A Comparative Study on Phytochemical Investigation and Antioxidant Activity of Poly herbal Mixture of Ocimum canum and Pongamia pinnata hydro-alcoholic Leaf Extracts

Saiprasanna Behera¹*, S. Manohar Babu², Y. Roja Ramani³, Prasanta Kumar Choudhury¹

¹Department of Pharmacology, Royal College of Pharmacy and Health Sciences, Berhmapur, Odisha- 760002

²Department of Pharmacology, SIMS College of Pharmacy, Mangaldas Nagar, Guntur- 522001

³Department of Pharmacology, MKCG Medical College, Berhampur, Odisha- 760004

ABSTRACT:

Free radicals are implicated for many diseases including diabetes mellitus, arthritis, cancer, aging, etc. In the treatment of these diseases, antioxidant therapy has gained utmost importance. Reactive oxygen Species (ROS) is a metabolic side product of oxidative stress process which causes several diseases like atherosclerosis, cancer, etc. In defense of ROS, anti-oxidants play a key role in combatting them. As the process of aging increases the level of anti-oxidant in our body decreases and thereby needs utmost attention for its repair process which is generally administered externally. Many herbal remedies individually or in combination have been recommended in various medical expositions for the cure of diﬀerent diseases. Plant products serve a best source for controlling these activities by its own metabolic pathway. Currently there has been an increased interest globally to identify antioxidant compound that are pharmacologically potent and have low or no side effects. As plants are source of natural antioxidants, much attention has been gain to plants. The quest for natural antioxidants for dietary, cosmetic and pharmaceutical uses has become a major industrial and scientific research challenges over the last two decades. A variety of free radical scavenging antioxidants exists within the body in which many of them are derived from dietary sources like fruits, vegetables and teas. Thus in this aspect three plants namely Pongamia pinnata (Karanj) and Ocimum canum (KalaTulsi) were taken up for the study. Both the plants have been recognized in diﬀerent system of traditional medicines for the treatment of diﬀerent diseases and ailments of human beings. In this study, Antioxidant activity of all the hydro-alcoholic extracts was determined by various antioxidant assays. In all the testing, a significant correlation existed between concentrations of the extract and percentage inhibition of free radicals. These findings suggest that the hydro-alcoholic extracts are able to scavenge free radicals, by either hydrogen or electron donating mechanisms, and can therefore act as primary antioxidants. The antioxidant property may be related to the phenols and flavonoids present in the extracts. When all three extracts were mixed together in equal proportion, in same concentration it gives synergistic effect in percentage inhibition with respect to antioxidant property.

KEYWORDS: Pongamia pinnata, Ocimum canum, Poly herbal mixture, phytochemical constituents, antioxidant activity

INTRODUCTION:

Ayurveda is the oldest healing system of medicine. Major formulations used in Ayurveda are based on herbs. The medicinal herbs are used as decoctions, infusions, tinctures, and powder

The drug formulation in Ayurveda is based on two principles: A. Use as single drug. B. Use of more than two drugs. When two or more herb is used in formulations, they are known as poly herbal formulations. Sometime herbs are combined with mineral preparations also. The herbs often exist in crude state and Ayurveda describes method of purification of toxic herbs. The concept of poly herbalism is peculiar to Ayurveda although it is difficult to explain in term of modern parameters. ‘Sanghar Samhita’ highlights the concept of synergism behind poly herbal formulations. Ayurveda has fundamental aspects for drug formulation. The herbs are selected according to the disease; other herbs are used to prevent the side effects arising from chief herb. Herbal medicine is making dramatic comeback and increasing number of patients are visiting alternative medicine clinics. Side effects of synthetic medicine are alarming and recent time has seen risk of herbal-herbal and herbal-synthetic drug interactions.

Today, chromatographic methods HPLC, GC, HPTLC are used for identifying active constituent of medicinal herbs. The scientific procedures have brought revolution in the field of herbal medicine, particularly in case of single plant based formulation. The concept of standardization is rapidly catching with herbal products based on active constituent.

