INTRODUCTION:

Naturally occurring bioactive molecules commonly found in vegetables, herbs, and other plants promotes various health benefits.¹ Antioxidants are mainly composed of phenolic acids, flavonoids, and catechins, that suppresses the lipid peroxidation, prevents DNA oxidative damage, and scavenge the reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and hydroxyl radicals.² ROS causes oxidative damage to cellular compartment, leads to cell injury and death. It leads to various chronic diseases like coronary heart disease, carcinogenesis, and other health problems related to advancing age.³ An increased antioxidant intake in humans through diet can reduce such oxidative damage. The commercially available synthetic antioxidants are known to exhibit serious toxicity. Hence, efforts are made in search of natural, economical, and effective antioxidants.

Hyptis suaveolens (L.) Poit commonly known as ‘‘Wilaiti tulsi’’ belongs to the family Lamiaceae and is a common weed of roadsides and waste grounds, is an ethnobotanically important medicinal plant. The plant is distributed throughout the tropics and subtropics. All parts of this plant are being used in traditional medicine to treat various diseases. Hyptis suaveolens has both medicinal as well as insecticidal properties. Literature of Hyptis indicates that leaf extracts cure swellings, abscesses and haemorrhoids. In India the plant is considered to be stimulant, carminative, sudorific and lactogogue. Infusion is used in infections of the uterus; leaf juice is taken in cases of colic and stomach ache.⁴ Based on the traditional knowledge and recent pharmacological studies, the objective of the present study was to investigate the chemical constituents and antioxidant potential of various extracts of H. suaveolens by various in vitro assays.

METHODS:

Collection of Plant material and extraction:

The whole plant of H. suaveolens was collected from the forests of Maisammaguda, Secunderabad situated in the state of Telangana (India) and shade dried and powdered mechanically. The plant specimen was authenticated by botanist of Osmania University and authenticated voucher specimen Number 276 of the plant has been preserved in department for future reference. The dried plant powder was extracted with various solvents based on polarity (Pet ether, Chloroform, Ethyl acetate, Methanol AND Aqueous) by hot continuous extraction in Soxhlet's apparatus and the extracts were evaporated to dryness under vacuum, dried in vacuum desiccators and stored in refrigerator.

Phytochemical Evaluation:

Phytochemical investigation of alkaloids, saponins, carbohydrates, tannins, phenolics, flavonoids, steroids and glycosides were carried out for various extracts of plant using standard protocols.

Total phenol content Estimation:

The total phenolic content of H.suaveolens was assessed using Foline Ciocalteau phenol reagent method described by Singleton et al.⁵ Briefly, 1.0mL of the extract at various concentrations was mixed with 2.5mL of 10% Foline Ciocalteau reagent and 2.5mL of 7.5% sodium carbonate. The contents were thoroughly mixed and allowed to stand for 30 minutes. The absorbance was read at 750nm in a spectrophotometer. The total phenol content was expressed as gallic acid equivalents in milligram per gram of the extract.

Total Flavonoid estimation:

The flavonoid content of H.suaveolens was determined using aluminum chloride colorimetric method described by Chang et al.⁶ Briefly, 0.5mL of the extract at various concentrations was mixed with 3mL of 95% methanol, 0.1mL of 10% (weight/volume) aluminum chloride, 0.1 mL of 1M potassium acetate, and 2.8mL of distilled water. The reaction mixture was allowed to stand at room temperature for 30 minutes and absorbance was measured at 415nm against a blank sample. A calibration curve was prepared using quercetin in methanol. The flavonoid content was expressed as quercetin equivalents in milligram per gram of the extract.

In-vitro Antioxidant activity:

DPPH antiradical capacity

The antiradical potential of H.suaveolens was determined spectrophotometrically as described by Ilahi et al.⁷ Six different concentrations of various extracts of plant material (100, 200, 400, and 800 and 1000µg/ml) were mixed with 100µL of DPPH radical solution in a 96-well microplate and incubated for 20 min at room temperature. The resultant mixture was read spectrophotometrically at 517nm against a methanol blank and the following equation was used to calculate the % inhibition of each extract:

% inhibition= (A_Control−A_Sample)/ (A_Control) ×100

where A_Controland A_Sample indicate the absorbance of the DPPH solution and the reaction mixture, respectively. The effective dose of plant extract needed to neutralize 50% of the DPPH radical solution (IC50) was obtained from a plot comparing percent inhibition to extract concentration.

Nitric oxide scavenging activity:

Nitric oxide radical scavenging activity was determined according to the method reported by Garrat.⁸ Sodium nitro prusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions, which can be determined by the use of the Griess Illosvoy reaction. 2 ml of 10mM sodium nitroprusside in 0.5ml phosphate buffer saline (pH 7.4) was mixed with 0.5ml of extract at various concentrations and the mixture incubated at 25°C for 180 min. From the incubated mixture 0.5ml was taken out and added into 1.0ml sulfanilic acid reagent (33% in 20% glacial acetic acid) and incubated at room temperature for 5 min. finally, 1.0 ml naphthylethylenediaminedihydrochloride (0.1% w/v) was mixed and incubated at room temperature for 30 min, the absorbance at 540nm was measured with a spectrophotometer. The nitric oxide radicals scavenging activity was calculated.

