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Selection Process of Phytochemicals and Efficacy of Thymol, Eugenol and Calcium Ferulate on Heterotrophic Plate Count Bacteria in Water.

Byline: Humayun Wali and Muhammad Zafar

Summary: Water samples of Lahore Canal have been tested for log reduction of heterotrophic plate count microorganisms in the presence of three selected phytochemicals: thymol, eugenol and freshly prepared calcium salt of ferulic acid. Thymol results in about 0.9 log10 reduction (at 80 min contact time), 1.2 log10 reduction (at 90 min contact) and 2.6 log10 reduction (contact time: 60 min) for 75, 150 and 300 ppm wt./wt. phytochemical in water sample respectively. Eugenol at 150 and 300 ppm is as good as thymol for log10 reduction, but not at 75 ppm. Calcium ferulate does not significantly reduce the heterotrophic plate count microorganisms at the three tested concentrations. Contact time is found crucial for optimum reduction of microorganisms. Further studies have been carried out for thymol at 50 ppm. It is observed that thymol results in 0.9 log10 reduction at pH 9.5 and 30AdegC.

At this concentration and at 13AdegC and 20AdegC, significant decrease in heterotrophic plate count is not observed at either pH 4.5, 7.0, and 9.5. Contact time is again important for inactivating the microorganisms. Statistical analysis is made to evaluate differences between the phytochemical-time in concentration treatment groups and pH-time in the temperature treatment groups. Two linear models of thymol reduction of heterotrophic plate count microorganisms are developed to study the relationships between the variables responsible for inactivation. Where experimental data is not available, software PASS, GUSAR, EPI Suite and Marvin Sketch are used to predict antimicrobial properties, toxicity and water solubility. Based on these criteria, affordability and aesthetics, the final selection of phytochemicals is made.

Key Words: Selection; Phytochemicals; Drinking water; Concentration; Temperature; Heterotrophic microorganisms.

Introduction

Since ages, herbs and their derivatives have been utilized by the common man to improve the quality of drinking water. Seeds [1] and stems [2] have been used for coagulation and sedimentation of colloidal impurities in water, extracts used for disinfection [3], [4], and dried herbs' organs used as adsorbent beds for colors, odors removal etc. [5].

The use of harmful chemical disinfectants for drinking water is decreasing day by day and the use of safer chemicals is on the increase as a less harmful alternative. One such class of chemicals is phytochemicals [6-8].

Aromatic plants develop certain primary and secondary metabolites during the course of their growth. These are generally concentrated in the essential oils which are extracted from these plants. They are believed to play role in growth of the plants and to defend against competitors, invaders (e.g. insects, animals, pathogens etc.) and to attract some insects for dispersion of pollens and seeds. Generally, these chemicals are extracted from these plants through various extraction techniques and used as extracts, essential oils, etc. Isolation of these chemicals is usually costly and thus undesirable.

However, with the advancement in new synthetic routes for their preparation, and plant cell and tissue culture technologies [9], it now seems possible to produce some of these phytochemicals in large amounts at an affordable cost.

Phytochemicals are not generally regarded as essential nutrients due to lack of research that would establish the link between human health and intake of phytochemicals. They are however, present in abundance in fruits and vegetables (both of which are highly recommended by health nutritionists).

Broadly, phytochemicals may be classified as: terpenoids (considered the largest group of naturally occurring compounds that are basically derived from five-carbon isoprene units), phenolic compounds (that contain one or more phenol groups), glucosinolates (that contain sulphur and nitrogen and are derived from glucose and an amino acid), organic acids, carbohydrates, etc. Furthermore, the terpenoids are subclassified based on the number of isoprene units e.g. mono, di, tri, tetra and polyterpenoids. Worth to be mentioned here are the phenolic compounds that amongst others also contain polyphenols. The polyphenols are: 1. flavonoids; that have 15 carbon atoms contained in two phenyl rings and a heterocyclic ring and 2. tannins; large polyphenolic compounds that contain sufficient hydroxyl, carboxyl groups etc. so that they can form strong bonds with various other macromolecules.

