FREE RADICAL SCAVENGING ACTIVITY OF VARIOUS EXTRACTS OF ANISOMELES MALABARICA (L)

FREE RADICAL SCAVENGING ACTIVITY OF VARIOUS EXTRACTS OF ANISOMELES MALABARICA (L)

A. Supriya * and Lali Growther

Department of Microbiology, Hindusthan College of Arts and Science, Coimbatore, Tamil Nadu, India.

ABSTRACT: The present study aimed to determine the antioxidant potential of different solvent extracts of A. malabarica. Drug discovery from traditional medicinal herbs has played a significant role in the therapeutic management of cancer and, undoubtedly, most novel clinical applications of medicinal plants and its secondary metabolites and derivatives. The leaves extract showed various folklore and traditional applications in India. The polar and non-polar solvents extracts such as hexane chloroform, ethyl acetate, methanol, and aqueous were subjected to various in-vitro antioxidant assays such as DPPH, ABTS, Superoxide, Nitric oxide, Hydroxyl radical and Ferrous ion chelation, Butylated Hydroxy Toluene was used as a standard. The percentage of scavenging and chelation values for antioxidant were obtained and tabulated. Effective concentration (EC₅₀) values are calculated using linear interpolation. The result clearly exhibited that the methanol leaves extract possesses more efficient antioxidant properties than the other extracts. This scavenging free radical capacity can be used as a good anti-oxidizing agent in various fields of medicine, the food industry etc.

Keywords: Anisomeles, Antioxidant, DPPH, Ferrous ion, Leaves extracts

INTRODUCTION: Natural products have long been implemented as alternative health care treatment and in discovering modern drugs. Medicinal plants refer to the class of plants applied for therapy or to possess pharmacological actions for humans and animals. Drug discovery from traditional medicinal herbs has played a significant role in the therapeutic management of cancer. Undoubtedly, most novel clinical applications of medicinal plants and their secondary metabolites and derivatives of the medicinal plants over the last five decades have been effectively applied against combating cancer and other diseases ¹.

Recently, much attention has been paid to the extracts obtained from plant species to analyze their biological activities. The discovery of novel secondary metabolites from medicinal plants ². The human body possesses numerous antioxidant defense systems. It comprises enzymatic and non-enzymatic pathways that could help maintain a steady equilibrium between prooxidants and antioxidants to ensure well-being ³. Free radicals initiate oxidative stress, which seeks stability through electron pairing with biological macromolecules such as proteins, lipids, and DNA in healthy human cells and cause protein and DNA damage along with lipid Peroxidation ⁴.

These changes contribute to cancer, atherosclerosis, cardiovascular diseases, aging and inflammation. In most cases, the mechanism of action and efficiency of herbal extracts are yet to be scientifically validated. Therefore, researchers have carried out significant work to focus their attention on traditional medicines to develop outstanding drugs against different varieties of microbial infections ⁵. Oxidative stress is involved in developing and secondary pathology of various human diseases. Several studies showed evidence that regular consumption of plant foods is associated with lowered risk of incidence of these diseases ⁶.

Similarly, the health-beneficial effect of plants' food stuff is due to several phenolic compounds and their ability to promote antioxidant effects. Recently, antioxidant activity has been primarily examined in common food plants, such as fruits and vegetables. However, recent studies indicate that other plant categories, such as medicinal plants, possess significant antioxidant efficacy ⁷.

Vijayalakshmi et al., 2012 ⁸ reported that the whole plant of Anisomeles malabarica contains higher amount of flavonoids and phenolic compounds, which correspond to greater antioxidant activity. In vitro assays indicate that this plant extract is a better source of natural antioxidants, which might be helpful. The human body has a characteristic barrier system to counter free radicals as proteins, such as catalase, superoxide dismutase, and glutathione peroxidase. Selenium, vitamin C, -carotene, vitamin E, lycopene, lutein, and different carotenoids have been utilized as supplementary antioxidants. Hence, the secondary metabolites of the plant, flavonoids and terpenoids, play an important role in the defense against free radicals ⁹.

Anisomeles malabarica (L) is a medicinal plant that has been used as a folk medicine for anorexia, fevers, swelling, and rheumatism. In-vitro radical scavenging assays such as hydroxyl, superoxide anion radicals, 2,2-diphenyl-1-picryl hydroxyl (DPPH) and 2,2’-azinobis-(3-ethyl-enzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assays ¹⁰. This study aimed to assess the free radical scavenging ability of A. malabarica using various in-vitro antioxidant assays. Thus, in this study, a number of in-vitro assays (DPPH, ABTS, superoxide, nitric oxide, hydroxyl and ferrous ion chelation) were performed to evaluate the antioxidant properties.

