|Year : 2021 | Volume
| Issue : 1 | Page : 13-18
Antituberculosis activity of polyphenols of Areca catechu
Archana Raju1, Suparna Subrata De1, Mukti Kanta Ray2, Mariam Sohel Degani1
1 Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, N. P. Marg, Matunga (E), Mumbai, India
2 Radiation Medicine Centre-BARC, Tuberculosis Immunology and Immunoassay Development Section, Tata Memorial Hospital- Annexe Building, Parel, Mumbai, Maharashtra, India
|Date of Submission||28-Oct-2020|
|Date of Acceptance||30-Nov-2020|
|Date of Web Publication||28-Feb-2021|
Mariam Sohel Degani
Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, N. P. Marg, Matunga, Mumbai - 400 019, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Polyphenols have been studied for their potential involvement in the prevention of various chronic diseases as well as for their antimicrobial potential. The crude extracts of arecanut have been reported to have antiinfective properties. We aimed to explore the endosperm of Areca catechu (arecanut) for the extraction of polyphenol components and to study the antituberculosis activity of these polyphenol against Mycobacterium tuberculosis H37Rv. Method: A comparative extraction was performed using microwave and Soxlet apparatus. High performance liquid chromatography (HPLC) technique was used for the estimation of the extracted polyphenols. The minimum inhibitory concentration (MIC) values against M.tuberculosis H37Rv stain, Staphylococcus aureus and Escherichia coli were estimated by resazurin microtiter assay. Results: There was a 11-fold increase in the total phenolic content by microwave assisted extraction compared to the Soxhlet extraction. The powdered extract was found to be active with MIC value of 0.975 ± 0.02 μg/mL. Fractionation and HPLC-based estimation of the extract revealed catechin, epicatechin, and epigallocatechin gallate to be the polyphenol components in the ethanol fraction. Conclusions: The bioactivity of these polyphenols confirmed their presence and complementary effect in the extract form. Because the toxic alkaloid arecoline, known to be present in arecanut, did not show any activity individually, the bioactivity of the extract was attributed to the nontoxic polyphenols present. This extract also showed selective inhibition of M. tuberculosis over other gram positive and gram-negative bacteria, thereby establishing that arecanut is an exploitable selective source of polyphenols acting against M. tuberculosis.
Keywords: Anti-tuberculosis activity, arecanut, microwave-assisted extraction, polyphenols
|How to cite this article:|
Raju A, De SS, Ray MK, Degani MS. Antituberculosis activity of polyphenols of Areca catechu. Int J Mycobacteriol 2021;10:13-8
| Introduction|| |
Over the course of the last 20 years, polyphenols have been studied for their potential involvement in the prevention of chronic diseases, such as cardiovascular diseases, cancer, osteoporosis, diabetes mellitus, and neurodegenerative diseases. Initially, their protective activity had been attributed to their antioxidant, free radical scavenger, and metal chelator properties, later to the capability of inhibiting or reducing different enzymes, such as telomerase, cyclooxygenase, or lipoxygenase, and in more recent years, to the interaction with signal transduction pathways and cell receptors. The antimicrobial activity of polyphenols occurring in vegetables and medicinal plants has been extensively investigated against a wide range of microorganisms. Among polyphenols, flavan-3-ols and flavonols received most attention due to their wide spectrum and higher antimicrobial activity in comparison with other polyphenols.
The crude extracts of many plant species, especially those with ethnomedical uses have been assessed for in vitro antimycobacterial properties; however, relatively few active compounds have been isolated. In most cases, the crude extracts showed better activity than the isolated individual components because synergistic association may exist between the components.
Arecanut is the seed of the areca palm (Areca catechu). The seed contains alkaloids, such as arecaidine and arecoline, which when chewed, are intoxicating and addictive. The major polyphenols reported in arecanut include catechin, epicatechin, and epigallocatechin gallate (EGCG). The crude extracts of arecanut have been reported to have anti-infective properties against small pox, dysentery, cholera, and leucorrhea. The extracts have also been reported to show activity against Mycobacterium tuberculosis H37Rv within infected cells at 1:40 in tube dilution test; however, this study included the ethanol extract and did not mention the complete removal of the alkaloids that are the toxic components of arecanut. Hence, our study focused on the extraction of the polyphenol components of arecanut to study their antituberculosis activity. Because microwave-assisted extraction (MAE) has been reported to show better extraction of polyphenols, this technique was explored for the extraction from arecanut using methanol as the solvent.
| Method|| |
The endosperm of A. catechu, Mangalore variety, was procured from the local market. The plant material was authenticated by Dr. Ganesh Iyer of Ramnarain Ruia College, L Nappo Road, Matunga, Dadar East, Mumbai, Maharashtra 400019 (Voucher specimen number: MSD2012P01). Catechin, epicatechin, EGCG, gallic acid, and arecoline, procured from Sigma-Aldrich Chemicals, Mumbai, India, were provided by Prof. Rekha S. Singhal of Institute of Chemical Technology. Folin–Ciocalteu reagent and all solvents were procured from HiMedia®.
