CN110632224B

CN110632224B - Establishment method of hemp plant fingerprint spectrum

Info

Publication number: CN110632224B
Application number: CN201910869191.8A
Authority: CN
Inventors: 占卓; 黄青; 单世斌; 苏丽辉; 黄丽婵; 赵萍
Original assignee: Fujian Sanan Sino Science Photobiotech Co Ltd
Current assignee: Fujian Sanan Sino Science Photobiotech Co Ltd
Priority date: 2019-09-16
Filing date: 2019-09-16
Publication date: 2022-02-15
Anticipated expiration: 2039-09-16
Also published as: CN110632224A

Abstract

The invention discloses a method for establishing a fingerprint of a hemp plant, which comprises the following steps: collecting inflorescence of the hemp plant, preparing a sample solution, detecting by using a high performance liquid chromatography and a diode array detector, establishing a fingerprint of the hemp plant, and analyzing the difference of main components of the hemp plant. The mobile phase detected by the high performance liquid chromatography comprises an organic phase and a water phase, wherein the organic phase is acetonitrile and gradient elution is adopted; the method has the advantages of high similarity of fingerprint spectra, good separation degree and repeatability, and chromatographic detection time within 60min, and provides a new method for evaluating the content and change of active ingredients of the hemp medicinal plants.

Description

Establishment method of hemp plant fingerprint spectrum

Technical Field

The invention relates to the technical field of traditional Chinese medicine component analysis, in particular to a method for establishing a fingerprint spectrum of a hemp plant.

Background

The traditional Chinese medicine fingerprint spectrum is a spectrum which is obtained by adopting a certain analysis means after a traditional Chinese medicine is properly processed and can mark a common peak of the characteristics of the traditional Chinese medicine. Is a spectrogram or chromatogram of the chemical components of the secondary metabolism of Chinese medicinal materials obtained by means of spectrum or chromatography. The fingerprint spectrum should have fingerprint properties, namely: (1) the specificity is strong. The finger print is unique to the medicinal materials, can be distinguished from other medicinal materials, and reflects chemical information with high selectivity; (2) the stability is good. The fingerprint spectrum of the traditional Chinese medicinal materials is the commonness induced from a plurality of batches of the traditional Chinese medicinal materials, and the common peak or the characteristic peak in the spectrum is relatively stable; (3) the reproducibility is good. The fingerprint map is prepared to reproduce fingerprint characteristics under specified conditions, and the error of the fingerprint map is within an allowable range.

At present, the fingerprint is the internationally accepted most effective method for controlling the quality of Chinese patent medicines and natural medicines. Hitherto, researchers at home and abroad mainly focus on the research on the biological fingerprint, namely the DNA fingerprint, of the hemp plant, the DNA fingerprint can only provide a basis for identifying the variety of the hemp plant and is used for evaluating the authenticity of the hemp plant, but the cultivation conditions and the like in the growth process of medicinal plants can greatly influence the chemical components of the hemp plant, so that the quality is influenced, the germplasm is far from insufficient, and a method for integrally analyzing the chemical components is provided.

Disclosure of Invention

In view of the above, the present invention provides a method for establishing a fingerprint of a hemp plant, which can provide a method for analyzing the overall chemical composition of the hemp plant.

The invention adopts the specific technical scheme that:

a method for establishing a bast fiber plant fingerprint comprises the following steps:

collecting inflorescences of the hemp plants, preparing a sample solution, detecting by using a high performance liquid chromatography and a diode array detector, and establishing fingerprint spectrums of the hemp plants;

the mobile phase detected by the high performance liquid chromatography comprises an organic phase and a water phase, the organic phase is preferably acetonitrile, gradient elution is adopted, in the gradient elution, the organic phase is used as an A phase, the water phase is used as a B phase, the mobile phase proportion is volume percentage, and the elution steps are as follows:

0-26 min, beginning: 8% -12% of mobile phase A and 88% -92% of mobile phase B, and stopping: 86-90% of mobile phase A and 10-14% of mobile phase B;

26-30 min, wherein the mobile phase A is 86-90%, and the mobile phase B is 10-14%;

30-40 min, beginning: the mobile phase A is 86% -90%, the mobile phase B is 10% -14%, and the process is terminated: 98-100% of mobile phase A and 0-2% of mobile phase B;

for 40-60 min, the mobile phase A is 98-100% and the mobile phase B is 0-2%.

