Effect of Solvent pH on Antioxidant and Phytochemical Activities of Mulhatti Aerial Parts (Glycyrrhiza glabra L.)

Mulhattiaerial parts (Glycyrrhiza glabra L.) were obtained from the experimental area of Medicinal, Aromatic & Potential Crops Section, Department of Genetics & Plant Breeding, Chaudhary Charan Singh Haryana Agricultural University, Hisar in 2017. Aerial parts of the herbarium were randomly collected from the field with a plant age of approximately 1.5 years and dried in a tunnel drying system during March. After drying, the samples were stored in a chemical laboratory. The sample was converted to powder form in a hammer mill.

Preliminary analysis of Mulhatti aboveground fractions is performed to determine water content, ash content, crude protein, total carbohydrate and calorific value according to standard methods specified in the Official Association of Analytical Chemists (AOAC) ⁷. The crude fibre content is estimated using a modified Maynard method ⁸.

Five grams of Mulhatti's aerial part powder sample was packaged in a thimble made from Whatman No. 1 and extraction was carried out using a classic Soxhlet apparatus. The apparatus was provided with a 500 mL round bottom flask and distilled water as solvent was added to about half a siphon (240-270 mL) of different pH (2, 4, 7 and 9) and the pH was adjusted using HCl (conc.) and a NaOH pellet. Then the extraction is carried out at the boiling point of water still continue up to 5 to 6 cycles of Soxhlet extraction completed. The solvent vapour rises to the column and condenses in the condenser section of the apparatus. After condensation, they flow into a chamber thimble filled with a sample of Mulhatti air passages and the respective extracts are filtered. This process is repeated three times. The resulting filtrate was stored in bottles for further experiments.

The phenolic compounds present in aqueous extracts of the Mulhatti aerial parts at different pH (2, 4, 7 and 9) were estimated using the Folin-Ciocalteu method ⁹. To estimate the total phenols in aqueous extracts at various pHs; 0.2 mL of extract from each tube was diluted with the respective solvent to adjust the absorbance within the calibration limits. One mL of Folin-Ciocalteu reagent was added and 2 mL of Na₂CO₃ (20%, w/v) were mixed and the volume was made up to 10 mL with distilled water. The mixture was allowed to stand for 8 minutes and centrifuged at 6000 rpm for 10 min. Similarly, a blank is prepared; respective solvents were used instead of extracts. The absorbance of the supernatant solution was measured against a blank made on a UV-VIS Dual Ray Spectrophotometer (Shimadzu 1900) at 730 nm. The total amount of phenol present in aqueous extracts of different pH was calculated from the standard curve and expressed as mg GAE/g (milligram gallic acid equivalent per gram).

The content of flavonoids present in aqueous extracts of the Mulhatti aerial parts at different pH (2, 4, 7 and 9) was estimated by the Aluminum chloride colorimetric method ¹⁰. To estimate the total flavonoids in aqueous extracts with different pH; 1 mL of each extract was taken into the tube and added 4 mL of distilled water, 0.3 mL of NaNO₂ (5%) and added 0.3 mL of AlCl₃ (10%) after 5 minutes and then 2 mL of NaOH (1 M) were added instantly and the final volume was made up to 10 mL with distilled water. Similarly, blank is prepared; respective solvents were used instead of the extracts. After the solution was thoroughly stirred, the absorbance was measured against a blank made on a UV-VIS dual beam spectrophotometer (Shimadzu 1900) at 510 nm. The quantity of flavonoids present in aqueous extracts of different pH was calculated from the standard curve and expressed as mg CE/g (milligram catechin equivalent per gram).

Evaluating the total antioxidant capacity of aqueous extracts with different pH (2, 4, 7 and 9) of Mulhatti root using the modified phosphomolybdenum method ¹¹. In glass vials, 0.3 mL of each extract was taken and 3 mL of phosphomolybdenum reagent was added and the solution was mixed thoroughly, the vial was closed with a cap. They were incubated for 90 minutes at 95°C. Next, the contents of the vial were allowed to cool and absorbance was measured at 695 nm on a UV-VIS model double beam spectrophotometer (Shimadzu 1900) against the prepared blanks. Similarly, blank is prepared; respective solvents were used instead of the extracts. The total antioxidant capacity in aqueous extracts of different pH was calculated from the standard curve and expressed as mg AAE/g (milligram ascorbic acid equivalent per gram).

