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

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).

Table 1. Yield and Physical Characteristics of the Plant Extracts.

As results of qualitative phytochemical screening tests, saponins, alkaloids, flavonoids, glycosides, and steroids were presented in both three samples. Tannins were presented in Moringa oleifera and the combination extract, while absented in Sauropus androgynus leaf extract (Table 2).

Table 2. Phytochemical Screening of Plant Extracts

1. 2. DPPH and ABTS assay

Table 3. Percentage Inhibition of DPPH Free Radical