For better implementation of the present invention, the flow phase ratios in the gradient elution are volume percentages:

0-26 min, beginning: mobile phase a 10%, mobile phase B90%, terminate: the mobile phase A is 88 percent, and the mobile phase B is 12 percent;

26-30 min, wherein the mobile phase A is 88% and the mobile phase B is 12%;

30-40 min, beginning: mobile phase a was 88%, mobile phase B was 12%, and terminated: the mobile phase A is 100 percent, and the mobile phase B is 0 percent;

and (3) 40-60 min, wherein the mobile phase A is 100% and the mobile phase B is 0%.

In order to better implement the invention, in the high performance liquid chromatography detection, the detection wavelength is 200-280nm, and is preferably 210 nm.

In order to better implement the invention, in the high performance liquid chromatography detection, the column temperature of the chromatographic column is 25-35 ℃, and preferably 25 ℃.

In order to better realize the invention, in the high performance liquid chromatography detection, the detection flow rate is 1.0 mL/min.

In order to better implement the invention, the sample amount in the high performance liquid chromatography detection is 10-20 μ L, and preferably 15 μ L.

In order to better implement the invention, the preparation method of the sample solution comprises the following steps: collecting inflorescence of hemp plant, decarboxylating at 100 deg.C, pulverizing, sieving, precisely weighing 0.1g powder, adding 15mL methanol, ultrasonic extracting, centrifuging at 4000r, and diluting the filtrate by 5 times to obtain sample solution.

In order to better implement the invention, the preparation method of the sample solution comprises the following steps: collecting 10 batches of flowers of the same variety of hemp plants, decarboxylating at 100 ℃ for 7h, crushing, sieving by a third sieve, respectively weighing 0.1g of powder, precisely weighing, adding 15mL of methanol, performing ultrasonic extraction at 45KHz for 20min, shaking up, centrifuging at 4000r for 5min, filtering by a 0.45 mu m microporous filter membrane, and diluting the subsequent filtrate by 5 times to obtain a sample solution.

In order to better realize the invention, the chromatographic column for high performance liquid chromatography detection takes octadecylsilane chemically bonded silica as a filler.

In order to better implement the invention, the comparison substances detected by the high performance liquid chromatography are Cannabidiol (CBDV), Cannabigerol (CBG), Cannabidiol (CBD), Tetrahydrocannabinol (THCV), Cannabinol (CBN), Tetrahydrocannabinol (THC), Cannabigerol (CBL).

Compared with the prior art, the invention has the following beneficial effects:

1. the method has the advantages of high similarity of the fingerprint, good separation degree and repeatability, and chromatographic detection time within 60min, and provides a new method for evaluating the quality of the hemp medicinal plants.

2. According to the invention, through research on 10 batches of decarboxylated flowers of the same hemp medicinal plant, a common pattern of a characteristic fingerprint of the decarboxylated flowers of the hemp medicinal plant is established, 51 common peaks are determined, 7 known components in the common peaks are calibrated, the gap of an original quality control method is filled, and the application value is high.

3. The invention compares the characteristic fingerprint spectrums of decarboxylated flowers of 18 different varieties of hemp medicinal plants, respectively establishes two different fingerprint spectrum common modes according to the similarity difference, analyzes the main component difference, can provide reference for experimental research and public security criminal investigation work, and has higher application value.

4. The high performance liquid chromatography method has the advantages of high precision, good reproducibility and certain specificity.

Drawings

FIGS. 1-11 are chromatograms of different mobile phase gradients;

FIGS. 12-14 are chromatograms at different column temperatures;

FIGS. 15-18 are chromatograms of different detection wavelengths;

FIGS. 19-21 are chromatograms of different sample volumes;

FIGS. 22-23 are chromatograms of different extraction times;

FIG. 24 is a stacked chromatogram of parallel sample solutions;

FIG. 25 is a control fingerprint of 10 batches of hemp flower decarboxylated samples;

FIGS. 26-27 are control fingerprints of decarboxylated flowers of different varieties of hemp plants;

in FIGS. 25-27, 1 is CBDV, 2 is CBG, 3 is CBD, 4 is THCV, 5 is CBN, 6 is THC, and 7 is CBL.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto, and various substitutions and alterations can be made without departing from the technical idea of the present invention as described above, according to the common technical knowledge and the conventional means in the field.