Ayurveda strongly recommends the use of the plant as a whole. Single plant based formulations may have better acceptance from quality control and standardization aspects, but still not ample amount of evidence has accumulated to prove concept of standardization of herbal drugs based on single constituent. Poly herbal formulations are difficult to standardize. The plant is a biosynthetic laboratory for chemical compounds like glycosides, alkaloids etc. These exert physiological and therapeutic effect. It can be concluded that poly herbal formulations should not be dismissed only on the basis that they do not withstand modern research. Ayurveda and herbal medicine has roots in medicinal herbs and they have been practiced for centuries¹.

The reactive oxygen species (ROS) is highly reactive side product of metabolic process in our body. These ROS plays key role in generation of the diseases like atherosclerosis, cancer, ischemic condition, cataracts, Alzeimer’s disease, arthritis, etc ². In defense against these ROS body have their own system including various enzymes, proteins and vitamins, which are known as Anti-oxidants ³. As the age increases the level of anti-oxidants declines in body which requires external source of anti-oxidants to defend against generated ROS ⁴. In Ayurveda, there are number of plants reported to possess anti-oxidant activity, which could be the best external source ⁵.Thus in this aspect few plants are selected which were subjected for determining their anti-oxidant activity, these are Pongamia pinnata- Karanja and Ocimum canum- Kala tulsi. Ocimum canum Sims (Hairy Basil) is a traditional medicinal plant distributes throughout Odisha. P. pinnata (Linn) Pierre is a medium sized glabrous tree popularly known as Karanja in Hindi, Indian Beech in English ⁶. In the traditional systems of medicines, such as Ayurveda and Unani, the P. pinnata plant is used for anti-inﬂammatory, anti-plasmodial, anti-nonciceptive, anti-hyperglycaemics, anti-lipidoxidative, anti-diarrhoeal, anti-ulcer, anti-hyperammonic and antioxidant. Its oil is a source of biodiesel. It has also alternative source of energy, which is renewable, safe and non-pollutant⁷. O. canum is used specially for treating various types of diseases and lowering blood glucose and also treats cold, fever, parasitic infestations on the body and inﬂammation of joints and headaches ⁸. Essential oil from the leaves of O. canum possesses antibacterial and insecticidal properties⁹. Ocimum canum has significant antioxidant properties and can partly prevent the consequences of ethanol-induced toxicity and to some extent reverse the consequences of ethanol toxicity¹⁰. Thus, our main objective for the present study was to determine the anti-oxidant activity of these extracts when given in combination (equi-proportional) to check the potency of the activity mentioned and also to find out whether they possess a synergistic effect or not.

MATERIALS AND METHOD:

Collection of Plant and Extraction:-

Leaves of Pongamia pinnata and Ocimum canum were collected in the month of December 2011 from its natural habitat from nearby Mohuda village, Berhampur, Ganjam district of Odisha. The plant was authenticated from Department of Botany, Khalikote College, Berhampur, Odisha. The leaves were cleaned and dried under the shade so that they do not lose their active constituents. The shade dried drugs were powdered by using a pulveriser. Then the powders were mixed with methanol-water mixture in ratio of 1:5 for maceration and were kept in dark for 4-5 days. After 5 days, they were filtered and macerates were collected. The collected macerates were concentrated at 40˚C in thermostat. These were dried and were used for further experiments¹¹.

File Photo: - Pongamia pinnata Linn. With permission from B and T world seeds

Chemicals:

All the chemicals used were of analytical grade. Kits used in the experiment were obtained from Crest Biosystems, Bambolim Complex. Goa, India. All drug solutions were freshly prepared in saline before each experiment. The extracts were dissolved in distilled water to carry out the experimental procedures.

