Headspace solid-phase microextraction method for rapidly identifying dalbergia odorifera and dalbergia cochinchinensis nursery stocks

Abstract

The invention discloses a headspace solid-phase microextraction method for rapidly identifying dalbergia odorifera and dalbergia cochinchinensis nursery stocks, and belongs to the technical field of plant identification. According to the method, the problem of high similarity of the dalbergia odorifera and the dalbergia cochinchinensis in leaf forms is well avoided, the difference of volatile components of the dalbergia odorifera and the dalbergia cochinchinensis in the leaves is fully utilized, and the problem that the dalbergia odorifera and the dalbergia cochinchinensis seedlings are difficult to accurately and quickly identify by traditional means such as leaf appearance is thoroughly solved. Compared with the traditional identification method, the method has the advantages of simplicity and convenience in operation, small sampling amount, short time consumption, high accuracy and the like, and is particularly suitable for quickly and accurately identifying some famous and ancient trees. The invention provides an efficient and reliable dalbergia odorifera and dalbergia cochinchinensis nursery stock rapid identification method for nursery stock transaction, famous and ancient tree protection, wood transaction, wood dealers, consumers, public security organizations, customs, scientific research institutions and the like, and has very high application value and wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of plant identification, and relates to a method for identifying Dalbergia odorifera and Dalbergia cochinchinensis seedlings, and in particular to a headspace solid phase microextraction method for rapidly identifying Dalbergia odorifera and Dalbergia cochinchinensis seedlings. Background Art

[0002] Dalbergia Dalbergia ) is a pantropical genus in the Papilionaceae family, with about 250 species worldwide, mainly distributed in tropical and subtropical regions such as Central and South America, Africa and southern Asia. Many species in the genus Dalbergia are classified as redwood species because of their unique wood texture, excellent material, special aroma and wide range of uses. Among them, Dalbergia odorifera ( Dalbergia odorifera ) is known as "fragrant rosewood", Dalbergia latifolia ( D. latifolia ) commonly known as "Indian rosewood", Dalbergia cochinchinensis ( D. cochinchinensis ) is called "Thai rosewood", Brazilian black rosewood ( D. nigro ) is called "Brazilian rosewood", Belize rosewood ( D. stevensonii ) calls it "Honduras rosewood". Due to differences in wood properties, structural characteristics and chemical composition, the economic value of different rosewood species varies greatly. However, the seedlings of these rosewood species have a high degree of similarity or overlap in appearance characteristics such as leaf shape, leaf color and leaf sequence, resulting in a large number of counterfeit and shoddy products in the seedling market. For example, it is not uncommon to sell Dalbergia cochinchinensis seedlings as Dalbergia odorifera seedlings, which has caused huge economic losses to consumers, planters and operators. This phenomenon not only affects the sustainable use of rosewood resources, but also aggravates market chaos. It is urgent to strengthen the identification and supervision of rosewood species through scientific means.

[0003] At present, the identification of seedlings of Dalbergia odorifera and Dalbergia cochinchinensis mainly relies on the differences in external morphological characteristics such as leaf size, branch shape and number of leaflets. However, in the actual identification process, these characteristic differences partially overlap and are difficult to define accurately. In addition, the professional and technical level of the appraiser is also required to be very high, and erroneous identification results often occur. The use of chemical composition analysis methods may make up for this defect. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a headspace solid phase microextraction method for rapidly identifying Dalbergia odorifera and Dalbergia cochinchinensis seedlings.

[0005] The present invention is based on the composition difference of volatile components of Dalbergia odorifera and Dalbergia cochinchinensis leaves, adopts a solid phase microextraction method to extract volatile substances from the leaves of the two plants respectively, and applies a gas chromatography-mass spectrometry to perform qualitative and quantitative analysis, and accurately identifies the two seedlings by comparing the types and relative percentage content ranges of the main characteristic components of the leaves of the two tree species. The main characteristic components of the volatile substances in the leaves of Dalbergia odorifera and Dalbergia cochinchinensis are obviously different, and the two seedlings can be effectively distinguished.

[0006] The purpose of the present invention is achieved through the following technical solutions: A headspace solid phase microextraction method for rapid identification of Dalbergia odorifera and Dalbergia cochinchinensis seedlings comprises the following steps: A. Sampling and pretreatment of leaf samples: Fresh leaf samples of Dalbergia odorifera and Dalbergia cochinchinensis were collected during the growing season, the leaves were washed, dried, withered, dried to constant weight, crushed, and sieved; B. Extraction of volatile components: Place the sample sieved in step A into a headspace injection bottle, balance at a constant temperature, and then insert the aged extraction fiber head into the headspace of the injection bottle for extraction; C. Composition analysis of volatile components: After the extraction in step B, the extraction fiber head is taken out and quickly inserted into a gas chromatography-mass spectrometer (GC-MS) for detection to obtain the total ion current chromatogram and mass spectrum data of the volatile components, and each component is matched and searched with the standard mass spectrum library (NIST20); D. Qualitative identification of characteristic components: perform pattern matching retrieval to obtain the main characteristic components, calculate the relative percentage of the main characteristic components using the area normalization method, and then identify Dalbergia odorifera and Dalbergia cochinchinensis based on the types of the main characteristic components and the relative percentage range.