Examples

Drying 7h of the hemp plant inflorescence at 100 ℃, powdering, and screening by a third sieve to establish a fingerprint spectrum determination method.

Chromatographic conditions

Weighing 0.1g of powder, precisely weighing, placing in a 15mL centrifuge tube, adding 15mL of methanol, performing ultrasonic extraction for 20min (45KHz), shaking up, centrifuging (4000r,5min), filtering with 0.45 μm microporous membrane, and diluting the filtrate by 5 times to obtain sample solution.

1. Gradient of mobile phase

The sample solutions were tested according to the chromatographic conditions of table 1 and the spectra at different elution gradients were compared to select the preferred elution gradient. The elution gradient is shown in Table 2, and the results are shown in FIGS. 1-6, and FIG. 1 is a chromatogram of gradient one; FIG. 2 is a chromatogram of gradient two; FIG. 3 is a chromatogram of gradient three; FIG. 4 is a chromatogram of gradient four; FIG. 5 is a chromatogram of gradient five; FIG. 6 is a chromatogram of gradient six; FIG. 7 is a chromatogram of gradient seven; FIG. 8 is a chromatogram of gradient eight; FIG. 9 is a chromatogram of gradient nine; FIG. 10 is a chromatogram of gradient ten; FIG. 11 is a chromatogram of gradient eleven; and the separation degree between main chromatographic peaks is better in the sixth gradient, and the detection time is shorter, so the sixth gradient is better.

TABLE 1 chromatographic conditions

TABLE 2 gradient of mobile phase

2. Column temperature

The sample solutions were measured at column temperatures of 25, 30, and 35 ℃ respectively according to the chromatographic conditions of Table 3 to select the preferred column temperature, and the results are shown in FIGS. 12-14, FIG. 12 is a chromatogram at 25 ℃; FIG. 13 is a chromatogram at 30 ℃; FIG. 14 is a chromatogram at 35 ℃. The separation degree difference between the main chromatographic peaks at the three column temperatures is small, which indicates that the influence of the column temperature on the detection result is small, so that the lower column temperature, namely 25 ℃, is selected as the best.

TABLE 3 chromatographic conditions

3. Detection wavelength

The sample solutions were measured according to the chromatographic conditions of table 4, and the chromatograms at detection wavelengths of 200, 210, 220, and 280nm were compared to select the preferred detection wavelength, and the results are shown in fig. 15-18, fig. 15 is the chromatogram at the detection wavelength of 200 nm; FIG. 16 is a chromatogram at a detection wavelength of 210 nm; FIG. 17 is a chromatogram for a detection wavelength of 220 nm; FIG. 18 is a chromatogram for a detection wavelength of 280 nm; the detection wavelength is 210nm, because the chromatographic peaks are more, the detection wavelength is 210nm better.

TABLE 4 chromatographic conditions

4. Sample volume

Sequentially injecting 10, 15 and 20 μ L of sample solution, determining according to the chromatographic conditions of Table 5 to select a preferred injection amount, and obtaining the result shown in FIGS. 19-21, wherein FIG. 19 is a chromatogram when 10 μ L of sample solution is injected; FIG. 20 is a chromatogram obtained when 15. mu.L of sample was injected; FIG. 21 is a chromatogram obtained when 20. mu.L of sample was injected; when the amount of sample is 15. mu.L, a large number of chromatographic peaks are detected, and therefore, the amount of sample is preferably 15. mu.L.

TABLE 5 chromatographic conditions

Second, the number of times of extraction

Weighing 0.1g of powder, precisely weighing, placing in a 15mL centrifuge tube, adding 15mL of methanol, performing ultrasonic extraction for 20min (45KHz), shaking, centrifuging (4000r,5min), filtering with 0.45 μm microporous membrane, diluting the filtrate by 5 times to obtain sample solution, and extracting the residue once by the same method for examining the extraction times.

