INTRODUCTION:

Antioxidants can be defined as molecules which interact with free radicals and terminate the chains before vital molecules are damaged. These compounds are synthesized in the body or obtained in the diet, the amount of protection provided by antioxidants therefore depends on its concentration, its reactivity towards the particular reactive oxygen species and status of antioxidants¹. Many plants contain substantial amounts of antioxidants, Vitamin C and E, carotenoids, tannins, flavonoids and thus can be utilized to scavenge the excess free radicals from the human body². Antioxidants prevent oxidative stress by free radical and ROS, and avoid the occurrence of disease, cancer and aging³.

During the process of oxygen utilization in a normal physiological and metabolic process, approximately 5% of oxygen gets univalently reduced to oxygen derived free radicals like superoxide, hydrogen peroxide, hydroxyl and nitric oxide radicals⁴. Excess production of these free radicals leads to cause cellular damage by reacting with various biomolecules such as membrane lipids, nucleic acid, proteins and enzymes⁵.

Ocimum basilicum L., commonly known as Sweet Basil, belongs to the family Lamiaceae. The term ‘Ocimum’ (from Greek ozo for smell) is appropriate for the genus since its various species are known for their peculiar strong odour. It is a small perennial, tropically growing shrub of Asian origin. It is popularly known as “Kali Tulsi” in Hindi, a widely grown plant of Hindus. Ocimum basilicum Linn. (Sweet basil in English) is an aromatic plant, nearly glabrous branching herb, 60-90 cm in height with hairy stems, branches green; leaves are 2.5-5 cm. long and 1.6-3.2 cm broad, elliptical, oblong obtuse. Flowers are in racemes 15-20 cm long in close whorls; fruits are ellipsoid nutlets, black and pitted^6,7,8. The present study compares the antioxidant activity of the chloroform and ethanolic extracts of leaves of Ocimum basilicum. Also the macroscopical, microscopical, physico-chemical standards which could be useful for the proper identification of the plant were studied.

MATERIALS AND METHODS:

Collection and authentication of plant material:

The leaves of Ocimum basilicum Linn. were collected from the surrounding area of Chennai and taxonomically identified by Botanist Dr. P. Jayaraman, Director, Plant Anatomy Research Centre, Chennai. A voucher specimen was deposited in the herbarium of the Department of Pharmacognosy, SRM University. Sections of leaves were studied for different microscopic characters and photographs of different magnifications of the sections were taken with Nikon Lab Photo 2 Microscopic unit. For normal observations, bright field was used. For the study of crystal, starch grains and lignified cells, polarized light was employed⁹. Physiochemical standard were done according to WHO guidelines¹⁰. The shade dried and powdered leaves were subjected to continuous hot extraction in Soxhlet apparatus using chloroform and ethanol. The extract was filtered and concentrated under vacuum. The concentrated extracts were used for further study. The drug powder, chloroform and ethanol extract was subjected to preliminary phytochemical screening for the detection of various phytoconstituents such as alkaloids, glycosides, tannins, phenolic compounds, flavonoids, steroids, saponins, proteins, amino acids, carbohydrates and triterpenoids¹¹.

In-vitro antioxidant activity:

Several concentrations ranging from 100-500 µg/ml of the chloroform and ethanolic extract were tested for the antioxidant activity using nitric oxide radical scavenging method.

Nitric oxide radical scavenging assay:

Nitric oxide was generated from sodium nitroprusside and measured by Griess reaction. Sodium nitroprusside (5mM) in standard phosphate buffer saline solution (0.025M, pH-7.4) was incubated with different concentrations of chloroform and ethanolic extract (100-500µg/ml) dissolved in phosphate buffer saline (0.025M, pH-7.4) and the tubes were incubated at 25^oC for 5 hr. Control experiments without the test compounds but equivalent amounts of buffer were conducted in an identical manner. After 5 hr, 0.5ml of incubation solution was removed and diluted with 0.5ml of Griess reagent (1% sulphanilamide, 2% ortho-phosphoric acid and 0.1% naphthyl ethylene diamine dihydrochloride). The absorbance of the chromophore formed during diazotization of nitrite with sulphanilamide and its subsequent coupling with naphthyl ethylene diamine was read at 546nm¹². All determinations were performed in 6 replicates.

Statistical analysis:

Level of significance of all the parameters was expressed as the arithmetic mean ± SEM and was analyzed by one-way analysis of variance (ANOVA), followed by Bonferroni post comparison test. P value less than 0.05 (P < 0.05) was the critical criterion for statistical significance.

RESULTS AND DISCUSSION:

Microscopic evaluation:

The leaf is dorsiventral and the midrib is prominent. It consists of short wide adaxial hump and wide and fairly thick abaxial part (Fig.2). The midrib is 720μm thick; the adaxial hump is 300μm wide; the abaxial part is 800 μm wide. The epidermal layer of the midrib is single-layered the cells being small elliptical and thin walled. The ground tissue of the midrib is parenchymatous, the cells being angular, thin walled and compact. The vascular strand is single, horizontally widened and collateral it includes several thin parallel lines of xylem elements; The lateral vein is similar to the midrib in general structure. The lateral vein has prominent adaxial hump, wide abaxial part and parenchymatous ground tissue. The vascular bundle is single, collateral with limited number of xylem strands.

