A rapid identification method for agarwood and common agarwood and application of new markers

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a rapid identification method for agarwood and common agarwood and the application of a new marker. The present invention first provides a rapid identification method, which is a pyrolysis-gas chromatography-mass spectrometry method. Take the agarwood sample to be tested, perform pyrolysis at 550-650°C, and then analyze and identify the characteristic peaks of the mass spectrum of the pyrolysis product. The method of the present invention is time-saving, the entire identification process only takes tens of minutes, and the method cost is low. In addition, the analysis of the identification results only requires determining the size of the characteristic peaks of the mass spectrum, and the results are easy to read and clear at a glance.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a rapid identification method for agarwood and common agarwood and the application of a new marker. Background Art

[0002] Agarwood is a classic raw material for both medicine and incense. As medicine, it has the effects of "promoting qi and relieving pain, warming the middle and stopping vomiting, and relieving asthma". As incense, it has a special fragrance composition, so it is known as the "first of the hundred fragrances" of "Agarwood, Sandalwood, Dragon and Musk". It is often used to process into incense powder, incense sticks, incense tablets and other health-preserving incense. Existing agarwood is divided into two types: agarwood type and ordinary type.

[0003] Common agarwood is a traditional medicinal agarwood, and its biological activity and pharmacological research have been clearly documented. It has an expensive market price due to its low yield. Kynam agarwood has a high yield and a low price, but it does not meet the current agarwood pharmacopoeia standards and cannot be used as medicinal agarwood. However, there are currently cases in the market where Kynam agarwood is used as a raw material for agarwood. Therefore, a method for identifying agarwood germplasm in agarwood and its products is of great significance for regulating the agarwood product market and protecting consumer rights.

[0004] The currently widely used agarwood germplasm identification method is mainly based on molecular identification, which is achieved by screening specific molecular biomarkers, and has the characteristics of high technical requirements and long time consumption. In addition, this method often requires a sufficient amount of agarwood extract to be implemented. For precious agarwood crafts, the destructive method of removing sufficient samples to prepare agarwood extracts for identification is obviously unrealistic. The patent with publication number CN117250298A and invention name "Construction method of liquid-mass fingerprint spectrum of agarwood and its fingerprint spectrum" discloses a UPLC-Q-TOF-MS / MS fingerprint spectrum of agarwood, which is used for quality detection and quality control of agarwood. However, this method has high technical requirements, is time-consuming, and requires extraction of agarwood samples.

[0005] Based on this, it is necessary to propose a more widely applicable method and strategy to quickly identify the types of agarwood germplasm. Summary of the invention

[0006] The purpose of the present invention is to provide a method for rapid identification of agarwood and common agarwood, as well as the application of a new marker, to partially solve or alleviate the above-mentioned deficiencies in the prior art. The present invention specifically adopts the following technical solutions.

[0007] A rapid identification method for agarwood and common agarwood, the rapid identification method is a pyrolysis-gas chromatography-mass spectrometry (Py-GC / MS) method, and the specific steps are as follows:

[0008] S01: Take the sample to be tested;

[0009] S02: setting reaction conditions, wherein the thermal cracking conditions are set as follows: the protective gas is air, the thermal cracking temperature is 550-650°C, and the cracking time is 2-5s; wherein the gas chromatography conditions are set as follows: the chromatographic column is a gas phase capillary column, the carrier is an inert gas, the initial temperature of the column oven is 40-60°C, and then the temperature is increased to no more than 180°C at a rate of 8-10°C / min; the mass spectrometry conditions are set as follows: the ion source temperature is 200-250°C, the quadrupole temperature is 150-200°C, and the mass spectrometry scanning range is 20-500Hz;

[0010] S03: Analyze the characteristic peaks of the mass spectrum of the thermal cracking product, and distinguish the agarwood from the common agarwood according to the area of ​​the characteristic peaks; the characteristic peaks are the characteristic peaks of benzyl acetone and 1,3-indanedione.

[0011] Furthermore, the sample to be tested includes agarwood extract, agarwood block, agarwood powder, agarwood extract, agarwood paste, agarwood dry paste, agarwood essential oil or agarwood raw material.

[0012] In some specific embodiments, the above-mentioned samples to be tested include agarwood extracts extracted using solvents such as ethanol or methanol; agarwood extracts, agarwood dry pastes, and agarwood powders extracted using extraction methods such as reflux and ultrasound; and agarwood essential oils extracted using subcritical or supercritical CO2 fluids.

[0013] Furthermore, the amount of the sample to be tested is 0.1-0.5 mg.

[0014] As a preferred implementation method, the gas phase capillary column is a HP-5MS or DB-5MS chromatographic column, as well as other chromatographic columns of the same carrier type.