The sample solutions extracted for the first and second times were measured according to the chromatographic conditions of table 6 to determine the number of times the sample needs to be extracted, and the results are shown in fig. 22-23, where fig. 22 is a chromatogram of the first extraction; FIG. 23 is a chromatogram for the second extraction; no new chromatographic peak appears in the second extraction, so that the extraction is only needed once.

TABLE 6 chromatographic conditions

Third, repeatability

Weighing 0.1g of powder, precisely weighing, placing in a 15mL centrifuge tube, adding 15mL of methanol, performing ultrasonic extraction for 20min (45KHz), shaking, centrifuging (4000r,5min), filtering with 0.45 μm microporous membrane, and diluting the filtrate by 5 times. 6 sample solutions were prepared in parallel and measured under the chromatographic conditions shown in Table 7 to examine the reproducibility of the measurement method, and the results are shown in FIG. 24 and Table 8, in which the similarity between the measurement results of the 6 sample solutions was 1, and the RSD values between the main peak areas were all less than 2.0%, and the reproducibility of the method was good.

TABLE 7 chromatographic conditions

TABLE 86 parallel sample solution chromatograms similarity to each other

	S1	S2	S3	S4	S5	S6
							S1		1.000	1.000	1.000	1.000	1.000
S2	1.000		1.000	1.000	1.000	1.000
							S3	1.000	1.000		1.000	1.000	1.000
S4	1.000	1.000	1.000		1.000	1.000
							S5	1.000	1.000	1.000	1.000		1.000
S6	1.000	1.000	1.000	1.000	1.000

Establishment of common mode of characteristic fingerprint spectrum

Firstly, collecting 10 batches of hop plant inflorescences of the same variety at different harvesting times, decarboxylating at 100 ℃ for 7 hours, powdering, sieving by a third sieve, weighing 0.1g of powder, precisely weighing, placing in a 15mL centrifuge tube, adding 15mL of methanol, performing ultrasonic extraction for 20min (45KHz), shaking up, centrifuging (4000r,5min), filtering by a 0.45 mu m microporous membrane, and diluting the subsequent filtrate by 5 times to obtain a sample solution. And (3) determining the sample solution by adopting the optimized chromatographic conditions, processing the determination result by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition), obtaining a characteristic fingerprint common mode of the decarboxylated flowers of the hemp plants, determining common peaks of the characteristic fingerprint common mode and calibrating known components. The comparison map and the calibrated known component obtained after 10 batches of hemp plant inflorescence decarboxylation sample fingerprint determination result software are processed is shown in figure 25, 51 common peaks are determined, and 7 known components are calibrated.

Collecting 18 hemp plant inflorescences of different varieties, decarboxylating at 100 ℃ for 7h, powdering, sieving by a third sieve, weighing 0.1g of powder, precisely weighing, placing in a 15mL centrifuge tube, adding 15mL of methanol, performing ultrasonic extraction for 20min (45KHz), shaking uniformly, centrifuging (4000r,5min), filtering by a 0.45 mu m microporous filter membrane, and diluting the subsequent filtrate by 5 times to obtain a sample solution. The sample solution is measured by adopting the optimized chromatographic conditions, and the measurement result is processed by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 edition).

According to the similarity result, 18 hemp plant inflorescences of different varieties are divided into two main categories, namely category A and category B. Wherein, A is

variety

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 16, 17, 18; the B types are

varieties

12, 13, 14, 15 and 16. Respectively obtaining the common mode of the characteristic fingerprint spectrum, determining common peaks and calibrating known components. The spectrum of class A is shown in FIG. 26 with 29 common peaks, and the spectrum of class B is shown in FIG. 27 with 35 common peaks.

The main component differences can be analyzed by fig. 26 and 27: the A type is mainly characterized by high THC content, wherein the THC content of the variety 16 is the highest, and the CBD content of the

varieties

2, 3, 5, 6, 7 and 9 is relatively high; class B is characterized primarily by high CBD content, with variety 15 having the highest CBD content and variety 12 having a relatively high THC content. In addition,

varieties

14 and 15 with higher CBDV content; variety 7 with higher CBG content; the

varieties

1 and 16 with higher THCV content;

varieties

1, 2, 3, 6 and 16 with higher CBN content;

varieties

11, 12, 13, 14, 15 with higher CBL content.

