阅读说明：本技术 一种提高甘草中查尔酮a含量的方法 (Method for improving content of chalcone A in liquorice ) 是由 李勇青 王瑛 曾江艺 周丽君 于 2021-09-22 设计创作，主要内容包括：本发明提供了一种提高甘草查尔酮A的方法,包括以下步骤：将甘草根或甘草种子苗置于含组蛋白去乙酰化酶抑制剂的培养基中培养,收集根部,提取甘草查尔酮A。本发明通过研究首次发现,使用组蛋白去乙酰化酶抑制剂处理甘草根或甘草种子苗,能有效提高甘草根部查尔酮A的含量。本发明提供的方法具有简单、快速、高效和成本低等优点,在医药等领域具有广阔的应用空间。(The invention provides a method for improving licochalcone A, which comprises the following steps: culturing Glycyrrhrizae radix or Glycyrrhrizae radix seed seedling in culture medium containing histone deacetylase inhibitor, collecting root, and extracting licochalcone A. According to the invention, the research discovers for the first time that the content of chalcone A in the root of liquorice can be effectively improved by treating the liquorice roots or liquorice seedlings with the histone deacetylase inhibitor. The method provided by the invention has the advantages of simplicity, rapidness, high efficiency, low cost and the like, and has wide application space in the fields of medicines and the like.)

1. A method for improving licochalcone A is characterized by comprising the following steps: culturing Glycyrrhrizae radix or Glycyrrhrizae radix seed seedling in culture medium containing histone deacetylase inhibitor, collecting root, and extracting licochalcone A.

2. The method of increasing licochalcone a according to claim 1, wherein the histone deacetylase inhibitor is selected from at least one of vorinostat, nicotinamide and sodium butyrate.

3. The method for increasing licochalcone A according to claim 2, wherein the concentration of vorinostat in the culture system is 80-120 μ M; and/or the concentration of nicotinamide in the culture system is 0.5-2 mM; and/or the concentration of sodium butyrate in the culture system is 0.5-2 mM.

4. The method for increasing licochalcone A according to claim 3, wherein the concentration of vorinostat in the culture system is 95-105 μ M; and/or the concentration of nicotinamide in the culture system is 0.8-1.2 mM; and/or the concentration of sodium butyrate in the culture system is 0.8-1.2 mM.

5. The method for increasing licochalcone A according to claims 1-4, wherein the cultivation time is at least 3 days.

6. The method for increasing licochalcone a according to claim 1, wherein the medium is a MS medium comprising the following components in the following concentrations: 4.43 +/-0.05 g/L MS culture medium, 20 +/-0.2 g/L cane sugar, 0.5 +/-0.01 g/L morpholine ethanesulfonic acid and pH5.7-6.0.

7. The method for enhancing licochalcone a according to claim 1, wherein the method for extracting licochalcone a comprises the following steps:

(1) grinding the collected roots of the culture with liquid nitrogen and freeze-drying to obtain a sample;

(2) according to the material-liquid ratio of 10: (0.8-1.2) mg/ml, adding methanol into the sample obtained in the step (1) for ultrasonic extraction, sucking supernatant, filtering, evaporating to dryness and concentrating to obtain licochalcone A.

8. Application of histone deacetylase inhibitor in improving licochalcone A content is provided.

9. The use according to claim 8, wherein the histone deacetylase inhibitor is selected from at least one of vorinostat, nicotinamide and sodium butyrate.

10. The use according to claim 9, wherein the vorinostat is present at a final concentration of 80-120 μ Μ; the final concentration of nicotinamide is 0.5-2 mM; the final concentration of the sodium butyrate is 0.5-2 mM;

preferably, the final concentration of the vorinostat is 95-105 mu M; the final concentration of nicotinamide is 0.8-1.2 mM; the final concentration of sodium butyrate is 0.8-1.2 mM.

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a method for improving the content of chalcone A in liquorice.