Antioxidant activity was estimated by DPPH free radical scavenging activity method in aqueous extracts of Mulhatti aerial parts of different pH, namely 2, 4, 7 and 9 ¹². The aqueous extract was taken and dried completely and the dry mass was recorded. Aqueous extract was redissolved in an appropriate amount of 50% (v/v, methanol:water) to obtain a stock solution of 1000 g/mL depending on the dry weight of the extract (because it is not completely soluble in methanol). Concentrations from 5 g/mL to 500 g/mL were prepared from stock solutions (1000 g/mL) at appropriate dilutions with 50% (v/v) water:methanol. To evaluate the free radical scavenging activity of DPPH, 1 mL of extract of each concentration was put into a glass vial covered with a lid and 2 mL of 2,2'diphenyl1picrylhydrazyl (DPPH; 0.1 mM was prepared in 50% (v/v) water: methanol) and covered with a lid, shake well for 5 minutes.Control was carried out with 1 mL of each solvent used as a sample substitute. They were incubated for 30 min in the dark and then the absorbance of the sample and control was measured against a blank containing pure methanol at 517 nm on a UV-VIS Double Beam spectrophotometer (Shimadzu 1900) and each sample was brought to three replicas. Using Microsoft Excel software, graphs were plotted by plotting the free radical scavenging activity of DPPH (%) on the y-axis versus the extract concentration (µg/mL) on the x-axis, then the quadratic regression equation (y = ax² + bx + C). The obtained equation is converted into the form (ax² + bx + c = 0) by setting y = 50%. Using the equation (ax² + bx + c = 0), the IC₅₀ value is calculated by applying the formula

x =

Where, x = IC₅₀ (µg/mL) Percentage of activity eliminated by DPPH (% DPPH * _SC) is calculated using

Where, A_control= controlabsorbance, A_sample= sampleabsorbance

All results are calculated in triplicate for their statistical study and expressed as mean ± SD. To assess a significant difference between the sample means in online statistical analysis (OPSTAT), one-way variances (ANOVA) were performed. IC₅₀ values ​​for the free radical activity of DPPH were calculated using a quadratic regression equation. The correlation between total phenolic compounds, total flavonoids and DPPH values ​​for free radical scavenging and total antioxidant capacity were calculated using the Karl Pearson method in Microsoft Excel and all other measurements were also performed in Microsoft Excel 2019.

The nutritional composition of Mulhatti aerial parts was determined by proximity method. This work was performed to evaluate the feasibility of using the above-ground parts of Mulhatti for medicinal purposes. Results of this study: Moisture, ash, crude fat, crude fiber, crude protein, total carbohydrate and calorific value were measured at 7.23 ± 0.15%, 6.82 ± 0.01%, 1.04 ± 0, 08% and 24.84 ± 0.54%, 15.84 ± 0.83%, 44.27 ± 1.19%, 249.96 ± 6.15 kcal in the aerial part of Mulhatti (Table 1). The results obtained for this analysis are similar to the reference data ¹³,¹⁴,¹⁵. The calorific value depends on the approximate composition of various plants.

The amount of phenolic compounds present in the aqueous extract at different pH, namely 2, 4, 7 and 9 of arial parts of Mulhatti was determined using the equation (y = 0.0104x + 0.0079, R² = 0.9989) obtained from the acid calibration standard. Standard curve was used for the determination of amount of phenol and the total amount of phenol in mg GAE/g was determined. The study data for the amount of phenol did not show a regular trend but showed wide variation in the yield of aqueous extracts with different pH. The maximum amount of total phenolic content was present at pH 7 (14.13) followed by pH 2 (3.77), pH 9 (3.22) and pH 4 (2.60 mg GAE/g). Research data for phenolic compounds, flavonoid content and free radical scavenging activity of DPPH and total antioxidant capacity showed uneven trends at different pHs. Other researchers have also reported various effects of pH on phytochemical and antioxidant activity. In 2019, Krungkree and Arikul studied the effect of solvent pH on total phenolic content (TPC), DPPH radical scavenging, and their results showed that more phenolic compounds were present at pH 5-7¹⁶. In general, TPC and antioxidant activity were higher as the pH increased. Therefore, the high amount of these phytochemicals at pH 5-7 indicates that the pH conditions have a significant effect on the stability of total phenols and the overall antioxidant capacity. Medina et al. (2007) also pointed out the reason for the high amount of total phenolic compounds at neutral pH, because hydrolysis of phenolic acids in alkaline and strongly acidic conditions reduces the amount of phenolic compounds ¹⁷. In sweet potato leaves, polyphenols exhibited high retention rates with an increase in acidity or alkalinity at pH 5-7, which had a significant effect on stability ¹⁸. Phenolic compounds extracted from Chamomile (Matricariapubescens) at different pHs namely: 3, 4, 5, 6 and 7 also showed variation, along with increasing pH from 3 to 5, the phenolic content also increased but after pH 5 the quantity decreased which indicated that the difference in the pH value of the extraction has a significant effect on the extraction of phenolic compounds ¹⁹.

