Extraction And Analysis Of Chemical Constituents Of Volatile Oil From Pericarpium Citri Reticulatae Viride

Pericarpium Citri Reticulatae Viride is the unripen fruit or its peel of the Citrus reticulata Blanco and other plants of family Rutaceae. The herb is pungent and bitter in flavour, warm in nature, and acts on the liver and stomach channels. Being pungent for dispersing, warming and clearing, and bitter for lowering and purging, the herb has strong effects of relieving Qi stagnation, soothing the liver, promoting digestion and resolving food retention. It is one of drugs for regulating Qi or promoting Qi circulation most in use.The volatile oil from Pericarpium Citri Reticulatae Viride was analyzed and studied in the paper. The volatile oils from Pericarpium Citri Reticulatae Viride were respectively extracted by hydrodistillation and supercritical CO₂ fluid extraction in a two-stage separation system. The each optimum extraction process for volatile oil obtained by hydrodistillation or supercritical CO₂ fluid extraction methods was investigated by the simple factor experiment and orthogonal design. The chemical constituents of volatile oil were separated and identified by gas chromatography-mass spectrometry (GC-MS). The relative content of each component was determined by area normalization. The efficiency and chemical constituents of supercritical CO₂ fluid extraction were compared with that of traditional hydrodistillation. The results showed that as follows:1. Hydrodistillation extraction of volatile oil from Pericarpium Citri Reticulatae Viride was carried out under different parameters of crush degree of herb (A), soak time (B) and extraction time (C). The effect of extraction time (C) on the extraction rate of volatile oil was highly significant (P<0.01), crush degree of herb (A) was significant (P<0.05) and soak time (B) was not significant (P>0.05). The sequence of factors according to the effects was as follows: extraction time (C)> crush degree of herb (A)> soak time (B).2. The optimum extraction process for volatile oil obtained by hydrodistillation was as follows: the proportion of material to solvent of 1:10, crush degree of herb of 80 mesh, soak time of 2 h and extraction time of 7 h.3. Supercritical CO₂ fluid extraction of volatile oil from Pericarpium Citri Reticulatae Viride was carried out under different parameters of extraction pressure (A), extraction temperature (B) and extraction time (C). The effect of extraction pressure (A) on the extraction rate of volatile oil was significant (P<0.05), extraction temperature (B) and extraction time (C) was not significant (P>0.05). The sequence of factors according to the effects was as follows: extraction pressure (A)>extraction temperature (B)>extraction time (C).4. The optimum extraction process for volatile oil obtained by supercritical CO2 fluid extraction was as follows: extraction pressure of 25MPa, extraction temperature of 35℃and extraction time of 1.5h, under the stationary extraction conditions of crush degree of herb of 80 mesh, first separator pressure of 8MPa, first separator temperature of 55℃, second separator pressure of 6MPa, second separator temperature of 35℃and flow of liquid CO2 of 20 L·h^-1.5. In the optimum extraction conditions, the extraction rate of volatile oil extracted by supercritical CO2 fluid extraction was 1.3197%, which was 3.4 times higher than that from hydrodistillation of 0.3823%. Compared with hydrodistillation, supercritical CO2 fluid extraction demonstrated better qualities, stronger fragrance and extraction time decreased by 78.57%.6. Twenty-two compounds were identified in the volatile oil extracted by hydrodistillation by GC-MS, represented 95.75% of the total volatile oil, which were terpenes(53.10%),alcohols (7.53%),esters (4.82%),alkanes (24.98%) and other components (5.32%). The major components of volatile oil were found to be d-Limonene (45.76%),n-Eicosane (5.90%),γ-Terpinene (4.52%),p-Cymene (4.22%),α-Terpineol (2.57%),Linalool (2.11%),(-)-Spathulenol (1.61%),α-Selinene (1.44%),Cyclohexane, 1-ethenyl-1-methyl- 2,4-bis (1-methylethenyl)-, [1S-(1à,2á,4á)]- (1.38%),p-Menth-1-en-4-ol, (R)-(-)- (1.24%),Eicosane, 7-hexyl- (1.22%),Benzene, 1-methyl- 4 -(1-methylethenyl)- (1.10%), etc.7. Nineteen compounds were identified in the volatile oil extracted by supercritical CO₂ fluid extraction by GC-MS, represented 89.53% of the total volatile oil, which were terpenes(3.69%),alcohols (8.62%),esters (19.45%) and alkanes (57.77%). The major components of volatile oil were found to be n-Eicosane (13.53%),9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester, (Z,Z,Z)- (9.09%),9-Octadecenoic acid (Z)-, methyl ester (5.05%),γ-Sitosterol (4.81%),d-Limonene (3.69%),1,2-Cyclohexanediol, 1-methyl-4-(1-methylethenyl)- (2.60%),Hexadecanoic acid, methyl ester (1.81%),Stearic acid, 3-(octadecyloxy)propyl ester (1.60%),1-Hexadecanol, 2-methyl- (1.21%), etc.8. The volatile oil extracted by supercritical CO₂ fluid extraction was found to be markedly different from the corresponding volatile oil obtained by hydrodistillation in the chemical constituents. More ten compounds in volatile oil from Pericarpium Citri Reticulatae Viride were reported for the first time, which were respectivelyα-Selinene,p-Menth-1-en-4-ol,(R)-(-),Benzene,1-methyl-4-(1-methylethenyl)-,1,2-Cyclohexanediol, 1-methyl-4-(1-methylethenyl)-,γ-Sitosterol,1-Hexadecanol, 2-methyl-,Hexadecanoic acid, methyl ester,9-Octadecenoic acid (Z)-, methyl ester,9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester, (Z,Z,Z)-,Stearic acid, 3-(octadecyloxy)propyl ester,n-Eicosane,Eicosane, 7-hexyl-,Docosane, 7-hexyl-,Heptadecane,2,6,10,15-tetramethyl-, etc.The experiment results provide profitable supplements and references for the further utilization and study on Pericarpium Citri Reticulatae Viride.