Comparative Analysis of Free Radical Scavenging in Moringa oleifera, Sauropus androgynus and Their Dynamic Synergy

Abstract

Background: Antioxidants play an important role in guarding the cells from oxidative damage due to free radicals. Moringa oleifera and Sauropus androgynus are known leafy plants which have satisfying nutritive value and medicinal properties. There is a lack of data on antioxidant activity of combination extract. Objective: This study aims to assess the antioxidant potential of Moringa oleifera, Sauropus androgynus methanolic leaf extracts, and the combination extract of the two plant leaves. Materials and Methods: 100g of each plant sample combination was extracted with 99% methanol. Preliminary qualitative phytochemical screening tests were performed for each extract. DPPH and ABTS assay were performed for three plant extracts and two standard solutions (ascorbic acid and BHT) at five concentrations. Percentage of inhibition and IC50 value were calculated. Results and Discussion: Kruskal-Wallis H test was performed for the non-normally distributed data. Significant difference in the percentage of free radical inhibition value between the five antioxidants was observed (P<0.05). Ascorbic acid showed the highest performance among the five antioxidants (mean rank=14.00) in both assays. Among the three plant samples, Moringa oleifera shown the highest free radical scavenging activity (mean rank=8.00). The combination extract shown superior performance at high extract concentration (1000?g/mL). The rich vitamin and lipid contents possessed by Sauropus androgynus leaves promised its nutritive value for dietary and herbal supplement purposes. Conclusion: The addition of Sauropus androgynus leaf extract in Moringa oleifera leaf herbal formulation is promising and considerable with its own nutritive value.

Keywords

Introduction

Antioxidants play an important role in guarding the cells from oxidative damage due to free radicals¹. Free radicals are molecules that are easily formed during daily activities, such as exercising. Apart from natural formation, environmental exposure to free radicals, including UV light, smoke, and contaminated air, are common in daily life. The free radicals are highly unstable, which can induce oxidative stress that leads to cell damage and diseases. Antioxidants act as a reducing agent to reduce the reactive oxygen species which are in the form of free radicals². In contrast with synthetic antioxidants which have higher potential to impair health, a lot of studies have been carried out to explore more natural oxidants from natural sources such as plants and animals³. Vegetables, fruits, and edible plants are the targets of researchers to study the antioxidant activity of the extracts of different plant parts such as roots, leaves, pods, seeds, fruits, and flowers. More and more researchers are putting efforts into exploring the ability of antioxidants obtained from natural sources in protecting consumers against diseases and aging^4.5. Moringa oleifera (Lam.) is a tropical tree of Moringaceae family that natively grown in northern India⁶. Some common names of Moringa oleifera include moringa, the miracle tree, drumstick tree and horseradish tree. It is commonly known as kelor or murungai in Malaysia. Moringa oleifera is rich in nutrients, especially vitamins, and its leaves contain all essential amino acids⁷. Its leaves, root, bark, pods, seeds, flowers and fruits are consumed as dishes and drinks traditionally to treat influenza, diabetes, used as antiepileptic, diuretic, stimulant and anti-paralytic⁸. Sauropus androgynus (L.) Merr. is a tropical shrub of Phyllanthaceae family⁹. It is widely cultivated in East Malaysia, to be served as dishes. It is commonly known as katuk or cekur manis in East Malaysia. Other common names include sweet leaf and star gooseberry. The vitamin C, B1 and B2 compositions are high in Sauropus androgynus leaves. Traditionally, the leaves and roots were extracted and consumed to treat dysuria, hypertension, diarrhoea, and increase breast milk production.The leaf extracts have also been consumed to reduce body weight ¹⁰.

AIM

There is a lack of data for the research on antioxidant activity assays for combination extract of Moringa oleifera and Sauropus androgynus leaves. Hence, this study aims to investigate the free radical scavenging potential of combination extract with that of the individual extracts by ABTS and DPPH free radical scavenging assays.

