PHENOLIC ACID DERIVATIVES AS PRESERVATIVES: SYNTHESIS, ANTIOXIDANT, ANTIMICROBIAL POTENTIAL AND THEIR PRESERVATIVE EFFECTIVENESS

PHENOLIC ACID DERIVATIVES AS PRESERVATIVES: SYNTHESIS, ANTIOXIDANT, ANTIMICROBIAL POTENTIAL AND THEIR PRESERVATIVE EFFECTIVENESS

Sumit Sigroha, Amit Lather and Anurag Khatkar *

Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak, Haryana, India.

ABSTRACT: Natural phenolic acids alkyl esters viz methyl, ethyl, propyl was synthesized and characterized by spectral means FTIR, 1HNMR, and 13CNMR. All the synthesized esters were examined for their antimicrobial potential, preservative efficacy and antioxidant potential. Among the synthesized ester derivative caffeic acid and gallic acid, derivatives were displayed excellent antioxidant and antimicrobial potential. They were further evaluated their preservative efficacy according to USP 2004 protocol for preservative effectiveness testing. Caffeic acid propyl ester and gallic acid ethyl ester exhibited promising preservative potential better than existing preservative agents. From the study, we can conclude that these caffeic and gallic acid derivatives can be used as lead compounds to further explore their application as preservative agents in pharmaceuticals and in the food industry.

Keywords: Phenolic acid derivative, Cellulose, Antioxidant, Antimicrobial, Preservative potential, Ester derivatives

INTRODUCTION: Natural phenolic acids are widely distributed in plants, generally found as amides, glycosidic conjugates, and esters in various fruits and vegetable cell walls ¹. Alkaline hydrolysis is used to extract these phenolic acids from nature. Phenolic acids are widely utilized antioxidants among food, pharmaceuticals, and beauty care products. Numerous scientific studies have reported biological activities of phenolic acids, and most of the studies confirmed that these phenolic acids possessed excellent antitumor, hypoglycaemic, antihypertensive, anti-inflammatory potential etc. ².

Biological properties of phenolic acids were conferred by benzene ring, carboxylic group, and the molecule structure bearing one or more hydroxyl and/or methoxyl groupings ⁴. Formation of resonance stabilized phenoxy radical attributed to the presence of side chain conjugation and phenolic nucleus upon UV absorption phenolic acids promotes the stable radical formation. Hence, their ability is enhanced to terminate free radical chain reactions.

Due to their ability to suppress radiation-induced oxidation reactions and excellent free radical scavenging potential various phenolic acids are being used to protect the physiological integrity of cells. Due to photoprotection action, phenolic acids are incorporated in many cosmetic lotions. The addition of natural phenolic acids in food products increases shelf life by preventing oxidation spoilage by inhibiting lipid peroxidation. By the same mechanism, phenolic acid may protect against various inflammatory diseases ⁵. According to the latest findings, position and a number of free phenolic hydroxyl groups significantly affect the strength of biological activities ⁶.

Natural phenolic acids have also been reported for their different biological activities, as represented in Table 1. Caffeic acid, which is a natural phenolic acid exhibits excellent antioxidant activity ⁷ and has been reported for its anti-inflammatory, antibacterial and anti-tumor activities ⁸. Another natural cinnamic acid: sinapic acid extracted from mustard seeds, was tested against E. coli (Gram-negative) and S. aureus (Gram-positive) and found to exhibit antimicrobial activity ⁹. Phenolic compounds are natural antioxidants widely distributed in plants and become a vital part of the human diet with decreased risk of cardiovascular and cancer diseases linked with oxidative stress ¹⁰. The possession of excellent biological properties of phenolic compounds has become an incentive for scientists to explore these natural compounds further, and it seems logical due to their availability in natural sources. Thus to extend their role in the pharmaceutical and food industries, the solubility and stability of these natural antioxidants must be improved ¹¹. An amphiphilic molecule can be obtained by esterification of carboxylic acid functional group with alcohols ¹². Moreover, the relationship between their antioxidant efficacy and hydrophobicity is well described by some studies ¹³. The present study has reported the synthesis, antimicrobial and antioxidant activity of the synthesized derivatives of phenolic acids along with the preservative efficacy of ethyl 3, 4, 5-trihydroxybenzoate, and propyl 3-(3,4-dihydroxyphenyl) acrylate.

