Extraction method and detection method suitable for terpene volatile substances of machilus pauhoi

Abstract

The invention discloses a method for extracting terpene volatile substances suitable for machilus pauhoi, which comprises the following steps: digging out a whole machilus pauhoi plant, timely cleaning the root with clear water, taking down the root, leaves, tender stems and old stems, and quickly freezing with liquid nitrogen to obtain tissue samples; grinding each tissue sample into powder by using liquid nitrogen, and extracting terpene volatile substances in each tissue sample by using an ethyl acetate solution to obtain a primary extracting solution of each tissue; respectively centrifuging each tissue primary extracting solution for 10 minutes at the rotating speed of 12,000 r / min, washing residues twice by using an ethyl acetate solution, centrifuging again, collecting supernate, diluting by five times, and filtering by using a 0.22 mu m filter membrane to obtain each tissue extracting solution. According to the method, ethyl acetate is used for extracting volatile chemical substances in different tissues of the machilus pauhoi, then the volatile substances are detected and analyzed through the GC-MS detection technology, and the composition and relative content difference of the volatile substances in the different tissues of the machilus pauhoi can be more sufficiently and accurately detected through the method.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant extraction, and particularly relates to a method for extracting and detecting terpene volatile substances suitable for Machilus pauhoi. Background Art

[0002] Machilus pauhoi is a rare evergreen broad-leaved native tree species in the subtropical region of China and belongs to precious afforestation tree species in China. The whole tree can be ground into powder and used as a blending agent for various incense, and is the main raw material for mosquito coils and sacrificial incense. Its branches, leaves, bark and trunk have complex chemical components and can be used to extract essential oils, tannins and make adhesives, hair conditioners, etc. In recent years, this tree species has been mainly sold at home and abroad as a raw material for incense powder, and the purchase price in its market production areas has been on the rise year by year. As an important raw material for incense powder and a precious afforestation tree species, the current research on the volatile aroma substances of Machilus pauhoi is still relatively weak, and there is only research on the detection of chemical substances in leaf essential oils. For the development and utilization of this precious tree species resource, especially the research on the differences and composition of volatile substances in different tissues, there are still blanks and further in-depth exploration is needed.

[0003] Chinese Patent with application number CN202111525493.7 discloses an application of Machilus pingii var. wilsonii extract in shampoo for promoting hair growth. Among them, it also discloses a preparation method for obtaining Machilus pingii var. wilsonii extract by extracting the roots, bark, trunk and branches of Machilus pingii var. wilsonii with water or ethanol solution, but the chemical substances extracted by this method are less and the extraction efficiency is low. In the prior art, it is also disclosed to use steam distillation method to obtain the leaf essential oil of Machilus pauhoi, and then use gas chromatography-mass spectrometry to accurately measure the chemical components in the essential oil. However, the steam distillation method needs to be carried out at high temperature, which will cause some thermosensitive components in the volatile oil to be destroyed. In addition, due to the open system and high temperature of the steam distillation method, in addition to obtaining the chemical components originally present in the plant, some volatile decomposition products generated during the distillation process may be contained in the extract. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a method for extracting and detecting terpene volatile substances suitable for Machilus pauhoi, using ethyl acetate, an organic solvent, to extract volatile chemical substances in different tissues of Machilus pauhoi, and then using GC-MS detection technology to detect and analyze the volatile substances. This method can more fully and accurately detect the composition and relative content differences of volatile substances in different tissues of Machilus pauhoi.

[0005] The object of the present invention can be achieved by the following technical solutions:

[0006] One of the objects of the present invention is to provide a method for extracting terpene volatile substances suitable for Machilus pauhoi, comprising the following steps:

[0007] S11: After digging out the whole 2-3-year-old Machilus pauhoi plant without diseases and pests, wash the roots with clean water in time, respectively remove the roots, leaves, young stems and old stems, and quick-freeze them with liquid nitrogen to obtain tissue samples of each part.

[0008] S12: Grind the tissue samples obtained in step S11 into powder with liquid nitrogen respectively, and extract the terpene volatile substances in the tissue samples with ethyl acetate solution with a purity of 99% to obtain the primary extraction solutions of each tissue.

[0009] S13: Centrifuge the primary extraction solutions of each tissue obtained in step S12 at a speed of 12000 r / min for 10 min, wash the residue twice with ethyl acetate solution and then centrifuge again, collect the supernatant, dilute it five times respectively, and filter it with a 0.22 μm filter membrane to obtain the extraction solutions of each tissue.

