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 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, where a voucher sample (Ref.No. NISCAIR/RHMD/Consult/-2016/3000-27-2) has been deposited. For further studies in Pharmacognostical manner, Phytochemical analysis and Extraction, leaves were collected, shade dried and converted them into fine powdered form
Pharmacognostical Study
The pharmacognostical 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 (Phlorogucinol + HCL) reagents such as chloral hydrate and glycerine to study the cells, fibre, xylem vessels, starch grains, and calcium oxalate crystals. Permanent slide of TS of Leaf was prepared to observe the presence and arrangement of cellular structures as per the procedure of Johansen[8] and the representative figures were taken with the help of microscopic image camera.
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 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:2.0µL
- 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) were done and evaluated for the characterization of the drug through below given pictures in their respective sequence. These pictures are critically evaluated with the specific microscopic features.
a) Characterization of OG
Gas Chromatography-Mass Spectrometry analysis
A Perkin-Elmer gas chromatograph (model 8700), with 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 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 area12-14.
Table 1
Chemical Composition of volatile oil extract from O. gratissimum analyzed by GC-MS.
RT
|
Compound Name
|
Probability
|
Peak Area
|
Area %
|
Molecular Weight
|
10.75
|
1,6-Octadien-3-ol, 3,7-dimethyl-
|
70.62
|
264140772.25
|
1.98
|
154
|
10.75
|
Linalyl acetate
|
5.63
|
264140772.25
|
0.36
|
196
|
12.36
|
Linalyl isobutyrate
|
1.68
|
199242845.32
|
1.50
|
224
|
13.49
|
1,5-Dimethyl-1-vinyl-4-hexenyl butyrate
|
1.95
|
200305251.13
|
1.50
|
224
|
15.17
|
Estragole
|
23.88
|
1042348110.23
|
7.38
|
148
|
17.97
|
2-Hexyl-1-octanol
|
3.08
|
27009602.76
|
0.20
|
214
|
b) Characterization of OS
Table 2
Chemical Composition of volatile oil extract from O. Sanctum analyzed by GC-MS.
RT
|
Compound Name
|
Probability
|
Peak Area
|
Area %
|
Molecular Weight
|
21.24
|
1-Dodecanol, 2-octyl-
|
3.46
|
136866142.39
|
0.54
|
298
|
21.24
|
1-Decanol, 2-octyl-
|
2.95
|
136866142.39
|
0.54
|
270
|
22.35
|
2-methyltetracosane
|
3.26
|
19645383.15
|
0.08
|
352
|
24.09
|
Tetracontane, 3,5,24-trimethyl-
|
4.27
|
358800273.33
|
1.41
|
604
|
26.82
|
Octatriacontyl pentafluoropropionate
|
2.09
|
29166462.68
|
0.11
|
696
|
38.29
|
Sulfurous acid, butyl octadecyl ester
|
4.07
|
1702077568.54
|
6.70
|
390
|
c) Characterization of O C
Table 3
Chemical Composition of volatile oil extract from O. Canum analyzed by GC-MS.
RT
|
Compound Name
|
Probability
|
Peak Area
|
Area %
|
Molecular Weight
|
13.47
|
1,6-Octadien-3-ol, 3,7-dimethyl
|
57.31
|
1415878189.55
|
4.08
|
154
|
13.47
|
Linalyl isobutyrate
|
1.82
|
1415878189.55
|
4.08
|
224
|
16.77
|
Estragole
|
43.16
|
10188160501.52
|
29.39
|
148
|
16.77
|
Benzene, (1-propynylthio)-
|
0.91
|
10188160501.52
|
29.39
|
148
|
18.68
|
10-Methylnonadecane
|
18.24
|
57180203.88
|
0.16
|
282
|
18.68
|
Sulfurous acid, 2-propyl undecyl ester
|
4.77
|
57180203.88
|
0.16
|
278
|
19.52
|
2-Hexyl-1-octanol
|
8.03
|
155688172.07
|
0.45
|
214
|
20.07
|
1-Octadecyne
|
5.13
|
55019667.38
|
0.16
|
250
|
20.07
|
1-Heptadecyne
|
5.34
|
55019667.38
|
0.16
|
236
|
22.07
24.54
|
Oxalic acid, cyclobutyl octadecyl ester
|
6.49
|
223103118.93
|
0.64
|
396
|
25.50
|
Levomenol
|
24.39
|
231862815.33
|
0.67
|
222
|