Free Radical Scavenging Activity of Majorana hortensis leaves.

Antioxidants are an essential defense mechanism to protect our body against free radical damage. They balance the production of free radicals and detoxify them when in excess. The objective of the study is to determine the free radical scavenging potential of the candidate plant leaves when subjected to a battery of free radical scavenging assays. The different leaf extracts used were aqueous, methanol and chloroform. The results suggest that Majorana hortensis (L.) H. Karst leaves could serve as a potential source of antioxidant and can be used in any preparation for combating free radical mediated damage to the body.

Introduction:

Oxygen is an indispensable part of aerobic life. However, under certain circumstances, it can seriously affect our well being through the formation of reactive oxygen species, representing both free radicals and non-free radical species1. Reactive oxygen species (ROS) are unstable and aggressive molecules, which have the tendency to give their unpaired electron to other cellular molecules or snatch electrons from other molecules to attain stability2. These ROS are capable of damaging biomolecules, provoking immune response, activating oncogenes and enhancing the aging process3. In the human and animal body, ROS can be neutralized by antioxidant defense systems including antioxidant enzymes and antioxidant compounds4. Antioxidants protect us from diseases such as cancer, Alzheimer's disease, diabetes and aging5.

Antioxidant supplements or foods rich in medicinal plants are used to help the human body in reducing oxidative damage by free radicals and active oxygen. Currently, research interest has been focused on the role of antioxidants as well as antioxidant enzymes, in the treatment and prevention of many diseases6. Antioxidants may guard against ROS toxicities by the prevention of ROS construction, by disruption of ROS attack, by scavenging reactive metabolites and converting them to less reactive molecules or by enhancing the resistance of sensitive biological target to ROS attack7. Herbs are widely exploited in the traditional medicine and their curative potentials are well documented. The demand for plant-based medicines, health products, pharmaceuticals, food supplement and cosmetics are increasing in both developing and developed countries, due to the growing recognition that the natural products are non-toxic, have lesser side effects and are easily available at affordable prices8.

Free radicals are small, diffusible molecules that differ from most biological molecules. They have an unpaired electron. Free radicals tend to be reactive and participate in chain reactions, in which a single free radical initiation event can be propagated to damage multiple molecules. Free radicals can be divided into Reactive Oxygen Species such as superoxide anion (O-2, hydroxyl radical (OH-), hydrogen peroxide (H2O2) and Reactive Nitrogen Species (RNS) such as nitric oxide (NO) and peroxynitrite (ONOO-)9.

Majorana hortensis (M. hortensis) is a perennial herb of the Mediterranean region commonly called as majoram, belonging to the Lamiaceae family (mint). It is an aromatic plant and due to its aroma, it has culinary uses. The plant extract has several therapeutic uses such as curing digestive disorders, treats fevers and is used as an expectorant. The fresh leaves of this candidate plant are used to study the free radical scavenging activity.

Materials and Methods:

The fresh, tender leaves were collected from the plants grown in pot cultures. The leaves grown throughout the year are used. The entire leaf was used for the study. Organic solvent extracts of methanol and chloroform were made and evaporated to dryness. The residue was dissolved in dimethysulphoxide (DMSO) at 20 mg/5 μl concentration. Apart from this, fresh aqueous extract was also used for the study. The scavenging effects of M. hortensis leaf extracts were evaluated against DPPH (1,1’-diphenyl-2-picryl hydrazyl), ABTS (2,2’-azino-bis-3-ethyl benzthiazoline-6-sulphonic acid), hydrogen peroxide, superoxide, nitric oxide and hydroxyl radicals.

DPPH Spectrophotometric Assay:

The scavenging ability of the natural antioxidants of the leaves towards the stable free radical DPPH was measured by the method of Mensor et al10.

ABTS Scavenging Effects:

The antioxidant effect of the leaf extracts was studied using ABTS radical cation decolourisation assay according to the method of Shirwaiker et al.11.

Hydrogen Peroxide Scavenging Effects:

The ability of the leaf extracts to scavenge hydrogen peroxide was assessed by the method of Ruch et al12.

Superoxide Scavenging Activity:

The superoxide scavenging ability of the extracts was assessed by the method of Winterbourn et al.13.

Nitric Oxide Scavenging Activity:

The extent of inhibition of nitric oxide radical generation in vitro was followed as per the method reported by Green et al.14.

Hydroxyl Radical Scavenging Activity:

The extent of hydroxyl radical scavenging from Fenton reaction was quantified using 2’-deoxyribose oxidative degradation as described by Elizabeth and Rao15.