Until now, more than 80,000 plant-derived chemicals have been scientifically classified as phytochemicals [10] and the number is increasing with the passage of time. Many accounts are available which describe their antibacterial, antiviral, antiprotozoal and antifungal characteristics [11-14].

One major advantage of the use of phytochemicals instead of chlorine in water treatment is that they inherently do not produce harmful chlorinated disinfection byproducts.

In the field of drinking water treatment, various indicator organisms have been used and their presence is interpreted to have different implications. For the general microbiological health of drinking water and to quantify the results of a certain disinfectant in water, the heterotrophic plate count microorganisms have been considered most appropriate [15]. These microorganisms are those that require organic carbon as their source of food and include a very diverse set of organisms including the Enterobacteriaceae family of which the total coliforms are also a part.

The present work involves the use of specific phytochemicals on heterotrophic plate count microorganisms in drinking water. In the first phase of research, herbal plants data were critically reviewed to find out those phytochemicals which may possess a potential application in drinking water. The phytochemicals were subsequently subjected to computer estimation tools. The selection was made based on the predicted probability of a phytochemical to act as an antimicrobial chemical, lower toxicity agent, and as an affordable product.

Based on these parameters, thymol, eugenol and calcium ferulate were selected. Samples of water were taken from Lahore Canal with the understanding that these contain a plethora of heterotrophic microorganisms. The heterotrophic plate count was performed for the water after application of the selected phytochemicals.

The results of this analysis revealed that thymol was the most effective of the three phytochemicals used. Of particular interest was the variation of heterotrophic plate count (HPC) microorganisms with time, which exhibited a minimum for each concentration of phytochemicals used.

Experimental

Reagents, chemicals and growth media

All reagents and chemicals used in this research work were of A.R. grade. Thymol (> 98%), and eugenol (> 98%) were from Daejung (Korea). Ca(OH)2 powder was from BDH (England), and trans-ferulic acid (99%) was from Sigma-Aldrich (Germany). KH2PO4, and MgCl2.6H2O used were from Reidel deHaen (Germany). Methanol (extra pure), used as solvent, was from Merck (Germany).

Standard Plate Count Agar (Oxoid, England, pH 7.0+- 0.2, 25AdegC) was used for the enumeration of heterotrophic plate count microorganisms.

Selection of phytochemicals

About 150 plant species including those which are indigenous to Pakistan were critically examined. According to literature [16]-[19], about 100 of these species possessed phytochemicals with antimicrobial tendencies. These phytochemicals were ranked on the basis of their tendency to act as antimicrobial agent, their relative toxicity to humans and environment and their cost.

The structures of the phytochemical molecules were drawn in Marvin Sketch(a) and checked with its structure checker function. These structures or the SMILES of the molecules were fed to PASS Online(b) [20], and GUSAR Online Acute Rat Toxicity Prediction(c) [21]. As a result, a list of about 4000 predicted biological activities, and toxicity data for each phytochemical molecule was obtained. PASS (prediction of activity spectra for substances) predicts the probability of an organic molecule to act as a biological agent. An examination of the probabilities of phytochemical molecules to act as anti-infective, antiseptic, antimicrobial, antibacterial, antiviral, antifungal, and antiprotozoal was made. Health and eco-toxicity data were obtained from TOXNET database(d), while cost data were taken from several (undisclosed) vendors.

Selection of phytochemical-metal complexes

Phytochemicals which can form stable metal complexes were searched from the literature. Metal complexes of citric and ferulic acids were selected and tested in-silico in the same manner as phytochemicals.

