Bioactive Compounds Extraction and Analysis of Ginger: A Study of Zingiber officinale

Fig.1 : Pharmacological Action of Ginger

SOXHLET EXTRACTION :

Numerous cutting-edge techniques, including rapid solvent extraction, Soxhlet extraction, supercritical fluid extraction, ultrasound-assisted extraction, and microwave-assisted extraction, have been developed to extract nutraceuticals from plants. These techniques seek to enhance extraction yield, decrease solvent usage, shorten extraction timeframes, and improve extract quality^.(10)Soxhlet extraction is a type of atmospheric liquid extraction in which specific compounds are extracted using solvents at low pressures (outside pressure) at boiling temperatures.⁽¹¹⁾  In 1879, Franz von Soxhlet created a particular kind of laboratory apparatus called the Soxhlet extractor. Its original purpose was to remove a lipid from a solid. Soxhlet extraction is usually used when the contaminant is insoluble and the desired molecule is poorly soluble in a solvent. It efficiently recycles a little amount of solvent to dissolve a larger amount of material, allowing for unsupervised and uncontrolled operation.^{(12, 13)}

Assembly:

• The source material containing the compound to be extracted is placed inside the thimble.
• The thimble is loaded into the main chamber of the Soxhlet extractor.
• The extraction solvent to be used is placed in a distillation flask.
• The flask is placed on the heating element.
• The Soxhlet extractor is placed atop the flask.
• A reflux condenser is placed atop the extractor.

                                                          (A)                                                             (B)

Fig.2: (A) Assembly of soxhlet apparatus For extraction. (B)Ginger powder kept inside thimble filter suspended inside Ethanol solution.

GAS CHROMATOGRAPHY – MASS SPECTRUM ANALYSIS :

For the analysis of unknown plant components, gas chromatography-mass spectrometry (GC-MS) is a crucial instrument.⁽¹⁴⁾ Gas Chromatography–Mass Spectrometry (GC-MS) is a hyphenated analytical technique that combines the separation and detection powers of gas-liquid chromatography and mass spectrometry to identify different compounds in a test sample. GC is used to distinguish the volatile and thermally stable alternatives in a sample, whereas GC-MS breaks apart the analyte to be identified based on its mass. A mass spectrometer is added to it to produce GC-MS/MS. Performance is better in single and triple quadrupole modes.

Working of GC-MS:

The size of the column and the phase characteristics (e.g., 5% phenyl polysiloxane) determine which capillary column is utilized in the gas chromatograph. Because different molecules in a mixture have different chemical characteristics and varying affinities for the stationary phase of the column, the molecules will separate as the sample travels along the length of the column. Since the molecules are kept by the column and then elute from the column at different times, the mass spectrometer downstream can independently collect, ionize, accelerate, and detect the ionized molecules. The mass spectrometer uses each molecule's mass-to-charge ratio to identify the ionized fragments that are produced. A description of its tools and capabilities is given below:

Fig .3 :Gas Chromatography Mass Spectroscopy of Shimadzu , Japan.

EXPERIMENTAL DESIGN:

1. Collection and preparation of plant material:

The ginger rhizomes were ground with a sanitized pestle and mortar after being allowed to dry at room temperature for ten to fifteen days. A sieve was then used to reduce the size of the powdered plant. After being placed in an airtight container to prevent the effects of humidity, the fine powder was kept at room temperature.

2. Preparation of Sample:

In the Soxhlet Extractor, the ginger sample is put in a porous thimble, which is often constructed of filter paper. Under the extractor, a flask containing hot methanol is used. Methanol rises through a tube to the condenser after evaporating. The sample in the thimble is hit by the condenser's condensation of the methanol vapor. Methanol dissolves the required components in the sample, filling the extractor with the solvent-solute mixture. Once the solvent level reaches the siphon tube, a new extraction cycle is started by sucking the solvent-solute mixture back into the flask. The desired compounds are extracted from the sample by repeating this cycle of boiling, condensation, and siphoning.