[0010] Further, in step S12, the ratio between the volume of the ethyl acetate solution and the mass of each tissue sample is 3 ml / g.

[0011] Further, in step S13, wash the residue twice with 1 ml of ethyl acetate solution.

[0012] The second object of the present invention is to provide a detection method for terpene volatile substances suitable for Machilus pauhoi. Detect and analyze the extraction solutions of each tissue obtained by any of the above extraction methods by GC-MS to obtain the composition and relative content of terpene volatile substances in each tissue of Machilus pauhoi.

[0013] Further, the chromatographic conditions for detection and analysis by GC-MS of the detection method for terpene volatile substances suitable for Machilus pauhoi are as follows: chromatographic column: Shimadzu RTX-5MS capillary chromatographic column, specification 30 m×0.25 mm×0.25 μm; column oven temperature: 70 °C; injection port temperature: 250 °C; programmed temperature rise: the initial temperature is 50 °C, hold for 2 min, then rise to 170 °C at a heating rate of 10 °C / min, hold for 2 min, and finally rise to 300 °C at a heating rate of 5 °C / min, hold for 2 min; carrier gas: He; flow control mode: pressure control, pressure is 51.2 kPa; total flow rate: 8.9 mL·min -1 ; column flow rate: 0.97 mL·min -1 ; linear velocity: 35.7 cm·s -1 ; purge flow rate: 5.0 mL·min -1 ; split ratio: 3:1; separation time: 44 min.

[0014] Further, the mass spectrometry conditions for the detection and analysis of the terpene volatile substances in Machilus pauhoi using GC-MS are as follows: ionization mode: electron impact ionization source; ion source temperature: 230 °C; interface temperature: 250 °C; solvent delay: 5 min; start time of mass spectrometry detection: 3 min, end time: 44 min; acquisition mode: Scan, scanning speed: 1666 amu·s -1 ; mass scanning range: 35 - 500 m·z -1 .

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The extraction method of terpene volatile substances in Machilus pauhoi provided by the present invention uses ethyl acetate, an organic solvent, to extract the volatile chemical substances in different tissues of Machilus pauhoi. The process operation is simple, fast, and has high extraction efficiency, and can extract 73 - 76 chemical substances from Machilus pauhoi. Since ethyl acetate is a medium-polarity solvent, it has a good balance between polar and non-polar substances, making it perform well in the separation and extraction process. Ethyl acetate also has strong solubility, can dissolve a variety of organic compounds, and has a low solubility in water. Moreover, ethyl acetate has a high volatility, and after extraction, it can be removed from the extract through a simple evaporation step, which is convenient for the recovery of subsequent target substances.

[0017] 2. A detection method for terpene volatile substances applicable to Machilus pauhoi provided by the present invention. In GC-MS analysis, based on previous experimental foundations, the present invention optimized the extraction conditions, mainly including conditions in two aspects: temperature and separation time. First, raise the temperature to 300 °C. High-temperature conditions contribute to the full desorption and detection of volatile substances because many volatile compounds can be more easily released from the sample at high temperatures. The final temperature of 300 °C enables the detection of even high-boiling-point compounds, which helps ensure the effective separation and detection of all volatile components. Second, a longer separation time allows compounds to have sufficient time to separate in the chromatographic column, thereby improving the separation effect and ensuring that different components can be clearly separated. This is particularly important for the analysis of complex samples as it can prevent peak overlap and interference, improving the accuracy of quantitative and qualitative analysis. Finally, in the part of the mass spectrometry detection conditions, setting the ion source temperature and interface temperature to 230 °C and 250 °C helps stabilize the performance of the ion source, improving the intensity and quality of the signal. Generally speaking, the purpose of these condition settings is to ensure that all volatile substances can be effectively separated and detected. The combined action of high temperature, long-time separation, and appropriate mass spectrometry conditions is to obtain detailed and accurate spectral information of volatile substances, thereby comprehensively analyzing the sample. These conditions are particularly applicable to the detection of multiple volatile compounds in complex samples, ensuring high-quality data. The present invention uses GC-MS detection technology to detect and analyze the extracted volatile substances, more fully and accurately detecting the composition and relative content differences of volatile substances in different tissues of Machilus pauhoi. Among them, the most types of volatile substances are detected in the roots. Among the volatile substances extracted from different tissues, the most types are terpene substances, and terpene substances also account for the largest proportion in roots, leaves, and young stems, but the relative content proportion of fatty acid derivatives is the largest in old stems. The present invention solves the problem that the research on volatile chemical substances in different tissues of Machilus pauhoi, especially in stems and roots, is not thorough at present, providing a theoretical basis for the subsequent development and utilization of Machilus pauhoi. Description of the Drawings

