2, 4-dihydroxy-6-benzyl formic acid polyol esters in gold brush as well as extraction method and application of 2, 4-dihydroxy-6-benzyl formic acid polyol esters

Abstract

The invention discloses a 2, 4-dihydroxy-6-benzyl formic acid polyol ester compound in a gold wire brush and an extraction and separation method and application thereof, the structure of the 2, 4-dihydroxy-6-benzyl formic acid polyol ester compound adopts 1H-NMR (nuclear magnetic resonance), 13C-NMR (nuclear magnetic resonance) and two-dimensional nuclear magnetic spectrum analysis methods to determine that compounds I-1-I-3 are new compounds, and the 2, 4-dihydroxy-6-benzyl formic acid polyol ester compound has the following structure. The 2, 4-dihydroxy-6-benzyl formic acid polyol ester compound can effectively inhibit the activity of acetylcholin esterase (AChE), so that the compound disclosed by the invention can be used as a lead compound for resisting the acetylcholin esterase, and a material basis and a theoretical basis are provided for developing new drugs and new components.

Description

Technical Field

[0001] This invention relates to the field of extraction and separation of traditional Chinese medicine, and to four types of 2,4-dihydroxy-6-benzylmethylformic acid polyol esters, extraction methods and uses. Background Technology

[0002] *Lethariella cladonioides* Nyl. Krog, also known as Golden Brush Handle, Red Snow Tea, and Deer Heart Snow Tea, is a branched lichen belonging to the genus *Lethariella* and subgenus *Chlorea* in the family Parmaliaceae. It is found only in East Asia, and in my country, its distribution covers six provinces and autonomous regions: Shaanxi, Gansu, Qinghai, Tibet, Yunnan, and Sichuan. Its main distribution centers are in southeastern Tibet, northwestern Yunnan, and western Sichuan. According to *Shaanxi Traditional Chinese Medicine*, it is bitter and neutral in nature, possessing sedative, anti-inflammatory, and analgesic properties; it is used to treat epilepsy, schizophrenia, neurasthenia, headaches, and dizziness.

[0003] Gold wire brushes mainly consist of a variety of chemical components, including condensed phenolic acids, phenolic acids, aromatic esters and aldehydes, steroids, polyols, and volatile oils, thus exhibiting a wide range of pharmacological activities. Modern pharmacological studies have shown that gold wire brushes possess antioxidant, lipid-lowering, anti-tumor, anti-fatigue, antibacterial, and anti-radiation effects.

[0004] Most of the chemical components isolated from gold wire brushes are known and have low structural novelty. However, the extraction of 2,4-dihydroxy-6-benzylcarboxylic acid polyol esters from gold wire brushes, the extraction method, and the uses have not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to provide 2,4-dihydroxy-6-benzylcarboxylic acid polyol ester compounds.

[0006] A second objective of this invention is to provide a method for extracting 2,4-dihydroxy-6-benzylcarboxylic acid polyol esters.

[0007] A third object of the present invention is to provide the use of 2,4-dihydroxy-6-benzylcarboxylic acid polyol ester compounds.

[0008] A fourth object of the present invention is to provide a gold brush extract comprising a polyol ester compound of 2,4-dihydroxy-6-benzylformic acid.

[0009] A fifth object of the present invention is to provide the use of the gold brush extract.

[0010] A sixth object of the present invention is to provide a pharmaceutical composition comprising a polyol ester of 2,4-dihydroxy-6-benzylcarboxylic acid.

[0011] A seventh object of the present invention is to provide the use of the above-described pharmaceutical composition.

[0012] The technical solution of this invention is summarized as follows:

[0013] 2,4-Dihydroxy-6-benzylcarboxylic acid polyol ester compounds having the structure shown in formula (I):

[0014]

[0015] The extraction method for the above-mentioned 2,4-dihydroxy-6-benzylcarboxylic acid polyol ester compounds includes the following steps:

[0016] Step 1: Using the dried medicinal materials from the gold wire brush as raw materials, extract with ethanol and water, filter, combine the filtrates and concentrate under reduced pressure until there is no alcohol smell, cool to room temperature, and obtain the extract for later use;

[0017] Step 2: Dissolve the extract from Step 1 in purified water, extract with petroleum ether and dichloromethane, and recover the solvent under reduced pressure to obtain a three-part extract;

[0018] Step 3: The aqueous layer from Step 2 was adsorbed onto a macroporous resin column and eluted with a gradient of water, 30% ethanol-water, 60% ethanol-water and 95% ethanol-water. The solution was concentrated to dryness under reduced pressure to obtain four fractions (water eluent, 30% ethanol-water eluent, 60% ethanol-water eluent and 95% ethanol-water eluent) for later use.