MATERIALS AND METHODS

1. 1. Chemicals

DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), potassium persulfate, ascorbic acid, BHT (butylated hydroxytoluene), 5% iron (III) chloride, sodium hydroxide, diluted hydrochloric acid, concentrated hydrochloric acid, concentrated sulphuric acid, chloroform, Dragendorff’s reagent, Wagner’s reagent, glacial acetic acid, 99% methanol.

1. 2. Sample preparation and extraction.

The fresh leaves of Moringa oleifera and Sauropus androgynus were collected from local wet market in Sarawak, Malaysia. The leaves were cleaned using tap water and shade dried on a dry cloth in an air-circulated place until complete drying. The leaves were then crushed and stored in plastic containers separately in fridge until needed. The study was conducted among three samples, namely Moringa oleifera leaves, Sauropus androgynus leaves, and the combination extract of the two plant leaves (1:1 w/w). Maceration extraction was performed. 100g of each plant sample combination was soaked in 99% methanol for two days with frequent mixing and stirring. The methanolic extracts obtained were filtered and concentrated to obtain dried extracts. The percentage yield was calculated, and the dry extracts were stored in the refrigerator (under 4?).

1. 3. Preliminary qualitative phytochemical screening

Extracts were screened for saponins, alkaloids, flavonoids, glycosides, phenols, steroids and terpenoids using appropriate methods with some modifications.^11,12&13.

1. 4. Preparation of diluted sample extract and standard solutions

The solid extracts were dissolved in methanol using DMSO. The two standards, L-ascorbic acid and BHT solutions served as positive control were prepared by dissolving in distilled water. Five concentrations which included 1mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL and 0.0626mg/mL were prepared by serial dilution for each sample and standard. Seven micrograms concentrations which included 100μg/mL, 50μg/mL, 25μg/mL, 12.5μg/mL, 6.25μg/mL, 3.125μg/mL and 1.5625μg/mL were prepared for the two standards to obtain IC50 value in free radical scavenging assays.

1. 5. Determination of Free Radical Scavenging Potential (DPPH•)

A solution of 0.3mM DPPH^• with violet colour was prepared by dissolving DPPH powder with methanol. 1mL of the solution was added to 2mL of each sample and standard concentrations, and to 2mL of methanol as negative control. The DPPH free radical solution decolorised upon addition of antioxidants. The absorbance was measured at 517nm using UV-vis spectrophotometer after 40 minutes. All the tests were done in triplicate and the percentage of inhibition for each sample and standard was calculated using the formula:

Abscontrol-Abssample Abscontrol×100%

 

1. 6. Determination of Free Radical Scavenging Potential (ABTS•+)

About 7mM ABTS⁺ solution and 2.45mM potassium persulfate solution were prepared separately by dissolving the powders with distilled water. ABTS^•+ solution with blue green colour was prepared by reacting the two stock solutions in 1:1 ratio in dark for 14 hours at room temperature. The radical solution was prepared only for two days use to ensure stability. Upon performing the assay, the radical solution was diluted with methanol to give an absorbance of 0.702 ± 0.002 at 734nm. Then, 3mL of the solution was added to 1mL of each sample and standard concentrations, and to 1mL of methanol as negative control. The ABTS free radical solution decolorised upon addition of antioxidants. The absorbance was measured at 734nm after 40 minutes. All the tests were performed in triplicate. The percentage of inhibition was calculated. Standard graph was formed from the data obtained, and the IC₅₀ value of each antioxidant was obtained from the graph.

RESULTS

Note: In all the following tables and figures, MO = Moringa oleifera; SA = Sauropus androgynus; MO+SA = combination extract

1. 1. Pharmacognostical parameters

The study was carried out to qualitatively determine the phytochemical contents in the three leaf extract samples. The highest percentage of yield was observed in Moringa oleifera leaf extract (18.86%) (Table 1).