File Photo:-Ocimum canum- Western Orissa Medicinal plant Gallery (photo by Surendra Hota Bargarh. Odisha. INDIA)

Phytochemical screening/: -

The freshly prepared crude extract was qualitatively tested for the presence of chemical constituents ¹².Phytochemical screening of the extract was performed using the following reagents and chemicals. These were identified by characteristic color changes using standard procedures¹³

1) Test for alkaloids: To the extract dilute hydrochloric acid will be added and filtered. The filtrate will be treated with various alkaloidal reagents

· Mayer’s test: The filtrate will be treated with Mayer’s reagent: appearance of cream colour indicates the presence of alkaloids.

· Dragendroff’s test: The filtrate will be treated with Dragendroffs reagent: appearance of reddish brown precipitate indicates the presence of alkaloids.

· Hager’s test: The filtrate when treated with Hager’s reagent, appearance of yellow colour precipitate indicates the presence of alkaloids.

2) Test for carbohydrates and reducing sugar: The small quantities of the filtrate will be dissolved in 4ml of distilled water and filtered. The filtrate will be subjected to

· Molisch’s test: A small portion of the filtrate will be treated with Molisch’s reagent and sulphuric acid. Formation of a violet ring indicates the presence of carbohydrates.

· Fehling’s test: The extract will be treated with Fehling’s reagent A and B. The appearance of reddish brown colour precipitate indicates the presence of reducing sugar.

· Benedict’s test: The extract will be treated with Benedict’s reagent; appearance of reddish orange colour precipitate indicates the presence of reducing sugar.

· Barfoed’s test: The extract will be treated with barfoed’s reagent and heated. Appearance of reddish orange colour precipitate indicates the presence of non-reducing sugars.

3) Test for steroids:

Libermann burchard’s test: The extract will be treated with 3ml of acetic anhydride, few drops of glacial acetic acid followed by a drop of concentrated sulphuric acid. Appearance of bluish green colour indicates the presence of steroids.

4) Test for proteins:

· Biuret test: The extract will be treated with copper sulphate solution, followed by addition of sodium hydroxide solution; appearance of violet colour indicates the presence of proteins.

· Millon’s test: The extract will be treated with Millon’s reagent; appearance of pink colour indicates the presence of proteins.

5) Test for tannins:

The extract will be treated with 10% lead acetate solution; appearance of white precipitate indicates the presence of tannins.

6) Test for phenolic compounds:

· The extract will be treated with neutral ferric chloride solution; appearance of violet colour indicates the presence of phenolic compounds.

· The extract will be treated with 10% sodium chloride solution; appearance of cream colour indicates the presence of phenolic compounds.

7) Test for flavonoids:

5ml of extract will be hydrolyzed with 10% sulphuric acid and cooled. Then, it will be extracting with diethyl ether and divided in to three portions in three separate test tubes. 1ml of diluted sodium carbonate, 1ml of 0.1N sodium hydroxide, and 1ml of strong ammonia solution will be added to the first, second and third test tubes respectively. In each test tube, development of yellow colour demonstrated the presence of flavonoids.

8) Shinoda’s test:

The extract will be dissolved in alcohol, to which few magnesium turnings will beaded followed by concentrated HCL drop wise and heated, and appearance of magenta colour shows the presence of flavonoids.

9) Test for gums and mucilage:

The extract was treated with 25 ml of absolute alcohol, and filtered. The filtrate will examine for its swelling properties.

10) Test for glycosides:

When a pinch the extract was treated with glacial acetic acid and few drops of ferric chloride solution, followed by the addition of conc. Sulphuric acid, formation of ring at the junction of two liquids indicates the presence of glycosides.

11) Test for saponins:

Foam test About 1 ml of the extract was diluted to 20 ml of with distilled water and shaken well in a test tube. The formation of foam in the upper part of test tube indicates the presence of saponins.

12) Test for Triterpenoids:

The substance was warmed with tin and thionyl chloride. Pink colour indicates the presence of triterpenoids.

