阅读说明：本技术 一种工业***花叶中***色烯（cbc）含量的测定方法 (Method for determining content of cannabichromene (CBC) in industrial cannabis sativa leaves ) 是由 王钲霖 刘胜贵 马海悦 付彬彬 李智高 孔令羽 于 2019-11-29 设计创作，主要内容包括：本发明涉及检测技术领域,特别涉及一种工业大麻花叶中大麻色烯(CBC)的定量检测方法。本方法包括以下步骤：将干燥后的大麻花叶粉碎,用甲醇进行超声提取,离心后收集上清液,残渣重复用甲醇超声提取一次,离心合并上清液,浓缩定容得到待测液,用高效液相色谱仪对待测液进行检测。高效液相色谱仪的流动相为水和乙腈,等度洗脱。采用外标法定量得到待测液中大麻色烯(CBC)的含量。本方法操作简单、分离度好,使大麻花叶中的大麻色烯(CBC)快速得到准确定量。(The invention relates to the technical field of detection, in particular to a quantitative detection method of cannabichromene (CBC) in industrial cannabis sativa leaves. The method comprises the following steps: pulverizing dried hemp flower and leaf, ultrasonic extracting with methanol, centrifuging, collecting supernatant, repeatedly ultrasonic extracting residue with methanol once, centrifuging, mixing supernatants, concentrating to desired volume to obtain solution, and detecting with high performance liquid chromatograph. The mobile phase of the high performance liquid chromatograph is water and acetonitrile, and isocratic elution is carried out. And (3) quantitatively obtaining the content of the cannabichromene (CBC) in the liquid to be detected by adopting an external standard method. The method is simple to operate and good in separation degree, and enables the cannabichromene (CBC) in the cannabis sativa leaves to be rapidly and accurately quantified.)

1. A method for measuring the content of cannabichromene (hereinafter referred to as CBC) in industrial cannabis sativa leaves is characterized by comprising the following steps:

(1) drying a fresh industrial hemp flower and leaf sample in an oven at 60 ℃, crushing by a crusher, and uniformly mixing to be tested;

(2) accurately weighing a flower and leaf sample in a centrifuge tube, adding an organic solvent, carrying out ultrasonic extraction, centrifuging to collect supernatant, repeatedly carrying out ultrasonic extraction on residues once, centrifuging, combining supernatants, and concentrating to a constant volume to obtain a solution to be detected;

(3) detecting a liquid to be detected by using a high performance liquid chromatograph, wherein a mobile phase of the high performance liquid chromatograph is water and acetonitrile, and isocratic elution is carried out;

(4) quantification using external standard: preparing series of reference solutions with CBC standard reference, and measuring content of CBC in the hemp flower and leaf solution by external standard method.

2. The assay method according to claim 1, wherein the hemp flower leaves are a leaf and bud composition of mature industrial hemp picked in 9 months, dried at 60 ℃ until the moisture is less than 5%, and pulverized to 20 mesh by a pulverizer.

3. The determination method according to claim 1, wherein the organic solvent is a chromatographic pure solvent, and is one or a mixture of two of methanol, ethanol, n-hexane and ethyl acetate, and the mass ratio of the volume amount of the organic solvent to the mosaic sample is 5-20: 1.

4. The method according to claim 1, wherein the ultrasonic time is 5 to 30min, the ultrasonic power is 250W, and the operating frequency is 40 kHz.

5. The method according to claim 1, wherein the centrifugation is performed at a rotation speed of 5000 to 10000 rpm for 5 to 10 minutes.

6. The assay method of claim 1, wherein the high performance liquid chromatograph is brand model number agilent 1260; the mobile phase acetonitrile is chromatographically pure, the water is deionized water, and the proportion of the acetonitrile to the water is (60% -80%): (20% -40%), isocratic elution is carried out, and the flow rate is 0.8 mL/min-1.2 mL/min; the sample injection amount of the high performance liquid chromatograph is 8 uL-12 uL; the column temperature of the high performance liquid chromatograph is 20-40 ℃; the detection wavelength of the high performance liquid chromatograph is 210 nm-230 nm.

7. The method according to claim 1, wherein the CBC standard control solution is purchased from Sigma company, usa, and prepared into a series of control solutions, and subjected to high performance liquid chromatography for detection, and a standard curve is drawn by using the substance concentration as abscissa and the peak area as ordinate.

Technical Field

The invention relates to the technical field of detection, and particularly relates to a method for determining content of cannabichromene (CBC) in industrial cannabis sativa leaves.

Background

The industrial hemp refers to the hemp with the content of Tetrahydrocannabinol (THC) lower than 0.3 percent, the industrial hemp is called hemp in China, the industrial hemp is an annual herbaceous plant in the cannabis genus of the cannabinaceae family, the main active ingredient of the hemp is a cannabinoids compound, and at present, 70 natural cannabinoids are known.

Among them, cannabichromene (hereinafter abbreviated as CBC) is the most common cannabinoid next to Cannabidiol (CBD) and Tetrahydrocannabinol (THC), is a non-psychoactive ingredient, and has good medical value: the CBC can be used as an antibiotic, has good antibacterial performance and shows bactericidal activity on methicillin-resistant staphylococcus aureus (MR-SA); CBC has light to moderate resistance to some yeasts, filamentous fungi, dermatophytes and other fungi; CBC has good anti-inflammatory effect, and research in 2010 finds that CBC is combined with THC to achieve better anti-inflammatory effect; after the CBC and the THC and other cannabinoids act together, the CBC also shows the fighting capacity for resisting breast cancer, and the anti-tumor effect of the CBC is not obvious as that of the CBD, the THC and the CBG, but the CBC is combined together to form a strong anti-tumor combination body; in addition, CBC also has the functions of resisting depression and promoting the neogenesis of brain cells, and has the medicinal value of reducing the risk of senile dementia and the like.