Note: (+) indicates presence and (-) indicates absence

Figure 1. Graphical comparison of Percentage Inhibition of DPPH Free Radical

Figure 2. Percentage inhibition of ABTS free radical

Assay	MO	SA	MO+SA	AA	BHT
DPPH	300.40 μg/mL	528.96 μg/mL	311.05 μg/mL	6.43 μg/mL	14.70 μg/mL
ABTS	149.77 μg/mL	154.24 μg/mL	171.18 μg/mL	2.94 μg/mL	7.70 μg/mL

Figure 3. IC₅₀ value in DPPH and ABTS scavenging

DISCUSSION

The study was carried out to compare the free radical scavenging activity between leaf extracts of Moringa oleifera, Sauropus androgynus, and the combination extract of the two plant leaves, with the standard antioxidants ascorbic acid and BHT. A higher percentage of free radical inhibition were detected in ascorbic acid and BHT than the three samples in both DPPH and ABTS assay (Table 3 - 4, Figure 1 - 2). The range was recorded from 1.36 ± 0.05 μg/mL to 97.11 ± 0.10 μg/mL for DPPH assay, and from 18.63 ± 0.00 μg/mL to 100.00 ± 0.00 μg/mL for ABTS assay at different concentrations. Among the five antioxidants, ascorbic acid possessed the lowest IC₅₀ value in both DPPH (6.43μg/mL) and ABTS (2.94μg/mL) free radical scavenging assay (Table 5, Figure 3). Among the three samples, Moringa oleifera recorded the lowest IC₅₀ value (300.40μg/mL for DPPH; 149.77μg/mL for ABTS). Sauropus androgynus and the combination extract possessed the highest IC50 value in DPPH (SA: 528.96μg/mL) and ABTS (MO+SA: 171.18μg/mL) free radical scavenging assay respectively. Normality test was performed for the data obtained. The results revealed some data were not normally distributed (i.e. significance value < 0.05 in Shapiro-Wilk test). Hence, a non-parametric test namely Kruskal-Wallis H test¹⁴ was performed for the determination of statistically significant difference between the sample groups. The choice of test was made provided more than three independent groups to be assessed¹⁵. The actual data were replaced with ranks for the test. The percentage of free radical inhibition was compared between the five antioxidants at each concentration. In both analysis of DPPH and ABTS free radical scavenging assay, a significant difference in the results between the five antioxidants was observed (P<0.05). Among the five antioxidants, ascorbic acid shown the highest mean rank value, which indicated the highest performance in free radical scavenging activity. On the other hand, comparison was made between the three samples in both DPPH and ABTS free radical scavenging assay. Two analyses shown a significant different free radical scavenging potential among the three antioxidants (P<0.05). Among the three samples, Moringa oleifera shown the highest performance in both free radical scavenging activity. Sauropus androgynus shown the lowest performance in DPPH assay, while the combination extract shown the lowest performance in ABTS assay. An exception was observed at 1000μg/mL in the analysis, where the combination extract shown a superior performance in DPPH assay, and common highest performance in ABTS assay. Overall, in the aspect of free radical scavenging potential, Moringa oleifera leaf extract possessed the highest antioxidant potential among the three samples. The combination extract shown a comparable high (in ABTS assay) or superior (in DPPH assay) antioxidant potential as compared to Moringa oleifera leaf extract at a higher extract concentration (1000μg/mL). In comparison with the combination extract, Sauropus androgynus leaf extract possessed lower DPPH radical scavenging potential, but higher ABTS radical scavenging potential. This could be due to the higher ability of Sauropus androgynus leaf extract to scavenge ABTS free radical than DPPH free radical. Nevertheless, the rich vitamin⁸, such as thiamin and riboflavin¹⁶, and lipid contents¹⁷ in Sauropus androgynus promised its nutritive value for dietary and herbal supplement. Hence, this could be its value to be added in the herbal formulation of Moringa oleifera. The combination formulation could possess a higher nutritive value than individual formulation. Safriani et. al., 2021¹⁸ reported significant amounts of total flavonoid content and total phenolic content possessed by both Moringa oleifera (15.44 ± 0.03mg QE/g; 5.27 ± 0.09mcg GAE/mg) and Sauropus androgynus (6.58 ± 0.01mg QE/g; 2.10 ± 0.12mcg GAE/mg). The phenolic compounds and flavonoids obtained from the Moringa oleifera and Sauropus androgynus extracts were found to be the main components to possess the antioxidant activity^18,19. The age of Moringa oleifera leaves and choice of extraction solvent shown impact on their DPPH^• activity as reported by Nobossé and colleagues. The results of the study recorded the highest total phenolic and flavonoid contents with highest DPPH^• scavenging potential in the highest maturity leaves (60-day-old) and ethanolic extracts (53.3% - 71.1%) ¹⁹. On the other hand, the concentration of solvent used for extraction of Sauropus androgynus leaves was reported by Hikmawanti et al to affect the acquisition of phytochemicals and hence affected its scavenging activity of DPPH free radical. 50% ethanol solvent was reported to extract the highest total phenolic and flavonoid content, and possessed the greatest antioxidant activity (IC₅₀: 88.33 ppm) as compared to extracts using 70% and 96% ethanol solvent²⁰.

