Effect of pH on Phytochemical and Antioxidant Potential of Satawar Tubers (Asparagus Racemosus Willd.)

Satawar (Asparagus racemosusWilld.) tubers were collected from the Medicinal, Aromatic & Potential Crops Section, experimental area of the Department of Genetics & Plant Breeding on the campus of Chaudhary Charan Singh Haryana Agricultural University, Hisar (figure 1). Tuber samples were randomly collected from sites with plant age of approximately 1.5 years and dried in a tunnel drying system during the month of March. After drying, the samples were stored in a chemical laboratory. The samples were converted to powder form in a hammer mill.

Figure 1.Dried tubers of Satawar (Asparaguaracemosus)

According to the standard method outlined in the Official Association of Analytical Chemists (AOAC) ⁶, proximate analysis of turnip roots is carried out to determine moisture content, ash content, crude protein, total carbohydrates and calorific value. The crude fiber content is estimated using a modified Maynard method ⁷. The method of Burns ⁸ Vanillin HCl was used to estimate the tannin content as a catechin equivalent. The method described by Obadoni and Ochuko ⁹ and Ezeonuet et al was used to estimate the saponin content ¹⁰. The minerals (Fe, Cu, Mn and Zn) were estimated using Atomic Absorption Spectrophotometer (AAS), in acid digested plant samples by Jackson ¹¹ and Ruig ¹².

A powder sample of 5 grams of Satawar tubers was packed into a thimble prepared from filter paper (Whatman No. 1) and extracted using a classic soxhlet extractor. In this apparatus equipped with a 500 mL round bottom flask and distilled water as solvent, half siphons (240-270 mL) of different pHs (2, 4,7and 9) were added and the pH was adjusted using conc. HCl and NaOH pellets. Extraction is carried out at the boiling point of the solvent. The solvent vapour condenses the heat in the rising condenser. After condensing, they overflow into a chamber containing a thimble containing a sample of Satawar tubers. When extraction was complete, each extract was filtered and this process was repeated 3 times, and then the obtained filtrate was stored in a storage container for further experiments.

Total phenolic content of aqueous extracts of different pHs, namely 2,4,7 and 9 Satawar tubers, was determined using the Folin Ciocalteu method. ¹³. For measurement of phenolic compounds; diluted each 0.2 mL extract with each solvent, adjust the absorbance within the correction range, added 1 mL Folin Ciocalteu reagent to the test tube, and add 2 mL Na₂CO₃ (20%, w /v). After mixing the final volume was made up to 10 mL with distilled water. The mixture was maintained for 8 minutes. Then centrifuge at 6000 rpm for 10 minutes.Similarly, blanks are prepared. Instead of samples, each solvent was taken. Thereafter, the absorbance of the supernatant was measured at 730 nm on a blank prepared by a UV-VIS double beam spectrophotometer model UV 1900 (Shimadzu Corporation). The total phenol content present in the aqueous extracts at different pHs was calculated from the calibration curve and expressed as mg GAE/g.

The flavonoid content of aqueous extracts of different pHs, namely 2, 4, 7 and 9 Satawar tubers, were determined using aluminum chloride colorimetric analysis ¹⁴. For the measurement of flavonoid compounds, 1 mL of each extract was taken and 4 mL of distilled water, 0.3 mL of NaNO₂ (5%), and 0.3 mL of AlCl₃ (10%) were added after 5 min. Then 2 mL of NaOH (1 M) was added immediately and the final volume was 10 mL with distilled water in a test tube. Similarly, blanks are prepared. Instead of samples, each solvent was used. After shaking the solution thoroughly, absorbance was measured at 510 nm on blanks prepared in UV-VIS double beam spectrophotometer model UV 1900 (Shimadzu Corporation). The amount of flavonoids that pH is present in other aqueous extracts is estimated from the standard curve and is expressed in mg CE / g.

