1. A2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound is characterized in that the structural formula of the compound is as follows:

2. a process for the preparation of 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound according to claim 1, comprising the steps of:

step 1, adding 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone into methanol, stirring until the methanol is dissolved clearly, then slowly adding sodium borohydride into the completely dissolved methanol solution in an ice bath, and stirring for 6-12 hours at a set temperature to obtain a mixture;

step 2, adding water into the mixture for quenching reaction, extracting by using dichloromethane after concentrating 80% (v/v) methanol, concentrating to obtain an organic phase, performing column chromatography on the organic phase to obtain a 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound,

in the step 1, the molar ratio of sodium borohydride to 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone is (1-5): 1.

3. The method for producing 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound according to claim 2, wherein:

wherein, in the step 1, the stirring time is 10 h.

4. The method for producing 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound according to claim 2, wherein:

in the step 1, the molar ratio of sodium borohydride to 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone is 3: 1.

5. The method for producing 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound according to claim 2, wherein:

in the step 2, silica gel column chromatography is adopted, and the eluant adopts petroleum ether and ethyl acetate in a volume ratio of 3: 1-1: 1.

6. Use of the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound of claim 1 for inducing antrodia camphorata ubiquinone as an active ingredient.

Background

Antrodia camphorata contains abundant active metabolites including Triterpenoids (Triterpenoids), Maleic acid and succinic acid derivatives (Maleic acid and succinic acid derivatives), Polysaccharides (Polysaccharides), ubiquinones derivatives (Ubiquinone derivatives), sterols (Steroids), Nucleic acids (Nucleic acids) and the like, and has various activities of resisting inflammation, resisting cancer, protecting liver, resisting oxidation, enhancing immunity and the like.

A plurality of ubiquinone derivatives are separated from the solid state fermentation product of antrodia camphorata and have good anti-inflammatory and anticancer activities. The Antrodia camphorata solid-state fermentation mainly uses grains as raw materials for culture, the culture period is long (about 30 days), and the fermentation process control and large-scale production cannot be well realized due to the limitation of the solid-state fermentation mode. However, the conventional antrodia camphorata liquid fermentation product basically does not contain ubiquinone active ingredients, so that the added value of the antrodia camphorata liquid fermentation product is greatly reduced.

At present, compounds such as p-hydroxybenzoic acid and coenzyme Q0 can be used for inducing antrodia camphorata to synthesize ubiquinone components in the liquid fermentation process, however, the induction yield is low, and the two compounds have a certain inhibiting effect on the growth of the antrodia camphorata thallus.

Disclosure of Invention

The present invention has been made to solve the above problems, and an object of the present invention is to provide a 2, 3-dimethoxy-5-methyl-1-hydroxy-4-benzoquinone compound, a preparation method and an application thereof.

The invention provides a 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound, which has the following characteristics: the structural formula of the compound is as follows:

the invention also provides a preparation method of the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound, which is characterized by comprising the following steps: step 1, adding 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone into methanol, stirring until the methanol is dissolved clearly, then slowly adding sodium borohydride into the completely dissolved methanol solution in an ice bath, and stirring for 6-12 hours at a set temperature to obtain a mixture; and 2, adding water into the mixture for quenching reaction, extracting by using dichloromethane after concentrating 80% (v/v) methanol, concentrating to obtain an organic phase, and performing column chromatography on the organic phase to obtain the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound, wherein in the step 1, the molar ratio of sodium borohydride to 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone is (1-5): 1.

The present invention also provides a method for producing a 2, 3-dimethoxy-5-methyl-1-hydroxy-4-benzoquinone compound, which comprises the steps of: wherein, in the step 1, the stirring time is 10 h.

The present invention also provides a method for producing a 2, 3-dimethoxy-5-methyl-1-hydroxy-4-benzoquinone compound, which comprises the steps of: wherein, in the step 1, the molar ratio of the sodium borohydride to the 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone is 3: 1.

The present invention also provides a method for producing a 2, 3-dimethoxy-5-methyl-1-hydroxy-4-benzoquinone compound, which comprises the steps of: wherein, in the step 2, silica gel column chromatography is adopted, and the eluant adopts petroleum ether and ethyl acetate in a volume ratio of 3: 1-1: 1.

The invention also provides application of the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound in inducing antrodia camphorata ubiquinone active ingredients.