Phytochemical investigation:-

Phytochemical investigation of Pongamia pinnata indicated the presence of abundant prenylated flavonoids such as furanoflavones, furanoflavonols, chromenoflavones, furanochalcones and pyranochalcones^14-16. The leaves of the plant consist of several flavone and chalcone derivatives such as Pongone, Galbone, Pongalabol, pongagallone A and B ^{17, 18}.

Flavonoids of Pongamia pinnata Linn

Two types of ﬂavonoids are present in Ocimum species. Lipophilic ﬂavonoid aglycones (external ﬂavonoids), often highly methylated, are found in glandular hairs on the surface of the leaves, stems and inﬂorescences. These have been the subject of two recent studies19, 20. The second type of ﬂavonoids is polar ﬂavonoid glycosides, which are stored in the vacuoles of aerial plant parts. Nevadensin and salvigenin are found in the leaves O. canum Sims²¹

Flavonoid glycosides of Ocimum canum Sims

Determination of antioxidant activity: -

A. DPPH radical scavenging assay²²

To the Methanol solution of DPPH (1 mM) an equal volume of the extract dissolved in alcohol was added at various concentrations from 250 to 2000 μg/ml in a final volume of 1.0 ml. An equal amount of alcohol was added to the control. After 20 min, absorbance was recorded at 517 nm. Experiment was performed in triplicate.

B. ABTS radical scavenging assay²²

To the reaction mixture containing 0.3 ml of ABTS radical, 1.7 ml phosphate buffer and 0.5 ml extract was added at various concentrations from 250 to 2000 μg/ml. Blank was carried out without drug. Absorbance was recorded at 734 nm. Experiment was performed in triplicate.

C. Nitric oxide radical scavenging²²

Sodium nitroprusside (5μM) in standard phosphate buffer solution was incubated with different concentration of the test extracts dissolved in standard phosphate buffer (0.025M, pH 7.4) and the tubes were incubated at 25 °C for 5 hr. After 5 h, 0.5 ml of incubation solution was removed and diluted with 0.5 ml Griess reagent (prepared by mixing equal volume of 1% sulphanilamide in 2% phosphoric acid and 0.1% naphthylethylene di-amine di-hydrochloride in water). The absorbance of chromophore formed was read at 546 nm. The control experiment was also carried out in similar manner, using distilled water in the place of extracts. The experiment was performed (in triplicate) and % scavenging activity was calculated using the formula:- 100 − [100/blank absorbance × sample absorbance] The activity was compared with ascorbic acid, which was used as a standard antioxidant.

D. Peroxide scavenging (NBT reduction assay)²²

Alkaline DMSO was used as a super oxide generating system. To 0.5 ml of different concentrations of the test compound, 1 ml of alkaline DMSO and 0.2 ml of NBT 20 mM in phosphate buffer pH 7.4 was added. The experiment was performed in triplicate.

E. Iron chelating activity assay²²

The reaction mixture containing 1 ml O-Phenanthroline, 2 ml Ferric chloride, and 2 ml extract at various concentrations ranging from 250 to 2000 μg/ml in a final volume of 5 ml was incubated for 10 minutes at ambient temperature. The absorbance at 510 nm was recorded. Ascorbic acid was added instead of extract and absorbance obtained was taken as equivalent to 100% reduction of all ferric ions. Blank was carried out without extract. Experiment was performed in triplicate.

F. Hydroxyl Radical Scavenging Activity (Deoxyribose degradation assay)

The scavenging capacity for hydroxyl radical was measured according to the modified method of Halliwell et al., (1987)²³. Stock solutions of EDTA (1mM), FeCl₃ (10 mM), ascorbic acid (1mM), H₂O₂ (10mM) and deoxyribose (10mM) were prepared in distilled deionized water. The assay was performed by adding 0.1 ml of EDTA 0.01 ml of FeCl3, 0.1 ml of H₂0₂, 0.36 ml of deoxyribose, 1.0 ml of plant extract (250-2000 μg/ml), 0.33 ml of phosphate buffer (50 mM, pH 7.4) and 0.1ml of ascorbic acid in sequence. The mixture was then incubated at 37°C for 1hr. About 1.0 ml portion of the incubated mixture was mixed with 1.0 ml of 10% TCA and 1.0 ml of 0.5% TBA to develop the pink chromogen, measured at 532 nm. The hydroxyl radicals scavenging activity was calculated using the following equation:-