Currently, the medical research on industrial cannabis is a new focus, and although the research on CBC is less than that on CBD, the current research shows that CBC also has higher medical value. In some hemp plants, the content of CBC is higher than that of CBD, so that the accurate determination of the content of cannabichromene in the flower and leaf raw materials is very important. However, there is no authoritative cannabichromene detection standard, so it is necessary to develop a detection method capable of accurately determining the content of cannabichromene in industrial cannabis sativa leaves.

Disclosure of Invention

According to the method, the cannabichromene (CBC) in the industrial cannabis sativa leaves is extracted by using methanol ultrasonic, the detection is carried out by using a high performance liquid chromatograph, the quantification is carried out by an external standard method, and the content of the cannabichromene (CBC) in the cannabis sativa leaves can be accurately and rapidly detected.

In order to achieve the purpose, the technical scheme of the invention is as follows:

(1) the invention provides a method for measuring content of cannabichromene (CBC) in industrial cannabis sativa leaves, which comprises the following steps:

① drying fresh industrial hemp flower and leaf sample in oven at 60 deg.C for 12 hr (until water content is less than 5%), pulverizing to 20 mesh, and mixing.

② accurately weighing the flower and leaf sample in a centrifuge tube, adding organic solvent, ultrasonic extracting, centrifuging, collecting supernatant, repeatedly ultrasonic extracting the residue once, centrifuging, mixing the supernatants, concentrating, and filtering to desired volume to obtain the final product.

③ detecting the liquid to be detected by high performance liquid chromatograph, wherein the mobile phase of the high performance liquid chromatograph is water and acetonitrile, and the liquid is eluted at equal rate.

④ and quantifying by external standard method, which comprises preparing series of control solutions with CBC standard control, and measuring CBC content in hemp flower and leaf solution to be measured by external standard method.

(2) Further, the hemp flower leaves in step ① are a leaf and bud composition of mature industrial hemp harvested at 9 months.

(3) Further, in the step ②, the organic solvent is a chromatographic pure solvent, and is one or a mixture of methanol, ethanol, n-hexane and ethyl acetate, and the mass ratio of the volume amount of the organic solvent to the flower and leaf sample is 5-20: 1.

(4) Preferably, the organic solvent in step ② is chromatographically pure methanol, and the ratio of methanol volume to cannabis sativa leaf mass is 10: 1.

(5) Further, in the step ②, the ultrasonic time is 5 min-30 min.

(6) Preferably, the ultrasonic time in step ② is 20min, the ultrasonic power is 250W, and the working frequency is 40 kHz.

(7) Further, in the step ②, the centrifugal rotation speed is 5000-10000 rpm, and the time is 5-10 minutes.

(8) Preferably, the centrifugation speed in step ② is 10000 rpm, and the centrifugation time is 5 minutes.

(9) Further, in the step ③, the brand model of the high performance liquid chromatograph is Agilent 1260, the mobile phase acetonitrile is chromatographically pure, the water is deionized water, the ratio of acetonitrile to water is (60% -80%) (20% -40%), isocratic elution is performed, the flow rate is 0.8 mL/min-1.2 mL/min, the sample injection amount of the high performance liquid chromatograph is 8 uL-12 uL, the column temperature of the high performance liquid chromatograph is 20-40 ℃, and the detection wavelength of the high performance liquid chromatograph is 210 nm-230 nm.

(10) Preferably, the ratio of the acetonitrile to the water in the step ③ is 65% to 35%, the flow rate is 1mL/min, the sample amount is 10uL, the chromatographic column is Agilent ZORBAX Eclipse Plus C18, the column temperature is 30 ℃, and the detection wavelength is 220 nm.

(11) Further, the CBC standard control solution in step ④ is purchased from Sigma company, usa, and the CBC standard solution with a concentration of 1mg/mL is diluted with chromatographically pure methanol to a series of control solutions of 1ug/mL, 2ug/mL, 4ug/mL, 8ug/mL, 10ug/mL, 20ug/mL, 40ug/mL, and 100ug/mL, respectively, and then the sample is subjected to high performance liquid chromatography under the above mentioned instrument conditions, and a standard curve is drawn with the substance concentration as abscissa and the peak area as ordinate.

The invention has the advantages that:

① the method uses methanol to carry out ultrasonic extraction of the cannabichromene (CBC) in the marihuana leaves, and has simple experimental operation, short processing time and accurate and stable detection result.

② the invention uses deionized water and acetonitrile as mobile phase, and has good peak shape and separation degree of the target object.

Drawings

FIG. 1 chromatogram of a standard control of cannabichromene (CBC).

FIG. 2 Cannabinochromene (CBC) standard curve.

FIG. 3 example 2 Cannabis mosaic sample chromatogram.

FIG. 4 example 3 Cannabis mosaic sample chromatogram.

FIG. 5 example 4 Cannabis sativa flower and leaf sample chromatogram.

Detailed Description

The following examples are merely illustrative of the present invention and do not limit the scope of the present invention in any way. It will be apparent to those skilled in the art that equivalent embodiments or modifications without departing from the technical spirit of the present invention are within the scope of the present invention.