CONCLUSION

This study reports the antioxidant potential of Moringa oleifera, Sauropus androgynus methanolic leaf extracts, and the combination extract of the two plant leaves by DPPH and ABTS free radical scavenging assay. The presence of phytochemicals including tannins, saponins, alkaloids, flavonoids, glycosides, and steroids are observed in most of the extracts. The addition of Sauropus androgynus leaf extract in Moringa oleifera leaf herbal formulation is promising and considerable with its own nutritive value and the high free radical scavenging potential of Moringa oleifera leaf extracts. Further research of combination extract in different aspects could be conducted to prove its value.

ACKNOWLEDGEMENT

This study was funded and supported by Faculty of Pharmacy, SEGi University, Kota Damansara, Malaysia.

REFERENCE

1. Saleem U, Sabir S, Niazi SG, Naeem M, Ahmad B. Role of oxidative stress and antioxidant defense biomarkers in neurodegenerative diseases. Crit Rev Eukaryot Gene Expr. 2020;30(4):311–322. DOI: 10.1615/critreveukaryotgeneexpr.2020029202.
2. Rak AE, Tharmadurai K, Khanam Z. Ethnobotanical uses, in-vitro total phenolic, flavonoidic content and antioxidant activity of plants consumed by Siamese community of Tanah Merah, Kelantan, Malaysia. Res J Pharm Biol Chem Sci. 2018;9(1):962–971. DOI: 10.1016/j.ejmech.2019.06.010.
3. Tekle EW, Sahu NP. Antioxidative and antimicrobial activities of different solvent extracts of Moringa oleifera: An in vitro evaluation. Int J Sci Res Publ. 2015;5(5):12. DOI: 10.29322/IJSRP.5.5.2015.
4. Neha K, Haider MR, Pathak A, Yar MS. Medicinal prospects of antioxidants: A review. Eur J Med Chem. 2019; 178:687–704. DOI: 10.1016/j.ejmech.2019.06.010.
5. Guo Q, Li F, Duan Y, Wen C, Wang W, Zhang L, Huang R, Yin Y. Oxidative stress, nutritional antioxidants and beyond. Sci China Life Sci. 2020;63(6):866–874. DOI: 10.1007/s11427-019-9591-5.
6. Dhakad AK, Ikram M, Sharma S, Khan S, Pandey VV, Singh A. Biological, nutritional, and therapeutic significance of Moringa oleifera Lam. Phytother Res. 2019;33(11):2870–2903. DOI: 10.1002/ptr.6475.
7. Mahmood KT, Mugal T, Haq IU. Moringa oleifera: A natural gift - A review. J Pharm Sci. 2010;7. DOI: 10.29322/IJSRP.7.2010.
8. Mishra G, Singh P, Verma R, Kumar S, Srivastav S, Jha K, Khosa RL. Traditional uses, phytochemistry and pharmacological properties of Moringa oleifera plant: An overview. Der Pharm Lett. 2011; 3:141–164. DOI: 10.29322/IJSRP.3.2011.
