Statistical evaluation of main extraction parameters in twenty plant extracts for obtaining their optimum total phenolic content and its relation to antioxidant and antibacterial activities

Abstract The main extraction parameters were statistically evaluated for 20 medicinal plants to obtain the optimum conditions for maximum extraction of total phenolic content (TPC) in each plant. Among various extraction parameters, pH, temperature, and concentration at different levels were studied. The results were analyzed using the analysis of variance to achieve the optimum conditions of phenolic extraction for all plants. Also, investigation of the optimum antioxidant (AnOX) activities using DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) method and antibacterial potential against common pathogenic bacteria of Staphylococcus aureus, Escherichia coli, Pseudomonas spp., and Candida spp. through disk diffusion method for the extract of all plants under the optimum total phenolic concentration of each plant extract confirmed a direct relation among bioactivity and TPC.


| INTRODUC TI ON
During the ancient times, plant resources are involving an integral part of human society as which after fulfilling the primary needs, man has sought for a suitable remedy among plants for curing vari-  and Hostettmann, 1991;Harrison, 1998;Ahmad Dar et al., 2017;Keskin, 2018;Dehdari and Hajimehdipoor, 2018. Following the latest scientific estimations, a very large spectrum of plant species including new ones is characterized each year. According to a report released by the World Health Organization (WHO), among of these numbers of the plants, those can be used for treatment is around 20,000. A statistic obtained based on the reports revealed that plant products used by patients of chronic medical conditions including cancers (2%), liver diseases (21%), HIV (22%), asthma (24%), and rheumatologic disorders (26%), Tolossa and Megersa, 2018;Varga et al., 2018;Anand et al., 2019. The historical development of the health and disease has led to the alternative medicines and its practices to be conveyed to today's societies. Therefore, phytotherapy as one of the alternative medicine practices has become basic subjects of many scientific studies about treatment with plants, Spiridon et al., 2011;Emad, 2013;Efferth and Kaina, 2011 can play an important role in the prevention of many diseases.

| Materials and instrumentations
All chemicals used in current study were purchased from Merck and Aldrich companies. UV-visible spectral analysis was recorded on a double-beam spectrophotometer (Super Aquarius) to monitor the TPC and An. Ox activity.

| Total phenolic content (TPC)
The Folin-Ciocalteu method was employed by Alhakmani, and his collaborators with some modifications were used to measure the TPC of each medicinal plant at studied conditions, Alhakmani et al. 2013. Generally, 1.0 ml of plant aqueous extract in 60 ml distilled water was mixed to 5.0 ml of Folin-Ciocalteu reagent. After some minutes, 15.0 ml Na 2 CO 3 (20%) was added to the mixture. Finally, the absorbance of the mixture was measured at 760 nm after 2h and the TPC was reported as mg of gallic acid equivalent (GAE) and the influence of each extraction parameters at a special range such as various pHs, temperatures, and concentrations were separately recorded on the rate of phenolic content of each plant, Table 1. The optimum conditions for achieving the maximum phenolic content of plant extracts then were evaluated using the statistical calculations.

| Calculation of the optimal antioxidant activity
After obtaining the optimal conditions to extract the maximum TPC for each plant, the antioxidant activity of each medicinal plant was monitored against 2,2-diphenyl-1-picrylhydrazyl (DPPH). Briefly, a 1:1 ratio of each plant optimum aqueous extract at different concentrations were mixed with freshly prepared DPPH solution, and after their keeping in the dark for 30 min, the absorbance at 517 nm was recorded using UV-vis spectrophotometer in contrast with the gallic acid as control. The optimal antioxidant activity for each plant extract using DPPH method was calculated as below: where A c and A t are the absorbance of control and sample, respectively. The results of DPPH method for each plant extract at optimal conditions to decrease the absorbance of DPPH radicals were reported as scavenging activity (%). The assay to monitor the antioxidant activity of each plant extract in optimal conditions was repeated during a time span ranging 24h to 1 month, Table S2.

| Investigation of the antibacterial activity
Following the obtain of total phenolic content (TPC) for each of the studied plant shown in Table 1, the antibacterial activity for each plant was monitored at mentioned optimal conditions. Briefly, the disk diffusion method was employed in which 100 µl of each plant aqueous extract and Chloramphenicol at the same condition were loaded on paper disks (6 mm D) and dried; then, they were placed on the Muller Hinton Agar plates including bacterial cultures (100 µl).
Each plate was inoculated at 37°C for 24 hr after incubation. Finally, the diameter of inhibition zones was measured and tabulated to find the MIC.