Background

The liquorice plays an important role in the treatment of diseases from ancient times to present as a long-standing traditional Chinese medicine. The main components of the liquorice comprise triterpenoid saponins (mainly glycyrrhizic acid), flavonoids, coumarins, alkaloids, polysaccharides, amino acids and the like, and the triterpenoid saponins (with the content of 4-24 percent) and the flavonoids (with the content of 1-5 percent) are the main active components. Licochalcone A (LCA) is a phenolic chalcone compound existing in liquorice and is considered as a characteristic component of glycyrrhiza inflata, and the content of LCA in chalcone compounds separated and identified in liquorice up to now is the highest, so that the research on LCA has more practical application value. However, the mechanism of regulation of the accumulation of LCA in glycyrrhiza inflata is not clear.

Epigenetic modifications are key factors in regulating the expression of genes involved in plant secondary metabolites, but few reports have been made on the use of epigenetic modifications to regulate plant secondary metabolism. Histone modification is an important modification mode in epigenetic inheritance, wherein Histone Deacetylase (HDAC) can catalyze deacetylation of lysine residues of Histone and non-Histone, resulting in inhibition of expression of various genes, and is an important epigenetic regulator of transcription inhibition. In contrast, histone deacetylase inhibitors (HDAC inhibitors, HDACI) can enhance the acetylation of histones and other target proteins by inhibiting their activity by binding to HDACs, thereby promoting the transcriptional activation of specific genes. The HDAC family in plants can be divided into three classes based on homology to histones in yeast: class I is the RPD3/HDA1 family, shuttles between the nucleus and cytoplasm during signal transduction; class II is the HD2 family, present only in the nucleus; class III is the SIR2 family, which is very different from the first two classes and whose activity is independent of Zn²⁺Rather, it is dependent on coenzyme I (NAD), homologous to yeast Sir2, and has at least 7 subtypes, which are not inhibited by I, II HDAC inhibitors. Accordingly, histone deacetylase inhibitors can also be classified into three types: short chain fatty acids, hydroxamic acids, aminobenzamides.

Therefore, the influence on the accumulation of LCA is explored through the apparent inhibitor, a reference can be provided for the utilization of LCA, a method for effectively improving the LCA content in glycyrrhiza inflata L.is provided, and a certain foundation is laid for better developing the clinical application of glycyrrhiza inflata L..

Disclosure of Invention

Based on the above, the invention aims to provide a method for improving licochalcone A, which can effectively improve the content of licochalcone A in boswellia.

The specific technical scheme is as follows:

a method for improving licochalcone A comprises the following steps: culturing Glycyrrhrizae radix or Glycyrrhrizae radix seed seedling in culture medium containing histone deacetylase inhibitor, collecting root, and extracting licochalcone A.

The licorice root of the present invention includes but is not limited to hairy root of licorice and root of licorice seedling.

In some of these embodiments, the licorice root is licorice hairy root.

In some of these embodiments, the licorice root is the root of a licorice seedling.

In some of these embodiments, the histone deacetylase inhibitor is selected from at least one of vorinostat, nicotinamide, and sodium butyrate. Vorinostat (Vorinostat, SAHA) and Sodium butyrate (NaB) are broad-spectrum HDAC inhibitors, which can inhibit HDAC activity of RPD3/HDA1 family and HD2 family; nicotinamide (NIC) is a specific form of HDAC inhibitor, an inhibitor of the SIR2 family.

Preferably, the histone deacetylase inhibitor is vorinostat.

In some embodiments, the concentration of the vorinostat in the culture system is 80-120 μ M; and/or the concentration of nicotinamide in the culture system is 0.5-2 mM; and/or the concentration of sodium butyrate in the culture system is 0.5-2 mM.

Preferably, the concentration of the vorinostat in the culture system is 95-105 mu M; and/or the concentration of nicotinamide in the culture system is 0.8-1.2 mM; and/or the concentration of sodium butyrate in the culture system is 0.8-1.2 mM.

