A method for extracting a galloyl pyranoglucoside from a lantana camara

Abstract

This invention discloses a method for extracting galloylpyranoside from Loropetalum chinense, belonging to the field of traditional Chinese medicine extraction technology. The extraction method of this invention is flexible and simple to operate, with mild reaction and processing conditions. Ultrasonic extraction technology offers advantages such as energy saving, environmental friendliness, and high efficiency. The entire process of this invention uses a single solvent system, allowing for solvent recovery and reuse, avoiding solvent waste and environmental pollution. Furthermore, the obtained galloylpyranoside has high purity, and the preparation process does not use toxic or harmful solvents, making it a green preparation suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extraction technology, and in particular to a method for extracting galloylpyranoside from Loropetalum chinense. Background Technology

[0002] Loropetalum chinense, an evergreen shrub or small tree belonging to the genus Loropetalum in the family Hamamelidaceae, has roots, leaves, flowers, and fruits that can all be used medicinally. It has long been used to treat traumatic bleeding and skin ulcers, exhibiting significant hemostatic, tissue-regenerating, anti-inflammatory, and analgesic effects. The branches and leaves of Loropetalum chinense contain various secondary metabolites with antibacterial, anti-inflammatory, antioxidant, and healing-promoting pharmacological effects. Among them, 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O-galloyl)-glucopyranoside (i.e., galloylglucopyranoside) is a phenolic acid substance with excellent antibacterial, antioxidant, and cell proliferation-promoting effects. However, this substance accounts for approximately 0.002% of Loropetalum chinense, making its isolation and purification difficult. Summary of the Invention

[0003] In view of this, the present invention provides a method for extracting galloylpyranoside from Loropetalum chinense. The process is simple, easy to operate, green, non-toxic, efficient, and low-cost, making it suitable for large-scale production.

[0004] The present invention relates to a method for extracting galloyl glucopyranoside from Loropetalum chinense, wherein the chemical structural formula of the extracted 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O-galloyl)-glucopyranoside (galloyl glucopyranoside) is as shown in formula (I):

[0005]

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] A method for extracting galloylpyranoside from Loropetalum chinense includes the following steps:

[0008] Step S1: Dry the branches and leaves of Loropetalum chinense in the shade, crush them, and sieve them to obtain powder with a particle size of 0.1-2.5 mm; extract the powder with a solvent using ultrasonic extraction, and concentrate the extract under reduced pressure to obtain Loropetalum chinense extract.

[0009] Step S2: Disperse the Loropetalum chinense extract obtained in step S1 with distilled water, and extract it using the first solvent. The mass ratio of the extract to the first solvent is 1:10 to 30. Concentrate the residue after extraction under reduced pressure to obtain the Loropetalum chinense extract residue.

[0010] Step S3: Add the extract residue of Loropetalum chinense obtained in step S2 to ethyl acetate for dissolution. The ratio of extract residue to ethyl acetate is 1:10-20 kg / L. Stir to dissolve completely, let stand for 30-100 min, pour the supernatant into another container, and repeat the above stirring and standing operation 3-5 times. Finally, concentrate the supernatant under reduced pressure to dryness.

[0011] Step S4, Macroporous Resin Chromatographic Separation: The packing material used is D101. The dried substance obtained in step S3 is dissolved in 10-20% ethanol (v / v). The ratio of dried substance to ethanol is 10-5:1 g / mL. The sample is loaded using 20-100% ethanol (v / v) as the mobile phase. 40% ethanol eluent is collected and concentrated to a specific gravity of 2.0-3.0 to obtain the Loropetalum chinense sample solution.

[0012] Step S5, High-Pressure Liquid Chromatography Separation: Connect the first and second chromatographic columns in series. The first column uses MCI packing material, and the second column uses C18 packing material. Equilibrate the columns with acetonitrile or methanol at a volume concentration of 5–30%. Use a UV detector with a wavelength of 200–400 nm. After the baseline stabilizes, load the sample solution from Step S4 onto the pre-packed column. Use acetonitrile or methanol at a volume concentration of 30–70% as the mobile phase. Collect the eluent based on UV detection at a wavelength of 250–300 nm. Collect the eluent, concentrate under reduced pressure, filter, and dry to obtain high-purity galloylpyranoside.