9. Zhang BD, Cheng JX, Zhang CF, Bai YD, Liu WY, Li W, Koike K, Akihisa T, Feng F, Zhang J. Sauropus androgynus L. Merr. - A phytochemical, pharmacological and toxicological review. J Ethnopharmacol. 2020; 257:112778. DOI: 10.1016/j.jep.2020.112778.
10. Arif T, G RS. Therapeutic potential and traditional uses of Sauropus androgynous: A review. J Pharmacogn Phytochem. 2020;9(3):2131–2137. DOI: 10.29322/IJSRP.9.3.2020.
11. Bargah RK. Preliminary test of phytochemical screening of crude ethanolic and aqueous extract of Moringa pterygosperma Gaertn. J Pharmacogn Phytochem. 2015;4(1):07–09. DOI: 10.29322/IJSRP.4.1.2015.
12. Das BK, Al-Amin MM, Russel SM, Kabir S, Bhattacherjee R, Hannan JMA. Phytochemical screening and evaluation of analgesic activity of Oroxylum indicum. Indian J Pharm Sci. 2014;76(6):571–575. DOI: 10.29322/IJSRP.76.6.2014.
13. Sandeep K, Singh BB, Narinder K. Physico-chemical and phytochemical investigation of plant Sesbania sesban. Res J Pharm Biol Chem Sci. 2014;5(1):110–117. DOI: 10.29322/IJSRP.5.1.2014.
14. Kruskal WH, Wallis WA. Use of ranks in one-criterion variance analysis. J Am Stat Assoc. 1952;47(260):583–621. DOI: 10.1080/01621459.1952.10483441.
15. Chan Y, Walmsley RP. Learning and understanding the Kruskal-Wallis one-way analysis-of-variance-by-ranks test for differences among three or more independent groups. Phys Ther. 1997;77(12):1755–1762. DOI: 10.1093/ptj/77.12.1755.
16. Beaulah A, Rajamanickam C, Swaminathan V. Nutritive values and importance of tropical green leafy vegetables in human diet – A review. Int J Curr Microbiol Appl Sci. 2020;9(9):656–669. DOI: 10.29322/IJSRP.9.9.2020.
17. Iwansyah AC, Damanik RM, Kustiyah L, Hanafi M. Relationship between antioxidant properties and nutritional composition of some galactopoietic’s herbs used in Indonesia: A comparative study. Int J Pharm Pharm Sci. 2016;8(12):236–243. DOI: 10.29322/IJSRP.8.12.2016.
18. Safriani N, Rungkat FZ, Yuliana ND, Prangdimurti E. Immunomodulatory and antioxidant activities of select Indonesian vegetables, herbs, and spices on human lymphocytes. Int J Food Sci. 2021; 2021:1–12. DOI: 10.1155/2021/1234567.
19. Nobossé P, Fombang EN, Mbofung CMF. Effects of age and extraction solvent on phytochemical content and antioxidant activity of fresh Moringa oleifera L. leaves. Food Sci Nutr. 2018;6(8):2188–2198. DOI: 10.1002/fsn3.2018.
20. Hikmawanti NPE, Fatmawati S, Asri AW. The effect of ethanol concentrations as the extraction solvent on antioxidant activity of katuk (Sauropus androgynus (L.) Merr.) leaves extracts. IOP Conf Ser Earth Environ Sci. 2021;755(1):012060. DOI: 10.1088/1755-1315/755/1/012060.