% Inhibition = [(A₀-A₁) / A₀× 100] Where, A₀ was the absorbance of the control (blank) and A₁ was the absorbance in the presence different concentrations of the extract

G. Total reduction capability²⁴

The Fe³⁺ reducing power of the extract was determined by the method of Oyaizu et al., with a slight modiﬁcation. Diﬀerent concentrations (250–2000 µg/mL) of extract (0.5 mL) were mixed with 0.5-mL Phosphate buﬀer (pH 6.6) and 0.5-mL 0.1% potassium hex-cyanoferrate, followed by incubation at 50°C in water bath for 20 min. After incubation, 0.5-mL 10% TCA was added to terminate the reaction. The upper portion of the solution (1 mL) was mixed with 1 mL of distilled water and 0.1 mL 0.01% FeCl₃ solution was added. The reaction mixture was left for 10 min at room temperature and the absorbance was measured at 700 nm against appropriate blank solution. All tests were performed three times. A higher absorbance of the reaction mixture indicated greater reducing power. Ascorbic acid was used as a positive control.

H. Scavenging Activity of Hydrogen Peroxide²⁵

This activity was determined according to a method suggested by B. Halliwell and group with minor changes. Aliquot of 50-mM H₂O₂ and various concentrations (0–2000 µg/mL) of samples were mixed (1: 1 v/v) and incubated for 30 minutes at room temperature. After incubation, 90μl of the H₂O₂-sample solution was mixed with 10 μl HPLC-grade methanol and 0.9-mL FOX reagent was added (previously prepared by mixing 9 volumes of 4.4-mM Ascorbic acid in HPLC -grade methanol with 1 volume of 1-mM xylenol orange and 2.56-mM ammonium ferrous sulfate in 0.25-M H₂SO₄). The reaction mixture was then vortexed and incubated at room temperature for 30 min. The absorbance of ferric-xylenol orange complex was measured at 560 nm. All tests were carried out three times and sodium pyruvate was used as the reference compound

Statistical analysis²⁶:

The experimental results were expressed as mean ± SEM of three replicates. Where applicable, the data were subjected to one way analysis of variance (ANOVA) and two way analysis of variance (ANOVA). All these analysis was done by Graph Pad Prism Software program (version 5). P values < 0.05 were regarded as significant.

RESULTS:

Yield of extraction:

Extractions of all three plants were carried out by using the solvent mixture of methanol (70%)-water (30%). The physical nature, color characteristic and percentage yield of each individual extracts are found as given in the Table-1

Table 1:- Yield of Extraction

Sr. No	Crude drug	Nature of Extract	Color	% Yield (w/w)
1	Ocimum canum	Semisolid	Greenish purple	28.35 %
2	Pongamia pinnata	Semisolid	Deep green	25.60 %

Table 2: Phytochemical analysis

Table 2a: Phytochemical analysis of the hydro-alcoholic extract of Pongamia pinnata leaves

Sl no.	Phyto Constituents	Methanol	Aqueous
1	Alkaloid	+ ++ +	+ - + +
2	Carbohydrate	+ - -	+ - -
3	Glycoside (cardiac glycoside)	+	+
4	Tanins and phenolics	+ + -	+ +
5	Protein and amino acid	- - -	- - -
6	Gum and mucilage	+++	+ + +
7	Flavones and flavonoids	++	+ +
8	Saponins	+	+
9	Steroids and sterols	+	+
10	Triterpinoids	+	+

+ = presence, – = absence.