RESULTS AND DISCUSSION

GAS CHROMATOGRAPHY- MASS SPECTRUM ANALYSIS

Sr. No.	Phytochemical Compounds	RT (Min.)	Formula	Molecular Weight	Chemical Structure
1	1-Propanol, 2-ethoxy-	4.611	C5H12O2	104
2	Propane, 1,1,3-triethoxy-	11.751	C9H20O3	176
3	endo-Borneol	14.446	C10H18O	154
4	L-.alpha.-Terpineol	15.147	C10H18O	154
5	Gingerol	18.799	C17H26O4	294
6	Tetradecane	20.696	C14H30	198
7	(1R,4R,4aS,8aR)-4,7-Dimethyl-1-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,8a-octahydronaphthalene	22.440	C15H24	204
8	1-Methyl-4-(6-methylhept-5-en-2-yl)cyclohexa-1,3-diene	22.844	C15H24	204
9	Benzene, 1-(1,5-dimethyl-4-hexenyl)-4-methyl-	22.928	C15H22	202
10	(1S,5S)-2-Methyl-5-((R)-6-methylhept-5-en-2-yl)bicyclo[3.1.0]hex-2-ene	23.235	C15H24	204
11	1H-Cyclopropa[a]naphthalene, decahydro-1,1,3a-trimethyl-7-methylene-, [1aS-(1a.alpha.,3a.alpha.,7a.beta.,7b.alpha.)]-	23.325	C15H24	204
12	Naphthalene, 1,2,3,4,4a,5,6,8a-octahydro-7-methyl-4-methylene-1-(1-methylethyl)-, (1.alpha.,4a.beta.,8a.alpha.)-	23.407	C15H24	204
13	.alpha.-Farnesene	23.409	C15H24	204
14	beta.-Bisabolene	23.560	C15H24	204
15	Cyclohexene, 3-(1,5-dimethyl-4-hexenyl)-6-methylene-, [S-(R*,S*)]-	23.944	C15H24	204
16	(E)-1-Methyl-4-(6-methylhept-5-en-2-ylidene) cyclohex-1-ene	24.140	C15H24	204
17	Nerolidyl acetate	24.834	C17H28O2	264
18	Hexadecane	25.556	C16H34	226
19	Guaiol	26.434	C15H26O	222
20	Butan-2-one, 4-(3-hydroxy-2-methoxyphenyl)-	26.894	C11H14O3	194
21	3-Cyclohexen-1-ol, 1-(1,5-dimethyl-4-hexenyl)-4-methyl-	27.356	C15H26O	222
22	6,10-Dodecadien-1-yn-3-ol, 3,7,11-trimethyl-	27.938	C15H24O	220
23	Phenol, 5-(1,5-dimethyl-4-hexenyl) -2-methyl-, (R)-	29.210	C15H22O	218
24	2(1H)-Naphthalenone, 4a,5,6,7,8,8a-hexahydro-7.alpha.-isopropyl-4a.beta.,8a.beta.-dimethyl-	30.727	C15H24O	220
25	1H-3a,7-Methanoazulen-5-ol, octahydro-3,8,8-trimethyl-6-methylene-	32.287	C15H24O	220
26	n-Hexadecanoic acid	34.293	C16H32O2	256
27	Glutaric acid, myrtenyl 3-methylbut-2-en-1-yl ester	34.731	C20H30O4	334
28	9,12-Octadecadienoic acid (Z,Z)-	37.961	C18H32O2	280
29	9-Octadecenoic acid, (E)-	38.064	C18H34O2	282
30	Octadecanoic acid	38.501	C18H36O2	284
31	Caryophyllene oxide	39.512	C15H24O	220
32	(E)-1-(4-Hydroxy-3-methoxyphenyl)dec-3-en-5-one	39.784	C17H24O3	276
33	3-Decanone, 1-(4-hydroxy-3-methoxyphenyl)	39.948	C17H26O3	278
34	1-(4-Hydroxy-3-methoxyphenyl) dec-4-en-3-one	41.059	C17H24O3	276
35	1-(4-Hydroxy-3-methoxyphenyl)decane-3,5-dione	41.745	C17H24O4	292
36	5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-3-one	42.449	C19H30O4	322
37	5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-3-one	42.738	C17H26O4	294
38	1-(4-Hydroxy-3-methoxyphenyl) dodec-4-en-3-one	44.594	C19H28O3	304
39	(3R,5S)-1-(4-Hydroxy-3-methoxyphenyl) decane-3,5-diyl diacetate	44.947	C21H32O6	380
40	(E)-1-(4-hydroxy-3-methoxyphenyl) dec-1-ene-3,5-dione	46.373	C17H22O4	290
41	5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl) dodecan-3-one	46.489	C19H30O4	322
42	2-Methylhexacosane	47.980	C27H56	380
43	1-(4-Hydroxy-3-methoxyphenyl) tetradec-4-en-3-one	48.519	C21H32O3	332
44	1-(4-Hydroxy-3-methoxyphenyl) tetradecane-3,5-dione	49.325	C21H32O4	348
45	Tetrapentacontane	49.935	C54H110	758

