Comparative Pharmacognostical, Phytochemical and Biological Evaluation of Five Ocimum Species

Medicinal properties of Ocimum species belongs to mint or lamiaceae family are known for thousand years to various civilizations of the world1-4 through Ayurveda, Siddha and Unani systems of medicine.5-9 In the Indian subcontinent, in Ayurveda and Indian mythology, it is commonly called as Tulsi means “matchless one” or “incomparable one” and considered as a sacred plant representing Holy Hindu Laxmi Goddess.10-12


Macroscopic evaluation
Macroscopy of whole plant was studied by observing the organoleptic characters such as color, odor, size, shape, taste and special features including touch and texture etc. Organoleptic evaluations can be done by means of organs of special sense which includes the above parameters and thereby define some specific characteristics of the material which can be considered as a first step towards establishment of identity. [25][26]

Microscopic evaluation
Microscopic evaluation is very helpful in the initial identification of herbs as well as in detection of adulteration by characteristics tissue features. Every plant possesses a characteristics tissue structure which can be demonstrated through study of tissue arrangement, cell walls and configurations, when properly mountain in stains, reagents and media. Thus it determines the size, shape, and relative structure of different cell Microscopical parameters

Powder microscopy evaluation
The powder of respective plant was used for powder microscopy study. Plain powder and powder treated with specific reagents like phloroglucinol -hydrochloric acid, iodine solution and Sudan red solution were spread as a thin layer on separate glass sides and observed under microscope. 26

Physicochemical evaluation
Ash values, extractive values and loss on drying determined as per standard procedures mentioned in Indian pharmacopoeia 2010. [26][27] These physicochemical standards are helpful in comparative determination quality and purity of the powder of plants of Ocimum genus.

Preliminary phytochemical evaluation
Preliminary phytochemical evaluations is the step to identify different classes of constituent that are primary constituents like carbohydrate, proteins, and lipids or secondary metabolite like glycosides, alkaloids, volatile oil, tannins etc. of great. The compounds that are responsible for medicinal efficacy are usually secondary metabolite. Hence plant material is subjected to preliminary phytochemical screening 26 for detection of various chemical constituents.

Extraction of essential oil
Extraction of oil was carried out by hydro distillation technique using Clevenger apparatus [26][27] and stored in dark glass vial in a refrigerator until further testing.

Chromatographic evaluation
Comparative TLC using pre-coated silica gel GF254plate as stationary phase, toluene: ethyl acetate (93:7) as mobile phase and Anisaldehydesulphuric acid as spraying reagent. Sprayed plate heated on hot plate at 110°C till color developed and intensified. 28

Antioxidant activity
Free radical generation due to oxidative stress is one of the major causes of many diseases in human body. Ocimum species are known to be a very good free radical scavenger hence it is decided to know comparative antioxidant potential of selected Ocimum species. DPPH (1, 1-diphenyl-2-picryl-hydrazyl) is a stable free radical and methanolic solution of it is used to evaluate the anti oxidant activity of several natural compounds. To 1ml of DPPH solution, 3ml of oil sample added. The same reaction mixture without sample but equivalent amount of standard phosphate buffer served as control. Well mixed solution allowed to stand at room temperature for 30 min. Absorbance of reaction mixture was measured at 517 nm. Percentage scavenging activity at different concentrations was calculated by using formula: % scavenging activity = 1-absorbance of test/ absorbance of control x 100. 26

Antibacterial activity
Pathogenic bacteria have always been considered as a major cause of morbidity and mortality in humans. Even though pharmaceutical companies have produced a number of new antibacterial in the last years, antimicrobial resistance has now become a global concern. The global emergence of multi-drug resistant bacteria is increasingly limiting the effectiveness of current drugs and significantly causing treatment failure. In vitro antibacterial potential of essential oils of selected Ocimum species against Escherichia coli was determined by agar well method using nutrient agar media. At the end of incubation, zone of inhibition formed measured in millimeter. Gentamicin is used as standard. [29][30] Antifungal activity In vitro antifungal potential of essential oils of selected Ocimum species using YEPD agar media against fungi Candida albicans was determined by using agar well method. All steps are same as procedure given in antibacterial activity, only the incubation period for antifungal activity was for 48 hours. At the end of incubation, zone of inhibition formed measured in millimeter. Fluconazole was used as standard. [29][30]

RESULT AND DISCUSSION
Medicinal use of Ocimum species is abundantly increasing due to its immunomodulator and antioxidant potential. It is commonly used in many marketed or even in homemade herbal tea formulas. There are about 150 species of Ocimum in the world and out of that 68 species are found in India. Comparative pharmacognostic study of these Ocimum species is unspecified. Hence, present research work generates comparative pharmacognostic data of selected five species of ocimum found in Vidarbha region of Maharashtra state.
Total five species of Ocimum i.e. Ocimum sanctum Linn, Ocimum americanum Linn, Ocimum basilicum Linn, Ocimum gratissimum Linn, Ocimum kilimandscharicum guerke are evaluated comparatively for their morphological, microscopical, physiochemical, and phytochemical parameters and also evaluated for their antioxidant and antimicrobial activity by in vitro methods. The whole plants are shade dried and powdered using grinding mill. The powder was stored in airtight container.
Comparative morphological evaluations of all selected five Ocimum species are summarized in Table 1. Microscopy of leaves and stem as well as powder of whole plant of all selected five Ocimum species studied in detail and comparisons are summarised in Table 2 and Table  3.
Microscopical examination of leaf O. americanum shows isobilateral lamina covered with cuticle; glandular trichomes with multi-cellular head and multicellular warty covering trichomes. Mid rib with arc shaped vascular bundle consisting of xylem and phloem, three to four layers of collenchymatous tissue present on upper side of vascular bundle whereas stem part shows cork, vascular bundle containing xylem & phloem, spongy tissue, collenchymatous cells, pith. Powder microscopy shows reticulate xylem vessel, stone cell, epidermal cell, collenchymas, cork cell.       Table 6.
For the extraction of essential oils, shaded dried powder of whole plant was used. Essential oil was extracted by using Clevenger apparatus and oil was stored in air tight amber colored bottle.
Comparative chromatographic evaluation (Table 7 and Figure 1) was carried out for methanolic extract and essential oils of all five species using silica gel GF 254 as a stationary phase and toluene: ethyl acetate (93:7) as mobile phase.