MATERIALS AND METHODS:

Reagents Required: ABTS, 1,1-diphenyl-2-picryl hydrazyl (DPPH), Butylated hydroxyl toluene (BHT), Methanol, Butylated hydroxyl toluene (BHT), Potassium persulphate solution, Nitroblue Tetrazolium Nicotinamide Adenine Dinucleotide, Phenazine Metho Sulphate, Griess reagent, Phosphate Buffer Saline (PBS), Sodium nitroprusside (NPS), Sulphanilic acid, Ascorbic acid, Dimethylsulfoxide (DMSO), Phosphate buffer saline, Tricarboxylic acetic acid, Ethylene diamine tetra acetic acid, Ammonium acetate, Acetyl acetone, Ferrous ammonium sulphate, Glacial acetic acid, NASH reagent, Ferric chloride (FeCl₂) and Ferrozine.

In-vitro Antioxidant Potential of A. malabarica:

DPPH Radical Scavenging Activity: The free radical scavenging ability of leaf extract of A. malabarica was determined by measuring the discoloration of DPPH radicals ¹¹. Different concentrations of samples (20, 40, 60, 80, and 100µg/mL) were added to 3 mL of DPPH solution (0.1 mM), mixed thoroughly, and incubated under dark conditions for 30 min at 20°C. After incubation, absorbance was measured at 517 nm using UV-Vis spectrophotometer by keeping DPPH in methanol solution as a negative control, whereas BHT was used as a positive control. The percentage of radical scavenging was calculated according to the formula:

Scavenging of DPPH (%) = Absorbance of Control – Absorbance of Sample × 100 / Absorbance of Control

ABTS Radical Scavenging Ability: The ability of leaves extracts to scavenge ABTS radicals (2, 20 -Azinobis-3-ethyl benzothiazolinee 6 sulfonic acid) was determined ¹². ABTS solution was prepared by mixing 3.75 mM ABTS di-ammonium salt with 1.225 mM potassium persulphate solution, and it was incubated overnight at 30°C for completion of the reaction. ABTS cationic free radical solution was adjusted to the absorbance of 0.6±0.05 at 734 nm. Standard ABTS solution 200 µL was mixed with different concentrations (20 – 100 µg/mL), and the absorbance was read at 734 nm for every 5 min up to 60 min. The ABTS scavenging ability was calculated;

Scavenging of ABTS (%) = Absorbance of Control – Absorbance of sample × 100 / Absorbance of control

Superoxide Radical Scavenging Activity of A. malabarica: This assay was based on the ability of leaves extracts to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) ¹³, with minor modifications. Briefly, 3.0 mL of each reaction mixture (0.05 M phosphate-buffered saline (pH 7.8), 13 mM methionine, 2 μM riboflavin, 100 μM EDTA, NBT 75 μM) and 1.0 mL of samples (20 – 100 μg/mL) was added. The tubes were kept in front of fluorescent light (725 lumens, 34 watts). The entire reaction was enclosed in a box dark condition. Identical tubes containing reaction mixtures were kept in the dark condition and served as a blank. The absorbance was read at 560 nm after 20 min. The percentage inhibition of superoxide generation was estimated by the following equation:

Scavenging of S02 (%) = Absorbance of Control – Absorbance of Sample × 100 / Absorbance of Control

Nitric oxide Radical Scavenging Ability of A. malabarica: Nitric oxide scavenging ability of leaves extracts was determined ¹⁴. The reaction mixture 2Ml (sodium nitroprusside 10 mM in 0.5mL phosphate buffer 0.5 M; pH7.4) was added to the different samples' concentrations (20–100µg/mL). After incubation for 60 min at 37°C, Griessreagent (α-napthyl-ethylene diamine (0.1%) and sulphanilic acid (1%) in H₃PO₄) was added. The pink chromophore generated during diazotization nitriteions was measured in a spectrophotometer at 540 nm. Ascorbic acid was used as a positive control. The scavenging ability of nitricoxide radical (%) was calculated by following formula:

Scavenging of N0₂ (%) = Absorbance of Control – Absorbance of sample × 100 / Absorbance of control