Similarly, the flavonoid content was also determined using the equation (y = 0.0018x + 0.0038, R² = 0.998) obtained from the calibration curve of the catechins used as standard and the amount of flavonoids was determined in mg CE/g. The results for flavonoids also show great variation. The maximum flavonoid content was found at pH 9 (4.90) followed by pH 7 (3.23), pH 2 (0.25) and pH 4 (0.24 mg EC/g). Flavonoids were determined from the top of the Schultz (Algerian MatricariaPubescens) and the results found to vary significantly at different pHs of the extraction solvent and increasing order were: pH 5 (6.36 ± 0.2 > pH 7 (4.88 ± 0.3) 12)> pH 6 (3.94 ± 0.17)> pH 4 (2.16 ± 0.17)> pH 3 (2.14 ± 0.19) ²⁰.

To calculate the IC₅₀ value of DPPH free radical scavenging activity, a quadratic equation was obtained by plotting the DPPH radical scavenging activity (%) on the y-axis and concentration (µg/mL) on the x-axis (Table 2) of Mulhatti aerial parts of aqueous extracts at different pH viz: 2, 4, 7 and 9.

The IC₅₀ value of the Mulhatti aerial part in aqueous extracts at different pH (2, 4, 7 and 9) was lowest at pH 7 (60.48) followed by pH 9 (79.82), pH 4 (104.53), pH 2 (115.32 g/mL) (Table 3). Dogãn and Salman 2007 reported that the pH-dependent increase in the antioxidant activity of phenolic compounds was due to the greater stabilization in alkaline solutions which led to the polymerization reaction of the polyphenol antioxidants and which could form new moieties of oxidizable hydroxyl groups in the polymers ²¹. The product produces greater antioxidant activity than phenolic compounds.

Similarly, the total antioxidant capacity was determined using the equation (y = 0.0066x + 0.0036, R² = 0.999) obtained from the calibration curve of ascorbic acid used as a standard and the total antioxidant capacity was determined in mg AAE/g. The maximum total antioxidant capacity was found at pH 9 (13.43) followed by pH 2 (8.19), pH 7 (7.51) and pH 4 (5.87 mg AAE/g). Total antioxidant capacity by phosphomolybdenum method on dates (Phoenix Dactylifera L .) in aqueous extracts of different pH was evaluated and reported that the results showed no regular trend, the maximum antioxidant capacity was present at pH 4 (68.34 ± 0.71) followed by pH 6 (64. 12 ± 1.08), pH 5 (63.62 ± 0.69), pH 7 (62.81 ± 0.73), pH 3 (55.92 ± 0.60) and pH 2 (55.11 ± 0.60) ^{22, 23}.

Free radical scavenging activities of phenol, flavonoid and DPPH in Thai curry paste extract at different pH (2 to 10) and the results showed a large variation as in this study and there was no correlation between the two. The number of phenolic compounds was higher in strong acids (pH 2) followed by slightly acidic (pH 6) and slightly alkaline (pH 10) and flavonoid compounds were present higher in strong acids (pH 2) and slightly acidic (pH 6). ) but the free radical scavenging activity of DPPH was found to be slightly higher in acid (pH 6) followed by pH 2, 3 and 10 respectively ²⁴. As we know, this large variation in yield may be due to the types of samples used and the different types of compounds extracted in each case and the different phytochemical compositions of the plants depending on their different properties such as waxy or non-waxy species, plant parts used i.e. leaves, stems, bark, roots, tops or whole plants for research and also depending on the selection of compounds such as carotenoids, non-polar compounds, polar compounds and simple phenolic compounds also act as determinants of factors causing differences in phytochemical composition. Pearson correlation coefficient analysis was carried out to study the significant and insignificant correlation between the total phenol content, flavonoid and IC₅₀ scavenging activity of DPPH and between the total phenol content, flavonoid and total antioxidant capacity. Pearson correlation coefficient is negative significant if 0.61 ≤ r ≤ -0.97 and significant positive if 0.61 ≤ r ≤ 0.97 ²⁵. Graphically, data for total phenolics, flavonoids and for total antioxidant capacity has shown below in figure 1 and Figure 2.

Figure 1.Image A of Mulhatti (Glycyrrhiza glabra) showed complete plant and B showed aerial parts

Figure 2.Effect on total phenolics, flavonoids and total antioxidant capacity of aerial parts of Mulhatti in aqueous extracts of different pH levels

This study showed that the total phenol and flavonoid content had a significant and negative correlation with IC₅₀ scavenging activity of DPPH (r = -0.782 and r = -0.794, P<0.05) and that phenolic compounds and flavonoids could be predicted as the main contributors in antioxidant activity by the DPPH method. This study also revealed that flavonoids had a significant and positive correlation with their total antioxidant capacity (r = 0.799, P < 0.05) and indicated that flavonoid compounds were the main contributors to total antioxidant capacity by the phosphomolybdenum method ²⁶.