Action and Effect of the invention

The preparation method of the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound is simple, low in cost, high in yield and easy to realize industrial production. In addition, the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound is used as a precursor, can efficiently induce antrodia camphorata to synthesize the ubiquinone active ingredients, has the induction effect higher than that of reported coenzyme Q0, and greatly improves the yield of the antrodia camphorata ubiquinone active ingredients. In addition, the addition of the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound has no inhibition effect on the growth of the cells of the antrodia camphorata.

Drawings

FIG. 1 is an HPLC chromatogram of a liquid fermentation product of Antrodia camphorata strain S-29 in an example of the present invention.

Detailed Description

In order to make the technical means and functions of the present invention easy to understand, the present invention is specifically described below with reference to the embodiments and the accompanying drawings.

The invention provides a 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound, which has the following structural formula:

the invention also provides a preparation method of the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound, which comprises the following steps:

step 1, adding 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone into methanol, stirring until the methanol is dissolved completely, then slowly adding sodium borohydride into the methanol solution completely dissolved in the ice bath, and stirring for 6 to 12 hours at a set temperature to obtain a mixture.

In the step 1, the molar ratio of sodium borohydride to 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone is (1-5): 1.

Preferably, the molar ratio of the sodium borohydride to the 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone is 3:1, and the stirring time is 10 hours.

And 2, adding water into the mixture for quenching reaction, extracting by using dichloromethane after concentrating 80% (v/v) methanol, concentrating to obtain an organic phase, and performing column chromatography on the organic phase to obtain the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound.

In the step 2, silica gel column chromatography is adopted, and the eluant adopts petroleum ether and ethyl acetate in a volume ratio of 3: 1-1: 1.

The invention also provides the application of the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound in inducing the active ingredients of antrodia camphorata ubiquinone,

specifically, there are two methods for performing the application:

the method comprises the following steps: the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound is added into the antrodia camphorata liquid state fermentation as an inducer, can effectively induce the antrodia camphorata ubiquinone active ingredients under the conditions of ventilation and stirring, and has no obvious inhibition effect on the growth of the antrodia camphorata thalli.

The method 2 comprises the following steps: the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound is added into antrodia camphorata liquid state fermentation as an inducer, a solvent is added, the antrodia camphorata ubiquinone active ingredients can be effectively induced under the conditions of ventilation and stirring, the ubiquinone active ingredients are highly enriched and concentrated in the solvent paraffin oil, and the antrodia camphorata thallus growth is not obviously inhibited.

Further, in the method 2, the solvent is any one or a mixture of any two or more of paraffin oil, long-chain alkane (C12-C16), linseed oil and linoleic acid.

Furthermore, the strain of Antrodia camphorata (Antrodia camphorata) S-29 is adopted, and the strain is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.9590 and the preservation time of 2014, 09 months and 09 days.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.

Slant medium (g/L): 60mL/L of soybean milk, 40 parts of glucose, 0.5 part of monopotassium phosphate, 0.5 part of magnesium sulfate heptahydrate, 0.5 part of citric acid and pH 5; sterilizing at 115 deg.C for 20 min.

Seed liquid culture medium (g/L): corn steep liquor 4, glucose 20, potassium dihydrogen phosphate 0.5, magnesium sulfate heptahydrate 0.5, citric acid 0.5 and pH 5.5; sterilizing at 115 deg.C for 20 min.

Fermentation broth medium (g/L): corn steep liquor 8, glucose 60, potassium dihydrogen phosphate 0.5, magnesium sulfate heptahydrate 0.5, citric acid 0.5 and pH 5.5; sterilizing at 115 deg.C for 20 min.

Example 1:

step 1, dissolving 1.822g (10mmol) of 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone in 100mL of methanol, stirring until the mixture is completely dissolved, placing the completely dissolved methanol solution in an ice bath condition, slowly adding 0.378g (10mmol) of sodium borohydride, wherein the molar ratio of the sodium borohydride to the 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone is 1:1, and stirring at 20 ℃ for 6 hours to obtain a mixture.

And 2, adding water into the mixture for quenching reaction, extracting by using dichloromethane after concentrating 80% (v/v) methanol, concentrating to obtain an organic phase, and purifying the organic phase by using silica gel column chromatography to obtain 1.166g of the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound with the yield of 64%. Wherein the eluent adopts petroleum ether and ethyl acetate in a volume ratio of 3: 1-1: 1.