Hydroxyl Radical Scavenging Ability of A. malabarica: The scavenging ability of hydroxyl radicals (OH•) was measured ¹⁵. Various concentrations of leaves extract (20 – 100µg/mL) was added to 1mL of ion-EDTA solution (0.13% ferrous ammonium sulphate and 0.26% EDTA), 0.5mLofEDTAsolution (0.018%) and 1Ml of DMSO (0.85%v/vin0.1M phosphate buffer, pH7.4). The reaction was initiated by adding 0.5mL of scorbic acid (0.22%) and incubated at 80–90°C for 15 min in a water bath. After incubation, the reaction was terminated by the addition of 1mL ice-cold TCA (17.5%w/v). In which, 3mL of Nash reagent (7.5g of ammonium acetate, 0.3mL of glacial acetic acid and 0.2 mL of acetyl acetone was mixed and made up to100mL with distilled water) was added and left at room temperature for 15 min. The intensity of the color formed was measured at 412 nm using UV-Visspectrophotometer. The reaction mixture without sample was used as a negative control whereas; BHT was used as a positive control. Percentage of hydroxyl radical scavenging ability (HRSA) was calculated by the following formula:

Scavenging of -OH (%) = Absorbance of Control – Absorbance of Sample × 100 / Absorbance of Control

Ferrous Inhalation Assay: The ability of leaves extract and bioactive compound to chelate the ferrous ion (Fe2+) was investigated ¹⁶. Briefly, 50 μL (2mM) FeCl₂ was added to 1 mL of different concentration of samples (20 – 100 μg/mL). The reaction was initiated by the addition of 0.2 mL of 5 Mm ferrozine solution. After which the reaction mixture was vigorously shaken and left at room temperature (28±2°C) for 10 min. The absorbance was measured at 562 nm, Na₂ EDTA was used as a positive control and percentage inhibition of ferrozine (Fe2+) complex formation was calculated.

Scavenging of -OH (%) = Absorbance of Control – Absorbance of sample × 100 / Absorbance of Control

RESULTS:

DPPH Free Radical Scavenging of Leaves Extracts of A. malabarica: The free radical scavenging of leaves extracts of A. malabarica such as hexane, chloroform, ethyl acetate, methanol, and aqueous was evaluated. Among all the extracts tested, the methanolic extract showed superior DPPH radical scavenging activity (92.90±0.36%) with an EC50 value of 34.85μg/mL, followed by ethyl acetate extract (71.78±0.31%) with an EC⁵⁰ value of 53.45, Aqueous (59.78±0.41%) with an EC₅₀ value of 60.04μg/mL. Chloroform extract showed only moderate activity at 56.67±0.44% (EC₅₀ of 59.34μg/mL), while; hexane extract exhibited the least radical scavenging activity (42.81±0.41%) with an EC₅₀ value of 94.03 μg/Ml Fig. 1. The standard BHT showed the optimal activity at 64.00μg/mL with an EC ₅₀ value 40.60 µg/mL. Overall, the crude extracts displayed a dose-dependent free radical scavenging activity.

FIG. 1: DPPH RADICAL SCAVENGING ACTIVITY OF A. MALABARICA

ABTS Radical Scavenging of A. malabarica: ABTS assay is a widely accepted model to determine the total antioxidant activity. Among the various solvent extracts, methanolic extract at 100 μg/mL was found to have the best scavenging activity, 89.21±0.17%, with an EC₅₀ value of 49.04μg/mL, followed by ethyl acetate 71.42±0.64% with an EC₅₀ value of 62.04 μg/mL, aqueous 69.04 ±0.57% with an EC₅₀valueof 79.09μg/mL, hexane 62.09±0.33% with an EC₅₀ value of 104μg/mL. In contrast, the chloroform extract showed lowest ABTS radical scavenging activity (57.90±0.17%) with an EC₅₀ value of 110.3μg/mL. The extent of reduction or decolorization of the reaction mixture is directly proportional to the increased concentration Fig. 2. BHT exhibited the best activity (91.64±0.33%), with an EC₅₀ value of 38.11μg/mL, respectively. This result implies that the radical scavenging activity of the extracts might be attributed to their strong proton–donating ability.

FIG. 2:  ABTS EFFECT ON VARIOUS EXTRACTS OF A. MALABARICA

Superoxide Radical Scavenging Assay: The superoxide radical scavenging of leaves extracts of A. malabarica are illustrated in Fig. 3. In which, ethyl acetate extract was the most effective superoxide scavenger (82.13±0.16%) at 100 µg/mL, followed by methanol (68.27±0.23%), Aqueous (44.38±0.11%), and chloroform (41.54±0.42%) with an EC₅₀ value of 59.04, 69.04, 100.02 and 103.4 µg/mL, respectively. But, hexane extract exhibited the least scavenging activity (29.91±0.14%), with an EC₅₀value 120 µg/mL. BHT exhibited best activity (87.37±0.30%), with an EC₅₀ value of 56.04µg/mL, respectively. These results indicated that ethyl acetate and methanol extracts of A. malabarica had a notable effect on the scavenging of superoxide radicals.