[0019] Step 4: The 30% ethanol-water eluent from the fraction in Step 3 was further separated by reversed-phase silica gel column chromatography, using methanol-water and methanol gradient elution to obtain several eluted fractions. These fractions were then detected by thin-layer chromatography, combined according to the colorimetric results, and concentrated to dryness under reduced pressure to obtain 6 fractions (Fr.w1-Fr.w6).

[0020] Step 5: Separate the fraction Fr.w2 obtained in Step 4 by polyamide column chromatography, using methanol-water and methanol gradient elution respectively, and detect by thin-layer chromatography. Combine and concentrate the fractions according to the colorimetric results for later use, to obtain 7 fractions (Fr.w21-Fr.w27) for later use.

[0021] Step 6: Separate the fraction Fr.w22 obtained in Step 5 using Sephadex LH-20, detect it by thin-layer chromatography, and combine and concentrate it according to the colorimetric results for later use;

[0022] Step 7: The concentrate obtained in Step 6 is separated by reversed-phase silica gel column chromatography, using methanol-water as the mobile phase for elution, and detected by thin-layer chromatography. The concentrates are then combined and concentrated based on the colorimetric results for later use.

[0023] Step 8: The fraction Fr.w22 obtained in Step 7 was separated and prepared by HPLC using methanol-water as the mobile phase to obtain compounds I-1, I-2, I-3 and I-4.

[0024] Step 1 is preferably as follows: using dried medicinal materials (such as those from the gold wire brush) as raw materials, adding 8-16 times the weight of the raw materials in a 95% ethanol solution, ultrasonically extracting 1-3 times, 30 minutes each time, then adding 8-16 times the weight of the raw materials in a 75% ethanol solution, ultrasonically extracting 1-3 times, filtering, combining the filtrates, recovering the ethanol under reduced pressure, and concentrating until no ethanol remains to obtain the extract; or using dried medicinal materials (such as those from the gold wire brush) as raw materials, adding 8-16 times the weight of the raw materials in a 95% or 75% ethanol solution, ultrasonically extracting 1-3 times, filtering, combining the filtrates, recovering the ethanol under reduced pressure, and concentrating until no ethanol remains. Alcohol is used to obtain an extract; or, using dried medicinal materials as raw materials, 8-16 times the weight of the raw materials in 95% ethanol solution is added, and the mixture is heated and extracted 1-3 times, followed by 8-16 times the weight of the raw materials in 75% ethanol solution, and the mixture is heated and extracted 1-3 times. The extracts are filtered, the filtrates are combined, the ethanol is recovered under reduced pressure, and the mixture is concentrated until no ethanol remains to obtain an extract; or, using dried medicinal materials as raw materials, 8-16 times the weight of the raw materials in 95% or 75% ethanol solution is added, and the mixture is heated and refluxed to extract 1-3 times, the extracts are filtered, the filtrates are combined, the ethanol is recovered under reduced pressure, and the mixture is concentrated until no ethanol remains to obtain an extract.

[0025] The above-mentioned 2,4-dihydroxy-6-benzylcarboxylic acid polyol esters are used in the preparation of anti-acetylcholinesterase drugs.

[0026] Gold brush extract containing the above-mentioned 2,4-dihydroxy-6-benzylcarboxylic acid polyol ester compounds.

[0027] Application of gold brush extract in the preparation of anti-acetylcholinesterase drugs.

[0028] A pharmaceutical composition characterized by comprising a 2,4-dihydroxy-6-benzylcarboxylic acid polyol ester compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0029] The above-mentioned pharmaceutical composition is used in the preparation of anti-acetylcholinesterase drugs.

