The authors have declared that no competing interests exist.
Medicinal plants have been used since the era of Vedic history for their health care system where herbal medicine has a long history of use. It is also a very popular medicinal plant belonging to the Leguminosae family and is commonly known as Mulhatti. It contains phytochemicals such as flavonoids, triterpene saponins and other constituents such as coumarins, sugars, amino acids, tannins, starch, choline, phytosterols etc. The present study was conducted for the estimation of phytochemicals (total phenols and flavonoids) and the evaluation of the total antioxidant capacity and DPPH free-radical scavenging activity in aqueous extracts of different pH (2, 4, 7 and 9) from aerial parts of
For their basic needs as a source of medicine, shelter, food, perfume, clothing, flavouring, fertilizer, and transportation, humans depend on nature. Medicinal plants have been used since the era of Vedic history. Especially in developing countries, large parts of the world's population depend on medicinal plants for their health care systems, where herbal medicines have a long history of use
Mulhattiaerial parts (
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)
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
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
Evaluating the total antioxidant capacity of aqueous extracts with different pH (2, 4, 7 and 9) of Mulhatti root using the modified phosphomolybdenum method
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
x =
Where, x = IC50 (µg/mL) Percentage of activity eliminated by DPPH (% DPPH * SC) is calculated using
Where, Acontrol= controlabsorbance, Asample= 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 (
|
|
---|---|
|
7.23 ± 0.15 |
|
6.82 ± 0.01 |
|
1.04 ± 0.08 |
|
24.84 ± 0.54 |
|
15.84 ± 0.83 |
|
44.27 ± 1.19 |
|
249.96 ± 6.15 |
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 5-7
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 (
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 (
p |
|
---|---|
2 | y = -0.0005x |
4 | y = -0.0006x |
7 | y = -0.0021x |
9 | y = -0.0008x |
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) (
p |
|
|
||||||
|
|
|
|
|
|
|
||
2 | 89.75 | 78.99 | 45.21 | 30.25 | 23.03 | 16.97 | A |
|
4 | 86.44 | 85.08 | 48.37 | 30.53 | 18.35 | 13.55 | 12.35 |
|
7 | A | 88.97 | 67.84 | 45.02 | 29.40 | 16.85 | 12.86 |
|
9 | 89.88 | 89.42 | 67.33 | 42.94 | 29.30 | 18.25 | 15.64 |
|
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 (
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
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
From this study, we can conclude that the phytochemical and free radical scavenging activities of DPPH and total antioxidant capacity were significantly affected by the aqueous extracts of different pH: 2, 4, 7 and 9 and the results of the research data clearly showed differences in the amount of phenolic compounds, flavonoids and antioxidants. Total antioxidants capacity and DPPH of free radical scavenging activity was shown by aqueous extracts with different pH levels. The highest phenol content was present at pH 7 and the highest flavonoid was present at pH 9. Therefore, pH 7 was the best for studying phenolic compounds and pH 9 was an excellent factor for determining flavonoid compounds in the aerial part of Mulhatti at different pH of aqueous extracts. The pH 9 is better to confirm the total antioxidant capacity of Mulhatti's aerial parts. The phenolic compounds and flavonoids present are responsible for the antioxidant activity and show a correlation with the free radical scavenging activity of DPPH and flavonoid compounds also show a correlation with the total antioxidant capacity. As the present study provides strong evidence of high total phenolic content and significantly higher free radical scavenging activity at pH 7 of the aqueous extracts.