Table 2b:- Phytochemical analysis of the hydro-alcoholic extract of Ocimum canum leaves

Sl no.	Phyto Constituents	Methanol	Aqueous
1	Alkaloid	+ ++ -	+ - + +
2	Carbohydrate	+ - -	+ - -
3	Glycoside (cardiac glycoside)	++	++
4	Tanins and phenolics	+ + -	+ +
5	Protein and amino acid	- - -	- - -
6	Gum and mucilage	- - -	+ + +
7	Flavones and flavonoids	++	+ +
8	Saponins	+	+
9	Steroids and sterols	+	+
10	Triterpinoids	+	+

+ = presence, – = absence.

Phytochemical screening of the hydro-alcoholic leaf extracts of Pongamia pinnata (Table 2a) revealed the presence of alkaloids, flavonoids, saponins, steroids and tannins whereas the hydro-alcoholic extracts of extracts Ocimum canum (Table 2b) leaves revealed the presence of carbohydrates, flavonoids and tannins. So the results obtained signify the presence of flavonoids in both the plants, which serves as a key antioxidant.

Antioxidant Activity:

DPPH scavenging activity

The proton radical scavenging action is known to be one of the various mechanisms for measuring antioxidant activity. The DPPH test provides information on their activity of the test compounds with a stable free radical. This assay determines the scavenging of stable radical species of DPPH by antioxidants. The degree of reduction in absorbance measurement by the hydro-alcoholic extracts is indicative of the radical scavenging (antioxidant) power of the plants (Table 3). The study showed that the hydro-alcoholic extracts of both the plants have the proton-donating ability and can serve as free radical inhibitors or scavenger, acting possibly as primary antioxidant. From figure-1 it is quite clear that the poly herbal mixture shows the maximum scavenging of stable radical species.

Fig 1:- Study on DPPH scavenging activity at 517 nm

Table 3:- Study on DPPH scavenging activity in Pongamia pinnata + Ocimum canum leaves

Concentration (µg/ml)	Ascorbic acid (% scavenging activity)	*P. pinnata* (% scavenging activity)	*O. canum* (% scavenging activity)	*P. pinnata + O. canum* (% scavenging activity)
0	0	0	0	0
250	90.2±0.004	43 ±0.005	46.8 ± 0.005	73.3 ± 0.004
500	91 ±0.009	68.7± 0.004	56.7 ± 0.003	76.9 ± 0.005
1000	92.4 ±0.005	71±0.003	72 ± 0.005	80.1 ± 0.003
2000	93±0.007	76±0.007	79.2 ± 0.004	81.3 ± 0.004

Values are mean ± SEM of three separate experiments; Statistical comparison has been done by student‘s t- test

Table 4 a:- Study on ABTS scavenging activity in Pongamia pinnata + Ocimum canum leaves

Concentration (µg/ml)	Ascorbic acid (% scavenging activity)	*P. pinnata* (% scavenging activity)	*O. canum* (% scavenging activity)	*P. pinnata + O. canum* (% scavenging activity)
0	0	0	0	0
250	68±0.014	62.3±0.013	52.65 ± 0.012	69.3 ± 0.014
500	73.4±0.016	80.2±0.015	70.8 ± 0.013	71.8 ± 0.013
1000	75±0.015	83.4±0.014	76.5 ± 0.014	72.7 ± 0.018
2000	79.2±0.017	84.5±0.014	86.5 ± 0.011	78.1 ± 0.016

Values are mean ± SEM of three separate experiments; Statistical comparison has been done by student‘s t- test

Fig 4:- Study on Superoxide ion scavenging activity at 560 nm

Table 5:- Study on Superoxide ion scavenging activity in Pongamia pinnata + Ocimum canum leaves

