|Year : 2020 | Volume
| Issue : 1 | Page : 13-17
Anti-fungal study on aqueous and ethanolic leaves extracts of Piper sarmentosum
Maizatul Akma Ibrahim, Aiesyaa Majdiena Emlee
Department of Plant Science, Kulliyyah of Science, International Islamic University Malaysia, Kuantan, Pahang, Malaysia
|Date of Submission||22-Apr-2020|
|Date of Acceptance||21-May-2020|
|Date of Web Publication||12-Jun-2020|
Dr. Maizatul Akma Ibrahim
Department of Plant Science, Kulliyyah of Science, International Islamic University Malaysia, Bandar Indera Mahkota, Kuantan 25200, Pahang
Source of Support: None, Conflict of Interest: None
Background: Piper sarmentosum or “Kaduk” had been utilized for treating human diseases in traditional medicine. Aim and Objectives: Previous studies provided evidence for its therapeutic properties such as antimicrobials, anticholesterolemic, and antioxidant. An antifungal agent is highly important to cure diseases caused by pathogenic fungi. Materials and Method: Thus, the aim of this study is to discover the antifungals activity of P.sarmentosum leaves& aqueous and ethanolic extracts against four pathogenic fungi, namely Candida albicans, Aspergillus niger, Fusarium verticillioides, and Microsporum gypseum. Result: The yield percentage of the aqueous extract was 17.72%, followed by ethanolic extract with 11.95% of yield. Phytochemical analysis by gas chromatography–mass spectrometer showed the presence of acetic acid, hydrocinnamic acid, oxazole, guanidine, pyranone, benzofuran, β-asarone, and 1-pentadecene in aqueous extracts, while it showed the presence of β-asarone, β-asarone, myristicin, apiol, isocaryophyllene, 1,1-dichlorocyclopentane, and 14-chloro-1-tetradecanol in ethanolic extract. Conclusion: The antifungal susceptibility by disc-diffusion test found that the inhibition growth on F.verticillioides by the aqueous extract occurred at a concentration of 100 mg/mL with an average diameter of 7.3 mm ± 0.06. C. albicans, A. niger, and M. gypseum were shown to be insusceptible to both the extracts.
Keywords: Aqueous, ethanolic, extracts antifungal, gas chromatography–mass spectrometer, Piper sarmentosum
|How to cite this article:|
Ibrahim MA, Emlee AM. Anti-fungal study on aqueous and ethanolic leaves extracts of Piper sarmentosum. Matrix Sci Pharma 2020;4:13-7
| Introduction|| |
Approximately 100 of 100,000 species of fungus are classified as pathogenic to human. The contamination of fungi heavily affects human daily life and environment. It was estimated globally from annual reports of fungal infections recorded 3,000,000 cases of chronic pulmonary aspergillosis, 700,000 cases of invasive candidiasis, 250,000 cases of invasive aspergillosis, and 10,000,000 cases of fungal asthma.
In recent years, demand to find new antifungi compounds have been increasing. Fungi have natural capability to produce toxic reactions which effectively deteriorate the effect of treatment against them. They are also capable to develop genetic resistance toward multiple drugs. Herbal plants are important sources for biological activity due to their beneficial phytochemical compounds. Plants with antifungi properties serve essential roles against diseases caused by fungal infections. An important local medicinal herb Piper sarmentosum (”kaduk”) was utilized in this study. P.sarmentosum that belongs to family Piperaceae possesses many therapeutic activities such as antioxidants, antimicrobials, anti-inflammatory, antituberculosis, anticancer, anticancer, and antiamebic. Moreover, the antifungal properties of P.sarmentosum were proven by the previous study conducted by Chanprapai and Chavasiri. The antimicrobial properties of P. sarmentosum are occurred mainly due to the presence of biologically active compounds such as guineensine, methyl piperate, hydrocinnamic acid, carotenes, and brachystamide.
Thus, it is important to understand more about the role of P.sarmentosum as antifungal agent in this study. The objectives of this research were to extract crude compounds of P.sarmentosum&;s leaves using aqueous and ethanol as solvent. Both the extracts were analyzed for their phytochemical compositions by gas chromatography–mass spectrometer (GC-MS). The extracts were tested for their antifungi activities.
| Material and Methods|| |
Preparation and extraction of plant materials
P.sarmentosum had been obtained from the Glasshouse and Nursery Complex in International Islamic University Malaysia (IIUM), Kuantan campus, Pahang. The fresh leaves of P.sarmentosum were harvested and washed with distilled water. Then, the leaves were oven-dried with the temperature at 45°C for 3 days. After that, the leaves were blended into powdered form and stored in a chiller at 4°C for future use. The powdered samples of P.sarmentosum were used for aqueous and ethanolic extraction.
Approximately 20 g of a powdered sample of P.sarmentosum was placed in a thimble and subjected to Soxhlet apparatus and extracted using 100% ethanol as a solvent. The extraction was carried out for 8 h. Then, the ethanolic extract of P.sarmentosum was evaporated using a rotary evaporator to separate the solvent and crude extract. The obtained crude extract was weighed and stored in the chiller at 4°C for further use.