| Statistical analysis
The test of examining normality was detected to conduct the comparison test. The data showed that the assumption of normality was met and ANOVA test was applied to find the difference between the groups at both aspects, and LSD post hoc test was implemented for the pairs. Plus, t test was used for comparing two groups. The statistical software SPSS v25 was used to generate all the results and outputs for this study with 0.05 significant value.

| Statistical analysis to monitor the optimal extraction parameters
To reach the objective of the study and explore, we have carried out several statistical techniques for the accuracy of the methods and the comparison between the groups of implanting. An intensive descriptive statistic including tables and graphs were generated to show the nature of the data and detect patterns among the dataset, Table 1. To see if there is any statistically significant among the degree of concentration percentage used in this study, ANOVA test was carried out since there are more than two groups to compare their mean values and Figure 1 proves that the data are normally distributed. Table S3 states that there is indeed not statistical difference between the three degrees as the p-value of ANOVA test is greater than 0.05.
Similar to previous technique, we are seeking to identify which degree of pH recorded the highest TPC. Mean value can lead us to find, and at pH7, the largest mean value was found with 9.43, and a very close to this was at pH 9 with 9.16, whereas the smallest value was at pH3 with 6.15 only. Again, we use ANOVA due to meeting our requirements, the very interested one is normality distribution as shown in Figure 2 assuming normality. Table S4 states that there is indeed statistical difference between the three degrees as the pvalue of ANOVA test is less than 0.05.
Since the p-value of ANOVA test in Table S5 was less than 0.05, we can report that there is statistically significant between the different types of pH degree provided to the plants in the study. Like before, LSD test was computed for each pair to find out the exact difference. Table S6 illustrates that they were all found to be statistically significant regarding to their mean values except between pH 7 and pH 9 as we already discussed in the descriptive phase where their mean values were quite close to each other.

| Investigation of the optimal TPC and AnOX activity
Through this study, some main extraction parameters were assessed to extract of phenolic compounds of twenty widely distributed medicinal plants and the results were defined as total phenolic contents of the plant extracts, Figure S1. Also, as the antioxidant activity shows a very closed relation to the TPC of the plants, it can be defined as TPC dependent factor. Thus, along with evaluation of the TPC for the plants at optimum conditions, the optimum AnOX activity was also reported.
The discussed parameters during this study influenced on the TPC and AnOX activity were pH, temperature, and concentration (plant ration (g)/ 100 ml DW) where all of them were considered during a time span from 24h to 1 month to study of the repeatability and accuracy of our results, Table S8. As the statistically evaluation of the results showed in previous sections, the optimum extraction parameters were pH 7, temperature 75°C, and concentration 20%.

| Investigation of the antimicrobial activity
As it was mentioned for investigation of the AnOX activities for studied plants after evaluation of the extraction parameters to extract F I G U R E 2 Box plot chart for pH data for each degree F I G U R E 3 Box plot of TPCs at different Temperature of phenolic compounds of twenty medicinal plant extracts, in this part, we try to evaluate the antibacterial activities of the mentioned plant extracts including optimal amount of total phenolic content (TPC), Figure 4. As it can be seen in antimicrobial activity is also improved, Figure 5. The result is probably referred to the considerable antimicrobial effects of phenolic compounds against a large spectrum of pathogenic bacteria comparing other bioactive phytochemicals.

| CON CLUS IONS
This study shows the relation of the main extraction parameters of phenolic compounds such as pH, temperature, and concentration to obtain conditions of the mentioned parameters and theoretical study using the assessment of examining normality using ANOVA and LSD post hoc tests. The results showed that the best condition to achieve the maximum TPC for plants were pH 7, temperature 75°C, and concentration 20% where employing these optimal conditions caused to minimum deformation and decomposition side processes. Furthermore, the antioxidant and antibacterial activities of the plant extracts including the optimal amounts of phenolics were investigated to demonstrate the effect of phenolic content on their biological activity in which the results showed that increasing the phenolic content in plant extracts caused to enhance the antioxidant and antibacterial activities.

ACK N OWLED G M ENT
The author is grateful to department of phytochemistry, SRC, Soran university, KRG, Iraq for some data information and instrumentation support of this work. Also, I strongly dedicate my thanks to Dr.
Farough Abbasi and Dr. Shahram Babadoust Demouchali for collection of some plants and their analysis in this work.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

O RCI D
Dler H. Kadir https://orcid.org/0000-0003-0180-5161 F I G U R E 6 Relation of antimicrobial activities of plant extracts and their optimal total phenolic content