Preparation of terpene phenolic acid substances in hemp and its application in anti-ultraviolet radiation

Abstract

The invention discloses a method for preparing terpene phenolic acid substances in hemp, and belongs to the field of extraction and separation of plant secondary metabolites. The method uses methanol as a solvent, adopts ultrasonic-assisted extraction to obtain an extract, and then concentrates, separates and purifies by silica gel column chromatography and UPLC-MS / MS technology. The obtained terpene phenolic acid substances can effectively protect plant leaves from ultraviolet damage without affecting the normal growth state of the plant, and can also be used to improve the anti-ultraviolet performance of cosmetics or chemical materials, so it has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of extraction and separation of plant secondary metabolites, and specifically relates to the preparation and application of terpene phenolic acid substances in hemp. Background Art

[0002] Sunlight is the main source of energy on Earth. Ultraviolet light has a short wavelength and high energy, so it has a great impact on organisms after reaching the ground. Ultraviolet radiation can cause DNA damage, membrane lipid oxidation, damage to the electron transport chain to produce reactive oxygen, destroy the photosynthesis system, and destroy biological macromolecules. It usually leads to genetic mutations and other physiological responses, which ultimately lead to a reduction in biomass and even cell death. Ultraviolet light can be divided into three categories according to its wavelength: UV-A (315-400 nm), UV-B (280-315 nm) and UV-C (200-280 nm). From the perspective of its biological effects, UV-A is generally non-lethal and is rarely absorbed by ozone, so it is a weak effect wave. UV-B can destroy biological macromolecules, and most of it can be absorbed by the ozone layer. About 10% of UV-B will reach the ground, which is a strong effect wave. Studies have confirmed that heterologous secondary metabolites can cause changes in plant cell membrane lipids, resulting in changes in cell membrane permeability, thereby destroying the basic functions of the membrane, leading to plant structural function and metabolic disorders and aging. Therefore, there are currently no reports on the application of heterologous secondary metabolites in plant UV resistance.

[0003] Terpene phenolic acid compounds are a special class of endogenous secondary metabolites of hemp plants, which are mainly divided into three categories: plant terpene phenolic acid compounds, endogenous terpene phenolic acid compounds and synthetic terpene phenolic acid compounds. Among them, the most well-known plant terpene phenolic acid compounds are tetrahydrocannabinol (THC) and cannabidiol (CBD). Previous studies have shown that plant terpene phenolic acid compounds can be used to treat various diseases, including inflammation, Parkinson's disease, diabetes, obesity, autism and several skin diseases. Tetrahydrocannabinolic acid (THCA) is the acidic precursor of THC. In plants, it is synthesized from its precursor cannabigerolic acid (CBGA) under the catalysis of the oxidative cyclase THCAS. However, in addition to its medicinal value, the UV absorption properties of terpene phenolic acid compounds and their applications in UV protection have rarely been reported. Summary of the invention

[0004] In order to solve the shortcomings and deficiencies in the prior art that it is impossible to effectively protect plant leaves against ultraviolet damage without affecting the normal growth state of the plants, the present invention provides a method for preparing terpene phenolic acid substances with ultraviolet absorption ability extracted from hemp, which is convenient and efficient.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A method for preparing terpene phenolic acid substances in hemp comprises the following steps: grinding hemp tissue, performing ultrasonic-assisted extraction with methanol at a volume of 1 L / 100 g, and leaving the mixture overnight; concentrating the obtained supernatant to dryness, performing separation and purification by silica gel column chromatography, and further performing separation by UPLC-MS / MS technology to obtain the terpene phenolic acid substances.

[0007] Furthermore, the hemp tissue includes hemp flowers.

[0008] Furthermore, the ultrasonic power of the ultrasonic-assisted extraction is 40 KHz and the time is 30 min.

[0009] Furthermore, the silica gel used in the silica gel column chromatography has a specification of 15-25 μm, 120 The eluent used was a methanol / water mixed solution, and the elution procedure was 100 vol% methanol elution for 30 min, 75 vol% methanol elution for 30 min, 50 vol% methanol elution for 30 min, 25 vol% methanol elution for 30 min, and pure water elution for 30 min.

[0010] Furthermore, the UPLC-MS / MS technology uses a YMC Pack ODS-A chromatographic column with a pore size of 12 nm, a particle size of 5 μm, and an injection volume of 1 μL; the mass spectrometry analysis uses a negative mode electrospray ionization (ESI-) mode with a capillary voltage of 2.80 kV; the cone gas is 50 L / h, the desolvation gas is 600 L / h, the source temperature is 120 ° C, and the desolvation temperature is 380 ° C; the scan time is 0.1 s, and the collision energy is 25 kV.