More preferably, the concentration of the vorinostat in the culture system is 100 μ M; and/or nicotinamide is present in the culture system at a concentration of 1 mM; and/or the concentration of sodium butyrate in the culture system is 1 mM.

In some of these embodiments, the histone deacetylase inhibitor is vorinostat and nicotinamide.

In some of these embodiments, the histone deacetylase inhibitor-containing medium further contains a DNA methyltransferase inhibitor; the DNA methyltransferase inhibitor is 5-azacytidine (5-azacytidine, 5-azaC), and the concentration of 5-azaC in the culture medium is 30. mu.M.

In some of these embodiments, the culturing period is at least 3 days.

In some of these embodiments, the culturing period is 3 days.

Further, the culture time was 5 days.

Further, the cultivation time was 7 days.

In some of these embodiments, the medium is liquid 1/2MS medium for licorice hairy roots, consisting of: 2.215 + -0.05 g/L MS culture Medium (Murashige & Skoog basic Medium with Vitamins), 20 + -0.2 g/L sucrose, 0.5 + -0.01 g/L morpholine ethanesulfonic acid, 250 + -2 mg/L cefadroxil (cef), and pH 5.7-6.0.

In some of these embodiments, the medium is a MS solid medium comprising the following components in concentrations: 4.43 plus or minus 0.05g/L MS culture medium, 20 plus or minus 0.2g/L cane sugar, 0.5 plus or minus 0.01g/L morpholine ethanesulfonic acid, pH 5.7-6.0.

In some of these embodiments, the method of extracting licochalcone a comprises the steps of:

(1) grinding the collected roots of the culture with liquid nitrogen and freeze-drying to obtain a sample;

(2) according to the material-liquid ratio of 10: (0.8-1.2) mg/ml, adding methanol into the sample obtained in the step (1) for ultrasonic extraction, sucking supernatant, filtering, evaporating to dryness and concentrating to obtain licochalcone A.

In some embodiments, the number of times of ultrasonic extraction in step (2) is 1-2, and after the supernatant is sucked and filtered, the filtrates are combined.

In some of these embodiments, the method for extracting licochalcone a further comprises the steps of:

(3) and (3) resuspending the product evaporated to dryness in the step (2) by using methanol, filtering, and collecting filtrate to obtain licochalcone A.

The invention also aims to provide the application of the histone deacetylase inhibitor in improving the licochalcone A content.

In some of these embodiments, the licorice is glycyrrhiza inflata.

In some of these embodiments, the histone deacetylase inhibitor is selected from at least one of vorinostat, nicotinamide, and sodium butyrate.

In some of these embodiments, the final concentration of vorinostat is 80-120 μ M; and/or nicotinamide is present in a final concentration of 0.5 to 2 mM; and/or the final concentration of sodium butyrate is 0.5-2 mM.

Preferably, the final concentration of the vorinostat is 95-105 mu M; and/or nicotinamide is used at a final concentration of 0.8-1.2 mM; and/or the final concentration of sodium butyrate is 0.8-1.2 mM.

More preferably, the final concentration of vorinostat is 100 μ Μ; and/or nicotinamide at a final concentration of 1 mM; and/or the final concentration of sodium butyrate is 1 mM.

According to the invention, researches show for the first time that the content of chalcone A in the root of liquorice can be effectively improved by treating the hairy roots or the seedlings of liquorice with the histone deacetylase inhibitor, and particularly when the histone deacetylase inhibitor vorinostat is used, the content of chalcone A can be improved by more than 30 times, so that the effect is obvious. The method provided by the invention has the advantages of simplicity, rapidness, high efficiency, low cost and the like, and has wide application space in the fields of medicines and the like.

Drawings

FIG. 1 shows the results of the detection of LCA content in 7 d-cultured Glycyrrhiza inflata seedlings cultured on MS medium containing 100. mu.M SAHA (left), and in 7 d-cultured Glycyrrhiza inflata seedlings (right).

