BACKGROUND
Ocimum sanctum L., commonly known as holy basil, is a plant known for its medicinal values, of the genus Ocimum belonging to the family Lamiaceae Ocimum is very important for its therapeutic potential among the most importantaromatic herbs for their enormous medicinal properties, such as anticancerous, antidiabetic, spasmolytic, carminative, cardioprotective, anthelmintic, and diaphoretic actions. Ocimum sanctum possesses various biological activities such as analgesic, antipyretic, antidiabetic, hepatoprotective, hypolipidemic, immunomodulatory, and anti-inflammatory. It is observed that some of its phytoconstituents such as eugenol, linoleic acid, luteolin, and β-sitosterol prevent skin, liver, oral, and lung cancers through increasing the anti-oxidant activity, inducing apoptosis, altering the gene expression, and inhibiting metastasis[11 Ocimum sanctum leaves showed inhibition of the growth of tumour cells[10 Ocimum sanctum would be an effective medicine so far in inhibiting all kinds of cancer[1 , 2 , 3 , 4
Ocimum sanctum is used as traditional medicine and possesses various biological activities because of the active components present in the plant such as eugenol, linoleic acid, oleic acid, rosmarinic acid, ocimarin, isorientin, orientin, aesculectin, aesculin, chlorogenic acid, galuteolin, gallic acid, citronellal, camphene, sabinene, dimethylbenzene, ethylbenzene, vitamin C, and calcium[5 , 6 , 7
MATERIAL & METHODS
Collection, identification, and authentication of selected plants
A collection of all three types of tulsi leaves named Rama Tulsi (Ocimum sanctum), Krishna Tulsi (Ocimum canum) and Vana Tulsi (Ocimum gratissimum) of the genus Ocimum were collected for one month from the local area of Haryana, India. Identification of this was confirmed by Dr. RS Jayasomu, Head, Raw material Herbarium and Museum Division (RHMD), NISCAIR, New Delhi, India where a voucher sample (Ref. No. NISCAIR/RHMD/Consult/-2016/3000-27-2) has been deposited. For further studies in a pharmacognostical manner, phytochemical analysis, and extraction, leaves were collected, shade dried, and converted into fine powdered form.
Pharmacognostical study
The pharmacognostic study has been done by performing morphological and microscopic analysis of Leaf as per WHO guidelines.
Microscopic studies
Transverse section (TS) of leaf and powder characteristics were identified with (Phloroglucinol + HCL) reagents such as chloral hydrate and glycerine to study the cells, fibres, xylem vessels, starch grains, and calcium oxalate crystals. A permanent slide of TS of Leaf was prepared to observe the presence and arrangement of cellular structures as per the procedure of Johansen[8
Experimental design for GC-MS
GC-MS analysis of the Rama Tulsi (Ocimum sanctum), Krishna Tulsi (Ocimum canum), and Vana Tulsi (Ocimum gratissimum) essential oil was performed using the below-given instruments information.
Thermo Trace 1300GC coupled with Thermo TSQ 800 Triple Quadrupole MS.
For GC - THERMO TRACE 1300 GC
For MS - THERMO TSQ 8000
Software used: XCalibur 2.2SP1 with Foundation 2.0SP1
Column: BP 5MS (30m X 0.25mm, 0.25μm)
Column Makeup: 5% Phenyl Polysilphenylene- siloxane
Injector: S (Split)
Injection volume:2μL
Split Ratio: 20:1
Injector temp: 250°C
MS transfer line temp:230°C
Ion source temp: 230°C
Mass Range:40-700
Carrier Flow:1.0ml/min
Oven Program:
Initial Temp: 50°C Hold time: 1.0 min
Temp 1: 220°C Hold Time: 5.0 min
Rate: 5°C/min
Detector: MS TSQ 8000
Library used: NIST 2.0
Microscopical evaluation
The powder microscopy of selected varieties of Tulsi such as Rama Tulsi (Ocimum sanctum ), Krishna Tulsi (Ocimum canum ), and Vana Tulsi (Ocimum gratissimum ) was done and evaluated for the characterization of the drug through pictures in their respective sequence. These pictures are critically evaluated with specific microscopic features (Fig. 1 ).
Figure 1: Powder Microscopy of Rama Tulsi (Ocimum sanctum) .