[0011] Furthermore, the obtained terpene phenolic acid substances include tetrahydrocannabinolic acid (THCA, ), cannabichromene acid (CBCA, ) and cannabidiolic acid (CBDA, ) at least one of the following.

[0012] The obtained terpene phenolic acid substance has the ability to absorb ultraviolet rays. When mixed with methanol and applied to the surface of plant leaves, it can protect the leaves from ultraviolet radiation and maintain the normal growth state of the plants. At the same time, the obtained terpene phenolic acid substance can also be added as an additive to cosmetics or chemical materials to improve the anti-ultraviolet radiation performance of the cosmetics or chemical materials.

[0013] The significant advantages of the present invention are:

[0014] The present invention extracts a terpene phenolic acid substance with ultraviolet absorption ability from hemp for the first time. The extraction method is simple. The obtained terpene phenolic acid substance can effectively protect plant leaves from ultraviolet damage without affecting the normal growth state of the plant. The terpene phenolic acid substance can also be used to improve the anti-ultraviolet performance of cosmetics or chemical materials, and thus has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The mass spectrum (a) and mass spectrum ion fragmentation peak diagram (b) of the terpene phenolic acid compound obtained in Example 1.

[0016] Figure 2 This is a high performance liquid chromatogram of the terpene phenolic acid compound THCA obtained in Example 1.

[0017] Figure 3 This is the UV absorption diagram of THCA, CBCA and CBDA in Example 2.

[0018] Figure 4 The phenotype of the terpene phenolic acid compound THCA obtained in Example 1 applied to the leaves of the moss plant Marchantia polyphylla and then treated with ultraviolet light (a), the results of the determination of the active oxygen level of the leaves (b), and the results of the determination of the photosynthetic rate of the leaves (c).

[0019] Figure 5 The phenotype of the terpene phenolic acid compound THCA obtained in Example 1 after being applied to the leaves of the monocot rice plant and then treated with ultraviolet light (a), the phenotype of nitroblue tetrazolium NBT staining (b) and the result of leaf photosynthetic rate measurement (c).

[0020] Figure 6 The phenotype of the terpene phenolic acid compound THCA obtained in Example 1 after being applied to the leaves of the monocotyledonous plant corn and then treated with ultraviolet light (a), the phenotype of nitroblue tetrazolium NBT staining (b) and the result of leaf photosynthetic rate measurement (c).

[0021] Figure 7 The phenotype of the terpene phenolic acid compound THCA obtained in Example 1 applied to the leaves of the dicotyledonous plant Arabidopsis thaliana and then treated with ultraviolet light (a), the phenotype of nitroblue tetrazolium NBT staining (b) and the result of leaf photosynthetic rate measurement (c).

[0022] Figure 8 The phenotype of the terpene phenolic acid compound THCA obtained in Example 1 after being applied to dicotyledonous tobacco leaves and then treated with ultraviolet light (a), the phenotype of nitroblue tetrazolium NBT staining (b) and the result of leaf photosynthetic rate measurement (c).

[0023] Fig. 9The phenotype of the terpene phenolic acid compound THCA obtained in Example 1 after being applied to dicotyledonous tomato leaves and then treated with ultraviolet light (a), the phenotype of nitroblue tetrazolium NBT staining (b) and the result of leaf photosynthetic rate measurement (c).

[0024] Fig.10 The phenotype of the terpene phenolic acid compound THCA obtained in Example 1 applied to dicotyledonous soybean leaves and then treated with ultraviolet light (a), the phenotype of nitroblue tetrazolium NBT staining (b) and the result of leaf photosynthetic rate measurement (c). DETAILED DESCRIPTION

[0025] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0026] Example 1 Extraction of terpene phenolic acid substances in hemp

[0027] 1) Crush 100 g of fresh hemp flower tissue, soak in 1 L of methanol for 2 h, then use 40 KHz ultrasonic assisted extraction for 30 min and soak overnight;

[0028] 2) Collect the resulting supernatant into a 500 mL beaker and concentrate using a rotary evaporator;

[0029] 3) The obtained concentrate was precipitated on a silica gel column (the silica gel column specification was 12-25 μm, 120 ) and gradient elution was performed using methanol / water. The elution procedure was 100 vol% methanol for 30 min, then reduced to 75 vol%, eluted for 30 min, then reduced to 50 vol%, eluted for 30 min, then reduced to 25 vol%, eluted for 30 min, and finally eluted with pure water for 30 min.