[0018] Figure 1 For Example 1 of the present invention, the total ion current of volatile organic substances in 4 tissues of Machilus pauhoi, from top to bottom are old stem, young stem, root, and leaf;

[0019] Figure 2 For Example 1 of the present invention, the classification statistics of volatile substances in 4 tissues of Machilus pauhoi;

[0020] Figure 3 For Example 2 of the present invention, the total ion current of volatile organic substances in 4 tissues of Machilus pauhoi, from top to bottom are old stem, young stem, root, and leaf;

[0021] Figure 4In Example 2 of the present invention, the classification statistics of volatile substances in four tissues of Machilus pauhoi

[0022] Figure 5 In Comparative Example 1 of the present invention, the total ion current of volatile organic substances in the stem tissue of Machilus pauhoi Detailed implementation manners

[0023] The following further describes the present invention in conjunction with preferred embodiments and with reference to the attached Figures 1-5 , in the present invention, the endpoints and any values within the disclosed ranges are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values; for numerical ranges, between the endpoint values of each range, between the endpoint values and individual point values of each range, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein; the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without special instructions; the experimental methods in the following embodiments are conventional methods without special instructions

[0024] Example 1

[0025] This example provides a method for extracting terpene volatile substances suitable for Machilus pauhoi, including the following steps

[0026] S11: After digging out the whole plants of 2-3-year-old healthy and disease-free Machilus pauhoi plants, wash the roots with clean water in time, take 0.5 g of roots, leaves, young stems, and old stems respectively, quickly freeze them with liquid nitrogen to obtain tissue samples of each part, and mark them

[0027] S12: Grind the tissue samples obtained in step S11 into powder with liquid nitrogen respectively, and extract the terpene volatile substances in each tissue sample with 1.5 ml of ethyl acetate solution with a purity of 99% to obtain the primary extraction solution of each tissue

[0028] S13: Centrifuge the primary extraction solutions of each tissue obtained in step S12 at a speed of 12,000 r / min for 10 min, wash the residue twice with 1 ml of ethyl acetate solution and then centrifuge again, collect the supernatant, dilute each by five times respectively, and filter with a 0.22 μm filter membrane to obtain the extraction solutions of each tissue. Each sample is extracted in parallel three times

[0029] This example also provides a method for detecting terpene volatile substances suitable for Machilus pauhoi. Use GC-MS to detect and analyze the extraction solutions of each tissue obtained by any of the above extraction methods to obtain the composition and relative content of terpene volatile substances in each tissue of Machilus pauhoi

[0030] In this embodiment, the chromatographic conditions for detection and analysis using GC-MS are as follows: chromatographic column: Shimadzu RTX-5MS capillary chromatographic column, with a specification of 30 m × 0.25 mm × 0.25 μm; column oven temperature: 70 °C; inlet temperature: 250 °C; programmed temperature rise: the initial temperature is 50 °C, after holding for 2 min, the temperature is raised to 170 °C at a rate of 10 °C / min, held for 2 min, and finally raised to 300 °C at a rate of 5 °C / min and held for 2 min; carrier gas: He; flow control mode: pressure control, with a pressure of 51.2 kPa; total flow rate: 8.9 mL·min -1 ; column flow rate: 0.97 mL·min -1 ; linear velocity: 35.7 cm·s -1 ; purge flow rate: 5.0 mL·min -1 ; split ratio: 3:1; separation time: 44 min. The total ion current chromatogram is obtained as shown Figure 1 below.

[0031] The mass spectrometry conditions are as follows: ionization mode: electron impact ionization source; ion source temperature: 230 °C; interface temperature: 250 °C; solvent delay: 5 min; start time of mass spectrometry detection: 3 min, end time: 44 min; acquisition mode: Scan, scanning speed: 1666 amu·s -1 ; mass scanning range: 35 - 500 m·z -1 . After sample separation, it is compared and qualitatively analyzed with the computer spectral library to obtain the chemical components. The relative content of volatile substances is expressed by the peak area ratio of different substances. The classification statistical chart of volatile substances in four tissues of Machilus pauhoi is as shown Figure 2 below.

