Spectrum of Emerging Sciences, 3 (1) 2023, 1-7

Synthesis of potassium salts derived from natural acids

Pankaj Patel¹, Shilpi Shrivastava*², Preeti Pandey³

¹MSc Chemistry IV Sem, Department of Chemistry, Kalinga University, Raipur 492101(C.G.)

²Professor & Head, Department of Chemistry, Kalinga University, Naya Raipur 492101(C.G.).

³Assistant Professor, Department of Chemistry, Kalinga University, Naya Raipur 492101(C.G.).

*Corresponding Author:

E-mail Address: shilpi.srivastava@kalingauniversity.ac.in

Article available online at: https://esciencesspectrum.com/AbstractView.aspx?PID=2023-3-1-1

Introduction

Phenolic acids display numerous biological effects as a result of their inherent properties of anti-oxidation. Phenolic acids are available abundantly in environment and are consumed orally by human beings through vegetables and fruits. The research and interest on the profile of phenolic acids is seen to increase gradually and is directly related to the activity of anti-oxidation and greatly aid in defending the body against formation of free radicals which affects the normal metabolic activity of cells. Biosynthesis of phenolic acids in plants is due to phenylpropanoid which is a metabolic activity [1]. The free radical scavenging of phenolic acids is primarily established on the redox properties of the existing hydroxyls and their relation with the other chemicals in the compound. Anti-oxidants are believed to have potential ability in fighting diseases such as neurological, cardiac-relation, cancers and various others [2]–[4]. In relevance to medicines, a lot of research has been dedicated on identifying and developing phenolic acids from natural sources such as plants.

Plants have acquired the capacity for producing numerous ancillary metabolites which will not contribute to the regular growth, development and reproduction but they assist in ecology, defense activities and in adaptation to the surrounding environment [5]–[8]. Plants that have known for such ancillary metabolism have been reported to be of great value in fields of food, textile, medicine and industries. Such plants are recognized as plant phenols [9]. In terms of structure, phenols have a ring with hydroxyl groups attached and they are aromatic compounds. The phenolic acids may have structures varying from simple acids to complex polymers [10]. A lot of research on phenolic’s from plants and their applications to commercial and medicinal purposes have been reported [11]–[15].

In this paper we have reported the extraction of phenolic acids and derived potassium salts from the phenols.  For identification and quantification of compounds gas chromatograph in with mass spectrometer (GC--MS), thin layer chromatography (TLC), high performance liquid chromatography (HPLC) were used. We have quantified the properties of these alkali salts and evaluated their anti-oxidation activity, anti-microbial activity, and their application as preservatives.

2. Materials and Methods

2.1 Materials

The compounds that were necessary for synthesizing sodium derivatives from phenolic acid and evaluating their anti-microbial activity, anti-oxidant activity, and efficiency of preservation was procured from CDH ltd, and Loba Chemicals. For performing TLC (thin layer chromatography) the plates of silica-gel were coated. Solvents which was used for extraction was Hexane and Ethyl acetate.

2.2   Methods

2.2.1. Extraction of phenolic acids

Extraction of phenolic acids can be done on frozen, fresh or dried plant mass. We have employed dried biomass as the cell are active in fresh biomass and can degrade the phenolic acids. So the dried biomass was washed, dried and powdered. For extraction of phenolic acids, a solution comprised of 20% to 50 % ethanol, methanol, acetone or an amalgam of these solvents in water was made. This extract in its crude form is hydrolyzed using 1 to 4 M sodium hydroxide for liberation of phenolic acids. The lipids present in in the crude extract are removed by liquid fractionation using hexane as solvent.

2.2.2. Deriving potassium salts from phenolic acids

Cells of plant possess phenolic acids which are integrated and these will be released upon alkali hydrolysis. Potassium salt of naturally occurring phenolic acids viz. cinnamic acid and benzoic acid were obtained by the neutralization of phenolic acid with KOH. Table 1 shows structure of synthesized alkali derivatives.

3. Characteristics

The alkali salts that were synthesized as preservatives were characterized by spectral means FTIR, ¹HNMR, ¹³CNMR, mass spectroscopy and elemental analysis.  The infrared spectra (IR) were carried out using KBr pellets method on Perkin Elmer spectrum II. The characterization of ¹³C NMR and ¹H NMR spectra was analyzed by CDCl₃, and DMSO. The separation was done at 400 MHz by employing standard, tetramethylsilane (TMS) and performed on NMR spectrometer, Bruker Advance II 400. The measurements of chemical shifts are documented in ppm and the coupling-constant (J) was documented in Hertz (Hz).

4. Results and Discussions

4.1.  Chemistry

Potassium salt of naturally occurring phenolic acids viz. cinnamic acid and benzoic acid were obtained by the neutralization of phenolic acid with KOH. Table 1 shows structure of synthesized alkali derivatives.

Table 1 Structures of synthesized Alkali derivatives

4.2.  Characterization of synthesized alkali derivatives of natural acids

KBR pellets were used for IR studies and the unit was cm-1.