[0030] 4) The collected eluent was detected by UPLC-MS / MS technology, that is, YMC Pack ODS-A column (column specifications: pore size 12 nm, particle size 5 μm) was used for ultra-high phase liquid chromatography separation, and the sample injection volume was 1 μL; mass spectrometry analysis was performed in negative mode electrospray ionization (ESI-) mode, and the capillary voltage was 2.80 kV. The cone gas was 50 L / h, the desolvation gas was 600 L / h, the source temperature was 120 ℃, and the desolvation temperature was 380 ℃; the data-dependent MS / MS model was used, the scan time was 0.1 s, and the collision energy was 25 kV. UNIFI software was used to collect UPLC-MS / MS data, and the retention time and reference mass spectrum were used for identification using authentic standards, and then the products were collected in 50 mL centrifuge tubes. The results confirmed that the terpene phenolic acid compounds obtained were tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA).

[0031] Figure 1 The mass spectrometry diagram of the obtained terpene phenolic acid compounds THCA, CBCA and CBDA is shown in the figure. As can be seen from the figure, the obtained compounds are THCA, CBCA and CBDA.

[0032] Figure 2 The HPLC chromatogram of the obtained terpene phenolic acid compound THCA is shown in Figure 1. As can be seen from the figure, the purity of the obtained THCA is 95.12%.

[0033] Example 2 Ultraviolet absorption capacity test

[0034] The ultraviolet absorption capacity of the terpene phenolic acid substances obtained in Example 1 was detected, and the detection method was as follows: the obtained standard stock solutions of THCA, CBDA and CBCA were diluted to 1 μM with methanol, and detected by UPLC-PDA; the detection conditions were: reverse phase column (2.1×100 mm, 1.7 μm, Waters Acquity UPLC Shield RP18), temperature of 40°C, and monitored by AcquityUPLC photodiode array detector; a binary solvent system consisting of methanol containing 0.1% formic acid (solvent A) and ultrapure water containing 0.1% formic acid (solvent B) was used for separation, and the elution gradient was as follows: solvent A was linearly eluted from 50% to 80% over 3 minutes, then increased to 100% over 11 minutes, and maintained for 1 minute, then decreased to 50% over 2 minutes, and then balanced for 3 minutes.

[0035] Figure 3The UV absorption diagram of THCA, CBCA and CBDA. It can be seen from the figure that the three terpene phenolic acid compounds THCA, CBCA and CBDA all have UV absorption ability, and the UV absorption ability of THCA in the UV-B band is stronger than that of the other three terpene phenolic acid compounds.

[0036] Example 3

[0037] The terpene phenolic acid substance THCA obtained in Example 1 was combined with methanol and applied to the surface of the moss plant liverwort, monocot rice, corn and dicot Arabidopsis, soybean, tomato and tobacco to investigate its role in protecting plant leaves from ultraviolet radiation. The specific operation is: 1, 2.5 and 4 mg of the terpene phenolic acid substance THCA were weighed, dissolved in 1 mL of 70% (v / v) methanol solution, mixed to obtain sample solutions of corresponding concentrations, and 200 μL of the above solution was pipetted and evenly coated on the leaf surfaces of the moss plant liverwort, monocot rice, corn and dicot Arabidopsis, soybean, tomato and tobacco, and 70% methanol solution was used as a blank control, and then various plants were exposed to UV-B lamp (wavelength 280 nm-320nm, intensity 53.6 mW·cm -2 ) were used for irradiation treatment, and the degree of leaf damage was measured by measuring the leaf photosynthetic rate and nitroblue tetrazolium (NBT) staining.

[0038] The Arabidopsis, tobacco, tomato, soybean, rice, and corn used were grown from seeds in soil under the following conditions: temperature of 21 ± 2 °C, photoperiod of 16 / 8 h, relative humidity of 60%, and white light intensity of 200 μmol·m -2 ·s -1 The female strain of Marchantia polymorpha Takaragaike-1 (Tak-1) was cultured on sterile half-strength Gamborg's B5 solid medium (Gamborg et al., 1968) containing 2% agar at 22°C and a white light intensity of 100 μmol m -2 s -1 The cells were cultured under the condition of 16 / 8 h photoperiod. The intensity and quality of visible light were measured by HR-350 spectrometer (HiPoint, Taiwan, China), and the intensity of wide-spectrum UV-B radiation (280 nm-320 nm) was measured by UV-254 UV-B ultraviolet radiometer (Beijing Normal University Optoelectronic Instrument Factory).