[0032] According to the total ion chromatogram of volatile substances in four tissues of Machilus pauhoi as shown Figure 1 below, spectral library analysis and retrieval are carried out to obtain the composition and relative content table of volatile substances in each tissue. Among them, R represents roots, Y represents leaves, NJ represents young stems, and LJ represents old stems. The specific table is as follows:

[0033] Table 1 Composition and relative content table of volatile substances in each tissue

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040] As shown in the above table, among the four tissues of Machilus pauhoi, five volatile compounds are contained in all four tissues, namely: 2-Palmitoyl-rac-glycerol, 4,6-Dimethyldodecane, Eicosyl acetate, 7,9-ditert-butyl-1-oxaspiro[4.5]deca-6,9-diene-2,8-dione, and 2,4-Di-tert-butylphenol. The relative content of 2-Palmitoyl-rac-glycerol in the old stem is as high as 34.59%; the relative contents of 4,6-Dimethyldodecane and Eicosyl acetate in the four tissues are both lower than 1%; the relative content of 7,9-ditert-butyl-1-oxaspiro[4.5]deca-6,9-diene-2,8-dione is the highest in the root, which is 1.53%. The relative contents of 2,4-Di-tert-butylphenol in the four tissues are all above 10%.

[0041] In the four tissues of the old stem, young stem, root, and leaf of this example, a total of 73 volatile compounds were detected, and the root contained the most types of volatile substances. Among them, 2-Methylhexacosane and N-Tetracontane are specifically present in the young stem. Six substances, namely 2-Methyltetracosane, δ-elemene, germacrene D, β-bisabolene, Hexatriacontane, and (+)-β-funebrene, are specifically present in the old stem. The root contains the most specific volatile substances, which is 22 kinds. 14 chemical substances are specifically present in the leaf.

[0042] In this example, statistics were also carried out using the four tissues of Machilus pauhoi as materials, and the detected volatile components can be mainly divided into several compounds such as benzenes / phenylpropanoids, terpenes, alkanes, fatty acid derivatives, steroid compounds, acetate esters, and pyrimidine derivatives, such as Figure 2As shown in the figure. The roots contain the most volatile substances, with 39 kinds, including 4 kinds of benzenes / phenylpropanoids, with a relative content of 32.39%; 2 kinds of acetate compounds, with a relative content of 2.82%; 26 kinds of terpene compounds, with a relative content of 58.55%; 3 kinds of alkanes account for 0.58%; 4 kinds of fatty acid derivatives account for 4.6%. The old stems have the fewest types of volatile substances, only 20 kinds. Among them, there are 2 kinds of benzenes / phenylpropanoids and 5 kinds of fatty acid derivatives, with relative content ratios of 33.38% and 48.85% respectively; 1 kind of steroid compound accounts for 3.04%, and 5 kinds of alkane compounds account for 5.51%; 7 kinds of terpene volatile substances account for only 5.96%. The number of volatile substances in the young stems and leaves only differs by one kind, being 31 and 32 kinds respectively. In the young stems, the terpene substances are the most in number, with 17 kinds, with a relative content ratio of 42.99%. There are 5 kinds of alkane compounds and fatty acid derivatives each, with ratios of 4.73% and 16.18% respectively. One kind of steroid compound with a relative content ratio of 3.85%, one kind of acetate compound with a relative content ratio of 1.16%, and 2 kinds of benzenes / phenylpropanoids with a ratio of 29.42% are also detected. In the leaves, there is only one kind each of steroid compounds, pyrimidine derivatives, and benzenes / phenylpropanoids, with relative mass ratios of 6.33%, 0.19%, and 15.12% respectively; the number of terpene substances accounts for 53.1% of the total number of substances, a total of 17 kinds, with a relative mass ratio of 56.06%; there are 5 and 7 kinds of alkanes and fatty acid derivatives respectively, with relative content ratios of 4.66% and 12.37% respectively. In short, among the four different tissues of Machilus pauhoi, the terpene substances are the most abundant in terms of the types of volatile substances. At the same time, the terpene substances in the roots, young stems, and leaves are also the main components. However, in the old stems, the fatty acid derivatives have a relatively higher content ratio.