Preparation and characterization of calcium salt of trans-ferulic acid

Calcium ferulate was prepared by direct neutralization of acid with base. For this purpose, 0.02 g of ferulic acid was poured into five test tubes each containing 4 mL of water, ethanol, methanol, acetone, and acetonitrile, respectively. To each tube, aqueous Ca(OH)2 (1.73 g/L) was added using a burette. Phenolphthalein was used as an indicator. The slight pink color of phenolphthalein indicated the end point. The best results were obtained with ethanol and methanol solvents. The synthesis of salt was confirmed by conducting FT-IR analysis with a JASCO FT-IR-4100. A pellet for FT-IR analysis was formed using 200-300 mg potassium bromide with a trace amount of sample pressed to 30-40 MPa for 10 min under vacuum.

Fig.-1 shows the FT-IR spectrum of calcium ferulate solid which was prepared in methanol used as a solvent. Of all the solvents used, major peaks were very similar in case of ethanol and methanol. For example, in the case of methanol, the major peaks of solid calcium ferulate were at 1638.23, 1515.78, 1404.89, and 1272.79 cm-1. These corresponded to v(C=C)-C=C-, vas(COO-), vs(COO-), and [beta](CH)-C=C- + [beta](CH)ar + v(C-O), respectively and signified that the synthesis was done correctly.

The flame test of the prepared solid complex in the presence of HCl yielded brick red coloration indicating the presence of Ca2+ ion.

Sampling and preparation of the water sample

Grab water samples were obtained from Lahore Canal during summer of 2016 to spring of 2017 in sterile containers. A few averaged parameters were as shown in Table-1:

Table-1: Averaged parameters of Lahore Canal water. "*" symbol is for the values measured using HANNA HI9811-5 portable meter.

###Temperature (AdegC)###21.35

###pH *###7.42

###Electrical Conductivity (S/cm) *###220.00

###Total Dissolved Solids (mg/L) *###101.36

###Density at 25 AdegC (g/cm3)###1.07

###BOD5 (mg/L as O2)###2.53

###COD (mg/L as O2)###10.33

###Total Coliforms (CFU/mL)###1076.7

###E. coli (CFU/mL)###100

###Total Hardness (mg/L as CaCO3)###255

###Total Fe (mg/L)###<1

###Cl - (mg/L)###11.74

###SO42 - (mg/L)###64.75

###Orthophosphate (mg/L)### 5000

###0.548 0.013 Antiprotozoal###irritative effect

###(Trypanosoma)###0.893 0.004 Non-

###mutagenic. Salmonella

###0.710 0.003 Antiviral (Rhinovirus)

###0.987 0.002 Skin

###0.615 0.008 Anti-parasitic

###irritation. moderate

###0.605 0.004 Anthelmintic

###Verbascum thapsus###0.972 0.002 Skin

19###Phytol###0.611 0.017 Antifungal###6559 *

###(mullein).###irritative effect

###0.601 0.014 Antiprotozoal

###0.967 0.002 Skin

###(Leishmania)

###irritation. Weak

###E. coli,

###E. coli O157:H7, S.

###0.930 0.003 Antiseptic###0.968 0.008 Toxic.

###Typhimurium, S. aureus, L.

###0.829 0.005 Anti-infective###respiration

###monocytogenes.

###0.765 0.003 Anthelmintic###0.929 0.003

20###Thymol###980

###(Nematodes)###Hematemesis

###Thymus vulgaris L. (garden

###0.588 0.009 Anti-parasitic###0.924 0.003 Ulcer.

###thyme) EO; Ptychotis

###0.551 0.005 Anthelmintic###aphthous

###ajowan (ajwain).

###0.782 0.004 Antiseptic

###0.678 0.004 Anthelmintic###0.902 0.004 Urine

###(Nematodes)###discoloration

###0.548 0.017 Antiviral (Influenza)###0.885 0.005

21###Trans-Caffeic acid###2386 *

###0.520 0.010 Anti-tuberculosic###Hematemesis

###0.515 0.022 Antiprotozoal###0.877 0.005 Ulcer.