FIG. 3: SUPEROXIDE RADICAL SCAVENGING OF VARIOUS EXTRACT OF A. MALABARICA

Nitricoxide Radical Scavenging of A. malabarica: The capability of A. malabarica on nitric oxide scavenging was increased with an increasing concentration (20 – 100 µg/mL). Among all the extracts, methanol extract possessed the highest radical scavenging activity 85.52 ± 0.28%, followed by ethyl acetate 67.40±0.41% and aqueous extract 62.23±0.39%, with an EC₅₀ value 52.50, 63.94 and 83.02 µg/mL, respectively.

However, chloroform and hexane extract showed moderate scavenging activity 58.69±0.17% and 53.43±0.12% with EC₅₀ values 93.46, 95.05 µg/mL when compared to standard (BHT 94.39±0.43%) with an EC₅₀ value 48.25 µg/Ml Fig. 4.

FIG. 4:   NITRIC OXIDE RADICAL SCAVENGING OF VARIOUS EXTRACT OF A. MALABARICA

Hydroxyl Radical Scavenging of A. malabarica: The antioxidant activity of A. malabarica was on the basis of their ability to scavenge hydroxyl radical Fig. 5. The consistent scavenging activity was obtained in methanolic extract (92.90±0.26%) followed by ethyl acetate (71.78±0.11%) and aqueous (59.78±0.31%), whereas chloroform and hexane extract showed the moderate activity (56.77±0.44 and 42.81±0.53%) at 100 µg/mL. The EC₅₀ values of methanol, ethyl acetate, aqueous, chloroform, and hexane ranged from 51.14, 79.10, 95.56, 112.03, and 118.4µg/mL, respectively. The standard BHT scavenged hydroxyl radical sup to 81.04±0.42% (EC₅₀58.25 µg/mL). The present study indicated that diterpenescontents were significantly concomitant with antioxidant activity.

FIG. 5: HYDROXYL RADICAL SCAVENGING OF VARIOUS EXTRACT OF A. MALABARICA

Ferrousion Chelation of Anisomeles malabarica: The chelating effect of crude extracts was estimated by measuring the iron–ferrozine complex. All the extracts revealed chelating activity in a dose-dependent manner Fig. 6. Methanol extract displayed the superior activity (85.52±0.26%), with an EC₅₀ value of 52.04 µg/mL, followed by ethyl acetate (67.40±0.24% µg/mL), with an EC₅₀ value of 75.89 µg/mL, aqueous extract 62.32±0.22% with an EC₅₀ value of 85.03 µg/mL and chloroform 58.69±0.65% with an EC₅₀ value 89.03 µg/mL. However, hexane extract displayed the least ferrous ion chelating efficacy (53.43±0.21%) with EC₅₀ values of 93.03 µg/mL. The standard BHT scavenged ferrous ions up to 59.12 µg/mL with EC₅₀ values of 75.25µg/mL. It was noteworthy that methanol extract displayed the highest antioxidant activity when compared to other crude extracts of A. malabarica. EC₅₀ values of antioxidant assays were tabulated based upon the ability Table 1.

FIG. 6: EFFECT OF FERROUS ION CHELATION ON VARIOUS EXTRACTS OF A. MALABARICA

TABLE 1: EC₅₀ VALUE OF ANTIOXIDANT ASSAYS

DISCUSSION: The DPPH reaction was very stable at room temperature, producing reliable values in repeated tests. Its odd electron gives a strong absorption at 515 nm in visible spectrophotometer ¹⁷. The decrease in absorbance of DPPH is caused by the reaction between antioxidant molecules and the radical, which results in the scavenging of the radical by hydrogen donation. A freshly prepared DPPH solution exhibits a deep purple color with an absorption maximum of 517 nm. As antioxidants donate protons to these radicals, the absorbance decreases. The purple color generally fades when an antioxidant is present in the medium ¹⁸. The antioxidant activity is expressed by EC₅₀ value, which is defined as the effective concentration of substrate that causes 50% loss of the DPPH & ABTS activity ¹⁹. The crude extracts exhibited highest EC₅₀ value (methanol extract 34.85 µg/mL) when compared with BHT standard (40.60µg/mL).