[0043] Example 2

[0044] This example provides a method for extracting terpene volatile substances applicable to Machilus pauhoi, including the following steps:

[0045] S11: After digging out the whole healthy and disease-free one-year-old Machilus pauhoi plant, wash the roots with clean water in time, take 1 g each of the roots, leaves, young stems, and old stems respectively, quickly freeze them with liquid nitrogen to obtain tissue samples of each part, and mark them with labels;

[0046] S12: Grind the tissue samples obtained in step S11 into powder with liquid nitrogen respectively, and extract the terpene volatile substances in each tissue sample with 3 ml of ethyl acetate solution with a purity of 99% to obtain the primary extraction solution of each tissue;

[0047] S13: Centrifuge each initial tissue extract obtained in step S12 at a rotational speed of 12,000 r / min for 10 min, wash the residue twice with 1 ml of ethyl acetate solution and then centrifuge again, collect the supernatant, dilute it five times respectively, and filter it through a 0.22 μm filter membrane to obtain each tissue extract. Each sample is extracted in parallel three times.

[0048] This embodiment also provides a method for detecting terpene volatile substances suitable for Machilus pauhoi. The GC-MS is used to detect and analyze each tissue extract obtained by any of the above extraction methods to obtain the composition and relative content of terpene volatile substances in each tissue of Machilus pauhoi.

[0049] In this embodiment, the chromatographic conditions for detection and analysis by GC-MS are as follows: chromatographic column: Shimadzu RTX-5MS capillary chromatographic column, specifications 30 m × 0.25 mm × 0.25 μm; column oven temperature: 70 °C; injection port temperature: 250 °C; programmed temperature rise: the initial temperature is 50 °C, after maintaining for 2 min, the temperature is raised to 170 °C at a rate of 10 °C / min, maintain for 2 min, and finally the temperature is raised to 300 °C at a rate of 5 °C / min and maintain for 2 min; carrier gas: He; flow control mode: pressure control, pressure is 51.2 kPa; total flow rate: 8.9 mL·min -1 ; column flow rate: 0.97 mL·min -1 ; linear velocity: 35.7 cm·s -1 ; purge flow rate: 5.0 mL·min -1 ; split ratio: 3:1; separation time: 44 min. Obtain the total ion current chromatogram, as Figure 3 shown.

[0050] The mass spectrometry conditions are as follows: ionization mode: electron impact ion source; ion source temperature: 230 °C; interface temperature: 250 °C; solvent delay: 5 min; start time of mass spectrometry detection: 3 min, end time: 44 min; acquisition mode is Scan, scanning speed is 1666 amu·s -1 ; mass scanning range is 35 - 500 m·z -1 . After sample separation, it is compared and qualitatively analyzed with the computer spectral library to obtain each chemical component. The relative content of volatile substances is represented by the peak area ratio of different substances. The classification statistical chart of volatile substances in 4 tissues of Machilus pauhoi is as Figure 4 shown.

[0051] According to the total ion chromatogram of volatile substances in 4 tissues of Machilus pauhoi as Figure 3 shown, perform spectral library analysis and retrieval to obtain the composition and relative content of volatile substances in each tissue as shown in the following table, where R is the root, Y is the leaf, NJ is the young stem, and LJ is the old stem:

[0052] Table 2 Composition and relative content of volatiles in each tissue

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] As shown in the above table, among the four Machilus pauhoi tissues in this example, eight volatiles are contained in all four tissues, namely: 2-Palmitoyl-rac-glycerol, (+)-DELTA-CADINENE, 4,6-Dimethyldodecane, Eicosyl acetate, 7,9-ditert-butyl-1-oxaspiro[4.5]deca-6,9-diene-2,8-dione, γ-Sitosterol, β-Caryophyllene, 2,4-Di-tert-butylphenol. The relative content of 2-Palmitoyl-rac-glycerol in the old stem is as high as (37.29%); the content of Δ-Cadinene in the leaves is 4.97%; the contents of 4,6-Dimethyldodecane and Eicosyl acetate in all four tissues are less than 1%; the relative contents of 7,9-ditert-butyl-1-oxaspiro[4.5]deca-6,9-diene-2,8-dione in all four tissues are relatively low; the relative contents of γ-Sitosterol, 2,4-Di-tert-butylphenol and β-Caryophyllene in the young stem are the highest, being 4.81%, 14.74% and 7.72% respectively.