###(Leishmania)###aphthous

###0.775 0.004 Antiseptic

###0.583 0.008 Anthelmintic

###0.839 0.007 Urine

###(Nematodes)

###discoloration

###0.554 0.018 Antiprotozoal

22###Trans-Ferulic acid###Effective against C. albicans.###0.819 0.004 Irritation###2754 *

###(Leishmania)

###0.812 0.004

###0.501 0.012 Anti-tuberculosic

###Hypercholesterolemic

###0.501 0.022 Antiviral (Influenza)

###0.496 0.019 Anti-mycobacterial

In PASS, the activities of 45 phytochemicals and 39 phytochemical-metal complexes were predicted. Out of a total of around 4000 different biological activities predicted for each phytochemical by PASS, only the first few are presented in Table-2. As could be seen from the table, the 'Pa value' (i.e. probability as predicted by PASS for a given phytochemical to be active for a certain biological effect) of thymol is very high at 0.930 as 'antiseptic' (i.e. thymol resembles up to 93%, a subgroup of compounds in PASS that are typically labelled antiseptics). If it is to be consumed, thymol's LD50 rat should be a large value, but at 980 mg/kg it is not as compared to many other phytochemicals presented in Table-2. However, thymol's high Pa value and affordability are enough compelling causes to select it for experimentation. The aromatic odor is another factor for choosing thymol. Thymol is also produced synthetically from p-cymene, and m-cresol.

Eugenol (though at lower Pa value 0.814) has relatively higher rat LD50 (1930 mg/kg) and fairly high water solubility (2460 mg/L), both favoring experimentation to establish its efficacy. Eugenol is isolated in sufficient amounts through extraction from various essential oils. Though, menthyl salicylate has high Pa value 0.938, however, being expensive we did not perform experimentation on it.

Apart from their low cost, these 22 phytochemicals (Table-2) were selected keeping in mind their reported activity against E. coli, coliforms and Enterobacteriaceae. These bacteria are almost ubiquitous in water bodies and are also indicative of the presence of pathogens. Many of these bacterial species are known to cause gastro-intestinal disorders. Therefore, Table-2 includes those phytochemicals that are present in herbs which are traditionally used in herbalism to treat gastro-intestinal disorders.

Selection of phytochemical-metal complexes

Not all phytochemicals have tendency to form complexes with metals. Both positive and negative effects have been reported on the antimicrobial activities of the organic compounds in the presence of metal ions [23]. The antimicrobial activities of citric and ferulic acid complexes were predicted in PASS. The citric acid complexes were shown to possess inferior antimicrobial properties as compared to ferulic acid complexes. The predicted general antimicrobial activities of ferulic acid complexes are presented in Table-3. Calcium ferulate possesses the highest Pa value of 0.959 as antiseptic (i.e. that prevents the growth of disease-causing microorganisms). Predicted rat oral LD50 of ferulic acid is 2754 mg/kg and water solubility is high, 5970 mg/L.

Table-3: Desired and side effects some of the metal complexes of ferulic acid presented as predicted activities in PASS.

###PHYTOCHEMICAL-METAL###DESIRABLE PASS PREDICTIONS###PASS PREDICTIONS (SIDE EFFECTS)

S####COMPLEX###Pa Pi EFFECTS###Pa Pi EFFECTS

###0.872 0.014 Diarrhea

1###Cadmium ferulate###0.807 0.004 Antiseptic###0.829 0.017 Hepatotoxic

###0.806 0.018 Weakness

###0.861 0.010 Neurotoxic

2###Calcium ferulate###0.959 0.003 Antiseptic###0.831 0.017 Hepatotoxic

###0.773 0.014 Dyspnea

###0.776 0.014 Dyspnea

3###Copper (II) ferulate###0.664 0.006 Antiseptic###0.752 0.010 Hypercholesterolemic

###0.752 0.014 Urine discoloration

###0.750 0.005 Antiseptic###0.858 0.005 Urine discoloration

4###Gold (III) ferulate###0.586 0.015 Antiprotozoal (Leishmania)###0.821 0.004 Irritation