According to the method, the characteristic fingerprint spectrums of decarboxylated flowers of 18 different varieties of hemp plants are compared, two different fingerprint spectrum common modes are respectively established according to the similarity difference of the characteristic fingerprint spectrums, and the main component difference of the fingerprint spectrums is analyzed, so that reference can be provided for experimental research and public security criminal investigation work, and the method has high application value.

Although the embodiments have been described, once the basic inventive concept is known, other variations and modifications can be made to the embodiments by those skilled in the art, so that the above embodiments are only examples of the present invention, and not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes that can be used in the present specification or directly or indirectly applied to other related fields are encompassed by the present invention.

Claims

1. A method for establishing a bast fiber plant fingerprint is characterized by comprising the following steps:

the preparation method of the sample solution comprises the following steps: collecting inflorescence of hemp plant, decarboxylating at 100 deg.C, pulverizing, sieving, precisely weighing 0.1g powder, adding 15mL methanol, ultrasonic extracting, centrifuging at 4000r, and diluting the filtrate by 5 times to obtain sample solution;

the chromatographic column for high performance liquid chromatography detection takes octadecylsilane chemically bonded silica as a filler;

the comparison substances detected by the high performance liquid chromatography are CBDV, CBG, CBD, THCV, CBN, THC and CBL;

the mobile phase detected by the high performance liquid chromatography comprises an organic phase and a water phase, the organic phase is acetonitrile, gradient elution is adopted, in the gradient elution, the organic phase is used as an A phase, the water phase is used as a B phase, the mobile phase proportion is volume percentage, and the elution steps are as follows:

0-26 min, beginning: 8-12% of mobile phase A, 88-92% of mobile phase B,

and (4) terminating: the mobile phase A is 86-90%, and the mobile phase B is 10-14%;

30-40 min, beginning: 86-90% of mobile phase A, 10-14% of mobile phase B,

and (4) terminating: 98-100% of mobile phase A and 0-2% of mobile phase B;

the time is 40-60 min, the mobile phase A is 98-100%, and the mobile phase B is 0-2%.

2. The method for establishing the fingerprint map of the hemp plant as claimed in claim 1, wherein the flow phase ratios in the gradient elution are volume percentages:

0-26 min, beginning: 10 percent of mobile phase A and 90 percent of mobile phase B,

and (4) terminating: the mobile phase A is 88 percent, and the mobile phase B is 12 percent;

26-30 min, wherein the mobile phase A is 88% and the mobile phase B is 12%;

30-40 min, beginning: 88% of mobile phase A and 12% of mobile phase B,

and (4) terminating: the mobile phase A is 100 percent, and the mobile phase B is 0 percent;

3. The method for establishing the fingerprint of the hemp plant as claimed in claim 1 or 2, wherein the detection wavelength is 200-280nm in the HPLC detection.

4. The method for establishing the fingerprint of the hemp plant according to claim 3, wherein the detection wavelength is 210nm in the high performance liquid chromatography detection.

5. The method for establishing the fingerprint of the hemp plant according to claim 1 or 2, wherein the column temperature of a chromatographic column in the high performance liquid chromatography detection is 25-35 ℃.

6. The method for establishing the fingerprint of the hemp plant as claimed in claim 5, wherein the column temperature of the chromatographic column is 25 ℃ in the HPLC assay.

7. The method for establishing the fingerprint of the hemp plant according to claim 1 or 2, wherein the detection flow rate in the high performance liquid chromatography is 1.0 mL/min.

8. The method for establishing the fingerprint of the hemp plant according to claim 1 or 2, wherein the sample size is 10-20 uL in the high performance liquid chromatography detection.

9. The method for establishing the fingerprint of the hemp plant according to claim 8, wherein the sample size is 15 uL in the HPLC detection.

10. The method for establishing the fingerprint of the hemp plant according to claim 7, wherein the preparation method of the sample solution comprises the following steps: collecting 10 batches of flowers of the same variety of hemp plants, decarboxylating at 100 ℃ for 7h, crushing, sieving by a third sieve, respectively weighing 0.1g of powder, precisely weighing, adding 15mL of methanol, performing ultrasonic extraction at 45KHz for 20min, shaking up, centrifuging at 4000r for 5min, filtering by a 0.45 mu m microporous filter membrane, and diluting the subsequent filtrate by 5 times to obtain a sample solution.