Krishna Tulsi (Ocimum canum) and Vana Tulsi (Ocimum gratissimum) were done and evaluated for the characterization of the drug through pictures in their respective sequence. These pictures are critically evaluated with specific microscopic features. For this, Freehand sections of the plant material were stained with Phloroglucinol and HCl reagents such as chloral hydrate and glycerine to study the cells, bre, xylem vessels, starch grains, and calcium oxalate crystals. A permanent slide of TS of Leaf was prepared to observe the presence and arrangement of cellular structures, the representative figures were taken with the help of a microscopic image camera (Fig. 2 ).
Figure 2: Powder Microscopy of Krishna Tulsi (Ocimum canum).
Krishna Tulsi (Ocimum canum ) and Vana Tulsi (Ocimum gratissimum) were done and evaluated for the characterization of the drug through the below-given pictures in their respective sequence. These pictures are critically evaluated with specific microscopic features. For this, Freehand sections of the plant material were stained with Phloroglucinol and HCl reagents such as chloral hydrate and glycerine to study the cells, bre, xylem vessels, starch grains, and calcium oxalate crystals. A permanent slide of TS of Leaf was prepared to observe the presence and arrangement of cellular structures, the representative figures were taken with the help of a microscopic image camera (Fig. 3 ).
Figure 3: Powder Microscopy of Vana Tulsi (Ocimum gratissimum ).
Characterization of Ocimum
Gas Chromatography-Mass Spectrometry analysis
A Perkin-Elmer gas chromatograph (model 8700), with a flame ionization detector (FID), was used for the chemical analysis of the Rama Tulsi (O. sanctum), Krishna Tulsi (O. canum) and Vana Tulsi (O. gratissimum) essential oil. The temperatures of the injector and detector were set at 220°C and 290°C, respectively. The column thermostat temperature was started from 80°C and raised to 220°C at the rate of 4°C min-1 , whereas initial and final temperatures were held for 3 and 10 min, respectively. The carrier gas was helium with a flow of 1.5 mL min-1 . A sample of 1.0 μL was injected (split ratio 100:1). For quantification purposes a built-in data-handling program of the equipment (Perkin-Elmer) was used. The essential oil composition was reported as a relative percentage of the total peak area[12 , 13 , 14 Fig. 4 , 5 & 6 ) (Table 1 , 2 & 3 ).
Table 1: Chemical Composition of volatile oil extract from O. gratissimum analyzed by GC-MS
Table 2: Chemical Composition of volatile oil extract from O. sanctum analyzed by GC-MS
Table 3: Chemical Composition of volatile oil extract from O. canum analyzed by GC-MS
Figure 4: Characterization of Ocimum gratissimum.
Figure 5: Characterization of Ocimum sanctum.
Figure 6: Characterization of Ocimum canum.
Ethical statement: Not applicable
RESULTS
The hydro-distillation of essential oil from leaf extract of Ocimum sanctum L. yielded pale yellow aromatic oil. GC-MS chromatogram of the Ocimum sanctum, Ocimum canum, and Ocimum gratissimum oil showed major peaks and has been identified after comparison of the mass spectra with the NIST library, indicating the presence of three phytocomponents. The GC-MS study suggested that anethole which is well reported antimicrobial compound is more in O. canum (2.66%) in comparison to O. sanctum (1,28%) but absent in O. gratissimum. The results indicated that the antimicrobial activity is more in O. canum due to the presence high amount of anethole in comparison to O. gratissimum, and O. sanctum (Table 4 ).
Table 4: Morphological Analysis
CONCLUSION
The summarized information has been focused on the microscopic character of O. canum, with the characteristic GC-MS analysis of the extracts[9 Ocimum plant. The retention time of each chemical constituent is reported for future identification of the plant and its variant. In the future, it will be a source of literature to identify variant plant species for the quality production of the plant. Ocimum sanctum L. and further experiments are required for better exploitation of essential oil for its commercial purposes.
ABBREVIATIONS
OS = Ocimum sanctum
O. = Ocimum genus
COX-1 = Cyclooxygenase-1
COX-2 = Cyclooxygenase-2
VEGF = Vascular endothelium growth factor
WHO = World Health Organization
COPD = Chronic obstructive pulmonary disease),
IBD = Inflammatory bowel syndrome
DMBA= 7, 12- dimethylbenz(a)anthracene
VEGFR-3 = Vascular Endothelial Growth Factor Receptor 3
VLA-4 = Very Late Antigen-4
Conflict of interest:
None
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