weather, and geographic location. In general, betel leaves comprised of alkaloids, phenol, tannins, reducing sugars, and saponins in the different solvents.[36] The GC-MS analysis of P. betle leaf extract revealed the presence of eleven compounds peak with representation of 33 active compounds and 27 compounds exposed by this instrumentation study. Similarly, Dwivedi et al.[37] identified with help of GC-MS analysis which revealed the presence of 19 compounds. The compounds were identified by comparing their retention time and covate indexes with that of literature and by interpretation of mass spectra. Many of them are used in industry for various applications such as perfumes, flavors, deodorants, antiseptic, and pharmaceutics.[39] From our analyses, we suggesting that P. betle possesses many phytochemicals based on the solvents that used in the study as well as by their botanical origin. Based on the molecular docking study, active site of the Candidapepsin-1 enzyme was correlated to the active site and compare to the docking score (−7.5 kcal/mol) which was higher than Trachyaspermum ammi (−5.75). Abhishek et al.[40] evaluated that Ligustilide has the lowest free binding energy of −5.75 kcal/mol against Candidapepsin-1 with three hydrogen bond interactions at Ile223, Tyr225, and Thr222 at the active site.

  Conclusion Top

The selected microorganism, C. albicans was subjected to elucidate against the organic phytochemicals of P. betle extracted using various solvents. It was found that methanol and ethanol extracts of P. betle have more antimicrobial, scavenging, and antioxidant activity than other extracts. The active phytochemicals were further analyzed from the methanol extracts using GC-MS and it reveals the presence of terpenoids, tannins, and saponins. The antifungal activity of methanol extract of P. betle extract showed comparatively more susceptible by C. albicans with 17 mm zone of inhibition. The molecular docking was performed for the C. albicans protein, Candidapepsin-1, and extracted bioactive compound of P. betle. 4-hydroxy-5-imino-3,4-dimethyl-1-(4-nitrophenyl)-2-imidazolidinone and ethyl 6,8-difluoro-4-hydroxyquinoline-3-carboxylate showed the highest docking activity against Candidapepsin-1 protein which compared with control drug, fluconazole. The current findings represent that betel leaves have to be elucidated more to explore its efficiency in the drug development and treatment of various microbial infections.

Limitation of study

No limitation in the study samples.

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Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2]


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