Example 2:

step 1, dissolving 1.822g (10mmol) of 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone in 100mL of methanol, stirring until the solution is clear, placing the completely dissolved methanol solution in an ice bath condition, slowly adding 1.135g (30mmol) of sodium borohydride, wherein the molar ratio of the sodium borohydride to the 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone is 3:1, and stirring at 20 ℃ for 10 hours to obtain a mixture.

And 2, adding water into the mixture for quenching reaction, extracting by using dichloromethane after concentrating 80% (v/v) methanol, concentrating to obtain an organic phase, and purifying the organic phase by using silica gel column chromatography to obtain 1.601g of the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound with the yield of 88%. Wherein the eluent adopts petroleum ether and ethyl acetate in a volume ratio of 3: 1-1: 1.

Example 3:

step 1, dissolving 1.822g (10mmol) of 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone in 100mL of methanol, stirring until the solution is clear, placing the completely dissolved methanol solution in an ice bath condition, slowly adding 1.892g (50mmol) of sodium borohydride, wherein the molar ratio of the sodium borohydride to the 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone is 5:1, and stirring at 20 ℃ for 12 hours to obtain a mixture.

And 2, adding water into the mixture for quenching reaction, extracting by using dichloromethane after concentrating 80% (v/v) methanol, concentrating to obtain an organic phase, and purifying the organic phase by using silica gel column chromatography to obtain 1.257g of the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound with the yield of 69%. Wherein the eluent adopts petroleum ether and ethyl acetate in a volume ratio of 3: 1-1: 1.

FIG. 1 is an HPLC chromatogram of a liquid fermentation product of Antrodia camphorata strain S-29 according to an embodiment of the present invention, FIG. 1(A) is an HPLC chromatogram of a fermentation blank control experiment of example 4, FIG. 1(B) is an HPLC chromatogram of an induction experiment of coenzyme Q0 according to example 5, and FIG. 1(C) is an HPLC chromatogram of an induction experiment of 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound according to example 6, wherein the abscissa represents retention time and the ordinate represents UV254nm absorption response value.

Example 4: antrodia camphorata strain S-29 fermentation blank control experiment

Preparing seed liquid from the Antrodia camphorata strain S-29, inoculating the seed liquid into a fermentation culture medium according to the inoculation amount of 15%, wherein the culture temperature is 28 ℃, the rotation speed is 150r/min, the culture time is 10d, and the ventilation volume is 1.2 v/(v.m). After the fermentation is finished, the thalli are collected, and the thalli amount and the content of ubiquinone active ingredients are measured.

Under the condition, the fermentation bacterial amount of the antrodia camphorata strain S-29 is 13.55 g/L. The Antrodia camphorata thallus does not contain ubiquinone active ingredients.

Example 5: induction action of compound coenzyme Q0 on antrodia camphorata ubiquinone active ingredients

Preparing seed solution from Antrodia camphorata strain S-29, inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 15%, wherein the culture temperature is 28 ℃, the rotation speed is 150r/min, and the ventilation volume is 1.2 v/(v.m). In the 3 rd fermentation period, 0.3g/L of coenzyme Q0 was added to the fermentation broth, and the fermentation was continued for 7 d. After the fermentation is finished, the thalli are collected, and the thalli amount and the content of ubiquinone active ingredients are measured.

Under the condition, the fermentation bacterial amount of the Antrodia camphorata strain S-29 is 8.74 g/L. Active components of ubiquinones are induced from the antrodia camphorata thallus, wherein the content of Antroquinonol reaches 35.77 mg/L.

Example 6: induction action of 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound on antrodia camphorata ubiquinone active ingredients

Preparing seed solution from Antrodia camphorata strain S-29, inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 15%, wherein the culture temperature is 28 ℃, the rotation speed is 150r/min, and the ventilation volume is 1.2 v/(v.m). And 3d, adding 0.3g/L of 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound into the fermentation liquor, and continuing to ferment for 7 d. After the fermentation is finished, the thalli are collected, and the thalli amount and the content of ubiquinone active ingredients are measured.

Under the condition, the fermentation bacterial amount of the Antrodia camphorata strain S-29 is 13.84 g/L. Active components of ubiquinones are induced from the antrodia camphorata thallus, wherein the content of Antroquinonol reaches 58.27 mg/L.