Antioxidant activity was assessed with aqueous extracts of Satawar tubers at different pHs, namely 2,4,7 and 9, using the DPPH free-radical scavenging activity method ¹⁵. Record the weight of the completely dry mass of 10 mL aqueous extracts and re-dissolve the dry mass of the aqueous extract in an appropriate amount of 50% (v/v methanol:water) to make a 10000 µg/mL stock solution. Concentrations ranging from 10 µg/mL to 5000 μg/mL were prepared from stock solutions with appropriate dilutions with 50% (v/v) water : methanol. To assess DPPH free radical scavenger activity ; extracts of 1 mL at each concentration were taken then added 2 Ml of 2, 2’-diphenyl-1-picrylhydrazyl (DPPH; 0.1 mM prepared in 50% (v/v) water: methanol) and glass tube covered with a lid and shake thoroughly for 5 min.A control was made by using solvent in place of the sample. After culturing in a dark place for 30 minutes, they measured the absorbance of a blank containing pure methanol at 517 nm with UV-VIS Double Beam Spectrophotometer ( UV1900 Shimadzu), and each sample was taken to replication three times. Using Microsoft Excel software, graph a quadratic regression equation (y = ax² + bx + c) between the y-axis DPPH free radical scavenging activity (%) and the x-axis extract concentration (μg / mL). The resulting equation was converted to the form (ax² + bx + c = 0) with y = 50%. The IC₅₀ value was calculated using the equation (ax² + bx + c = 0) by applying the following formula

Where, A_control= control absorbance, A_sample= sample absorbance

For pH 2, 4, 7, and 9, the total antioxidant capacity of Satawar tubers from aqueous extracts was determined using the modified phosphomolybdenum method given by ¹⁶. For assessment of total antioxidant capacity; 1 mL of each extract was taken and 3 mL of phosphomolybdenum reagent was added to a glass vial covered with a lid and the solution was mixed thoroughly. They were cultured for 90 minutes. Absorbance was measured at at 95 ° C, then the contents of the vial were cooled and a blank prepared with UV-VIS Double Beam Spectrophotometer Model UV 1900 (Shimadzu) was measured at 695 nm. Similarly, blank was prepared and respective solvent was used instead of the sample. The pH is calculated from the standard curve and the total antioxidant capacity of other aqueous extracts is expressed in mg AAE / g.

The nutritional composition of Satawar tubers was determined by proximity analysis. This work was conducted to evaluate the potential for nutritional and therapeutic usefulness of Satawar tuber rhizomes. In the present investigation, moisture, ash, crude fat, crude fiber, crude protein, total carbohydrate and calorific values ​​were measured at the levels of 3.67 ± 0.33%, 7.67 ± 0.33%, 2.23 ± 0.15%, and 42.67 ± 1.20 %, 6.43 ± 0.35%, 43.00 ± 0.58%, 213.83 ± 1.59 kcal in tubers of Satawar (Table 1). The results obtained for compositional analyzes are comparable with Wagh et al ¹⁷. Bhakuni and Jain ¹⁸ found similar results for moisture, ash and total carbohydrates in Satawar tubers.

In the current study, tubers analyzed by chemical analysis have a 19.48 ± 0.77 mg CE / g tannin content and a 3.51 ± 0.23% saponin content (Table 2). The results are consistent with previous studies such as Raval et al. ¹⁹ and Bhakuni and Jain ¹⁸ who reported the amounts of tannins and saponins in the range of 0.83 ± 0.9% and 5.4 ± 0.4%, respectively, However, the findings of Singla and Jaitak ²⁰ were slightly different from current studies concerning Mn, Fe, Cu and Zn 5.0 to 62.0 mg/kg, 211.0 to 1493.0 mg/kg, 14.0to 23.0 mg/kg and 44.0 to 148.0 mg/kg respectively. These differences are due to the different effects of environmental factors on plants in different places.

Using the formula (y = 0.0104x + 0.0079, R² = 0.9989), the calibration curve for gallic acid used as a standard dose of total phenol was determined in mg GAE/g of aqueous extracts at different pHs 2,4,7,9 of Satawar tubers. The highest phenol content was found at pH 9 (18.88), followed by pH 4 (9.32), pH 7 (2.41) and pH 2 (2.30 mg GAE / g). These results showed that wide variations in data at different pH and other researcher also find this type of variation. The phenol content first increased by raising the pH to 2-4, then decreased to pH 7, and then the pH increased.Phenolic compounds were extracted from Chamomile (Matricariapubescens) with pH 3,4,5,6,7 showing same type of trend, raising the pH to 3-5 also increases the phenol content, but decreases after pH 5. This shows that different extraction pH values ​​significantly affect the extraction of phenolic compounds ²¹. In Tulsi leaf extract (Ocimum sanctum), the estimated phenols and their values ​​varied with pH 2 (120 mg GAE/g), pH 7.2 (135 mg GAE/g) and pH 7.6 (75 mg GAE/g). It is clear that this may not be necessary, since increasing pH increases the amount of phenolic compounds and vice versa and they conclude that pH has a significant effect on the antioxidant activity of the methanolic O. sanctum leaf extract ²².