Results:

The total antioxidant activities of different leaf extracts were measured on the basis of their ability to scavenge stable free radial. The three different leaf extracts were screened for their antioxidant activity in the DPPH antioxidant assay and the results are depicted in the table 1 which revealed that the methanolic extract exhibited the maximum scavenging activity, followed by the aqueous extract. Chloroform extract expressed the least ability to quench DPPH. The maximum extent of both DPPH and ABTS radical scavenging was elicited by the methanolic extract, followed by the aqueous extract. Following this, the extracts were individually tested on the in vitro generation of SO-Radical and NO-Radical. Table 2 shows the per cent inhibition of SO-Radicals and NO generation in vitro by the leaf extracts of M. hortensis. The results of the study showed that the maximum extent of inhibition of SO-Radical and NO-Radical was mediated by the methanolic extract, followed by the aqueous extract. Among various oxygen-derived free radicals, the hydroxyl radical is one of the most highly reactive and harmful oxygen-derived free radicals in living organisms. The extent of thiobarbituric acid reactive substance (TBARS) produced is a measure of hydroxyl radical formation. The effects of M. hortensis leaf extracts on H2O2-induced damage to deoxyribose was quantified as the amount of TBARS formed, and the results are represented inTable 3. The value of H2O2 treated group was fixed as 100 per cent and the other groups were calculated relative to this. H2O2 exposure resulted in a steep increase in TBARS formation. The efficiency of the inhibition of TBARS formation was better in methanolic extract treated group. Aqueous and chloroform extracts also strongly inhibited the formation of TBARS. The efficiency of M. hortensis leaf extracts to scavenge H2O2 in an in vitro system was also performed and the results obtained are depicted in table 1. The maximum scavenging of H2O2 was exhibited by the methanolic extract followed by the aqueous extract. The H2O2 scavenging ability was lower in the chloroform extract.

Table 1

DPPH, ABTS, and H2O2 scavenging activities of M. hortensis leaf extract

Table 2

Percent inhibition of nitric oxide and Superoxide generation by M. hortensis leaf extract

Table 3

Hydroxyl radical scavenging activity of M. hortensis leaf extract

Discussion

DPPH radical scavenging ability was found to be more in fresh juices of orange and grape fruit than their concentrated form16. The methanol extract of the leaves of Mimusops elengi Linn. exhibited a dose-dependent scavenging of DPPH radical17. Several studies provide evidence to the fact that DPPH-scavenging ability is a reliable index of antioxidant potential. Thus, our results showed that the M. hortensis leaves possess good antioxidant activity, which gets maximally extracted into methanol. The four different solvent extracts, namely chloroform, ethyl acetate, methanol and ethyl acetate /methanol extracts, and natural substances from Rhus tripartitum showed differential ABTS radical scavenging effect18. The leaf extracts of M. hortensis were tested for their scavenging effect on the in vitro generation of SO-Radical. The results of our study indicated that the methanolic extract exhibited highest SO-Radical scavenging activity than the other five extracts tested. Sreelatha and Padma19 demonstrated that the extracts of both mature and tender leaves of Moringa oleifera Lam. have potent superoxide radical scavenging activity. The aqueous extract of Wasabia japonica (Miq.) Matsum. expressed their antioxidant property by strong scavenging activity towards NO in a cell free system22. Nitric oxide scavenging assay was adopted by Deepa et al.23 to compare the antioxidant effect of the ethanolic extracts of the leaves of Commiphora caudate (Wight & Arn.) Engl. and Commiphora varpubescens (Wight & Arn.). With these available research findings, the ability of the Majorana hortensis leaf extracts to effectively scavenge superoxide and nitric oxide radicals reveals the strong radical scavenging potential of the leaves. The presence of antioxidants can block the initiation of OH-Radical formation and break free radical chain reactions24. Jimenez et al.25 demonstrated that the raw and processed apricots (Prunus armeniaca v. bulida) samples showed very good scavenging effect against OH-Radical, which was better than the standard antioxidants BHA and BHT. An aqueous extract of Melothria maderaspatana (Linn.) was capable of scavenging H2O2 in a dose-dependent manner and the scavenging activity was better than the standard antioxidant α-tocopherol26. In the context of these literature reports, the observation made in the present study of M. hortensis leaf extracts exhibiting strong H2O2- scavenging activity gains significance in strengthening the antioxidant potential of the leaves.

Conclusions

Majorana hortensis proved to be a good scavenger of free radicals and can be used as an effective antioxidant to prepare any formulation which needs to be enhanced with antioxidants. The easy availability and perennial growth of this plant is an added advantage in traditional Indian medicine.