[0039] The NBT staining method follows the following steps: weigh 0.066 g NBT, dissolve it in 50 mL 50 mM phosphate buffer (pH = 7.5), and prepare a 0.15% NBT staining working solution; take the treated fresh plant leaves and put them in a 15 mL centrifuge tube, add NBT staining working solution to fully immerse the leaves; let the staining stand for 12 hours, discard the staining working solution, add anhydrous ethanol, and heat in a water bath at 80 °C to remove chlorophyll; after decolorization, immerse in 60% glycerol solution and wait for the tissue to soften, and take pictures.

[0040] Figure 4 The phenotype of the terpene phenolic acid compound THCA applied to the leaves of the moss plant Marchantia polyphylla after ultraviolet treatment (a), the results of the determination of the active oxygen level of the leaves (b) and the results of the determination of the photosynthetic rate of the leaves (c). As can be seen from the figure, in terms of phenotype, with the increase of the concentration of the terpene phenolic acid solution, the growth state of the moss plant Marchantia polyphylla after high-intensity UV-B irradiation is better, and the growth state of the Marchantia polyphylla applied with a high concentration of 4 mg / mL terpene phenolic acid is the best; the determination of the photosynthetic rate of the leaves found that with the increase of the concentration of the terpene phenolic acid solution, the photosynthetic rate of the leaves of the Marchantia polyphylla after high-intensity UV-B irradiation decreased more slowly; the determination of the active oxygen (ROS) level of the leaves found that the ROS level of the leaves applied with a high concentration of 4 mg / mL terpene phenolic acid was significantly lower than that of other treatments, and was close to the ROS level of the healthy Marchantia polyphylla leaves.

[0041] Figure 5 The phenotype of THCA compound applied to monocot rice leaves after ultraviolet treatment (a), nitro blue tetrazolium NBT staining phenotype (b) and leaf photosynthetic rate measurement results (c). As can be seen from the figure, in terms of phenotype, with the increase of the concentration of terpene phenolic acid solution, the growth state of rice after high-intensity UV-B irradiation is better, and the growth state of rice with 4 mg / mL high concentration of terpene phenolic acid is the best; the photosynthetic rate measurement of leaves found that with the increase of the concentration of terpene phenolic acid solution, the photosynthetic rate of rice leaves after high-intensity UV-B irradiation decreases more slowly; NBT staining of leaves found that the blue-black precipitation area of ​​NBT in leaves with 4 mg / mL high concentration of terpene phenolic acid is significantly smaller than that of other treatments.

[0042] Figure 6The phenotype of THCA applied to the leaves of monocot maize after ultraviolet treatment (a), nitro blue tetrazolium NBT staining phenotype (b) and leaf photosynthetic rate measurement results (c). As can be seen from the figure, in terms of phenotype, with the increase of the concentration of terpene phenolic acid solution, the growth state of maize after high-intensity UV-B irradiation is better, and the growth state of maize with 4 mg / mL high concentration of terpene phenolic acid is the best; the photosynthetic rate measurement of the leaves found that with the increase of the concentration of terpene phenolic acid solution, the photosynthetic rate of maize leaves after high-intensity UV-B irradiation decreases more slowly; the leaves were stained with NBT and found that the blue-black precipitation area of ​​NBT in the leaves with 4 mg / mL high concentration of terpene phenolic acid was significantly smaller than that in other treatments.

[0043] Figure 7 The phenotype of the dicotyledonous plant Arabidopsis thaliana after the terpene phenolic acid compound THCA was applied to the leaves and then treated with ultraviolet light (a), the phenotype of nitro blue tetrazolium NBT staining (b), and the result of leaf photosynthetic rate measurement (c). As can be seen from the figure, in terms of phenotype, with the increase of the concentration of terpene phenolic acid solution, the growth state of Arabidopsis thaliana after high-intensity UV-B irradiation is better, and the growth state of Arabidopsis thaliana with a high concentration of 4 mg / mL terpene phenolic acid is the best; the photosynthetic rate measurement of the leaves found that with the increase of the concentration of terpene phenolic acid solution, the photosynthetic rate of Arabidopsis thaliana leaves after high-intensity UV-B irradiation decreased more slowly; the leaves were stained with NBT and found that the blue-black precipitation area of ​​NBT in the leaves with a high concentration of 4 mg / mL terpene phenolic acid was significantly smaller than that in other treatments.