[0059] In the four tissues of old stems, young stems, roots, and leaves in this example, a total of 76 volatile compounds were detected, and the roots contained the largest number of volatile substances. Among them, 1,6-Cyclodecadiene,1-methyl-5-methylene-8-(1-methylethyl)-,(1E,6E)-, N-tetratetracontane (n-tetratetracontane), and sec-Butyl nitrite are unique compounds in old stems. Ethyl Acetate, (E)-beta-farnesene, δ-elemene, β-bisabolene, α-Humulene, and (+)-β-funebrene are specifically present in young stems. The roots contain 26 specific volatile substances. γ-Elemene, Ethyl caprate, 10-Undecenal, n-Nonadecanol-1, Phytol, 1,5,9-Cyclododecatriene,1,5,9-trimethyl-, bicyclogermacrene, 1,3,6-Octatriene,3,7-dimethyl-,(Z)-Ocimene isomer mixture, 1-Chlorooctadecane, and 3-HEXENAL are unique volatile chemical substances in leaves.

[0060] In this example, four tissues of Machilus pauhoi were used as materials for statistics, and the detected volatile components could be mainly divided into six categories: benzenes / phenylpropanoids, terpenes, alkanes, steroids, fatty acid derivatives, and acetate esters ( Figure 5)。The root contains the most volatile substances, with 44 kinds. Among them, there are 6 kinds of benzenes / phenylpropanoids, with a relative content of 31.67%; 27 kinds of terpenoid compounds, with a relative content of 58.7%; 2 kinds of alkanes account for 0.47%; 8 kinds of fatty acid derivatives account for 6.86%, and a steroid compound is also detected, accounting for 1.22%. The old stem has the fewest types of volatile substances, only 22 kinds. Among them, there are 2 kinds of benzenes / phenylpropanoids and 6 kinds of fatty acid derivatives, with relative contents accounting for 30.59% and 54.83% respectively; 1 kind of steroid compound accounts for 2.96%, 5 kinds of alkane compounds account for 4.38%; 8 kinds of terpenoid volatile substances account for only 5.55%. The young stem and leaves contain 31 and 30 kinds of volatile substances respectively. In the young stem, the terpenoid substances are the most numerous (18 kinds), with a relative content accounting for 40.83%. There are 4 and 5 kinds of alkane compounds and fatty acid derivatives respectively, accounting for 3.23% and 19.63%. A steroid compound with a relative content of 4.81%, an acetate compound with a relative content of 1.17%, and 2 kinds of benzenes / phenylpropanoid compounds with a ratio of 28.28% are also detected. In the leaves, there is only one kind of steroid compound and benzenes / phenylpropanoid compound respectively, with relative mass ratios of 5.23% and 14.11%; the types of terpenoid substances account for 60% (18 kinds) of the total substance types, with a relative mass ratio of 59.99%; both alkanes and fatty acid derivatives contain 5 kinds, with relative contents accounting for 4.03% and 11.22% respectively. In short, among the four different tissues of Machilus pauhoi, the most abundant volatile substances are terpenoid substances. At the same time, terpenoid substances in the root, young stem and leaves are also the main components. However, in the old stem, the fatty acid derivatives have a relatively higher content ratio.

[0061] Comparative Example 1

[0062] In this comparative example, three reagents, namely ethyl acetate solution with a purity of 99%, methanol, and n-hexane, were used to extract the volatile compounds in the stem tissue sample respectively to obtain the preliminary extract of the stem tissue.

[0063] After the sample was separated, it was compared and qualitatively analyzed with the computer spectral library to obtain each chemical component. The relative content of the volatile substances was expressed by the peak area ratio of different substances. Figure 5 This is the total electron flow of the volatile organic substances in the stem tissue of Machilus pauhoi in this comparative example. As Figure 5 shown, the extraction reagents from top to bottom are methanol, ethyl acetate, and n-hexane. According to the total ion chromatogram of the volatile components in the stem tissue of Machilus pauhoi as Figure 5 shown, spectral library analysis and retrieval were carried out to obtain the composition and relative content table of the volatile components in the stem tissue. The composition and relative content of the volatile substances in the stem tissue of Machilus pauhoi co-detected by the three extraction solutions are shown in the following table:

[0064] Table 3 Composition and relative content of volatile compounds in the stem

[0065]

[0066]