Similarly, it was determined in mg CE/g using the formula (y = 0.0018x + 0.0038, R² = 0.998) obtained from a calibration curve for catechins used as standard doses of flavonoids. The flavonoid content was highest at pH 9 (2.83), followed by pH 2 (2.05), pH 4 (1.70), and pH 7 (0.55) mg CE/g, respectively. It was reported that the flavonoid content significantly changed the pH of the extraction solvent (water) and the order of the flavonoids measured in aerial parts of Schultz (Algerian MatricariaPubescens) as follows: pH 5 (6.36 ± 0.2 > pH 7 (4.88 ± 0.12)) > pH 6 (3.94 ± 0.17) > pH 4 (2.16 ± 0.17) > pH 3 (2.14 ± 0.19) ²³. They also studied the effect of pH on the extraction yield of flavonoids in Citron (Citrus medica) bark extracts prepared in various organic solvents (methanol, ethanol, ethyl acetate, water) and observed that the extraction yield of flavonoids was also found to change at different pH values ​​(3, 4, 5, 6, 7 and 8)²⁴.

The DPPH free radical scavenging activity (%) and IC₅₀value (µg/mL) of aqueous extractsof Satawar tubers at different pH levels of Satawar tubers are presented in (Table 3). Using the quadratic equation the IC₅₀value for DPPH free radical scavenging activity was calculated in µg/mL in aqueous extracts with different pH, namely: 2, 4, 7 and 9 of the Satawar tubers. IC50 values ​​were found to be lowest at pH 7 (2582.85), followed by pH 4 (3183.83), pH 2 (3246.50), pH 9 (4598.77 µg/mL) respectively. DPPH radical scavenging activity in methanol extract of peanut shells, studies and results obtained show higher activity at neutral and acidic pH ²⁵. The alkaline pH of the coca by-product yielded the opposite result ²⁶.

Calculated in mg AAE / g using the equation (y = 0.0066x + 0.0036, R² = 0.999) obtained from the ascorbic acid calibration curve used for standard total antioxidant capacity. Total antioxidant capacity was found to be highest at pH 2 (15.96), followed by pH 7 (15.03), pH 9 (9.32) and pH 4 (8.52) mg AAE / g respectively. Total antioxidant capacity by phosphomolybdenum method at different pH of in Date palm (Phoenix Dactylifera L.) was estimated and obtained results showing the difference in data and higher antioxidant capacity found 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) ²⁷. Graphically, the total phenolics, flavonoids and total antioxidant capacity data have been given below in figure 2.

Figure 2.Effect of different pHs on total phenolics, flavonoids and total antioxidant capacity of Satawar tubers in aqueous extract

Variations in the results of this phytochemical and antioxidant activity and total antioxidant capacity are likely due to changes in the pKa values ​​of the reactions, and are related to the degree of ionization and deprotonation of functional group of the compound. Deprotonation of phenolic compounds can affect the thermodynamics of hydrogen atom delivery. ^{28, 29}. All researchers have found significant differences in total antioxidant capacity at different pHs, as in current studies with data on phenol, flavonoids and antioxidant activity. The correlation coefficient was also calculated by Karl Pearson's method in Microsoft Excel, and Pearson's correlation coefficient was significantly negaeive when 0.61 ≤ r ≤ 0.97 and significantly positive when 0.61 ≤ r ≤ 0.97 ³⁰. Its values ​​indicated a significant and positive correlation (r = 0.911; 0.925, P & lt; 0.01) between phenol and flavonoid content with IC₅₀ of DPPH scavenging activity in Satawara tubers and had It was possible to predict that phenolic and flavonoid compounds are the main components of the antioxidant activity of Satawar tubers by the DPPH method. The total content of phenols and flavonoids along with their total antioxidant capacity was also significantly and negatively correlated (r = 0.817; 0.964, P & lt; 0.01) and could also predict that the compounds Phenolics and flavonoids are the main components of the total anti oxidant capacity of Satawar tubers by the phosphomolybdenum method.