Defattening of plant material
The endosperm of arecanut was ground in a stainless steel mixer grinder to obtain 10.0 g of fine powder. In order to separate the fat-based components from the ground samples, petroleum ether treatment using Soxhlet apparatus was performed at 65°C for 2 days. Treatment was followed by filtration, and the residual material was collected. The plant material was then freeze dried in amber-colored vials in small approximately 2.0 g quantities and packed and stored at 4°C in the same vials.
For the comparative study, two techniques, Soxhlet and MAE were employed for the arecanut plant material. The concentration of methanol was optimized for Soxhlet extraction and then used for the other extraction technique. Soxhlet extraction was performed at 65°C ± 5°C for 2 days for 1.0 g of the arecanut plant material. MAE was operated in a conventional microwave at 140 W for 15 min (60°C, three cycles) for 1.0 g of arecanut plant material. The protocol employed for the comparative study of the two extraction protocols was as summarized in [Table 1].
|Table 1: Comparison of conventional extraction and microwave-assisted extraction|
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Determination of total phenolic content
The quantitative assays used for the analysis was Folin–Ciocalteu assay for the determination of total phenolic content (TPC). In total, 1.0 mg/mL of methanol extracts was used for the analysis. The assay mixture was prepared by mixing 0.5 mL of methanol extracts, 2.5 mL of 10% Folin–Ciocalteu reagent, and 2.5 mL of 7.5% sodium bicarbonate. A control reaction was concomitantly prepared, which contained 0.5 mL methanol, 2.5 mL of 10% Folin–Ciocalteu reagent, and 2.5 mL of 7.5% sodium bicarbonate. Samples were thereafter incubated for 30 min at the room temperature. Absorbance was determined using spectrophotometer at λmax 765 nm. Samples were analyzed in triplicate, and the mean value was calculated. A standard calibration plot was generated using the known concentrations of gallic acid. The concentrations of phenols in the test samples were calculated from the calibration plot and expressed as mg gallic acid equivalent (GAE) per g of sample.
Biological activity estimation (whole cell assay)
The crude extract of arecanut was screened against M. tuberculosis H37Rv using Middlebrook 7H9 medium (HiMedia®) to determine the minimum inhibitory concentration (MIC) using resazurin microtiter assay (REMA) (Palamino et al., 2002). The first line drug ethambutol (HiMedia®) was used as the standard drug. The testing was performed at Radiation Medicine Center -Bhabha Atomic Research Center, Mumbai. Mid-log phase M. tuberculosis (H37Rv) cells were used for testing. Resazurin (HiMedia®), 0.02% in sterile distilled water, filter sterilized through 0.22 μ filter, was used as the indicator dye. Stock solution of compounds was prepared in dimethyl sulfoxide (HiMedia®), and at the time of testing, fresh dilutions of the stock solutions were prepared in Middlebrook 7H9 media to achieve the concentrations required.
Bioactivity guided fractionation
To concentrate and obtain polyphenol rich fractions before analysis, crude extract of arecanut obtained after MAE was subjected to fractionation by sequential extraction or liquid–liquid partitioning using solvents with increasing polarity (hexane, chloroform, ethyl acetate, and ethanol). Aqueous solution of extract and equivalent volume of respective solvent was separated using a separating funnel. The different fractions were collected, and the solvents were evaporated using a rotary evaporator.
High-performance liquid chromatography analysis
High-performance liquid chromatography (HPLC) analysis of the ethanol fraction of the arecanut extract (0.1% aqueous solution) was performed on an HPLC system consisting of Agilent Technologies 1200 Series instrument combined with a fixed wavelength ultraviolet (UV)–visible detector. The separation was performed on a Hibar® 250 Purospher® RP-18 encapped (5 μm) Merck column. The mobile phase was optimized to 90:10 methanol: water (100% methanol for first 10 min). The flow rate was maintained at 1.0 mL/min. Detection was performed by the measurement of UV absorbance at 280 nm. Retention times were compared with the standards solutions comprising of 0.1% weight/volume of catechin, epicatechin, and EGCG in water.