Potassium vanilate: 1735 (C=O str., ester), 3349 (OH str. Phenol), 1059.37(C-O-C str., -OCH3), 2363(C-H str., OCH3), 1346 (C=C str., aromatic) 1H NMR (400 MHz, DMSO-d6) δ: 7.34 (m, 3H, ArH), 5.40 (s, 1H, OH of phenolic hydroxyl), 3.63 (s, 3H, -OCH3)

Potassium veratrate: 1648 (C=O str., ester), 1180 (C-O-C str., -OCH3), 2363(C-H str., OCH3), 1388 (C=C str., aromatic)  1H NMR (400 MHz, DMSO-d6) δ: 8.01 (m, 3H, ArH), 3.83 (s, 3H, -OCH3)

Potassium gallate: 1637 (C=O str., ester), 3424 (OH str. Phenol), 1406 (C=C str., aromatic) 1H NMR (400 MHz, DMSO-d6)  δ: 7.87 (m, 3H, ArH), 4.71 (s, 1H, OH of phenolic hydroxyl)

Potassium syringate: 1738(C=O str., ester), 3394 (OH str. Phenol), 1057(C-O-C str., -OCH3), 2345 (C-H str., OCH3), 1287 (C=C str., aromatic)1H NMR (400 MHz, DMSO-d6) δ: 7.80 (m, 3H, ArH), 4.65 (s, 1H, OH of phenolic hydroxyl), 3.84 (s, 3H, -OCH3)

Potassium protocatechuate: 1636 (C=O str., ester), 3422 (OH str. Phenol), 1458 (C=C str., aromatic) 1H NMR (400 MHz, DMSO-d6)  δ: 8.37 (m, 3H, ArH), 4.80 (s, 1H, OH of phenolic hydroxyl)

Potassium sinapate: 1743 (C=O str., ester), 1113 (C-O-C str.,-OCH3), 2363 (C-H str., OCH3), 1369 (C=C str., aromatic)1H NMR (400 MHz, DMSO-d6)  δ: 8.28(m, 3H, ArH), 4.48 (s, 1H, OH of phenolic hydroxyl), 7.53 (s, 2H C=C), 3.60 (s, 3H, -OCH3)

Potassium ferulate: 1633 (C=O str., ester), 3368 (OH str. Phenol), 1116 (C-O-C str., -OCH3), 2363 (C-H str., OCH3), 1489 (C=C str., aromatic) 1H NMR (400 MHz, DMSO-d6) δ: 6.99 (m, 3H, ArH), 5.79 (s, 1H, OH of phenolic hydroxyl), 3.29 (s, 3H, -OCH3)

Potassium p-coumarate: 1747 (C=O str., carboxylic acid), 3186 (OH str. Phenol), 1388 (C=C str., aromatic); 1H NMR (400 MHz, DMSO-d6) δ: 7.421 (m, 3H, ArH), 5.167 (s, 1H, OH of phenolic hydroxyl), 3.88 (s, 3H, -OCH3)

Potassium gentisate: 1749 (C=O str., carboxylic acid), 3421 (OH str. Phenol), 1450 (C=C str., aromatic) 1H NMR (400 MHz, DMSO-d6) δ: 7.379 (m, 3H, ArH), 3.54 (s, 2H, OH of phenolic hydroxyl)

Potassium caffate: 1738 (C=O str., carboxylic acid), 3377 (OH str. Phenol), 1256 (C=C str., aromatic)1H NMR (400 MHz, DMSO-d6) δ: : 8.04 (m, 3H, ArH), 4.71 (s, 2H, OH of phenolic hydroxyl), 7.29 (s, 2H, CH=CH), 3.83 (s, 3H, -OCH3)

Potassium anisate: 1746 (C=O str., ester), 1165 (C-O-C str., -OCH3), 2362 (C-H str., OCH3), 1340 (C=C str., aromatic);1H NMR (400 MHz, DMSO-d6) δ: : 7.56 (m, 3H, ArH), 3.39 (s, 3H, OH of OCH3)

4.3.  Antimicrobial evaluation of the synthesized alkali salts of natural acids:

The synthesized chitosan derivatives were tested for antimicrobial susceptibility using tube dilution method against gram-positive (S. aureus), gram-negative (K. pneumoniae, E. coli, P. mirabilis and P. aeruginosa) and fungal stains (A. niger and C. albicans). Stock standards of antibiotics and standard preservatives viz. sstreptomycin, ciprofloxacin, ampicillin, fluconazole, sodium benzoate, methyl paraben and propyl paraben were obtained as gift samples from pharmaceutical companies. The synthesized esters were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 µg/mL, which was further diluted to get the concentrations of 50, 25, 12.5, 6.25, 3.125 and 1.562 µg/mL.