FIG. 2 shows the results of the measurement of LCA content in the cultured seedlings of Glycyrrhiza inflata 7 days old (left) and the cultured seedlings of Glycyrrhiza inflata 7 days old (right) on MS medium containing 1mM NIC.

FIG. 3 shows the results of the LCA content measurement of hairy roots of Glycyrrhiza inflata cultured for 7d (left) and the hairy roots of Glycyrrhiza inflata cultured for 7d (right) on 1/2MS liquid medium containing 1mM NIC.

FIG. 4 shows the results of the LCA content measurement in 3d, 5d, and 7d seedlings of Glycyrrhiza inflata (top) cultured on MS medium containing 1mM NIC and 3d, 5d, and 7d seedlings of Glycyrrhiza inflata (bottom).

FIG. 5 shows the results of the LCA content measurement in the cultured Glycyrrhiza inflata seedlings (left) for 7d and the cultured Glycyrrhiza inflata seedlings (right) for 7d on the MS medium containing 1mM NaB.

FIG. 6 shows the results of measurement of LCA content in 7 d-cultured seedlings of Glycyrrhiza inflata (top) cultured on MS medium containing 30. mu.M 5-azaC, 100. mu.M SAHA +1mM NIC, and 100. mu.M SAHA +1mM NIC + 30. mu.M 5-azaC (bottom).

Detailed Description

Experimental procedures according to the invention, in which no particular conditions are specified in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.

The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.

The materials used in the present invention are as follows:

the solid MS culture medium comprises the following components: 4.43g/L MS Medium (Murashige & Skoog 15 basic Medium with Vitamins), 20g/L sucrose and 0.5g/L MES (morpholine ethanesulfonic acid), and adjusting the pH to 5.7-6.0.

Liquid 1/2MS medium comprising the following components: 2.215g/L MS Medium (Murashige & Skoog basic Medium with Vitamins), 20g/L sucrose (sucrose) and 0.5g/L MES (morpholine ethanesulfonic acid), and adjusting the pH to 5.7-6.0.

The preparation of the SAHA mother liquor comprises the following steps: 6.61mg of SAHA powder was weighed and dissolved in 1ml of DMSO to obtain the final product.

The preparation of the NIC mother solution comprises the following steps: 39.7mg of NIC powder was weighed, dissolved in 1.3ml of sterile deionized water, and sterilized by filtration through a 0.22 μm filter membrane.

The NaB mother liquor preparation method comprises the following steps: 35.8mg NaB powder is weighed, dissolved in 1.3ml sterile deionized water and filtered and sterilized through a 0.22 mu m filter membrane to obtain the NaB powder.

The preparation of the 5-azaC mother liquor comprises the following steps: 2.38mg of 5-azaC powder was weighed, dissolved in 1.3ml of sterile deionized water, and filtered through a 0.22 μm filter membrane for sterilization to obtain the final product.

A solid MS medium comprising an HDAC inhibitor and a DNA methyltransferase inhibitor formulated to comprise the steps of: the solid MS medium was autoclaved and SAHA, NIC, NaB, 5-azaC stock solution was added to final concentrations of 100. mu.M, 1mM, 30. mu.M before cooling and coagulation.

Example 1

1. Planting of glycyrrhiza inflata seedlings

300 seeds of the glycyrrhiza inflata are taken and placed in a 50ml centrifuge tube, 10ml of concentrated sulfuric acid is added, and the seeds are soaked for 15 to 20 minutes. The concentrated sulfuric acid is sucked out, and the pure water is rapidly washed for 5 times with 15ml each time until no concentrated sulfuric acid is left. Adding 20ml of 2% NaClO, and sterilizing for 15-20 minutes. Then, the NaClO was poured out again, and washed 5 times with 15ml of sterile pure water, each time for 10 min. After the cleaning, 20ml of sterile pure water is added, shaking culture is carried out for 24h under the conditions of 28 ℃ and 200rpm, and the liquorice seeds begin to germinate, so as to obtain the just germinated liquorice seed seedlings.