TABLE 1 BIOLOGICAL PROPERTY OF NATURAL PHENOLIC ACIDS

MATERIALS AND METHODS: All the required chemicals for the synthesis of phenolic acid drivatives and their antimicrobial, antioxidant, and preservative efficacy test were bought from CDH Pvt. Ltd. (New Delhi, India) and Loba Chemie Pvt. Ltd. (Mumbai, India). The reaction monitoring was done by thin-layer chromatography on pre-coated silica gel plates for TLC obtained from Merck (Mumbai, India) and was inspected in UV chambers, Hexane: Ethyl acetate (7:3) was utilized as a solvent for TLC. Sonar melting point apparatus was used to record the melting points.

KBr pellets procedure was used to record Infrared (IR) spectra using Perkin Elmer spectrum II FTIR spectrophotometer. 13C NMR and 1H NMR spectra were affirmed in deuterated CDCl3, and DMSO separated at 400 MHz downfield using tetramethylsilane standard on Bruker Advance II 400 NMR spectrometer. Coupling constants (J) and chemical shifts were recorded in Hertz (Hz) and in parts per million respectively.

Procedure for the Synthesis of Phenolic Acid Chlorides: The selected phenolic acid (20mmol) was stirred at 80 °C for 2-4 h with 10ml thionyl chloride in presence diethyl ether as solvent and pyridine as catalyst. The excess of thionyl chloride was distilled off and the corresponding product was dried and weight as acid chloride. The single spot TLC under UV light confirmed the purity of acid chloride formed.

General Procedure for the Synthesis of Esters of Phenolic Acids Esters: Phenolic acid esters were synthesised by refluxing respective alcohols with the corresponding synthesised acid chlorides (0.05 mol) in 50 ml ether for 8-10 h at 70-80 °C as per Scheme 1, 2 and 3.

The reaction mixture was subjected to reflux on the water bath until the liberation of HCl gas was ceased, and the end of the reaction was confirmed with the appearance of single spot TLC. At the final stage, the ester was extracted with ether, and recrystallization was done with acetone. The percentage yield was recorded at room temperature after drying.

In-vitro Antimicrobial Activity: The prepared esters were examined for antimicrobial susceptibility using the tube dilution method against bacterial strains K. pneumoniae, E. coli, P. mirabilis, S. aureus, P. Aeruginosa, and fungal strains A. niger, and C. albicans. Stock standards of antibiotics viz. streptomycin and fluconazole were obtained as gift samples from pharmaceutical companies. The synthesized compounds were dissolved in DMSO (Dimethyl sulfoxide) to a concentration of 100 µg/mL and diluted further to concentrations of 50, 25, 12.5, 6.25, 3.125, and 1.562 µg/mL ²².

Double strength nutrient broth media I.P. for antibacterial study and Sabouraud dextrose broth media I.P. for the antifungal study were used ²⁴. The test tubes were examined after 24 h of incubation at 37±1°C for bacterial stains and after 2 days of incubation at 25±1°C for C. albicans and after 7 days of incubation for A. niger. Tubes were scanned for any visible turbidity or sediment, and tubes with no visible growth at least amount of test compound were reported as MIC (Minimal Inhibitory Concentration).

Preservative Efficacy: The selected most active antimicrobial compounds were tested for their preservative efficacy where the E. coli, P. aeruginosa, S. aureus, C. albicans, and A. nigerwere used as challenge microorganisms. The results were noted on 14th and 28th day. Pulp-based slurry of cellulose was used to evaluate the preservative effectiveness of synthesized compounds. The test compound and standard preservative compounds were incorporated by adding 0.5% of the test compound and standard compound in a mixture of 100 mg cellulose in 2 mL of 0.1M sodium phosphate buffer and 4 mL of dimethyl sulfoxide ²³. After 24 h stirring of the above mixture at 25 °C the sample was mixed with 1.0 M sodium chloride for 24h and washed with distilled water. The washing was done with DMSO and diethyl ether and distilled water in case of antifungal compounds, and the preparations were stored in sterile containers ²⁴. Preservative efficacy testing was performed according to the standard protocol as per USP-2004. The inoculum was prepared from recently grown stock culture on agar plates. The sterile test tubes with 10 mL of cellulose slurry were incubated with an inoculum of microorganisms at an optimal temperature under aseptic conditions. Each test tube was scanned at 7, 14, 21, and 28 days to determine the CFU by using the plate count procedure. The number of CFU was noted for each sample and calculated as logarithm values of the number of CFU/mL compared according to the standard protocol of USP 2004. Criteria for the test include not less than 1.0 log reduction from the initial count at 14 days and no increase from the 14 days count at 28th days ²⁵.