[0007] Preferably, in step A, the leaf samples are mature leaves.

[0008] Preferably, in step A, The washing is washing with sterile water; the drying is drying with filter paper; and the pulverizing is pulverizing with a high-speed pulverizer; The fixing is carried out at 100-110°C for 30-60 min; further, the fixing is carried out at 105°C for 1 h.

[0009] The drying to constant weight is to place the mixture at 70-80° C. and dry it to constant weight.

[0010] The sieving is through a 60-80 mesh sieve.

[0011] Preferably, in step B, The extraction fiber head was aged by gas chromatography-mass spectrometry (GC-MS) at 260°C for 30 min.

[0012] The extraction fiber head is a 50 / 30 μm DVB / CAR / PDMS extraction head.

[0013] The constant temperature balance condition is 70-80° C. constant temperature balance for 20-30 min; further, 70° C. constant temperature balance for 30 min.

[0014] The extraction time is 20 to 30 min; further 30 min.

[0015] Preferably, in step C, The chromatographic analysis conditions were as follows: chromatographic column: HP-5 MS, 30 m×0.25 mm×0.25 μm; temperature program: initial column temperature 30 °C, maintained for 5 min, and then increased at 7 °C·min -1 Raise to 260 °C and maintain for 5 min; injection port temperature 250 °C, carrier gas helium, flow rate 1.0 mL min -1 ; Splitless injection, injection volume 1.0 μL; Mass spectrometry conditions: EI ion source, electron energy 70 eV, ion source temperature 230 ℃, quadrupole temperature 150 ℃, GC / MS interface temperature 280 ℃, mass scanning range (m / z): 50-550 amu.

[0016] Preferably, in step D, the following steps are included: a. Identification of Dalbergia odorifera: The main characteristic components are methyl salicylate, benzyl alcohol, β-bisabolene and phenylethanol; among them, methyl salicylate is Methyl salicylate, CAS number is 119-36-8; benzyl alcohol is Benzyl alcohol, CAS number is 100-51-6; β-bisabolene is β -Bisabolene, CAS No. 495-61-4; Phenylethylalcohol, CAS No. 60-12-8. When the proportion of these four main characteristic components in the total volatile components exceeds 30%, it is determined to be Dalbergia odorifera; b. Identification of Dalbergia cochinchinensis: The main characteristic components are α-ionone, 1-(1-methylcyclopent-2-en-1-yl)ethan-1-one, (3E,5E)-oct-3,5-dien-2-one and β-ionone; among them, α-ionone is α-Ionone, with a CAS number of 127-41-3; 1-(1-methylcyclopent-2-en-1-yl)ethan-1-one is 1-(1-Methyl-2-cyclopentenyl)ethanone, with a CAS number of 68752-16-9; (3E,5E)-oct-3,5-dien-2-one is 3,5-Octadien-2-one, with a CAS number of 38284-27-4; β-ionone is β-Ionone, with a CAS number of 79-77-6. When the proportion of these four main characteristic components in the total volatile components exceeds 30%, it is determined to be Dalbergia cochinchinensis.

[0017] Compared with the prior art, the present invention has the following advantages and effects: The present invention provides a headspace solid phase microextraction method for rapidly identifying Dalbergia odorifera and Dalbergia cochinchinensis seedlings, which preferably avoids the problem of high similarity of Dalbergia odorifera and Dalbergia cochinchinensis in leaf morphology, makes full use of the difference in the composition of volatile components of Dalbergia odorifera and Dalbergia cochinchinensis in leaves, and thoroughly solves the problem that it is difficult to accurately and rapidly identify Dalbergia odorifera and Dalbergia cochinchinensis seedlings by traditional means such as leaf appearance. Compared with the traditional identification methods, the method has the advantages of simple operation, small sampling amount, short time consumption, and high accuracy, and is particularly suitable for the rapid and accurate identification of some famous trees and ancient trees. The present invention will provide an efficient and reliable method for rapidly identifying Dalbergia odorifera and Dalbergia cochinchinensis seedlings for seedling trading, famous tree and ancient tree protection, timber trading, timber dealers, consumers, public security organs, customs and scientific research institutions, etc., which has strong application value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the total ion current chromatogram of the volatile components of the leaves of sample 1; among them, 1: Benzyl alcohol; 2: Phenylethyl alcohol; 3: Methyl salicylate; 4: β-Bisabolene.