Reducing power Determination:

The Fe²⁺ reducing power of plant extract was determined by the method of Oyaizu⁹with slight modification. Various concentrations of plant extract (0.75mL) was mixed with 0.75ml of phosphate buffer (0.2 mole, pH 6.6) and 0.75mL of potassium ferricyanide K₃Fe(CN)₆ (1%w/v), followed by incubating at 50°C for 20 mins. The reaction was stopped by adding 2.5mL of 10% (w/v) trichloroacetic acid followed by centrifugation at 3000 rpm for 10min. Finally, 1.5mL of the upper layer was mixed with 1.5mL of distilled water and 0.5mL of FeCl₃ (0.1%) and the absorbance was measured at 700nm. Higher the absorbance of reaction mixture indicated greater the reducing power. Ascorbic acid is used as reference compound.

RESULTS AND DISCUSSIONS:

Phytochemical Constituents:

Phytochemical screening was carried out for various extracts and revealed the presence of the following constituents shown in Table 1. ‘+’ indicate the presence and ‘-’ indicates the absence of phytoconstituents.

Table 1: Phytochemical Constituents in various extracts of H. suaveolens:

	Pet. Ether	Chloroform	Ethyl Acetate	Methanol	Aqueous
Alkaloids	+	+	+	+	+
Saponin	-	-	-	-	+
Glycosides	-	-	+	+	+
Carbohydrates	+	+	+	+	+
Tannins and Phenolics	+	+	+	+	+
Flavonoids	₊	+	-	+	+
Steroids	₊	+	_	_	_
Proteins and Amino acids	₊	+	+	+	+

Total phenol content Estimation:

Phenolic compounds are the key phytochemicals with high free radical scavenging activity. It has generated a great interest among the scientists for the development of natural antioxidant compounds from plants. In the current work, phenolic content of the various extracts of H. suaveolens were measured and listed in (Table2). The Chloroform extract of H.suaveolens showed higher amount of phenolic compounds when compared to other extracts. The concentration of the phenolic compounds was increased with an increase in the dose. The results are described as Gallic acid equivalents (GAE) (Figure 1).

Figure 1. caliberation Curve of gallic acid.

Total Flavonoid estimation:

The flavonoid content of various extracts of H.suaveolens was determined using aluminum chloride colorimetric method. In the current study, the total flavonoid content were measured and listed in (Table 2). The total flavonoid content in Chloroform extract was found to be high when compared to other extracts. The flavonoid content was expressed as quercetin equivalents in milligram per gram of the extract (Figure 2).

Table 2: Total Phenolic and Total Flavonoid content in various extracts of H.suaveolens

S. No	Extract	Total Phenolic Content (Gallic acid equivalents in mg /gm extract)	Total Flavonoid Content (Quercetin equivalents in mg/ gm extract)
1	Pet Ether	17.66 ±0.381	11.22±0.838
2	Chloroform	86.16±0.877	64.66±1.201
3	Ethyl Acetate	30.66±0.629	--------
4	Methanol	69.33±0.144	21.44±0.509
5	Aqueous	76.08±0.629	34.22±1.170

Figure 2: Calibration curve of Quercetin

DPPH antiradical capacity:

DPPH antioxidant assay is based on the ability of DPPH, a stable free radical, to decolorize in the presence of anti oxidants. The DPPH radical contains an odd electron which is responsible for the absorption at 517nm and also for visible deep purple colour. When DPPH accepts an Electron, donated by an antioxidant compound, results in decolorisation which can be quantitatively measured from changes in the absorbance. DPPH radical scavenging activity of various extracts of H. suaveolens in comparison with ascorbic acid are reported in (Figure 3). IC₅₀values for the various extracts were mentioned in (Table 3). Chloroform Extract showed better antiradical activity than the remained extracts.

Figure 3: DPPH radical scavenging activity of various extracts of H. suaveolens

Figure 4: The nitric oxide scavenging activity of various extracts of H. suaveolens

Nitric Oxide scavenging activity:

Nitric Oxide is a potent pleiotropic mediator of physiological processes playing vital role in various biological systems. The nitric oxide scavenging activity of various extracts of H. suaveolens in comparison with ascorbic acid are reported in (Figure 4). IC₅₀values for the various extracts were mentioned in (Table 3).

Table 3: IC₅₀values for the various extracts of H. suaveolens:

S. No	Extract	DPPH Method	Nitric oxide scavenging activity
1	Pet Ether	311.334 ±5.138	765.324±3.605
2	Chloroform	204.759±1.005	217.616±0.583
3	Ethyl Acetate	253.794±1.669	310.629±1.361
4	Methanol	223.743±0.521	254.182±3.246
5	Aqueous	218.456±0.635	237.152±0.536
6.	Ascorbic acid	194.99±1.258	193.420±2.099

Reducing Power:

Reducing power experiment is a good reflector of antioxidant activity of the plant. The plant having high reducing power generally reported to carry high antioxidant potential too. In this experiment, Ferric ions are reduced to ferrous ions which is identified by colour change from yellow to bluish green. The results for ferric reducing power activity of various extracts of H.suaveolens in comparison with ascorbic acid are reported in (Figure 5). Chloroform extract showed high reducing power than that of other extracts. Reducing power potential of extracts increase with the dose, however the extracts exhibited low reducing power than that of ascorbic acid.

Figure 5: Reducing power of Various Extracts of H. suaveolens:

CONCLUSION:

Plant source is considered to be the good source of natural antioxidants for curing the diseases. In the present study, it is revealed that the plant H.suaveolens is rich in phenolic and flavonoid content in it. Chloroform extract has shown better antioxidant profile when compared to the other extracts and less potent than the synthetic antioxidants like ascorbic acid and quercetin.

ACKNOWLEDGEMENT:

The authors wish to thank the Management of Malla Reddy Institute of Pharmaceutical sciences, TS, India for providing necessary facilities to carry out this study.

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