###0.573 0.008 Anthelmintic (Nematodes)###0.833 0.025 Shivering

###0.832 0.007 Urine discoloration

###0.664 0.006 Antiseptic

5###Platinum (III) ferulate###0.785 0.005 Hypercholesterolemic

###0.531 0.012 Anthelmintic (Nematodes)

###0.808 0.034 Shivering

###0.832 0.007 Urine discoloration

6###Silver ferulate###0.912 0.003 Antiseptic###0.785 0.005 Hypercholesterolemic

###0.808 0.034 Shivering

###0.872 0.014 Diarrhea

7###Zinc ferulate###0.807 0.004 Antiseptic###0.829 0.017 Hepatotoxic

###0.806 0.018 Weakness

Effect of time and phytochemicals at 75 ppm concentration, on HPC

Fig.-2 shows the HPC results for 75 ppm wt./wt. of phytochemical in sample at different time intervals. The results of ANOVA depict that changing the phytochemical does not have a significant impact on HPC at this low concentration (P = 0.099). On the other hand, time has a crucial influence (P = 5.05 X 10-9). As the interactions are very weak (P = 0.866), this means that the additive model holds statistically, and we can conduct the Tukey's multiple comparisons test. From this test we found that there were no significant differences between the three different phytochemicals used. However, there were significant differences between all the time points, 0, 20, 50, and 90 min from the time point 140 min (P = 0.000 for each of the four intervals). We deduced from the trends that there was an optimum time at which the HPC number decreased to a minimum value.

Effect of time and phytochemicals at 150 ppm concentration, on HPC

The results are shown in Fig.-3. The ANOVA test did not highlight any significant differences between the three phytochemicals (P = 0.279), but there were significant differences between time points (P = 7.49 X 10-9). The interactions effects were very weak (P = 0.209), and Tukey's test results revealed that there were no significant differences between the three phytochemicals. However, there were significant differences of the initial time point from all other time points (P 0.000 for all) and between the time intervals 20 - 50, 20 - 90, and 20 - 140 min (P values 0.044, 0.0003, and 0.0004 respectively). Fig.-3, however, shows that the maximum reduction of the HPC microorganisms (1.22 log10) occurred with thymol after about 90 min in the water sample.

Effect of time and phytochemicals at 300 ppm concentration, on HPC

Fig.-4 shows the HPC results for 300 ppm wt./wt. of phytochemical in water sample at different intervals of time. Here, significant effects of both the phytochemicals (P = 7.46 x 10-5), and time (P = 5.74 x 10-10) have been observed. Though the additive model does not hold (P = 0.00019) yet we can observe from Fig.-4 that both thymol and eugenol caused appreciable reduction in HPC, especially at 60 min. In fact, the results of Tukey's test also indicate significant differences between the thymol-eugenol and thymol-calcium ferulate especially between the time zero to 15, and 60 min intervals. It can be observed from the trends that there was an optimum time at which the HPC number decreased to a minimum value. Thymol showed the greatest reduction at 60th min interval, (about 2.6 log10).

Since small amount of methanol was used as solvent for the phytochemical stock solution, therefore we checked the effect of this amount on HPC. It was found that growth of HPC was the same in case of raw sample and sample + methanol (within tolerable variation due to error). So, we concluded that the results represent the effects produced primarily by phytochemicals.

Effect of varying temperature and pH on HPC microorganisms in presence of 50 ppm thymol

Due to significant effect of thymol on HPC, it was desired to study the effect at lower concentration of thymol and at variable temperature and pH. Figs. 5a, 5b and 5c show the results for 50 ppm of thymol at pH values 4.5, 7.0, and 9.5 for three sets of experiments conducted at 13, 20 and 30AdegC respectively. Blank values (i.e. sample without addition of 50 ppm thymol) at pH 7.0 were also taken to compare the actual inactivation at various time points.