Anti-oxidant Activity: DPPH free radical procedure depends on the movement that turns violet coloration in ethanol. Antioxidant action depended on free radical scavenging potential towards stable DPPH radical on spectrophotometric reaction as per the technique detailed in literature ²⁶. Solutions of synthesized esters in methanol were made in 50 ml quantity at 25, 50, 75, and 100 μg/mL concentrations. A stock solution was prepared by 0.1 mM DPPH dissolved in methyl alcohol and 1 mL from this solution was added to 3 mL of test or standard and incubated at 30 °C for 30 min in the dark. The ascorbic acid was used for differentiation or as a positive control. At first, the clear range for ethanol/water was recorded in this manner; the spectrophotometric titration was done at different concentrations of synthesized compounds, and absorbance was estimated at 517 nm. Absorbance at a lower wavelength of the reaction mixture demonstrated higher free radical scavenging potential. The tests were performed in triplicate, and IC₅₀ values were determined by utilizing the equation:

IC₅₀ = (Ac-As) × 100/Ac

Here, as is the absorbance of the sample, and Ac is the absorbance of the control

RESULTS AND DISCUSSION:

Chemistry: A series of esters of cinnamic acid and benzoic acid derivatives were synthesised. The methyl, ethyl, and propyl esters were synthesized per the reaction outlined in Scheme 1, 2 and 3. The final structures of all the synthesized compounds were confirmed using IR, 1HNMR, 13CNMR and were found in close conformity with their molecular structures. The phenolic acid chlorides were initially prepared by stirring the respective phenolic acids (20 mmol) with 10ml thionyl chloride at 80°C for 1-4 h in the presence of diethyl ether as solvent using pyridine as a catalyst.

The reaction completion was examined by thin-layer chromatography (TLC). The single spot TLC under UV light observation confirmed the purity of acid chloride formed. Alkyl esters of phenolic acids were further synthesized by refluxing with the respective alcohols with corresponding acid chlorides (0.05 mol) in ether (50 ml) for 8–10 h at 70-80 °C. The reaction mixture was refluxed, and the end of the reaction was affirmed by single spot TLC. Finally, the ester was extracted with ether, and recrystallization was done with acetone to obtain the final product. Physicochemical properties and spectral data of the synthesized compounds have been shown in Table 2. Further, the synthesized products were studied for their spectral data viz. FTIR, 1H-NMR, 13C-NMR, wherein the shift in FTIR peaks from 1700 cm^-1 in phenolic acids for carbonyl group to 1730-1745 cm^-1 confirmed the corresponding formation ester derivatives.

1H-NMR was performed for the above-synthesized structures, and the appearance of chemical shift near δ 4.1-4.6 ppm for the ester linkage and disappearance of the peak near δ 11 ppm for OH functional group of carboxylic acid confirmed the formation of esters of the given phenolic acids. The 13CNMR ester peaks were recorded at 165-180 ppm for synthesized ester derivatives.

SCHEME 1: SYNTHESIS OF METHYL ESTERS

SCHEME 2: SYNTHESIS OF ETHYL ESTERS

SCHEME 3: SYNTHESIS OF PROPYL ESTERS

TABLE 2: PHYSICOCHEMICAL PROPERTIES AND SPECTRAL DATA OF SYNTHESIZED COMPOUNDS

In-vitro Antimicrobial Activity: The synthesized alkali esters showed good antimicrobial activity against the selected bacterial and fungal strains. The MIC values of the test, as well as standard compounds, have been summarised in Table 3. The results of antibacterial screening indicated that compound M1 (MIC=6.25 µg/mL), compound E1 (MIC = 6.25µg/mL) containing a hydroxyl group and methoxy group in adjacent position on ring and compound P10 (MIC=6.25µg/mL) containing two hydroxyl group at adjacent position exhibited promising activity against K. pneumonia. Compound E3 (MIC=12.5µg/mL) and P10 (MIC=12.5 µg/mL) having a hydroxyl group at ortho position effectively inhibited the growth of P. mirabilis. However, compound P10 (MIC 12.5 µg/mL) having a long alkyl chain was found to inhibit the growth of E. coli to a lesser extent than a short alkyl chain containing E3 (MIC = 6.25 µg/mL). Moreover, compound P10 and compound E3 were also found to be better antimicrobials than standard compounds against S. aureus  (MIC = 25 µg/mL) and against P. aeruginosa (MIC = 6.25 µg/mL). The results of antifungal activity indicated that compound P10 exhibited better activity than standard compound against the tested strains and exhibited better activity against C. albicans (MIC= 6.25 µg/mL) however, the E3 exhibited the comparable activity to that of the standard antifungal drug (25 µg/mL). The antifungal activity against A. niger for the compound P10 was found to be (MIC= 12.5 µg/mL), compound E3 (MIC= 12.5 µg/mL), while compound E8 (MIC=12.5 µg/mL) with a single electron-donating group was also found potent against A. niger. The standard drugs for comparison of antibacterial results were streptomycin, while fluconazole was used as a standard for antifungal evaluation.