[0015] As a preferred embodiment, the inert gas is high-purity helium.

[0016] As a preferred embodiment, the gas phase capillary column has a specification of 30m×250μm×0.25μm.

[0017] Furthermore, in the gas chromatography conditions, the transmission line temperature is further set to 280° C., the post-operation temperature is set to 300° C., and the operation time is set to 5 min.

[0018] Furthermore, the ion source temperature is 225-235°C, and the quadrupole temperature is 145-155°C.

[0019] Further, the characteristic peaks of the mass spectrum of the thermal pyrolysis product were analyzed. The characteristic peak of benzyl acetone was smaller than the characteristic peak of 1,3-indanedione, and the sample to be tested was determined to be agarwood; the characteristic peak of benzyl acetone was greater than the characteristic peak of 1,3-indanedione, and the sample to be tested was determined to be ordinary agarwood.

[0020] On the other hand, the present invention also provides an application of a new type of marker.

[0021] Application of benzyl acetone and 1,3-indanedione in the preparation of identification markers of agarwood and common agarwood.

[0022] Furthermore, the application is a thermal cracking identification marker.

[0023] Furthermore, the thermal cracking temperature is 550-650°C.

[0024] Application of benzyl acetone and 1,3-indanedione in the preparation of identification kits for agarwood and common agarwood.

[0025] Furthermore, the identification kit may also include other reagents commonly used in gas chromatography.

[0026] Furthermore, the identification kit may also include other reagents commonly used in mass spectrometry.

[0027] Beneficial technical effects:

[0028] (1) The present invention proposes a method for quickly identifying agarwood from agarwood. The present invention thermally cracks the agarwood sample to be tested at a specific temperature to obtain specific thermal cracking products, and further compares the mass spectrum peaks of the thermal cracking products to distinguish whether the sample to be tested is agarwood from agarwood or agarwood from agarwood. The identification result is reliable. The whole method is time-saving, the whole identification process only takes tens of minutes, and the method cost is low. In addition, the analysis of the identification result only needs to determine the size of the characteristic peak of the mass spectrum, and the result is easy to read and clear at a glance.

[0029] (2) The method of the present invention requires a small amount of sample to be tested, and can be directly tested and identified with powdered or block samples without the need to prepare agarwood extracts. For precious agarwood handicrafts, only a small amount of powder is needed for identification. Therefore, this method has a wide range of applications and high economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0031] Figure 1This is a mass spectrum of pyrolysis of 2-(2-phenylethyl) chromone at 550°C in one of the embodiments of the present invention;

[0032] Figure 2 This is a thermal decomposition mass spectrum of agarwood tetraol at 550°C in one of the embodiments of the present invention;

[0033] Figure 3 This is a mass spectrum of the agarwood extract in one of the embodiments of the present invention;

[0034] Figure 4 This is a mass spectrum of common agarwood powder in one of the embodiments of the present invention;

[0035] Figure 5 This is a mass spectrum of the agarwood slice product in one embodiment of the present invention. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0037] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0038] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0039] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0040] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0041] Definition of noun:

[0042] The "germplasm" mentioned in the present invention refers to plants that belong to the same species in terms of taxonomic status, but have obvious differences in traits.

[0043] Example 1

[0044] This embodiment provides a pyrolysis-gas chromatography-mass spectrometry (Py-GC / MS) method for analyzing the contents of agarwood tetraol and 2-(2-phenylethyl) chromone in agarwood.

[0045] Existing studies have found that in common agarwood, the content of agarwood tetraol is significantly higher than that of 2-(2-phenylethyl) chromone; the content of agarwood tetraol is about 4.3-37 times that of 2-(2-phenylethyl) chromone. In agarwood, the content of 2-(2-phenylethyl) chromone is higher than that of agarwood tetraol; the content of 2-(2-phenylethyl) chromone is about 28-170 times that of agarwood tetraol.

[0046] The present invention firstly thermally cracks agarwood tetraol and 2-(2-phenylethyl) chromone at 200-850°C, and finds that the two components can be respectively cracked into small molecular aromatic substances represented by benzyl acetone and 1,3-indanedione at a specific temperature, and can better reflect the content relationship of the two original components in agarwood. And after analyzing multiple samples, it is found that it has good stability.

[0047] The method operates as follows:

[0048] S01: Sampling. The samples include agarwood blocks, agarwood powder or agarwood extracts. The sampling amount is 0.1-0.5mg.

[0049] S02: Py conditions: air as protective gas, thermal cracking temperature of 550-650℃, cracking time of 2-5 s.