[0019] Figure 2 This is the total ion current chromatogram of the volatile components of the leaves of sample 2; among them, 1: 1-(1-Methyl-2-cyclopentenyl)ethanone; 2: 3,5-Octadien-2-one; 3: α-Ionone; 4: β-Ionone. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. The experimental methods in the following examples where specific experimental conditions are not specified are usually carried out under conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents, etc. used are reagents and materials obtained from commercial sources unless otherwise specified.

[0021] Example 1 In November 2023, 5 10-year-old Dalbergia trees of the same source and good growth condition were randomly selected in the planting base. Five mature and strong branches from the inner, middle and outer layers of the upper and middle crowns were cut from the southeast, northwest, northeast and southwest directions respectively with high-branch shears. All the leaves were removed and mixed evenly according to the tree species, then put into kraft paper bags, numbered and preserved, and quickly brought back to the laboratory for processing; the leaves of Dalbergia odorifera were recorded as sample 1; the leaves of Dalbergia cochinchinensis were recorded as sample 2.

[0022] Take Dalbergia odorifera or Dalbergia cochinchinensis leaf sample 1 and sample 2 to identify the authenticity. The identification is carried out according to the steps of the present invention, and the specific steps are as follows: 1. Sampling and pretreatment of leaf samples: Select 20 g of mature leaves from sample 1 and sample 2 respectively, wash them with sterile water and dry them with filter paper, dry them in an oven at 105 °C for 1 h, and then dry them at 70 °C for 12 h to constant weight, grind them with a high-speed grinder, and pass them through a 60-mesh sieve.

[0023] 2. Aging of the extraction fiber head: The extraction fiber head (50 / 30μm DVB / CAR / PDMS extraction head, Shanghai Anpu) was inserted into the gas chromatography-mass spectrometry (GC-MS) for aging. The aging condition was 260 °C for 30 min.

[0024] 3. Extraction of volatile components: Take 0.5 g of the sample after screening in step 1 and put it into a 15 mL PE headspace injection bottle (Shanghai Anpu), equilibrate at a constant temperature of 70 ℃ for 30 min, and insert the extraction fiber head aged in step 2 into the headspace part of the injection bottle for extraction. The extraction time is 30 min. After the extraction is completed, the component composition is analyzed; set up 3 replicates for each sample.

[0025] 4. Composition analysis of volatile components: After the extraction in step 3, the extraction fiber head was taken out and quickly inserted into the gas chromatography-mass spectrometry (GC-MS) for detection. The chromatographic detection and analysis conditions were as follows: Chromatographic column: HP-5 MS, 30 m×0.25 mm×0.25 μm. Heating program: Initial column temperature 30 ℃, maintained for 5 min, 7 ℃·min -1 Raise to 260 °C and maintain for 5 min; injection port temperature 250 °C, carrier gas helium, flow rate 1.0 mL min-1 ; Splitless injection, injection volume 1.0 μL. Mass spectrometry conditions: EI ion source, electron energy 70 eV, ion source temperature 230 ℃, quadrupole temperature 150 ℃, GC / MS interface temperature 280 ℃, mass scanning range (m / z): 50~550 amu. After detection, the total ion current chromatogram of essential oil components was obtained (see Figure 1 , Figure 2 ), and matched with the standard mass spectrum library (NIST20); 5. Qualitative identification of ingredients: According to the matching search of mass spectra, the main characteristic components of leaf volatiles were obtained, and then the relative percentage of each peak area (i.e. relative percentage content) was calculated by area normalization method. Through the search, the main component names and corresponding relative percentage contents of the volatile components of the leaves of sample 1 are methyl salicylate (14.52±2.25%), benzyl alcohol (11.64±1.12%), β-bisabolene (6.08±0.95%) and phenylethanol (5.52±0.95%); the sum of the relative percentage contents of the four main characteristic components of methyl salicylate, benzyl alcohol, β-bisabolene and phenylethanol is 37.76±3.45%>30%, which can be determined to be Dalbergia odorifera.

[0026] The main characteristic components of the volatiles in the leaves of sample 2 and their corresponding relative percentage contents were α-ionone (17.12±1.98%), 1-(1-methylcyclopent-2-en-1-yl)ethan-1-one (16.45±3.47%), (3E,5E)-octa-3,5-dien-2-one (16.34±2.18%), β-ionone (14.89±4.15%), 3,4-dimethylcyclohexanol (7.18±0.75%) and dihydroactinol (6.35±1.15%). Among them, the total relative percentage content of α-ionone, 1-(1-methylcyclopent-2-en-1-yl)ethane-1-one, (3E,5E)-octa-3,5-dien-2-one and β-ionone is 64.80±11.48%>30%, which can be determined to be Dalbergia cochinchinensis.