[0044] Figure 8 The phenotype of THCA applied to dicot tobacco leaves after ultraviolet treatment (a), nitro blue tetrazolium NBT staining phenotype (b) and leaf photosynthetic rate measurement results (c). As can be seen from the figure, in terms of phenotype, with the increase of the concentration of terpene phenolic acid solution, the growth state of tobacco after high-intensity UV-B irradiation is better, and the growth state of tobacco with 4 mg / mL high concentration of terpene phenolic acid is the best; the photosynthetic rate measurement of leaves found that with the increase of the concentration of terpene phenolic acid solution, the photosynthetic rate of tobacco leaves after high-intensity UV-B irradiation decreased more slowly; NBT staining of leaves found that the blue-black precipitation area of ​​NBT in leaves with 4 mg / mL high concentration of terpene phenolic acid was significantly smaller than that of other treatments.

[0045] Fig. 9The phenotype of THCA applied to dicotyledonous tomato leaves after ultraviolet treatment (a), nitro blue tetrazolium NBT staining phenotype (b) and leaf photosynthetic rate measurement results (c). As can be seen from the figure, in terms of phenotype, with the increase of the concentration of terpene phenolic acid solution, the growth state of tomatoes after high-intensity UV-B irradiation is better, and the growth state of tomatoes applied with 4 mg / mL high concentration of terpene phenolic acid is the best; the photosynthetic rate measurement of leaves found that with the increase of the concentration of terpene phenolic acid solution, the photosynthetic rate of tomatoes after high-intensity UV-B irradiation decreased more slowly; NBT staining of leaves found that the blue-black precipitation area of ​​NBT in leaves with 4 mg / mL high concentration of terpene phenolic acid was significantly smaller than that of other treatments.

[0046] Fig.10 The phenotype of THCA compound applied to dicotyledon soybean leaves after ultraviolet treatment (a), nitro blue tetrazolium NBT staining phenotype (b) and leaf photosynthetic rate measurement results (c). As can be seen from the figure, in terms of phenotype, with the increase of the concentration of terpene phenolic acid solution, the growth state of soybean after high-intensity UV-B irradiation is better, and the growth state of soybean with 4 mg / mL high concentration of terpene phenolic acid is the best; the photosynthetic rate measurement of leaves found that with the increase of the concentration of terpene phenolic acid solution, the photosynthetic rate of soybean leaves after high-intensity UV-B irradiation decreased more slowly; NBT staining of leaves found that the blue-black precipitation area of ​​NBT in leaves with 4 mg / mL high concentration of terpene phenolic acid was significantly smaller than that of other treatments.

[0047] from Figures 4 to 10 The results shown show that the strong UV-B absorption ability of the terpene phenolic acid compound THCA can be extended to mosses, monocots and dicots, which can enhance the resistance of plant leaves to ultraviolet rays and reduce the degree of stress.

[0048] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for preparing terpene phenolic acid substances in hemp, characterized in that: The hemp tissue is crushed, and ultrasonic-assisted extraction is performed with methanol at a volume of 1 L / 100 g, and the mixture is left overnight. After the obtained supernatant is concentrated to dryness, it is separated and purified by silica gel column chromatography, and then further separated by UPLC-MS / MS technology to obtain the terpene phenolic acid substance; The silica gel column chromatography uses silica gel with specifications of 15-25 μm, 120 The eluent used was a methanol / water mixed solution, and the elution procedure was 100 vol% methanol for 30 min, 75 vol% methanol for 30 min, 50 vol% methanol for 30 min, 25 vol% methanol for 30 min, and pure water for 30 min.

2. The method for preparing terpene phenolic acid substances in hemp according to claim 1, characterized in that: The hemp tissue includes hemp flowers.

3. The method for preparing terpene phenolic acid substances in hemp according to claim 1, characterized in that: The ultrasonic power of the ultrasonic assisted extraction is 40 KHz and the time is 30 min.

4. The method for preparing terpene phenolic acid substances in hemp according to claim 1, characterized in that: The UPLC-MS / MS technology uses a YMC Pack ODS-A chromatographic column with a pore size of 12 nm and a particle size of 5 μm. The mass spectrometry analysis uses a negative mode electrospray ionization mode with a capillary voltage of 2.80 kV. The conical gas is 50 L / h, the desolvation gas is 600 L / h, the source temperature is 120 °C, and the desolvation temperature is 380 °C. The scan time is 0.1 s and the collision energy is 25 kV.

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