Twenty gram of powdered sample of P.sarmentosum was put into a conical flask containing 200 mL distilled water as a solvent for extraction. The extraction was carried out using a hot maceration method that was subjected to the shaker inside a hot water bath for 8 h. Then, the extract was filtered using Whatman No. 1 filtered paper. The filtered extract was concentrated using a rotary evaporator. The aqueous crude of the extract of P.sarmentosum was weighted and store inside a chiller at 4°C for further use.
The percentage yields of the ethanolic and aqueous crude extracts of P.sarmentosum were calculated using Equation 1. The weight of the dried crude extracts was measured and recorded in the table after the evaporation phase was complete, living no liquid state.
Phytochemical analysis by gas chromatography–mass spectrometer
The determination of the phytochemical compound of aqueous and ethanolic crude extract of P.sarmentosum was carried out using PerkinElmer Gas Chromatograph (Clarus™ 680) equipped with an autosampler for sample injection. Each of the samples was diluted by absolute ethanol, making the concentration of 100 mg/mL and filtered to avoid unnecessary impurity. GC-MS analysis was performed with an Elite-5MS Capillary Column. The carrier gas was helium set at a flow rate of 1.5 mL/min throughout the program. The following oven temperature program was used: 50°C (hold 1 min), ramped up at 5°C/min to 250°C (hold 5 min). The oven program run time was 45 min.
Then, the chromatogram including the retention times and area of both the extracts were obtained in PerkinElmer TurboMass software. The expected compound of extracts from the internal library was indicated according to its retention time. The concentration percentage of the expected compound was calculated using Equation 2.
Antifungal susceptibility by disc-diffusiontest
All fungal strains were obtained from Microbiology Laboratory, Kulliyyah of Science, IIUM Kuantan, Pahang. The fungi were grown in the potato dextrose agar (PDA) plate and subcultured for growth maintenance. The fungi were inoculated into potato dextrose broth and maintained at 4°C for antifungal susceptibility testing. The fungal strains used in this study listed in [Table 1].
Approximately 100 mg each of the extracts was diluted in 1 mL each of dimethyl sulphoxide (DMSO) making a stock concentration of 100 mg/mL. The other two different concentrations of 50 mg/mL and 20 mg/mL were also prepared. Nystatin antibiotic (10 μg/mL) was used as a positive control, while sterile DMSO was used as a negative control.
The fungal suspension was standardized according to 0.5 McFarland standard. The fungi suspension (100 μL) was spread onto the surface of PDA agar in Petri dish More Detailses. Ten microliter of the extracts of P.sarmentosum with three concentrations (20, 50, and 100 mg/mL), nystatin, and DMSO was poured onto sterile paper disc. After 10 min, the paper discs containing P.sarmentosum extracts and controls were placed onto PDA plates containing fungal suspensions. The plates were incubated at 30°C for 72 h. The susceptibility tests were conducted with three replicates. The diameter of the inhibition zone of fungi growth was later measured after to indicate the sensitivity of fungi to the P.sarmentosum& s extracts.
| Results and Discussion|| |
Aqueous and ethanolic extracts
The yields for the aqueous and ethanolic crude extract of P.sarmentosum were for the average weight and the percentage of yield. The mean weight of P.sarmentosum extracts was found to be the highest (3.544 g ± 0.09) in an aqueous extract with yield percentage 17.72%, followed by ethanolic extract (2.389 g ± 0.14) with yield percentage 11.95%. Therefore, the yield of aqueous crude extract was higher compared to ethanolic extract. The aqueous extract was brownish-green with solid but nonsticky consistency, while ethanolic extract was dark green with a solid sticky consistency.
The difference yield percentage of ethanolic and aqueous extracts on the same family of P.sarmentosum was observed by the previous study. The yield of ethanolic extracts of Piperguineense was higher than aqueous extracts with the percentage 11.2% and 9.0%, respectively, with the maceration method. In addition, the weight of aqueous and ethanolic extracts of Piper cubeba and Piper nigrum using Soxhlet extractor was almost similar, with the difference about 0.05 g only. The variation in yield of this study as compared to previous research might be due to the polarity of the solvents used in the extraction process as well as the method of extraction. The solubility of the desired materials is depended on the type of solvent, temperature, the size of raw samples, and time taken for extraction to be completed. These factors will affect the extraction efficiency and yield.
The polarity, safety, selectivity, and solubility of the solvents need to be considered. The polarity of solvent and solute should be approximately similar. Water has high polarity compared to ethanol and both are considered as universal polar solvents for extraction. However, the selectivity and solubility tend to be different depends on certain plants or extraction methods. Besides, the temperature also plays a big role. Some active compounds are thermally unstable and have a high chance to be degenerated. Zhang et al. suggested that the solubility can be increased using high temperature but not too high as to avoid the deterioration of the thermal unstable compound. Furthermore, the long duration of extraction in certain times will contribute to higher extraction productivity.