For antibacterial study double strength nutrient broth media I.P. was used and Sabouraud dextrose broth media I.P. was used for antifungal study. The test tubes were examined after 24 hours of incubation at 37±1οC for bacterial stains and after 2 days of incubation at 25±1οC for C. albicans and after 7 days of incubation for A.niger.

The 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) Table 2 and Fig. 2.

The in vitro antimicrobial evaluation of the synthesized alkali salts showed excellent activity against bacterial and fungal species; among the synthesized alkali salts Potassium Gallate and Potassium sinapate were found to be most active antimicrobial agents against E.coli. (MIC = 6.25 µM). Potassium anisate was found most active antimicrobial agents against K. pneumonia ( MIC = 6.25 µM). Antifungal results indicated that potassium syringate (MIC = 12.5 µM) exhibited better activity against A. niger. The antibacterial results were compared to ciprofloxacin, Streptomycin and fluconazole is used as antifungal standard drug.

4.4.  Antioxidant activity

Antioxidant potential of all the synthesized derivatives were evaluated by DPPH radical scavenging assay method and result were summarized in Table 3. Further, the results revealed that the compound P3 (IC50 value 6.09±0.001μM) and P10 (IC50 value 06.48 ± 0.042μM) were found more potent antioxidants than reference l-ascorbic acid (IC50 value 8.5.18±0.009 μM). Due to presence of hydroxyl group at meta position where hydroxyl group act as electron withdrawing thus facilitates hydrogen release from acid derivatives. While compound P5 (IC50 value 19.19 ± 0.021μM) exhibited 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 release of hydrogen ion.

Table 3 DPPH radical scavenging activity of synthesized derivatives

^{a Value are expressed as mean ± SEM, n = 3}                 

4.5 Preservative efficacy

4.5.1 Criteria of acceptance for preservative system:

Selected Synthesized compounds were tested for their preservative efficacy and E. coli, P. aeruginosa, S. aureus, C. albicans and A. niger were used as challenge microorganisms. The results were noted on 14^th and 28^th day.

Pulp based slurry of cellulose was used to evaluate the preservative effectiveness of synthesized compounds. As per USP 2004 criteria to pass the preservative efficacy test for the category of test compounds preservative effectiveness is met when there is not less than 2.0 log reduction in bacteria from the initial level at 14th day and no increase at 28th day from second week with no further increase, if value is not more than 0.5 log10 higher than the previous value, it was considered as no increase [16]. The results have been shown in Table 4, Table 5, Table 6, Table 7, Table 8 and Fig 3, Fig 4, Fig 5, Fig 6, Fig 7.

Table 4: Preservative effectiveness of synthesized compounds against E. coli (Log₁₀ CFU/mL)

Fig.  3: CFU in Log₁₀ Values against E. coli

Table 5: Preservative effectiveness of synthesized compounds against P. aeruginosa (Log₁₀ CFU/mL)

Fig. 4 Log₁₀ CFU/mL against P. aeruginosa

Table 6: Preservative effectiveness of synthesized compounds against S. aureus (Log₁₀ CFU/mL)

Fig. 5 Log₁₀ CFU/mL against S. aureus

Table 7- Preservative effectiveness of synthesized compounds against C.albicans  (Log₁₀ CFU/mL)

Fig. 6 Log₁₀ CFU/mL against C. albicans

Table 8-Preservative effectiveness of synthesized compounds against A. niger (Log₁₀ CFU/mL)

Fig 7: Log₁₀ CFU/mL against A. niger

Conclusions

This study is devoted to deriving potassium salts from natural phenolic acid that have displayed great potential to be employed as preservatives. The compounds which were derived were evaluated to study various parameters such as anti-oxidant potential, antimicrobial efficiency and preserving characteristics.

The demonstration on antimicrobial behavior of synthesized potassium salts revealed the salts had outstanding antimicrobial characteristics. With the increment in chain length, there was improvement in the antimicrobial action. The potassium salts which are synthesized were Potassium vanilate, Potassium veratrate, Potassium gallate, Potassium syringate, Potassium protocatechuate, Potassium sinapate, Potassium ferulate, Potassium p-coumarate, Potassium gentisate, Potassium caffeate, and Potassium anisate. It was observed that the salts Potassium Gallate and Potassium sinapate exhibited good antimicrobial activity against E.coli. (MIC = 6.25 µM). Similarly Potassium anisate and potassium syringate exhibited good potential against K. pneumonia and A. niger respectively. The antibacterial results were compared to ciprofloxacin, Streptomycin and fluconazole is used as antifungal standard drug.

The examination on evaluating the efficiency of preservation showed that the potassium derivatives had good potential for preservation and the results were relatable to the regular preservatives that are being used such as streptomycin, ciprofloxacin, ampicillin, fluconazole, sodium benzoate, propyl paraben, and methyl paraben.

Hence, it can be seen that the potassium salts that were synthesized in this study can be used for understanding the employment of these for additional applications in pharma and food. The synthesized salts can be applied as an alternative to the present preservative.

.Conflict of interest:

Authors declares no conflicts of interest.