Concentration (µg/ml)	Ascorbic acid (% scavenging activity)	*P. pinnata* (% scavenging activity)	*O. canum* (% scavenging activity)	*P. pinnata + O. canum* (% scavenging activity)
0	0	0	0	0
250	68.4±0.024	68.1±0.029	62.9 ± 0.026	76.7 ± 0.025
500	72.7±0.022	70.9±0.028	68.2 ± 0.027	80.1 ± 0.026
1000	75.1±0.021	71.7±0.027	76.3 ± 0.028	83.9 ± 0.021
2000	80.5±0.027	73.1±0.026	81.7 ± 0.029	85.0 ± 0.020

Values are mean ± SEM of three separate experiments; Statistical comparison has been done by student‘s t- test

Table 6:-Study on Iron chelating activity in Pongamia pinnata + Ocimum canum leaves

Concentration (µg/ml)	Ascorbic acid (% scavenging activity)	*P. pinnata* (% scavenging activity)	*O.canum* (% scavenging activity)	*P. pinnata + O.canum* (% scavenging activity)
0	0	0	0	0
250	85.1±0.012	48.2±0.025	45.9 ± 0.010	81.3 ± 0.025
500	87.4±0.019	52.7±0.027	51.4 ± 0.021	84.1 ± 0.022
1000	88.1±0.018	56.8±0.024	62.9 ± 0.023	86.3 ± 0.020
2000	89.7±0.021	63.9±0.023	71.4 ± 0.026	87.2 ± 0.025

Values are mean ± SEM of three separate experiments; Statistical comparison has been done by student‘s t- test

Total reduction capability²⁵

Reducing power assay measures the electron-donating capacity of an antioxidant. The reduction of the ferric ion (Fe³⁺) to ferrous ion (Fe²⁺) is measured by the intensity of the resultant blue-green solution which absorbs at 700 nm, and an increased absorbance is indicative of higher reducing power. The reducing power of the extract increased progressively over the concentration range studied. Extract solutions at 1000µg/ ml had comparable reducing power to Ascorbic acid at 250 µg/ ml (Table-8). These findings suggest that the poly herbal hydro-alcoholic extract is capable of donating electrons, and could therefore react with free radicals or terminate chain reactions.

Scavenging Activity of Hydrogen Peroxide²⁵

The scavenging of hydrogen peroxide by the standard (ascorbic acid) and extract after incubation for 10 minutes increased with increased concentration. Poly herbal hydro-alcoholic extracts exhibited higher hydrogen peroxide scavenging activity than ascorbic acids and individual extracts at similar concentrations. While hydrogen peroxide itself is not very reactive, it can generate the highly reactive hydroxyl radical (OH) through the Fenton reaction (Equation 1).Thus, the scavenging of hydrogen peroxide is an important antioxidant defense mechanism.

Fe²⁺ + H²O² →Fe³⁺ +OH + OH-(Equation 1)

The decomposition of hydrogen peroxide to water involves the transfer of electrons as in Equation 2.

H₂O₂ + 2H+ + 2e- → 2H₂O (Equation 2)

The scavenging of hydrogen peroxide by phenolic compounds has been attributed to their electron-donating ability. Poly herbal extracts have high electron-donating abilities, and 76.31 ± 0.37 scavenging was achieved with concentrations of hydro-alcoholic extracts at 2000 µg/ ml. In comparison, the hydrogen peroxide scavenging activity of ascorbic acid at 2000 µg/ ml were found to be 98.79 ± 0.28 (Table-9)

DISCUSSION:

In plant materials, generally phenolic compounds and flavonoids are present which are responsible for antioxidant activity, which can be maximally extracted in the hydro-alcoholic solvent mixture. Antioxidant compounds like phenolic acids, polyphenols and flavonoids scavenge free radicals such as peroxide, hydroperoxide or lipid peroxyl and thus inhibit the oxidative mechanisms that lead to degenerative diseases. The use of herbal drugs as medicines for the treatment of a wide range of diseases can be traced back since ancient time’s i.e during the Vedic period in India²⁷. Being the outcome of therapeutic experiences of generations of practicing physicians of indigenous systems of medicine for over hundreds of years medicinal plants have played a key role in world health^{28, 29}. Hence, in spite of the great advances observed in modern medicine in recent decades, herbal medicines still make an important contribution to health care³⁰. Plants phenolics, in particular phenolic acid^31-33 tannins^{34, 35} and flavonoids³⁶ are known to be potent antioxidant and occur in vegetables, fruits, nuts, seeds, roots, barks and leaves.