The results of ANOVA depict significant differences in the time intervals for 20 and 30AdegC (P = 0.0008). No significant differences were observed between the pH values at P [greater than or equal to] 0.05 for any of the three temperature treatments. The results of Tukey's multiple comparisons of means showed significant differences for all time intervals that began from time zero, for only 20 and 30AdegC treatments but not for 13AdegC treatment (all P values 0.01). No significant differences were observed between the various pH treatments by the Tukey's multiple comparisons test. As seen from the trends in Figs. 5a, 5b and 5c, the slightly higher log reduction values at lower and higher values of temperature depict the possible additional stress of temperature on HPC microorganisms along with the phytochemical thymol.

Mathematical models for thymol inactivation of HPC in prepared water sample

The trends of log (N/No) vs time show a minimum value for each curve drawn. Therefore, our case does not represent the conventional disinfection model curves (convex, sigmoid, concave with shoulder, concave without shoulder). Neither do the curves solely represent growth models (three-phase linear, logistic, Von Bertalanffy, Richards, Morgan, Weibull, France, Gompertz, and Baranyi). Rather the trends show that initially the phytochemicals kill or inactivate the microorganisms appreciably but later the microorganisms flourish (probably utilizing the remaining phytochemical or its products as a feed for multiplying their numbers). As the initial concentration of the phytochemical was known, an attempt was made to model our system under phytochemical demand free conditions. Multiple linear regressions were carried out for thymol and it was found that the following equation (R2 = 0.956, at 25AdegC, pH 7.4, and concentration 0.075 to 0.3 g/L) represented the results:

log(N/No) = -7.940c + 109.876c2 - 285.228c2 + 0.111t2 - 9.918ct + 17.377c2t2

Similarly, using multiple linear regression results, the following equation was obtained after optimization for 50 ppm thymol (R2 = 0.855, pH 4.5 to 9.5, and temperature 13 to 30AdegC):

log(N/No) = -0.042T + 0.004T2 - 7.83 x 10-5T2 + 0.149t2 - 0.010Tt - 0.054(pH)t + 0.001(pH)2t2

N = Number of HPC microorganisms at time.

No = Number of HPC microorganisms at initial time.

t = Time the phytochemical has been in sample (hrs.).

c = Initial concentration of phytochemical in sample (g/L).

T = Temperature of water (AdegC).

Conclusion

The results of the present study establish that phytochemicals may be effective antimicrobial agents against HPC microorganisms in water. Therefore, it is thought that they can be utilized to help combat water-borne illnesses. Many of these illnesses relate to the gastrointestinal system. In the sub-continent, phytochemicals have been utilized extensively as herbs by hakims to treat gastrointestinal disorders. However, for the treatment to be effective, the water should be prefiltered, of low organic carbon content, and it is suggested that they are used in combination with some established water treatment technique. The present work selected and used only those phytochemicals that have low cost, are relatively non-toxic, and have low molecular masses. As the search for plant extracted chemicals continues with newer areas of applications, low molecular size phytochemicals of higher efficacy than thymol could find utilization in water treatment.

Presently, it is recommended that phytochemicals (or plant-extracts) should only be used for water disinfection and treatment where there is a dire need, for example in far-flung remote areas.

Acknowledgements

We gratefully acknowledge the contribution of Mr. Wali Muhammad for writing this research paper in correct English.

We also acknowledge the help and support of the Department of Polymer and Process Engineering during the course of present work and of Mr. Javed Bukhtawer for providing us the facility for carrying out FT-IR analysis.

Funding: The complete funding for this research work was provided by the Office of Research, Innovation and Commercialization of the University of Engineering and Technology, Lahore, Pakistan. The funding source has no involvement in completing this research work.

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Author:Wali, Humayun; Zafar, Muhammad
Publication:Journal of the Chemical Society of Pakistan
Article Type:Technical report
Geographic Code:9PAKI
Date:Apr 30, 2019
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