TABLE 3 MIC OF SYNTHESIZED ESTERS OF NATURAL ACIDS

Preservative Efficacy: The results of the preservative efficacy study of the selected synthesized compounds in cellulose slurry were performed and summarized in Table 4. The log CFU/mL for ethyl 3, 4, 5-trihydroxybenzoate (E3), and propyl 3-(3, 4-dihydroxyphenyl) acrylate (P10) revealed that the values were within the prescribed limit of USP standard criteria. The selected compounds E3 and P10 reduced the growth of microbes on the 14^th and 28^th day from the initial count, and there is more than 1.0 log reduction from the initial count. The results of preservative efficacy were also found comparable to standard preservatives taken.

TABLE 4 LOG CFU/ML VALUES OF THE SELECTED COMPOUNDS IN CELLULOSE SLURRY

*Initial microbial count present in inoculums 1x105-1x106 CFU/mL

Anti-oxidant Activity: Antioxidant potential of all the synthesized esters were evaluated by DPPH radical scavenging assay method, and result were summarized in Table 5. Further, the results revealed that the compound E3 (IC₅₀ value 6.48±0.001μM) and P10 (IC₅₀ value 06.09 ± 0.042μM) were found more potent antioxidants than reference l-ascorbic acid (IC₅₀ value 8.5.18±0.009 μM). Compound M3 and P7 also showed comparable antioxidant potential to reference with IC₅₀ values as 08.39 ± 0.007 μM and 08.22 ± 0.012 μM, respectively. Both had hydroxyl groups at meta position where hydroxyl groups act as electron-withdrawing, thus facilitating hydrogen release from acid derivatives. While compound M5 (IC₅₀ value 18.80 ± 0.003 μM) and E5 (IC₅₀ value 13.73 ± 0.045μM) exhibited the lowest antioxidant activity because of the presence of hydroxyl group at adjacent positions on the phenolic ring, the adjacent arrangement leads to stabilization of molecule against the release of hydrogen ion.

TABLE 5: DPPH RADICAL SCAVENGING ACTIVITIES OF SYNTHESIZED DERIVATIVES

^aValue are expressed as mean ± SEM, n = 3

CONCLUSION: This study has ascertained that the derivatives of phenolic acids possessed the excellent preservative ability. Based on the antimicrobial results of the present study, methyl 4-hydroxy-3-methoxybenzoate and ethyl 4-hydroxy-3-methoxybenzoate have demonstrated better antimicrobial activity that is comparable to standard compounds, and both the compounds showed better antioxidant potential than standard l-Ascorbic acid. Further, the preservative efficacy test clearly showed that ethyl 3, 4, 5-trihydroxy-benzoateand propyl 3-(3, 4-dihydroxyphenyl) acrylate were effective against all selected strains used during the study and even better than reference compounds in the case of E. coli and S. aureus.

Data Availability: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

ACKNOWLEDGMENT: Authors are thankful to the Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak, Haryana (India), for providing the necessary laboratory facilities to carry out this research work.

COMPETING INTERESTS: The authors declared that they have no competing interests.

Ethics Approval and Consent to Participate: Not Applicable

Funding: No funding was received for this research work from outside sources.

Author Contributions: The author's SS, AL, and AK have designed, synthesized, and carried out the work in equal contribution. All authors have read and approved the final manuscript.

Author Statement: All the methods were carried out in accordance with relevant guidelines and regulations.

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    How to cite this article:

    Sigroha S, Lather A and Khatkar A: Phenolic acid derivatives as preservatives: synthesis, antioxidant, antimicrobial potential and their preservative effectiveness. Int J Pharm Sci & Res 2021; 12(10): 5526-37. doi: 10.13040/IJPSR.0975-8232.12(10).5526-37.

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