[0030] Advantages of this invention:

[0031] The 2,4-dihydroxy-6-benzylcarboxylic acid polyol ester compounds of the present invention can effectively inhibit the activity of acetylcholinesterase (AChE), suggesting that the 2,4-dihydroxy-6-benzylcarboxylic acid polyol ester compounds of the present invention can serve as lead compounds for anti-acetylcholinesterase. Detailed Implementation

[0032] The technical solution of the present invention will now be described with reference to specific embodiments. These embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0033] Example 1

[0034] The extraction method for 2,4-dihydroxy-6-benzylcarboxylic acid polyol esters includes the following steps:

[0035] Step 1: Using 4.0 kg of dried whole herb of Lethariella cladonioides Nyl.Krog as raw material, cut it into small pieces and extract it three times (30 min) with 14 times the amount of 95% ethanol and 75% ethanol respectively using ultrasonic extraction. After filtration, the filtrates were combined and the solvent was recovered under reduced pressure at 40℃~45℃ to obtain 340 g of extract.

[0036] Step 2: Take the above extract, dissolve it in pure water, extract it with equal amounts of petroleum ether and dichloromethane, and recover the solvent from the extract under reduced pressure to obtain an aqueous extract.

[0037] Step 3: After the aqueous extract is dissolved in water, it is adsorbed in macroporous resin overnight and then separated by macroporous resin column chromatography. Gradient elution is performed using 30% ethanol-water, 60% ethanol-water and 95% ethanol-water as eluents to obtain 4 fractions (water eluent, 30% ethanol-water eluent, 60% ethanol-water eluent and 95% ethanol-water eluent) for later use.

[0038] Step 4: The 30% ethanol-water eluent was mixed with an equal volume of reversed-phase silica gel and then separated by reversed-phase silica gel column chromatography. Gradient elution was performed using methanol-water volume ratios of 1:19, 1:9, 3:7, 1:1, 4:1, and 9:1, with methanol as the eluent, to obtain 6 fractions (Fr.w1-Fr.w6).

[0039] Step 5: Fr.w2 was eluted using polyamide column chromatography with a gradient elution of methanol-water volume ratios of 1:9, 3:7, 2:3, 1:1, 3:2, 7:3, 4:1, 9:1 and methanol as eluent to obtain 7 fractions (Fr.w21-Fr.w27).

[0040] Step 6: Fr.w22 was purified using Sephadex LH-20 CH2C12-MeOH (1:1, v / v), and then subjected to ODS column chromatography with gradient elution using methanol-water volume ratios of 1:19, 1:4, 2:3, 1:1, 3:2, 4:1, 9:1 and methanol as eluent.

[0041] Step 7: Fr.w22 was separated and prepared by HPLC. The mobile phase was methanol-water with a volume ratio of 15:85 and isocratic elution was performed to obtain compounds 1-4.

[0042] The physicochemical properties and constants of the new compounds are as follows:

[0043] Compound I-1: White powder; (c = 0.021 g / 100 mL, methanol); UV (methanol) λmax (logε) 204 (4.07), 264 (3.73), 299 (3.32) nm; IR (potassium bromide) vmax 3391, 3009, 2837, 1681, 1646, 1437, 1407, 1317, 1266, 1204, 1173, 1139, 1019, 952 cm⁻¹ -1 High-resolution mass spectrometry (cation) m / z 303.1078 [M+H] + (Calculate the molecular formula as C) 13 H 19 O8, 303.1080); C-H spectral data are shown in Table 1.

[0044] Compound I-2: white powder; (c = 0.021 g / 100 mL, methanol); UV (methanol) λmax (logε) 205 (4.09), 265 (3.68), 301 (3.37) nm; IR (potassium bromide) vmax 3403, 3010, 2921, 1681, 1647, 1437, 1407, 1317, 1265, 1204, 1175, 1139, 1021, 953 cm⁻¹ -1 High-resolution mass spectrometry (cation) m / z 303.1083 [M+H] + (Calculate the molecular formula as C) 13 H 19 O8, 303.1080); C-H spectral data are shown in Table 1.

[0045] Compound I-3: white powder; (c = 0.016 g / 100 mL, methanol); UV (methanol) λmax (logε) 205 (4.16), 264 (3.87), 303 (3.49) nm; IR (potassium bromide) vmax 3383, 2919, 1647, 1621, 1457, 1400, 1318, 1264, 1203, 1169, 1106, 1016, 951 cm⁻¹ -1 High-resolution mass spectrometry (cation) m / z 303.1082 [M+H] + (Calculate the molecular formula as C) 13 H19 O8, 303.1080); C-H spectral data are shown in Table 1.