[0067] As shown in the above table, among the three reagents used in this comparative example for extracting Machilus pauhoi stem tissues, 5 volatile compounds could be detected in all three extraction methods, namely: 2,4-Di-tert-butylphenol, Germacrene D, Caryophyllene, alfa.-Copaene, and Phenol, 2,2'-methylenebis[6-(1,1-dimethylethyl)-4-methyl-. The relative contents of these 5 volatile compounds were the highest when extracted with ethyl acetate, being 14.24%, 5.67%, 2.42%, 1.92%, and 18.87% respectively. There were 5 volatile compounds that could be detected in the extraction methods using ethyl acetate and methanol, namely gamma.-Sitosterol, gamma.-Elemene, Naphthalene, 1,2,4a,5,8,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-,

[0068] [1S-(1.alpha.,4a.beta.,8a.alpha.)]-(β-Cadinene), n-Hexadecanoic acid (Palmitic acid), Cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)-, [1S-(1.alpha.,2.beta.,4.beta.)]-(β-Elemene). The extraction effect of these 5 volatile substances with ethyl acetate is the best, and the contents are 1.97%, 1.27%, 1.87%, 2.90%, and 1.46% respectively. Among them, Germacrene, β-Caryophyllene, α-Copaene, γ-Elemene, β-Cadinene, and β-Elemene all belong to terpene compounds. Benzene, 1,3-dimethyl- (m-Xylene) and 1,5,5-Trimethyl-6-methylene-cyclohexene can be detected by the extraction methods of ethyl acetate and n-hexane, and the relative content extracted by ethyl acetate is the highest. Therefore, the extraction effect of ethyl acetate used in the present invention is the best, the types of detected compounds are the most, and the relative content is the highest.

[0069] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A method for extracting terpene volatile substances from wood shavings, characterized in that: The following steps are involved: S11: dig out healthy and disease-free 2-3 year-old Phoebe philadelphica plants and wash the roots with clean water in time, remove roots, leaves, young stems and old stems respectively, and freeze them with liquid nitrogen to obtain tissue samples; S12: Grind each tissue sample obtained in step S11 into powder using liquid nitrogen, and extract the terpene volatile substances in each tissue sample using 99% pure ethyl acetate solution to obtain primary extracts of each tissue; S13: The primary extracts of each tissue obtained in step S12 were centrifuged at a speed of 12000 r / min for 10 min, and the residue was washed twice with ethyl acetate solution and centrifuged again, and the supernatant was collected, diluted five times, and filtered with a 0.22 μm filter membrane to obtain the extracts of each tissue.

2. The method for extracting terpene volatile substances suitable for wood shavings according to claim 1, characterized in that: In step S12, the ratio between the volume of the ethyl acetate solution and the mass of each tissue sample is 3 ml / g.

3. The method for extracting terpene volatile substances suitable for wood shavings according to claim 2, characterized in that: In step S13, the residue was washed twice with 1 ml of ethyl acetate solution.

4. A method for detecting terpene volatile substances in wood shavings, characterized in that: GC-MS is used to detect and analyze the tissue extracts obtained by any extraction method described in claims 1-3 to obtain the composition and relative content of terpene volatile substances in each tissue of Pseudostellaria baicalensis.

5. The method for detecting terpene volatile substances in wood shavings according to claim 4, characterized in that: The chromatographic conditions for GC-MS detection and analysis are as follows: chromatographic column: Shimadzu RTX-5MS capillary column, specification 30m×0.25mm×0.25μm; column box temperature: 70℃; injection port temperature: 250℃; program heating: starting temperature is 50℃, after holding for 2min, it is increased to 170℃ at a heating rate of 10℃ / min, held for 2min, and finally increased to 300℃ at a heating rate of 5℃ / min, held for 2min; carrier gas: He; flow control mode: pressure control, pressure is 51.2kPa; Total flow rate: 8.9 mL min -1 ; Column flow rate: 0.97mL·min -1 ; Line speed: 35.7cm·s -1 ; Purge flow rate: 5.0mL·min -1 ; Split ratio: 3:1; Separation time: 44min.

6. The method for detecting terpene volatile substances in wood shavings according to claim 4, characterized in that: The mass spectrometry conditions for GC-MS detection and analysis are as follows: ionization mode: electron bombardment ion source; ion source temperature: 230°C; interface temperature: 250°C; solvent delay: 5 min; mass spectrometry detection start time: 3 min, end time: 44 min; acquisition mode: Scan, scanning speed: 1666 amu·s -1 ; Mass scan range 35-500m·z -1 .

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