[0050] S03: GC conditions: The chromatographic column uses HP-5MS, the carrier gas is high-purity helium, the initial temperature of the column oven is 60°C, after maintaining for 1 min, the heating rate is 8-10°C / min, and it is increased to no higher than 180°C (preferably 148°C), the transfer line temperature is 280°C, and the post-operation temperature is 300°C for 5 min.

[0051] S04: MS conditions: ion source temperature was 230 °C, quadrupole temperature was 150 °C, and mass spectrometry scanning range was 20-500 Hz.

[0052] Results Figure 1 and Figure 2, two characteristic peaks can be seen in the figure, of which peak 1 is benzyl acetone, which is a representative thermal cracking product of agarwood tetraol at a specific temperature; peak 2 is 1,3-indanedione, which is a representative thermal cracking product of 2-(2-phenylethyl) chromone at a specific temperature. The two characteristic cracking peaks in the mass spectrum obtained by sample detection are integrated and the area size is compared to determine the type of agarwood germplasm in the test sample. The judgment standard is: if the area of ​​peak 1 (benzyl acetone) is smaller than that of peak 2 (1,3-indanedione), the analyzed sample is determined to be agarwood from Qinnan, otherwise it is ordinary agarwood.

[0053] This example further analyzes the products of thermal cracking of agarwood tetraol and 2-(2-phenylethyl) chromone at different temperatures, as shown in Table 1.

[0054] Table 1

[0055]

[0056] From the results in Table 1, it can be seen that 1,3-indanedione and benzyl acetone can be cracked in the temperature range of 450℃-650℃. Further verification found that although the peak of the target compound can be detected in the temperature range of 450℃-550℃, the peak area is very small; and the oxidative cracking products of the two target compounds will be detected above 650℃. Therefore, the present invention finally selects the thermal cracking temperature range of 550℃-650℃.

[0057] Example 2

[0058] This embodiment provides a specific example of the detection of agarwood ethanol extract.

[0059] S01: Sampling: Use tweezers to pick up about 0.4 mg of the sample to be tested and put it into a stainless steel sampling cup, and soak it in ethanol to obtain the agarwood alcohol extraction sample.

[0060] S02: Analysis: Py conditions: air as protective gas, thermal cracking temperature of 550°C, cracking time of 5 s.

[0061] The GC strips were: chromatographic column HP-5MS, carrier gas was high-purity helium, the initial temperature of the column oven was 60°C, after maintaining for 1 min, the heating rate was 8°C / min, rising to 148°C, the transfer line temperature was 280°C, and the post-operation temperature was 300°C for 5 min.

[0062] MS conditions: ion source temperature was 230 °C, quadrupole temperature was 150 °C, and mass spectrometry scanning range was 20-500 Hz.

[0063] S03: Result reading: The mass spectrum obtained by analysis and detection is as follows: Figure 3As shown, the two chromatographic peaks were integrated to obtain the area of ​​Peak 1 (benzyl acetone) 2072836.31, and the area of ​​Peak 2 (1,3-indanedione) 10336326.00; the area of ​​Peak 1 was smaller than that of Peak 2, so the extract sample was determined to be Aquilaria sinensis.

[0064] Example 3

[0065] This embodiment provides a specific example of detecting agarwood slices.

[0066] S01: Sampling: Use a blade to scrape an appropriate amount of sample, use tweezers to pick up about 0.13 mg of the sample to be tested, and place it in a stainless steel injection cup.

[0067] S02: Analysis: Py conditions: air as protective gas, thermal cracking temperature of 600 °C, cracking time of 5 s.

[0068] The GC strips were: chromatographic column HP-5MS, carrier gas was high-purity helium, the initial temperature of the column oven was 60°C, after maintaining for 1 min, the heating rate was 8°C / min, rising to 148°C, the transfer line temperature was 280°C, and the post-operation temperature was 300°C for 5 min.

[0069] MS conditions: ion source temperature was 230 °C, quadrupole temperature was 150 °C, and mass spectrometry scanning range was 50-300 Hz.

[0070] S03: Result reading: Analyze the mass spectrum obtained by the test, integrate the two chromatographic peaks, and obtain the area of ​​Peak 1 (benzyl acetone) as 1573486.20, and the area of ​​Peak 2 (1,3-indanedione) as 11384376.17; the area of ​​Peak 1 is smaller than that of Peak 2, so the agarwood slice sample is determined to be agarwood.

[0071] Example 4

[0072] This embodiment provides a specific example of detecting common agarwood powder.

[0073] S01: Sampling: Use a medicine spoon to take about 0.25 mg of the sample to be tested and place it in a stainless steel sampling cup.

[0074] S02: Analysis: Py conditions: air as protective gas, thermal cracking temperature of 550°C, cracking time of 5 s.