[0027] Identification results: Sample 1 is Dalbergia odorifera, and Sample 2 is Dalbergia cochinchinensis.

[0028] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A headspace solid phase microextraction method for rapid identification of Dalbergia odorifera and Dalbergia cochinchinensis seedlings, characterized in that: The steps include: A. Sampling and pretreatment of leaf samples: Fresh leaf samples of Dalbergia odorifera and Dalbergia cochinchinensis were collected during the growing season, the leaves were washed, dried, withered, dried to constant weight, crushed, and sieved; B. Extraction of volatile components: Place the sample sieved in step A into a headspace injection bottle, balance at a constant temperature, and then insert the aged extraction fiber head into the headspace of the injection bottle for extraction; C. Composition analysis of volatile components: After the extraction in step B is completed, the extraction fiber head is taken out and quickly inserted into a gas chromatography-mass spectrometer for detection to obtain the total ion current chromatogram and mass spectrum data of the volatile components, and each component is matched and searched with a standard mass spectrum library; D. Qualitative identification of characteristic components: Spectrum matching retrieval was performed to obtain the main characteristic components, and the relative percentage content of the main characteristic components was calculated using the area normalization method. Then, Dalbergia odorifera and Dalbergia cochinchinensis were identified based on the types and relative percentage content ranges of the main characteristic components; In step C, the chromatographic detection and analysis conditions are as follows: chromatographic column: HP-5 MS, 30 m×0.25 mm×0.25 μm; temperature program: initial column temperature 30 °C, maintained for 5 min, at 7 °C·min -1 Raise to 260 °C and maintain for 5 min; injection port temperature 250 °C, carrier gas helium, flow rate 1.0 mL min -1 ; Splitless injection, injection volume 1.0 μL; Mass spectrometry conditions: EI ion source, electron energy 70 eV, ion source temperature 230 ℃, quadrupole temperature 150 ℃, GC / MS interface temperature 280 ℃, mass scan range: 50~550 amu; In step D, the following steps are included: a. Identification of Dalbergia odorifera: The main characteristic components are methyl salicylate, benzyl alcohol, β-bisabolene and phenylethyl alcohol; when the proportion of these four main characteristic components in the total volatile components exceeds 30%, it is identified as Dalbergia odorifera; b. Identification of Dalbergia cochinchinensis: The main characteristic components are α-ionone, 1-(1-methylcyclopent-2-en-1-yl)ethane-1-one, (3E,5E)-octa-3,5-dien-2-one and β-ionone. When the proportion of these four main characteristic components in the total volatile components exceeds 30%, it is determined to be Dalbergia cochinchinensis.

2. The headspace solid phase microextraction method for rapid identification of Dalbergia odorifera and Dalbergia cochinchinensis seedlings according to claim 1, characterized in that: In step A, the leaf samples are mature leaves.

3. The headspace solid phase microextraction method for rapid identification of Dalbergia odorifera and Dalbergia cochinchinensis seedlings according to claim 1 or 2, characterized in that: In step A, the fixing is carried out at 100-110° C. for 30-60 min.

4. The headspace solid phase microextraction method for rapid identification of Dalbergia odorifera and Dalbergia cochinchinensis seedlings according to claim 1 or 2, characterized in that: In step A, the drying is carried out at 70-80°C.

5. The headspace solid phase microextraction method for rapid identification of Dalbergia odorifera and Dalbergia cochinchinensis seedlings according to claim 1 or 2, characterized in that: In step A, the sieving is through a 60-80 mesh sieve.

6. The headspace solid phase microextraction method for rapid identification of Dalbergia odorifera and Dalbergia cochinchinensis seedlings according to claim 1 or 2, characterized in that: In step A, the washing is washing with sterile water; the drying is drying with filter paper; and the pulverizing is pulverizing with a high-speed pulverizer.

7. The headspace solid phase microextraction method for rapid identification of Dalbergia odorifera and Dalbergia cochinchinensis seedlings according to claim 1 or 2, characterized in that: In step B, the extraction fiber head is aged using a gas chromatography-mass spectrometer.

8. The headspace solid phase microextraction method for rapid identification of Dalbergia odorifera and Dalbergia cochinchinensis seedlings according to claim 1 or 2, characterized in that: In step B, the constant temperature balance condition is 70-80°C for 20-30 min.

9. The headspace solid phase microextraction method for rapid identification of Dalbergia odorifera and Dalbergia cochinchinensis seedlings according to claim 8, characterized in that: In step B, the constant temperature equilibrium condition is 70° C. for 30 min.

10. The headspace solid phase microextraction method for rapid identification of Dalbergia odorifera and Dalbergia cochinchinensis seedlings according to claim 1 or 2, characterized in that: In step B, the extraction time is 20 to 30 min.

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