Phytochemical analysis by gas chromatography–mass spectrometer
GC-MS analysis of the aqueous and ethanolic crude extract of P. sarmentosum produced the chromatogram shown in [Figure 1] and [Figure 2], respectively. From the figure, the most significant peaks were observed, and the retention times were recorded in [Table 2] and [Table 3]. The compounds were hit results obtained by the internal library search available with the Perkin Elmer Turbomass software (PerkinElmer, Inc., Waltham, USA).
|Figure 1: Chromatogram of gas chromatography–mass spectrometer analysis of active fraction from the aqueous crude extract of Piper sarmentosum|
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|Figure 2: Chromatogram of the gas chromatography–mass spectrometer analysis of active fraction from the ethanolic crude extract of Piper sarmentosum|
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|Table 2: The retention time of chromatogram and the expected compound in aqueous crude extract of Piper sarmentosum|
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|Table 3: The retention time of chromatogram and the expected compound in ethanolic crude extracts of Piper sarmentosum|
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From the chromatogram in [Figure 1], it was observed that the first compound to elute was identified as acetic acid followed by guanidine, oxazole, pyranone, benzofuran, asarone, 1-pentadecene, and hydrocinnamic acid, as seen in [Table 2]. Hydrocinnamic acid showed a high relative abundance and percentage of concentration compared to other compounds.
Based on the chromatogram in [Figure 2], seven compounds were identified which are isocaryophyllene, myristicin, β-asarone, α-asarone, apiol, 1,1-dichlorocyclopentane, and 14-chloro-1-tetradecanol, as seen in [Table 3]. β-asarone was the highest of concentration among all the identified compounds.
The aqueous and ethanolic extract of P.sarmentosum depicted different types of compounds based on the GC-MS analysis. β-asarone was present in both the extracts, but it exhibited lower concentration in aqueous extracts with 2.98% only compared to ethanolic extract. The major compounds in aqueous extracts were hydrocinnamic acid (33.53%), acetic acid (23.19%), and oxazole (15.91%). Hussain et al. recorded from their study that hydrocinammic acid and myristicin were present in P.sarmentosum. Meanwhile, the most abundant compounds in ethanolic extract were β-asarone (37.37%), α-asarone (30.39%), and myristicin (24.26%). A study conducted by Sakilan et al. indicated the presence of asarone (73.72%) in leaf ethanolic extract of P.sarmentosum using GC-MS analysis. Similarly, the presence of α-asarone in the ethanolic extract of P. sarmentosum's leaves was also been proven by Sim et al.
The antifungal susceptibility testing was performed using a disc-diffusion method. The result is illustrated in [Table 4]. The average diameter of inhibition of zone was recorded in [Table 5].
|Table 4: Antifungal susceptibility test of aqueous and ethanolic extracts after 72 h|
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|Table 5: Average diameter of inhibitory zone for antifungal susceptibility testing|
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Based on the results, the aqueous extracts of P.sarmentosum at the concentration of 100 mg/mL showed a slight inhibition toward Fusarium verticilloides, whereas other concentrations as well as ethanolic extracts exhibited no inhibition towards the same fungi. The aqueous and ethanolic extracts of P. sarmentosum showed no inhibition at all concentrations toward Candidaalbicans, Aspergillusniger, and Microsporum gypseum. The inhibition only occurred from aqueous extracts of P.sarmentosum at the concentration 100 mg/mL against F.verticilloides with an average diameter of 7.3 mm ± 0.06. According to Khusnul et al., the ethanolic extracts of P.sarmentosum at 100% concentration exhibited strong inhibition against M.gypseum and C.albicans with inhibition zone diameter of 34.2 mm and 16.3 mm, respectively. Meanwhile, there is no previous research recorded on the antifungal activity of P.sarmentosum against F.verticillioides.
| Conclusion|| |
This study concludes that the difference between solvent of used for extraction will yield different compositions and yield of crude extracts. The aqueous crude extract of Psarmentosum has high yield as compared to ethanolic crude extracts. The chemical analysis using GC-MS resulted in the identification of acetic acid (23.19%), hydrocinnamic acid (33.53%), oxazole (15.91%), guanidine (5.13%), pyranone (7.38%), benzofuran (6.68%), β-asarone (2.98%), and 1-pentadecene (5.20%) in aqueous extracts, and β-Asarone (37.37%), α-Asarone (30.39%), myristicin (24.26%), apiol (1.16%), isocaryophyllene (2.33%), 1,1-dichlorocyclopentane (3.24%), and 14-chloro-1-tetradecanol (1.25%) in ethanolic extract. C.albicans,A.niger, M.gypseum, and F.>verticillioides were found to be insusceptible toward P.sarmentosum's ethanolic extract. However, the aqueous extract showed inhibition on the growth of F.verticillioides at the concentration of 100 mg/mL with an average diameter of 7.3 mm ± 0.06. To conclude, the finding of this study indicates that the aqueous extract of P.sarmentosum possesses antifungal activity against F.verticilloides.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]