Fig.4: chromatogram of zingiber officinale by GC-MS

Fig. 1: Structure of 1-Propanol, 2-ethoxy- present in Zingiber officinale with retention time=4.611 using GC-MS analysis

Fig. 2: Structure of Propane, 1,1,3-triethoxy- present in Zingiber officinale with retention time=11.751using GC-MS analysis

Fig. 3: Structure of endo-Borneol present in Zingiber officinale with retention time=14.446 using GC-MS analysis

Fig. 4: Structure of L-.alpha.-Terpineol present in Zingiber officinale with retention time=15.147using GC-MS analysis

Fig. 5: Structure of Gingerol present in Zingiber officinale with retention time=18.799 using GC-MS analysis

Fig .6: Structure of Tetradecane present in Zingiber officinale with retention time=20.696 using GC-MS analysis

Fig .7: Structure of (1R,4R,4aS,8aR)-4,7-Dimethyl-1-(prop-1-en-2-yl)-1,2,3,4,4a,5,6,8a-octahydronaphthalene present in Zingiber officinale with retention time=22.440 using GC-MS analysis

Fig .8: Structure of 1-Methyl-4-(6-methylhept-5-en-2-yl)cyclohexa-1,3-diene present in Zingiber officinale with retention time=22.844 using GC-MS analysis

Fig .9: Structure of Benzene, 1-(1,5-dimethyl-4-hexenyl)-4-methylpresent in Zingiber officinale with retention time=22.928 using GC-MS analysis

Fig .10: Structure of (1S,5S)-2-Methyl-5-((R)-6-methylhept-5-en-2-yl)bicyclo[3.1.0]hex-2-ene present in Zingiber officinale with retention time=23.235 using GC-MS analysis

Fig .11: Structure of 1H-Cyclopropa[a]naphthalene, decahydro-1,1,3a-trimethyl-7-methylene-, [1aS-(1a.alpha.,3a.alpha.,7a.beta.,7b.alpha.)]-present in Zingiber officinale with retention time=23.325 using GC-MS analysis

Fig .12: Structure of Naphthalene, 1,2,3,4,4a,5,6,8a-octahydro-7-methyl-4-methylene-1-(1-methylethyl)-, (1.alpha.,4a.beta.,8a.alpha.)- present in Zingiber officinale with retention time=23.407 using GC-MS analysis

Fig .13: Structure of alpha.-Farnesene present in Zingiber officinale with retention time=23.490 using GC-MS analysis

Fig .14: Structure of  beta.-Bisabolene present in Zingiber officinale with retention time=23.560 using GC-MS analysis

Fig .15: Structure of  Cyclohexene, 3-(1,5-dimethyl-4-hexenyl)-6-methylene-, [S-(R*,S*)]-  present in Zingiber officinale with retention time=23.944 using GC-MS analysis

Fig .16: Structure of  (E)-1-Methyl-4-(6-methylhept-5-en-2-ylidene)cyclohex-1-ene present in Zingiber officinale with retention time=24.140 using GC-MS analysis

Fig .17: Structure of  Nerolidyl acetate present in Zingiber officinale with retention time=24.834 using GC-MS analysis

Fig .18: Structure of  hexadecane present in Zingiber officinale with retention time=25.556 using GC-MS analysis

Fig .19: Structure of  Guaiol presgent in Zingiber officinale with retention time=26.443 using GC-MS analysis

Fig .20: Structure of Butan-2-one, 4-(3-hydroxy-2-methoxyphenyl)- presgent in Zingiber officinale with retention time=26.894 using GC-MS analysis