There is an increasing trend to replace synthetic antioxidants, which are of safety concern, with the natural antioxidants available from plant extracts or isolated products of plant origin ²⁰. The therapeutic benefits of medicinal plants are often related to their antioxidant properties attributed to the extracts' phenolics and flavonoid content ²¹. Flavonoids are naturally occurring in plants and are thought to positively affect human health ²². The perceived antioxidant activity of extracts may be due to the neutralization of ABTS radical ²³. All the extracts of A. malabarica possessed stronger ABTS scavenging activity than other research ²⁴.

The hydroxyl radicals are the most toxic among all the reactive oxygen species (ROS) and easily can cross cell membranes at specific sites, react with most biomolecules, and cause tissue damage and cell death ²⁵. Therefore, it is important to remove the hydroxyl radicals to protect the biological systems ²⁶.

Thus, hydroxyl radical scavenging activity is very important for evaluating the antioxidant activity of various extracts. In the present study, antioxidant activity was determined for the leaves crude extracts showed the highest EC₅₀ value in methanol 51.14 µg/mL, and ethyl acetate 79.10 µg/mL, respectively, which is lower than the standard BHT (58.25 µg/mL). Thus A. malabarica has a higher ability to scavenge even at lower concentrations, which shows that it is a good candidate for the preparation/additive in the formulation of functional food or nutraceutical. Reactive oxygen species (ROS), including superoxide radicals, have gained significant attention due to the progression of several human diseases and carcinogenesis. So, it is important to eliminate excessive O₂- in-vivo to prevent important oxidative stress originated diseases ²⁷.

Superoxide radicals are harmful to cellular components and are considered the primary ROS that can further interact with other molecules to generate secondary ROS such as hydroxyl radicals, hydrogen peroxide, and singlet oxygen ^{28, 29}. Overproduction of ROS leads to an imbalance in intracellular redox status and further, it is associated with oxidative stress. In this study, leaf extracts of A. malabarica showed higher EC₅₀value in ethyl acetate 59.04 µg/mL and methanol 69.04 µg/mL and standard BHT showed 56.04 µg/mL. It could be useful in drug formulation. Nitric oxide (NO) is a signaling molecule that plays a significant role in the prolongation of inflammation and immunological responses. The mechanism of action of crude extracts or their constituents may be via the inhibition of the activity of inducible nitric oxide synthase (iNOs) or by free radical scavenging activities ^{30, 31}. The recent study of nitric oxide scavenging of Desmodium gangiticum showed 30.18% at 200 µg/mL of crude extract (Usha & Muthu Krishnan, 2012) whereas it was 85.52% at 100 µg/mL of methanolic extract. The results clearly denoted that A. malabarica possesses superior scavenging ability than the D. gangiticum. Generally, iron can stimulate lipid peroxidation by the Fenton reaction and accelerates peroxidation by decomposing lipid hydroperoxides into peroxyl and alkoxyl ³².

In addition, other studies have indicated classes of compounds like polysaccharides ³³, some peptides, and proteins ³⁴. Exhibit significant chelating effects. Various authors have claimed that metal chelation plays a minor role in the overall antioxidant activities of some polyphenols ³⁵. In the present study, crude extracts showed the highest EC₅₀ value in methanol (52.04 µg/mL) and ethyl acetate (75.89 µg/mL), respectively, and it is compared with the standard BHT (75.25 µg/mL). Measurement of color reduction allows estimation of the chelating activity of the coexisting chelator ³⁶.

CONCLUSION: Anisomeles malabarica is an important traditional medicinal plant that possesses a rich source of phytocompounds. The present study results revealed that the in vitro antioxidant activities of A. malabarica confirmed that methanolic extract exerted the maximum antioxidant activity than the other extracts. The activity was due to highly soluble bioactive metabolites in plant extracts of A. malabarica. Based on the results, it may be proposed that the naturally obtained from the test plant may play a major role in the antioxidant defense mechanism.

ACKNOWLEDGEMENT: I K. A. Supriya would like to thank Prof. Dr. Lali Growther, Assistant Professor and Head, Department of Microbiology, Hindusthan College of Arts and Science, Coimbatore – 641004, for providing the laboratory facility to carry out this research work as successful.

CONFLICTS OF INTEREST: The authors declare no conflict of interest.

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    How to cite this article:

    Supriya KA and Growther L: Free radical scavenging activity of various extracts of Anisomeles malabarica (L). Int J Pharm Sci & Res 2022; 13(9): 3675-82. doi: 10.13040/IJPSR.0975-8232.13(9).3675-82.

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