2. Hairy root induction of glycyrrhiza inflata

Impregnating the liquorice explant by an explant impregnation method: and (3) germinating 8-day liquorice bloat seed seedlings on an MS solid culture medium, and cutting off hypocotyls and cotyledons of the liquorice seed seedlings in a sterile ultra-clean workbench for infecting agrobacterium rhizogenes. The impregnated liquorice explant is placed on 1/2MS culture medium containing 250mg/L of cephalosporin, and after the hairy root of the swollen glycyrrhiza fruit grows out 3-5cm from the explant, the cut hairy root of the swollen glycyrrhiza fruit is transferred to 1/2MS culture medium containing 250mg/L of the cephalosporin. The hairy roots of the glycyrrhiza inflata capable of rapidly growing on the culture medium are selected and transferred to 1/2MS culture medium containing 250mg/L of cephalosporins for suspension culture. And (3) taking a part of the hairy roots of the glycyrrhiza inflate fruit cultured in a suspension manner, extracting DNA, and screening positive hairy roots by PCR. And subculturing the hairy root of the glycyrrhiza inflata which grows rapidly into a plurality of hairy roots of the glycyrrhiza inflata.

HDAC inhibitor treatment of Glycyrrhiza inflata seedlings or hairy roots

After 2 days of germination of the glycyrrhiza inflata seeds on the MS culture medium, the glycyrrhiza inflata seeds are transferred to a new MS culture medium for vertical culture, the culture is recovered for 3 days, the glycyrrhiza inflata seeds are transferred to MS culture media respectively containing HDAC inhibitor SAHA (the final concentration is 100 mu M), NIC (the final concentration is 1mM), NaB (the final concentration is 1mM) and no HDAC inhibitor (Control group Control) for culture for 7 days, and then the roots of the glycyrrhiza inflata seeds are collected.

Selecting multiple bottles of hairy roots of the glycyrrhiza inflata fruits from the same strain and having the same growth state, transferring the bottles to a liquid 1/2MS culture medium, adding NIC mother liquor into a 1/2MS culture medium after two days, and adding the NIC mother liquor to a final concentration of 1 mM. Part of the culture medium is not added with any histone deacetylase inhibitor (Control group), and is respectively cultured for seven days, and then the hairy root of the glycyrrhiza inflata dunn is collected.

4. Extraction of licochalcone A

(1) Grinding the root of the glycyrrhiza inflata seedlings or the hairy roots of the glycyrrhiza inflata obtained by culturing each group by using liquid nitrogen, and freeze-drying to obtain a sample.

(2) Pure methanol extraction: adding 1ml of methanol into each 10mg of sample, performing ultrasonic treatment for 1 hour, preparing a new centrifugal tube, marking, and absorbing the supernatant into the new centrifugal tube by using a pipette after the ultrasonic treatment is finished; adding 1ml of pure methanol into the sample again, performing ultrasonic treatment for 1 hour, combining the filtrates twice, evaporating and concentrating.

(3) Dissolving: the extracted compound was resuspended in 300. mu.l of pure methanol, the resulting extract was filtered through a 0.22 μm filter and the filtrate (containing LCA) was collected.

4. Content determination of licochalcone A

(1) A chromatographic system: a C18 column (150 mm. times.4.6 mm,5 μm, Shimadzu, Kyoto, Japan) was used, the column temperature was 40 ℃, the flow rate was 1ml/min, and the sample volume was 10. mu.l; mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid water; gradient elution: t is 0min, 30% a; t is 25min, 55% a; t is 27min, 95% a; t is 30min, 95% a; t is 31min, 30% a; t 35min, detection wavelength of 30% a. lca 370 nm. The number of theoretical plates is calculated according to LCA peak and should be not less than 8000.