The biological, pharmacological and medicinal properties of bioﬂavonoids and proanthocyanidins have been extensively reviewed^37,38. The structure of flavonoid consists of two hydroxy substituted aromatic rings joined by a three carbon link (a C6-C3-C configuration) which renders them hydrogen and electron donors. Thus, they are effective scavengers of free radicals, which are intermediate products of lipid peroxidation, and they slow down oxidation reactions. The antioxidant activity of flavonoids has been studied in lipid substrates, as well as in relation to human health, in particular with regards to retarding the aging of cells and protecting against certain illnesses such as cardiovascular or coronary disease and cancer³⁹. Pongamia pinnata, rich in flavonoids (Pongagallone A and B), is known to be a strong antioxidant, breaking up free radicals. The main phenolics reported in basil are phenolic acids and flavonol-glycosides^40,41. Nevadensin and salvigenin are the two flavonol-glycosides found in O. canum Sims, which exert their antioxidant activity.

Antioxidant activity was carried out by using eight in-vitro models. All the activities were checked and hence mixed in equal (1:1) proportions and were checked for maximum inhibition in the same concentration as that of the individual extracts. Thus it can be concluded that in equal-proportional mixture, the extract gave synergistic effect. Thus this extract mixture gave a promising result for antioxidant activity and in future can be subjected for treatment of disease where the basis of disease has oxidative stress mechanism.

ACKNOWLEDGEMENTS:

Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors/ editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed

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Concentration (µg/ml)	Ascorbic acid (% scavenging activity)	*P. pinnata* (% scavenging activity)	*O. canum* (% scavenging activity)	*P. pinnata* + O. canum (% scavenging activity)
0	0	0	0	0
250	56.2±0.04	52.4±0.02	56.4 ± 0.02	76.6 ± 0.02
500	57.8±0.02	59.1±0.01	68.2 ± 0.03	80.1 ± 0.08
1000	61.7±0.04	60.2±0.06	78.1 ± 0.01	84.6 ± 0.01
2000	64.9±0.01	63.1±0.05	86.2 ± 0.07	90.9 ± 0.06

Concentration (µg/ml)	Ascorbic acid (Absorbance)	*P. pinnata* (Absorbance)	*O. canum* (Absorbance)	*P. pinnata + O. canum* (Absorbance)
0	0	0	0	0
250	0.335 ± 0.002	0.0545 ± 0.001	0.0844 ± 0.001	0.0984 ± 0.001
500	0.4159 ± 0.004	0.1183 ± 0.002	0.1683 ± 0.002	0.2083 ± 0.002
1000	0.5679 ± 0.003	0.2082 ± 0.001	0.2582 ± 0.001	0.2982 ± 0.004
2000	0.7404 ± 0.003	0.3120 ± 0.001	0.3620 ± 0.001	0.4220 ± 0.001

Concentration (µg/ml)	Ascorbic acid (% scavenging activity)	P. pinnata (% scavenging activity)	O. canum (% scavenging activity)	P. pinnata + O. canum (% scavenging activity)
0	0	0	0	0
250	52.74 ± 0.22	25.70 ± 0.23	30.70 ± 0.23	37.70 ± 0.22
500	62.63 ± 0.34	37.27 ± 0.38	41.27 ± 0.38	48.92 ± 0.36
1000	78.57 ± 0.32	49.24 ± 0.28	58.54 ± 0.28	64.71 ± 0.25
2000	98.79 ± 0.28	62.19 ± 0.35	68.21 ± 0.35	76.31 ± 0.37