[0046] Table 1. Proton and carbon spectral data of compounds I-1 to I-4

[0047] a Meamred at 1 H-NMR (500MHz) and 13 C-NMR (125MHz) in CD3OD b Measored at 1 H-NMR (600MHz) and 13 C-NMR (150MHz) in CDD3OD

[0048] The structure of the compounds was identified using physicochemical constants and modern spectroscopic techniques (MS and NMR), combined with relevant literature data. Compounds I-1 to I-3 are novel compounds not previously reported in the literature. See below:

[0049]

[0050] Example 2

[0051] Step 1: Using 10.0 kg of dried whole herb of the golden thread brush as raw material, cut it into small pieces, and extract it three times (30 min) by heating and reflux with 10 times the amount of 95% ethanol and 75% ethanol respectively. After filtration, the filtrates were combined and the solvent was recovered under reduced pressure at 30℃~45℃ to obtain the extract.

[0052] Steps 2-7 are the same as steps 2-7 in Example 1, to prepare compounds I-1-I-4.

[0053] Example 3

[0054] Assay for acetylcholinesterase (AChE) inhibitory activity of 2,4-dihydroxy-6-benzylcarboxylic acid polyol esters

[0055] Preparation of AChE solution: Dissolve in 10 mL of PBS buffer to prepare a 0.05 U / mL AChE solution. Preparation of ATCI solution: Accurately weigh 21.7 mg ATCI into a centrifuge tube, add 10 mL of PBS buffer and dissolve thoroughly to prepare a 7.5 mmol / L solution. Store at 4°C protected from light for later use. Preparation of DTNB solution: Accurately weigh 39.6 mg DTNB into a centrifuge tube, then add 10 mL of phosphate buffer and dissolve thoroughly to prepare a 10 mmol / L solution (prepare fresh before use). Preparation of compound solution: Accurately weigh the compound and dissolve it in DMSO. Dilute the compound to different concentrations with PBS buffer as needed. Similarly, prepare galantamine solution as a positive control. Add 140 μL of phosphate buffer, 20 μL of sample solution, and 20 μL of AChE solution sequentially to a 96-well plate, shake to mix thoroughly, and then incubate at 30°C for 15 min. Next, add 10 μL of DTNB and 10 μL of ATCI, vortex until homogeneous, and incubate at 37°C for 30 min. Finally, measure the absorbance (OD) of each well at 412 nm using a microplate reader. The positive control used galantamine instead of the test sample solution, the standard group used 20 μL of PBS instead of the sample solution, and the blank group used 40 μL of PBS instead of the test compound solution and AChE solution. Each group's data were repeated three times, and the inhibition rate was calculated using the following formula:

[0056] Inhibition rate (%) = (OD standard - OD sample) / (OD standard - OD blank) × 100%

[0057] Finally, SPSS software was used to calculate the half-maximal inhibitory concentration (IC50) of the test solution and the positive control drug against AChE. 50 )value.

[0058] Table 2 Results of the compounds inhibiting AChE activity

[0059]

[0060] Acetylcholinesterase inhibitors containing the compounds or compositions described in this invention can be available in forms suitable for oral or injectable applications, such as tablets, capsules, granules, injections, pills, syrups, powders, etc.

[0061] The above description of the embodiments is only for the purpose of helping to understand the method and central idea of ​​the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principle of the present invention, and these improvements and modifications are also included in the protection of the claims of the present invention.

Claims

1. 2,4-Dihydroxy-6-benzylcarboxylic acid polyol esters extracted and isolated from gold wire brushes, their extraction methods, and their uses. The chemical names of these compounds are: (2R,3S,4R)-2,3,4,5-tetrahydroxypentyl 2,4-dihydroxy-6-methylbenzoate, (2S,3S,4R)-2,3,4,5-tetrahydroxypentyl 2,4-dihydroxy-6-methylbenzoate, (2R,3R,4R)-2,3,4,5-tetrahydroxypentyl 2,4-dihydroxy-6-methylbenzoate, and (2R,3S,4R,5S)-2,3,4,5,6-pentahydroxyhexyl 2,4-dihydroxy - The chemical structural formula of 6-methylbenzoate is as follows:

2. The extraction method of the compound according to claim 1, characterized in that... Includes the following steps: Step 1: Using the dried medicinal materials from the gold wire brush as raw materials, add ethanol-water solution, extract, filter, combine the filtrates, recover ethanol under reduced pressure, concentrate until no ethanol remains, cool to room temperature, and obtain the extract for later use; Step 2: Dissolve the extract from Step 1 in purified water, extract with petroleum ether and dichloromethane, and recover the solvent under reduced pressure to obtain a three-part extract; Step 3: The aqueous layer from Step 2 was subjected to macroporous resin column chromatography for adsorption, and eluted with a gradient of water, 30% ethanol-water, 60% ethanol-water and 95% ethanol-water. The solution was concentrated to dryness under reduced pressure to obtain four fractions (water eluent, 30% ethanol-water eluent, 60% ethanol-water eluent and 95% ethanol-water eluent) for later use. Step 4: The 30% ethanol-water eluent from the fraction in Step 3 was further separated by reversed-phase silica gel column chromatography, using methanol-water and methanol gradient elution to obtain several eluted fractions. These fractions were then detected by thin-layer chromatography, combined according to the colorimetric results, and concentrated to dryness under reduced pressure to obtain 6 fractions (Fr.w1-Fr.w6). Step 5: Separate the fraction Fr.w2 obtained in Step 4 by polyamide column chromatography, using methanol-water and methanol gradient elution respectively, and detect by thin-layer chromatography. Combine and concentrate the fractions according to the colorimetric results for later use, to obtain 7 fractions (Fr.w21-Fr.w27) for later use. Step 6: Separate the fraction Fr.w22 obtained in Step 5 using Sephadex LH-20, detect it by thin-layer chromatography, and combine and concentrate it according to the colorimetric results for later use; Step 7: The concentrate obtained in Step 6 is separated by reversed-phase silica gel column chromatography, using methanol-water as the mobile phase for elution, and detected by thin-layer chromatography. The concentrates are then combined and concentrated based on the colorimetric results for later use. Step 8: The fraction obtained in Step 7 was separated and prepared by HPLC, using methanol-water as the mobile phase, to obtain compounds I-1, I-2, I-3 and I-4.

3. The method as described in claim 2, characterized in that step 1 comprises: using the dried medicinal material of *Gynostemma pentaphyllum* as raw material, adding 8-16 times the mass of the raw material to a 95% ethanol solution, ultrasonically extracting 1-3 times for 30 minutes each time, then using 8-16 times the mass of the raw material to a 75% ethanol solution, ultrasonically extracting 1-3 times, filtering, combining the filtrates, recovering ethanol under reduced pressure, and concentrating until no ethanol remains to obtain an extract; or using the dried medicinal material of *Gynostemma pentaphyllum* as raw material, adding 8-16 times the mass of the raw material to a 95% or 75% ethanol solution, ultrasonically extracting 1-3 times, filtering, combining the filtrates, and recovering ethanol under reduced pressure. Ethanol is concentrated until no ethanol remains to obtain an extract; or, using the dried medicinal material as raw material, 8-16 times its weight of 95% ethanol solution is added, and the mixture is heated and extracted 1-3 times, followed by 8-16 times its weight of 75% ethanol solution, and heated and extracted 1-3 times. The extract is then filtered, the filtrates are combined, ethanol is recovered under reduced pressure, and the mixture is concentrated until no ethanol remains to obtain an extract; or, using the dried medicinal material as raw material, 8-16 times its weight of 95% or 75% ethanol solution is added, and the mixture is heated and extracted 1-3 times, the extract is filtered, the filtrates are combined, ethanol is recovered under reduced pressure, and the mixture is concentrated until no ethanol remains to obtain an extract.

4. The use of the 2,4-dihydroxy-6-benzylcarboxylic acid polyol ester compound of claim 1 in the preparation of anti-acetylcholinesterase drugs.

5. A gold brush extract comprising the 2,4-dihydroxy-6-benzylcarboxylic acid polyol ester compound of claim 1.

6. The use of the gold brush extract of claim 5 in the preparation of anti-acetylcholinesterase drugs.

7. A pharmaceutical composition, characterized in that... This includes the 2,4-dihydroxy-6-benzylcarboxylic acid polyol ester compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

8. The use of the pharmaceutical composition of claim 7 in the preparation of an anti-acetylcholinesterase drug.

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