Bioactivity of individual components of arecanut
The polyphenol components identified using HPLC were tested for individual MIC values against M. tuberculosis H37Rv by using the protocol mentioned in before.
Bioactivity against other micro-organisms
The crude extract from arecanut was tested against Staphylococcus aureus and Escherichia coli as per the earlier mentioned protocol for REMA. Ciprofloxacin obtained from HiMedia® was used as a reference standard.
| Results|| |
Defattening of plant materials
In total, 10.0 g of powdered material from the plant material of arecanut, when subjected to petroleum ether-based defattening, yielded approximately 8.0 g of powder. This reduction in quantity may be attributed to the removal of lipids and fats into the petroleum ether layer, which on filtration resulted in the defattened material. In general, freeze drying retains the higher levels of phenol content in plant samples than air drying. Hence, freeze drying was performed to protect the plant material from moisture, and this was followed by cold storage.
Extraction of phenol components
The defattened material of arecanut was studied using extraction and fractionation techniques to establish the most efficient technique for extraction.
In total, 1.0 g of defattened arecanut seed material was extracted by varying the hydromethanol solvent ratio and using Soxhlet apparatus, as shown in [Table 2]. Crude extraction with 90% methanol resulted in an optimum TPC. An increase in the methanol content beyond this value conferred no additional extraction efficiency. It is well-known that most polyphenols are more soluble in methanol than in water; however, the extraction of these compounds was found to be better with a small percentage of water. This is probably due to the increase in effective swelling of the plant sample by water, which helped to increase the surface area for solute–solvent contact. This theory is supported by the literature. Hence, a hydromethanol mixture of 90% methanol and 10% water was used for all further extraction processes. Soxhlet extraction yielded TPC of 40.6 ± 0.05 mg GAE/g of extract.
|Table 2: Total phenolic content obtained with various hydromethanol mixtures as solvents|
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In order to optimize the most efficient extraction technique, 1.0 g of defattened arecanut was also extracted employing MAE with 90% methanol in water as the solvent. TPC of 363.0 ± 0.13 mg GAE/g of extract was achieved with MAE.
This comparative analysis indicated that MAE resulted in an 11-fold increase in the polyphenol extraction in comparison with traditional techniques.
Minimum-inhibitory concentration determination
In the REMA method, resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide), which is blue colored in its oxidized state, turns to pink color on reduction by viable cells to resorufin. The antituberculosis testing was performed on mid-log phase cells of Mycobacterium because during this phase the mycobacteria are in the most active growing form. Middlebrook 7H9 medium was selected for cultivation and whole cell assay because it contains many inorganic salts that help in the growth of mycobacteria. Citric acid formed from sodium citrate helps in retaining inorganic cations in solution. Glycerol supplies carbon and energy. Oleic acid and other long-chain fatty acids are essential for the metabolism of mycobacteria. Middlebrook ADC Growth Supplement (FD019) contains bovine albumin, dextrose, and sodium chloride. Some free fatty acids are toxic to mycobacteria, and albumin binds to those fatty acids and prevents toxic action on mycobacteria. Dextrose serves as an energy source. The MIC value for the arecanut crude extract is as depicted in [Table 3]. This extract showed better activity than the first-line drug ethambutol.
|Table 3: Minimum-inhibitory concentration values for the arecanut crude extract|
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Fractionation was performed for the crude arecanut extract using solvents hexane, chloroform, ethyl acetate, and ethanol. [Table 4] shows the polyphenol content obtained in the various fractions. It was observed that the ethanol fraction of the extract showed the maximum TPC.
|Table 4: Total phenolic content and minimum inhibitory concentration values of various fractions of the arecanut extract|
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The MIC value of the ethanol fraction was maximum and the same as MIC of the crude extract [Table 4]. Hence, it may be established that the ethanol fraction contains the bioactive polyphenols.
High-performance liquid chromatography analysis of arecanut extract
HPLC analysis was performed at 280 nm for the ethanol fraction of the arecanut extract and compared with standards. The graphs indicated the presence of catechin or epicatechin or both as a merged peak along with EGCG. The percentages of area under the two peaks were 60.43% and 39.57% as depicted by the graphs shown in Supporting information.
A literature study showed that the catechins in arecanut mainly comprised of catechin and EGCG. This had been established in further studies by other groups., The other constituents extracted vary based on the solvent utilized for extraction. Because methanol was used as the solvent for the extraction by Wang et al., and ethanol by the other three groups, it may be established based on our study that methanol extraction helps extract EGCG as an additional constituent present in the fraction.