Figure 1: Entire Plant of Ocimum basilicum

Figure 2: T.S of leaf through midrib La- Lamina; AdG – Adaxial; MR- Midrib; GT –Ground tissue

Lamina:

The lamina is bifacial with well differentiated adaxial part and abaxial part. It is amphistomatic (stomata occur on both upper and lower sides).The lamina is 170μm thick. The adaxial epidermis is slightly thicker and the cells are narrow and cyclindrical. The abaxial epidermis is thin; the cells are narrow and oblong. The mesophyll tissue consists of a single adaxial band of cylindrical, less compact palisade cells. Below the palisade zone is the spongy parenchyma where the cells are small and form thin filaments around the air-chambers. (Fig.3)

Figure 3: T.S of Lamina PM – Palisade mesophyll; SM- Spongy mesophyll; AdE – Adaxial epidermis; AbE – Abaxial epidermis

Figure 4: Paradermal section of the Lamina (40X) SC-Subsidiary cells; St-Stomata; AW- Anticlinal walls; EC-Epidermal Cells

Physicochemical Parameters:

The physico chemical constants are important parameters for detecting adulteration or improper handling of drugs. Various physicochemical parameters ash value and loss on drying were determined. The results were summarized in Table 1.

Table 1: Physico-chemical analysis of Ocimum basilicum leaves

S. No	Parameters	Values (%w/w)
1	Ash values
	Total ash	10
	Water soluble ash	12
	Acid insoluble ash	1.28
2	Loss on drying	10

Preliminary Phytochemical Screening:

The chloroform shows the presence of alkaloids, steroids, tannins and triterpenoids. Ethanol extract showed the presence of carbohydrates, flavonoids, glycosides, phenolic compounds, tannins and triterpenoids (Table.2).

Table 2: Preliminary phytochemical screening of Ocimum basilicum leaves

S. No	Phytoconstituents	Drug Powder	*Chloroform extract of Ocimum basilicum* leaves**	*Ethanolic extract of Ocimum basilicum* leaves**
1.	Carbohydrates	Present	Absent	Present
2.	Flavonoids	Present	Absent	Present
3.	Glycosides	Present	Absent	Present
4.	Alkaloids	Present	Present	Absent
5.	Phenolic compounds	Absent	Absent	Present
6.	Tannins	Present	Present	Present
7.	Triterpenoids	Present	Present	Present
8.	Saponins	Absent	Absent	Absent
9.	Proteins	Absent	Absent	Absent
10.	Lipids	Absent	Absent	Absent
11.	Steroids	Present	Present	Absent

Anti-oxidant activity:

Inhibition of Nitric oxide radical:

Nitric oxide (NO), physiologically being a potent pleiotropic mediator and in pathological conditions a diffusible free radical, reacts with superoxide anion and form a potentially cytotoxic molecule, the 'peroxynitrite (ONOO‐)'. Its protonated form, peroxynitrous acid (ONOOH), is a strong oxidant¹². The major damage is due to the nitration or hydroxyl‐ lation of aromatic compounds, particularly tyrosine. Under physiological conditions, peroxynitrite also forms an adduct with carbondioxide dissolved in body fluid and responsible for the oxidative damage of proteins in living system^10,11.

Reactive Oxygen species (ROS) generated endogenously or exogenously are associated with the pathogenesis of various diseases such as atherosclerosis, diabetes, cancer, arthritis and aging process. Thus antioxidants which can scavenge ROS are expected to improve these disorders¹². Oxidative stress occurs when the production of harmful molecules called free radicals is beyond the protective capability of the antioxidant defenses¹³.

The reactive oxygen species (ROS) and the reactive nitrogen species (RNS) are at the center of every physiological disease in plants and animals. The mitochondrial electron transport system (ETS) is a relatively well-investigated source of reactive oxygen species. Therefore, the presence of antioxidants in plants and specie help saved biological systems from diseases associated with free radicals¹⁴.

Table 4: Nitrite radical scavenging activity of Ascorbic acid, Chloroform and Ethanolic extracts of Ocimum basilicum Linn.,

Concentration (µg/ml)	Percentage inhibition (%)
Concentration (µg/ml)	Standard	*Chloroform extract of Ocimum basilicum* leaves**	*Ethanolic extract of Ocimum basilicum* leaves**
100	53.69± 0.216	1.377± 0.160^***	8.890± 0.359^***
200	53.55± 0.157	3.507± 0.141^***	19.58± 0.202^***
300	61.38± 0.282	50.66± 0.151^***	65.85± 0.147^***
400	69.53± 0.190	67.31± 0.296^***	83.45± 0.210^***
500	97.82± 0.174	84.63± 0.203^***	96.70± 0.698^***
IC₅₀	247.83	300.84	255.45

Values are expressed as Mean ± SEM, n=3, ^***P<0.001 vs. Standard, ^**P<0.01 vs. Standard, ^*P<0.05 vs. Standard; one way ANOVA followed by Bonferroni post comparison test all pairs of column.

CONCLUSION:

ll antioxidants have a chemical element referred to as a “redox” potential, which is the measurement of their ability to be oxidized. Considering the fact that the redox equilibrium is important to the body’s coping mechanism, it follows that antioxidants can influence many health conditions¹⁵. In the present investigations, the pharmacognostical and physicochemical characteristics of the leaves of Ocimum basilicum were studied. Phytochemical parmeters will help in controlling the standards and quality of the raw material of leaves of Ocimum basilicum. The study adds a systematic comparative record on the relative free radical scavenging activity in chloroform and ethanolic extract of the leaves of Ocimum basilicum. Our findings suggest that both the extracts have the property to counteract the effect of NO formation in turn may be of considerable interest in combating the ill effects of excessive NO generation. Also the ethanolic extract showed an edge better activity than chloroform extract even at very low concentrations which can be attributed to the flavonoid and phenolic compounds present in the extract.

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Received on 21.07.2017 Modified on 11.09.2017

Res. J. Pharmacognosy and Phytochem. 2017; 9(4): 215-218.

DOI: 10.5958/0975-4385.2017.00039.5