[0075] The GC strips were: chromatographic column HP-5MS, carrier gas was high-purity helium, the initial temperature of the column oven was 60°C, after maintaining for 1 min, the heating rate was 8°C / min, rising to 148°C, the transfer line temperature was 280°C, and the post-operation temperature was 300°C for 5 min.

[0076] MS conditions: ion source temperature was 230 °C, quadrupole temperature was 150 °C, and mass spectrometry scanning range was 20-500 Hz.

[0077] S03: Result reading: The mass spectrum obtained by analysis and detection is as follows: Figure 4 As shown, the two chromatographic peaks were integrated to obtain the area of ​​Peak 1 (benzyl acetone) of 13942490.33 and the area of ​​Peak 2 (1,3-indanedione) of 1957691.88; the area of ​​Peak 1 was greater than that of Peak 2, so the agarwood powder sample was determined to be ordinary agarwood.

[0078] Example 5

[0079] This embodiment provides an example of identifying the source of agarwood germplasm in agarwood slices.

[0080] S01: Sampling: Crush the sample, pick up about 0.36 mg of the sample to be tested with tweezers, and place it in a stainless steel sampling cup.

[0081] S02: Analysis: Py conditions: air as protective gas, thermal cracking temperature of 650°C, cracking time of 5 s.

[0082] The GC strips were: chromatographic column HP-5MS, carrier gas was high-purity helium, the initial temperature of the column oven was 60°C, after maintaining for 1 min, the heating rate was 8°C / min, rising to 176°C, the transfer line temperature was 280°C, and the post-operation temperature was 300°C for 5 min.

[0083] MS conditions: ion source temperature was 230 °C, quadrupole temperature was 150 °C, and mass spectrometry scanning range was 50-300 Hz.

[0084] S03: Result reading: The mass spectrum obtained by analysis and detection is as follows: Figure 1 As shown, the two chromatographic peaks were integrated to obtain peak 1 (benzyl acetone) area 5262215.01, peak 2 (1,3-indanedione) area 78528.91; the area of ​​peak 1 is smaller than that of peak 2, so it is determined that the agarwood used in the agarwood slice sample is ordinary agarwood.

[0085] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A rapid identification method for agarwood and common agarwood, characterized in that: The rapid identification method is a pyrolysis-gas chromatography-mass spectrometry method, and the specific steps are as follows: S01: Take the sample to be tested; S02: setting reaction conditions, wherein the thermal cracking conditions are set as follows: the protective gas is air, the thermal cracking temperature is 550-650°C, and the cracking time is 2-5s; wherein the gas chromatography conditions are set as follows: the chromatographic column is a gas capillary column, the gas capillary column includes an HP-5MS chromatographic column or a DS-5MS chromatographic column, the carrier gas is an inert gas, the initial temperature of the column oven is 40-60°C, and then the temperature is increased to no more than 180°C at a rate of 8-10°C / min; the mass spectrometry conditions are set as follows: the ion source temperature is 200-250°C, the quadrupole temperature is 150-200°C, and the mass spectrometry scanning range is 20-500Hz; S03: Analyze the characteristic peaks of the mass spectrum of the pyrolysis product, and determine whether it is agarwood or common agarwood according to the area of ​​the characteristic peaks; the characteristic peaks are the characteristic peaks of benzyl acetone and 1,3-indanedione; The characteristic peak of benzyl acetone is smaller than the characteristic peak of 1,3-indanedione, and the sample to be tested is determined to be agarwood; the characteristic peak of benzyl acetone is larger than the characteristic peak of 1,3-indanedione, and the sample to be tested is determined to be common agarwood.

2. The rapid identification method according to claim 1, characterized in that: The samples to be tested include agarwood extract, agarwood blocks, agarwood powder, agarwood extract, agarwood dry paste, agarwood essential oil or agarwood raw material.

3. The rapid identification method according to claim 1, characterized in that: The amount of the sample to be tested is 0.1-0.5 mg.

4. The rapid identification method according to claim 1, characterized in that: The gas phase capillary column comprises an HP-5MS chromatographic column or a DB-5MS chromatographic column.

5. The rapid identification method according to claim 1, characterized in that: The inert gas is high-purity helium.

6. The rapid identification method according to claim 1, characterized in that: In the gas chromatography conditions, the transmission line temperature was further set to 280° C., the post-operation temperature was set to 300° C., and the operation time was set to 5 min.

7. The rapid identification method according to claim 1, characterized in that: The ion source temperature is 225-235°C, and the quadrupole temperature is 145-155°C.

8. Application of benzyl acetone and 1,3-indanedione in the preparation of identification markers of agarwood and common agarwood.

9. Application of benzyl acetone and 1,3-indanedione in the preparation of identification kits for agarwood and common agarwood.

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