Fig .21: Structure of  3-Cyclohexen-1-ol, 1-(1,5-dimethyl-4-hexenyl)-4-methyl- presgent in Zingiber officinale with retention time=27.356 using GC-MS analysis

Fig .22: Structure of  6,10-Dodecadien-1-yn-3-ol, 3,7,11-trimethyl- present in Zingiber officinale with retention time=27.938 using GC-MS analysis

Fig .23: Structure of  Phenol, 5-(1,5-dimethyl-4-hexenyl)-2-methyl-, (R)- present in Zingiber officinale with retention time=29.210 using GC-MS analysis

Fig .24: Structure of  2(1H)-Naphthalenone, 4a,5,6,7,8,8a-hexahydro-7.alpha.-isopropyl-4a.beta.,8a.beta.-dimethyl-present in Zingiber officinale with retention time=30.727 using GC-MS analysis

Fig .25: Structure of  1H-3a,7-Methanoazulen-5-ol, octahydro-3,8,8-trimethyl-6-methylene- present in Zingiber officinale with retention time=32.287 using GC-MS analysis

Fig .26: Structure of  n-Hexadecanoic acid present in Zingiber officinale with retention time=34.293 using GC-MS analysis

Fig .27: Structure of Glutaric acid, myrtenyl 3-methylbut-2-en-1-yl ester  present in Zingiber officinale with retention time=34.731 using GC-MS analysis

Fig .28: Structure of  9,12-Octadecadienoic acid (Z,Z)-  present in Zingiber officinale with retention time=37.961 using GC-MS analysis

Fig .29: Structure of  9-Octadecenoic acid, (E)- present in Zingiber officinale with retention time=38.064 using GC-MS analysis

Fig .30: Structure of  Octadecenoic acid  present in Zingiber officinale with retention time=38.501 using GC-MS analysis

Fig .31: Structure of  Caryophyllene oxide present in Zingiber officinale with retention time=39.512 using GC-MS analysis

Fig .32: Structure of  (E)-1-(4-Hydroxy-3-methoxyphenyl)dec-3-en-5-one present in Zingiber officinale with retention time=39.784 using GC-MS analysis

Fig .33: Structure of  3-Decanone, 1-(4-hydroxy-3-methoxyphenyl)- present in Zingiber officinale with retention time=39.948 using GC-MS analysis

Fig .34: Structure of  1-(4-Hydroxy-3-methoxyphenyl)dec-4-en-3-one present in Zingiber officinale with retention time=41.059 using GC-MS analysis

Fig .35: Structure of  1-(4-Hydroxy-3-methoxyphenyl)decane-3,5-dione present in Zingiber officinale with retention time=41.742 using GC-MS analysis

Fig .36: Structure of  5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-3-one present in Zingiber officinale with retention time=42.449 using GC-MS analysis

Fig .37: Structure of  5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-3-one  present in Zingiber officinale with retention time=42.738 using GC-MS analysis

Fig .38: Structure of  1-(4-Hydroxy-3-methoxyphenyl)dodec-4-en-3-one present in Zingiber officinale with retention time=44.594 using GC-MS analysis

Fig .39: Structure of  (3R,5S)-1-(4-Hydroxy-3-methoxyphenyl)decane-3,5-diyl diacetate  present in Zingiber officinale with retention time=44.947 using GC-MS analysis

Fig .40: Structure of  1-(4-hydroxy-3-methoxyphenyl)tetradec-1-ene-3,5-dione present in Zingiber officinale with retention time=46.373  using GC-MS analysis

Fig .41: Structure of  5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecan-3-one present in Zingiber officinale with retention time=46.489 using GC-MS analysis

Fig .42: Structure of  2-Methylhexacosane present in Zingiber officinale with retention time=47.980 using GC-MS analysis

Fig .43: Structure of  1-(4-Hydroxy-3-methoxyphenyl)tetradec-4-en-3-one present in Zingiber officinale with retention time=48.519 using GC-MS analysis

Fig .44: Structure of  1-(4-Hydroxy-3-methoxyphenyl)tetradecane-3,5-dione  present in Zingiber officinale with retention time=49.325 using GC-MS analysis

Fig .45: Structure of  Tetrapentacontane present in Zingiber officinale with retention time=49.935  using GC-MS analysis