The structures of the polyphenols present as peaks in the HPLC graphs when compared with the standards are as shown in [Figure 1].
Minimum-inhibitory concentration of polyphenol components of arecanut
The components identified from the HPLC of the ethanol extract were procured and analyzed for biological activity. The MIC values obtained are shown in [Table 5].
|Table 5: Minimum inhibitory concentration values of polyphenol components of arecanut|
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The comparison of the activity of the ethanol fraction (0.91 ± 0.02 μg/mL) and the individual components [Table 5] confirms the presence of these polyphenols because the activity of the component EGCG is equivalent to that of the ethanol fraction (considering the standard deviation). Hence, these individual components act together to give the overall MIC of the ethanol fraction of the arecanut extract. The activity of the extract may be attributed to the activity of the EGCG component. Because arecoline individually showed an activity >125 μg/mL against M. tuberculosis H37Rv, the overall activity of the extract may be attributed to the presence of the polyphenols. In our earlier published work, these individual polyphenols were found to be active against M. tuberculosis dihydrofolate reductase (DHFR). Hence, it may be concluded that the overall activity of the ethanol fraction of the arecanut extract was mainly due to inhibition of DHFR enzyme of M. tuberculosis.
Anti-infective activities against other bacteria
Along with activity against M. tuberculosis, the crude extract of arecanut obtained by MAE with 90% methanol was screened against one Gram-positive (S. aureus) and one Gram-negative (E. coli) bacteria. The results are shown in [Table 6].
| Discussion|| |
The endosperm of arecanut has been exploited earlier for its antituberculosis activity. However, the crude extraction techniques that were performed earlier did not ensure that the activity was due to the polyphenols and not due to the alkaloid components. Because the alkaloid components are toxic in nature, the current study confirmed that the polyphenols of arecanut have potential activity against the tubercle bacilli.
MAE was found to be a better extraction process than the use of Soxlet apparatus, leading to higher yields of the polyphenols. This may be due to the following reasons. Although dried plant material is used for the extraction in most cases, plant cells contain minute microscopic traces of moisture that serve as the target for microwave heating. The moisture, when heated up inside the plant cell, due to microwave effect, evaporates and generates tremendous pressure on the cell wall due to swelling of the plant cell. The pressure pushes the cell wall from inside, stretching and ultimately rupturing it, which facilitates leaching out of the active constituents from the ruptured cells to the surrounding solvent, thereby improving the yield of the phytoconstituents. This phenomenon can be more intensified if the plant matrix is impregnated with solvents with higher heating efficiency under microwave. Higher temperature attained by microwave radiation can hydrolyze ether linkages of cellulose, which is the main constituent of plant cell wall and can convert it into soluble fractions within 1–2 min. The higher temperature attained by the cell wall during MAE enhances the dehydration of cellulose and reduces its mechanical strength, and this aid the solvent to access the compounds inside the cell easily. Due to all these reasons, MAE was established to be extraction technique of choice for polyphenols in this study.
These results indicate the selective activity of the extract against M. tuberculosis. This lowers the chance of development of cross resistance among the bacteria. Further, as reported in our previous study, the said polyphenols have additive or synergistic activity with para-amino salicylic acid (PAS), a well-known first-line drug for tuberculosis. These results make arecanut an exploitable source of polyphenols against M. tuberculosis.
| Conclusion|| |
In conclusion, the bioactivity of the ethanol extract of A. catechu against M. tuberculosis was attributable to the polyphenolic components and not to the arecolin-based toxic components, thereby warranting further exploration of this source as an antituberculosis agent. Moreover, because these polyphenols have been reported to have additive or synergistic activity with PAS, if further explored in detail along with other first-line and second-line drugs, they may help in lowering the doses of the existing drugs. Studies should also be conducted to explore if these polyphenols have the potential to reverse the resistance against the existing drugs, which would be beneficial to the patients suffering from multi-drug resistant and extremely-drug resistant tuberculosis.
The authors would like to thank Prof. Rekha S. Singhal for providing the polyphenol standards.
Financial support and sponsorship
Author Raju A is thankful to Indian Council of Medical Research, India (PHA-BMS-45/94/2012), and All India Council for Technical Education, India (RPS fund Ref no.: 8-2014/RIFD/RPS/POLICY-1/2014-15), for financial assistance. De S. S. is thankful to University Grants Commission, India (F.25-1/2014-15[BSR]/No.F.5-63/2007[BSR]) for financial support.
Conflicts of interest
There are no conflicts of interest.
| Supporting Information|| |
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]