[0013] Preferably, the solvent in step S1 is ethanol or methanol with a volume concentration of 95%.

[0014] Preferably, the ultrasonic extraction conditions in step S1 are as follows: the ratio of powder to solvent is 1:5 to 10 kg / L, the extraction temperature is 40 to 80°C, the ultrasonic power is 300 to 600 W, the extraction time is 30 to 90 min, and the number of extractions is 1 to 4.

[0015] More preferably, the ultrasonic extraction conditions in step S1 are: the ratio of powder to solvent is 1:5 to 7 kg / L, the extraction temperature is 40 to 60°C, the ultrasonic power is 400 to 600 W, the extraction time is 30 to 60 min, and the number of extractions is 2 to 4.

[0016] Preferably, the first solvent mentioned in step S2 is petroleum ether, n-hexane, dichloromethane, or chloroform;

[0017] Preferably, the chromatographic column in step S4 has a packing particle size of 0.3–1.25 mm, a column length of 900–1000 mm, and an inner diameter of 40–60 mm.

[0018] Preferably, in step S5, the first chromatographic column has a packing particle size of 10-100 μm, a column length of 310-620 mm, and an inner diameter of 15-100 mm; the second chromatographic column has a packing particle size of 5-50 μm, a column length of 310-620 mm, and an inner diameter of 15-100 mm.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention discloses a method for extracting 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O-galloyl)-glucopyranoside from Loropetalum chinense. The method is flexible and simple to operate, with mild reaction and processing conditions. Ultrasonic extraction technology offers advantages such as energy saving, environmental friendliness, and high efficiency. The first solvent extraction effectively removes pigments and some gum-like substances, while the second solvent (ethyl acetate) further extracts and removes viscous pectin-like substances from the extract, facilitating subsequent sample loading. The use of macroporous resin not only removes pigments from the sample but also effectively removes some viscous insoluble substances, better protecting the subsequent column chromatography packing material and facilitating reuse. The method of tandem first and second chromatographic columns achieves effective separation of galloyl glucopyranoside. The entire process uses a single solvent system, allowing for solvent recovery and reuse, avoiding solvent waste and environmental pollution. The obtained galloyl glucopyranoside has high purity, and the preparation process does not use toxic or harmful solvents, making it a green preparation suitable for large-scale industrial production. Attached Figure Description

[0021] Figure 1 This is the mass spectrum (in negative ion mode) of 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O-galloyl)-glucopyranoside.

[0022] Figure 2 This is the 1H NMR spectrum of 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O-galloyl)-glucopyranoside.

[0023] Figure 3 This is the carbon NMR spectrum of 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O-galloyl)-glucopyranoside. Detailed Implementation

[0024] The present invention will be further described below with reference to the embodiments.

[0025] Example 1

[0026] A method for extracting 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O-galloyl)-glucopyranoside (galloylglucopyranoside) from Loropetalum chinense comprises the following steps:

[0027] Step S1: The raw material, Loropetalum chinense branches and leaves, is air-dried, pulverized, and sieved to obtain a powder of 0.1–2.5 mm. The powder is then subjected to ultrasonic extraction with 95% ethanol as the solvent. The extract is concentrated under reduced pressure to obtain Loropetalum chinense extract. The ultrasonic extraction conditions are as follows: the ratio of powder to solvent is 1:5 kg / L, the extraction temperature is 60℃, the ultrasonic power is 500 W, the extraction time is 45 min, and the number of extractions is 3.

[0028] Step S2: Disperse the Loropetalum chinense extract obtained in step S1 with distilled water, and extract it with petroleum ether. The mass ratio of extract to petroleum ether is 1:14. Concentrate the residue after extraction under reduced pressure to obtain the Loropetalum chinense extract residue.