(2) Calculating the LCA content: by detecting peak areas on HPLC with LCA standards of different concentrations, a standard curve between the concentration and the peak area is established, and good linearity is achieved between the mass concentration of the standard and the absorption value intensity.

The results are shown in FIGS. 1 to 5. In this example, the glycyrrhiza inflata seedlings or hairy roots are used as experimental materials, and are treated with 3 types of HDAC inhibitors (SAHA, NIC, NaB) for 7 days, and then the LCA content in the roots of the culture is measured. If the sample of the glycyrrhiza inflata seedlings treated by 100 mu M SAHA for 7 days has the LCA detection result: the peak areas were 307941, 317790, 315485, respectively. The calculation formula for obtaining the concentration according to the standard curve between the LCA concentration and the peak area is as follows: and y is 2E-08 x. The LCA concentration of the liquorice bloat seedlings treated by 100 mu M SAHA for 7 days is calculated by the formula as follows: 157.92ng/mg dry weight, 174.93ng/mg dry weight, 131.45ng/mg dry weight.

As shown in fig. 1, treatment of glycyrrhiza inflata seedlings with 100 μ M SAHA for 7 days increased LCA content by about 31-fold compared to the control group without inhibitor treatment.

As shown in fig. 2, 1mM NIC treated licorice distending seed seedlings with 1mM NIC increased LCA content by about 4-fold compared to the control group without inhibitor treatment for 7 days.

As shown in fig. 3, treatment of hairy roots of glycyrrhiza inflata with 1mM NIC increased LCA content by about 2-fold for 7 days compared to the control group without inhibitor treatment.

As shown in fig. 4, treatment of glycyrrhiza inflata seedlings with 1mM NIC increased LCA content by about 2, 3, and 5 times for 3d, 5d, and 7d, respectively, compared to the control group without inhibitor treatment.

As shown in fig. 5, treatment of glycyrrhiza inflata seedlings with 1mM NaB increased LCA content by about 5-fold for 7 days, compared to the control group not treated with the inhibitor.

Histone deacetylase inhibitors (HDAC inhibitors) can enhance protein acetylation by inhibiting histone deacetylase activity, thereby promoting transcriptional activation of specific genes. The results show that the culture medium containing the HDAC inhibitor is used for culturing the glycyrrhiza inflata seedlings or the hairy roots, the acetylation degree of histone and non-histone of the glycyrrhiza inflata seedlings or the hairy roots can be improved, and further the gene expression related to licochalcone A accumulation is promoted, so that the licochalcone A accumulation is quickly and effectively promoted.

Example 2

1. Planting of glycyrrhiza inflata seedlings

The procedure is as in example 1

Treatment of Glycyrrhiza inflata seedlings with HDAC inhibitors and DNA methyltransferase inhibitors

After 2 days of germination of the glycyrrhiza inflata seeds on the MS culture medium, transferring the glycyrrhiza inflata seeds to a new MS culture medium for vertical culture, recovering for 3 days, and transferring the glycyrrhiza inflata seeds to the MS culture medium containing the following inhibitors (the following concentrations are the final concentrations of the inhibitors in the culture medium) for culture: 30 μ M5-azaC, 100 μ M SAHA +1mM NIC,100 μ M SAHA +1mM NIC +30 μ M5-azaC, and a Control group (Control) not treated with an inhibitor were set, and roots of Glycyrrhiza inflata seedlings were collected after culturing each group for 7 days.

3. Extraction of licochalcone A

The procedure is as in example 1

4. Content determination of licochalcone A

The procedure is as in example 1

The results are shown in FIG. 6, where 30 μ M5-azaC treatment increased LCA content by about 5-fold for 7 days compared to the control group not treated with inhibitor; the content of LCA is improved by about 32 times by treating 100 mu M SAHA and 1mM NIC for 7 days; treatment with 100. mu.M SAHA +1mM NIC + 30. mu.M 5-azaC for 7 days increased the LCA content by about 35-fold.

The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.