Example 7: inducing effect of coenzyme Q0 on active ingredients of antrodia camphorata ubiquinone in presence of paraffin oil solvent

Preparing seed solution from Antrodia camphorata strain S-29, inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 15%, wherein the culture temperature is 28 ℃, the rotation speed is 150r/min, and the ventilation volume is 1.2 v/(v.m). At fermentation 3d, 0.3g/L of coenzyme Q0 and 20% (v/v) paraffin oil were added to the fermentation broth and fermentation was continued for 7 d. After the fermentation is finished, the thalli are collected, and the thalli amount and the content of ubiquinone active ingredients are measured.

Under the condition, the fermentation bacterial amount of the Antrodia camphorata strain S-29 is 10.26 g/L. Active components of ubiquinones are induced from the antrodia camphorata thallus, wherein the content of Antroquinonol reaches 135.82 mg/L.

Example 8: induction action of p-hydroxybenzoic acid on antrodia camphorata ubiquinone active ingredients in the presence of paraffin oil solvent

Preparing seed solution from Antrodia camphorata strain S-29, inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 15%, wherein the culture temperature is 28 ℃, the rotation speed is 150r/min, and the ventilation volume is 1.2 v/(v.m). At fermentation 3d, 0.3g/L p-hydroxybenzoic acid and 20% (v/v) paraffin oil were added to the fermentation broth, and fermentation was continued for 7 d. After the fermentation is finished, the thalli are collected, and the thalli amount and the content of ubiquinone active ingredients are measured.

Under the condition, the fermentation bacterial amount of the Antrodia camphorata strain S-29 is 11.36 g/L. Active components of ubiquinones are induced from the antrodia camphorata thallus, wherein the content of Antroquinonol reaches 73.16 mg/L.

Example 9: inducing effect of 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound on antrodia camphorata ubiquinone active ingredient in presence of paraffin oil solvent

Preparing seed solution from Antrodia camphorata strain S-29, inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 15%, wherein the culture temperature is 28 ℃, the rotation speed is 150r/min, and the ventilation volume is 1.2 v/(v.m). In fermentation 3d, 0.3g/L of 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound and 20% (v/v) paraffin oil were added to the fermentation broth, and fermentation was continued for 7 d. After the fermentation is finished, the thalli are collected, and the thalli amount and the content of ubiquinone active ingredients are measured.

Under the condition, the fermentation bacterial amount of the Antrodia camphorata strain S-29 is 13.13 g/L. Active components of ubiquinones are induced from the antrodia camphorata thallus, wherein the content of Antroquinonol reaches 238.57 mg/L.

Example 10: inducing effect of 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound on antrodia camphorata ubiquinone active ingredient in presence of linseed oil solvent

Preparing seed solution from Antrodia camphorata strain S-29, inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 15%, wherein the culture temperature is 28 ℃, the rotation speed is 150r/min, and the ventilation volume is 1.2 v/(v.m). In the fermentation stage 3d, 0.3g/L of 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound and 20% (v/v) linseed oil are added to the fermentation broth, and the fermentation is continued for 7 d. After the fermentation is finished, the thalli are collected, and the thalli amount and the content of ubiquinone active ingredients are measured.

Under the condition, the fermentation bacterial amount of the Antrodia camphorata strain S-29 is 15.63 g/L. Active components of ubiquinones are induced from the antrodia camphorata thallus, wherein the content of Antroquinonol reaches 277.48 mg/L.

Effects and effects of the embodiments

From examples 1 to 3, it is understood that the yield of the 2, 3-dimethoxy-5-methyl-1-hydroxy-4-benzoquinone compound prepared by the preparation method of the present invention is high, and the maximum yield reaches 88% when the molar ratio of sodium borohydride to 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone is 3: 1.

From examples 4 to 10, it is clear that when the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound of the present invention is used to induce antrodia camphorata ubiquinone type active ingredients, the Antroquinonol content is high, and can reach 58.27mg/L under the conventional liquid state fermentation condition, and the yield is 238.57mg/L after the addition of paraffin oil solvent for fermentation, which is far superior to the induction effect of coenzyme Q0.

Therefore, the preparation method of the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound is simple, low in cost, high in yield and easy to realize industrial production. In addition, the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound is used as a precursor, can efficiently induce antrodia camphorata to synthesize the ubiquinone active ingredients, has the induction effect higher than that of reported coenzyme Q0, and greatly improves the yield of the antrodia camphorata ubiquinone active ingredients. In addition, the addition of the 2, 3-dimethoxy-5-methyl-1-hydroxy-4 benzoquinone compound has no inhibition effect on the growth of the cells of the antrodia camphorata.

The above embodiments are preferred examples of the present invention, and are not intended to limit the scope of the present invention.