[0029] Step S3: Add the extract residue of Loropetalum chinense obtained in step S2 to ethyl acetate for dissolution. The ratio of extract residue to ethyl acetate is 1:15 kg / L. Stir to dissolve completely, let stand for 50 min, pour the supernatant into another container, and repeat the above stirring and standing operation 4 times. Finally, concentrate the supernatant under reduced pressure to dryness.

[0030] Step S4, Macroporous Resin Chromatographic Separation: The packing material used is D101. The dried product obtained in step S3 is dissolved in 10% ethanol (v / v), with a solid-liquid ratio of 5:1 g / mL. For sample loading, 20% ethanol (v / v) is used as the mobile phase for elution for 3.5 h to remove low-polarity impurities. Then, 40% ethanol (v / v) is used as the mobile phase for elution for 4.5 h. The 40% ethanol eluent is collected and concentrated to a specific gravity of 2.5 to obtain the Loropetalum chinense sample solution. The chromatographic column has a packing material particle size of 0.3–1.25 mm, a column length of 950 mm, and an inner diameter of 50 mm.

[0031] Step S5, Medium- and High-Performance Liquid Chromatography (HPLC) Separation: Connect the first and second chromatographic columns in series. The first column uses MCI packing material with a particle size of 30 μm, a column length of 310 mm, and an inner diameter of 30 mm. The second column uses C18 packing material with a particle size of 10 μm, a column length of 380 mm, and an inner diameter of 35 mm. Equilibrate the column with 5% methanol (v / v). Use a UV detector at 275 nm. After baseline stabilization, load the sample solution from step S4 onto the pre-packed column at a volume of 150 mL. The temperature was room temperature, and 35% methanol (v / v) was used as the mobile phase at a flow rate of 20 mL / min. The eluent was collected by UV detection at a wavelength of 250 nm. The eluent containing the target analyte was collected, concentrated under reduced pressure, filtered, and dried to obtain high-purity 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O-galloyl)-glucopyranoside. HPLC analysis showed a purity of 98.9% and a yield of 1.3%. The pressure of the high-pressure liquid chromatography separation was less than 0.8 MPa.

[0032] The packing materials used in the examples were all commercially available products.

[0033] The mass spectrum (in negative ion mode) of 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O-galloyl)-glucopyranoside extracted in Example 1 is as follows. Figure 1 ; 1H NMR spectrum as shown Figure 2 ; Carbon NMR spectrum as shown Figure 3 .

[0034] Combination Figure 1 , Figure 2 , Figure 3Spectroscopic data of 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O-galloyl)-glucopyranoside: ESI-MS m / z: 497.1298 [MH]-; 1H NMR(400MHz, Methanol-d4)δ:7.07(s,2H,H-2',6'),6.41(s,2H,H-3,5),4.84(d,J=7.2Hz,1H,H-1'),4.64(dd,J=12.0,2.0Hz,1H,H -6'a),4.44(dd,J=11.9,6.6Hz,1H,H-6'b),3.68-3.67(9H,s,2,4,6-OMe),3.54–3.42(m,4H,H-2'-5'); 13CNMR(101MHz,MeOD)δ:13 3.31(C-1),153.35(C-2,6),94.98(C-3,5),154.47(C-4),101.88(C-1'),73.50(C-2'),76.28(C-3'),70.36(C-4'),74.41(C-5'), 63.71(C-6′),120.00(C-1″),108.76(C-2″,6″),145.19(C-3″,5″),138.50(C-4″),166.88(C-7″),59.85(4-OMe),55.12(2,6-OMe).

[0035] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A process for the extraction of 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O-galloyl)- glucopyranoside from Murraya exotica characterized in that, It comprises the following steps: Step S1, dry, crush and sieve the raw material Miltaria cordata branches and leaves to obtain powder with a size of 0.1-2.5 mm; extract the powder with a solvent by ultrasonic extraction, and concentrate the extract under reduced pressure to obtain Miltaria cordata extract; Step S2, disperse the Miltaria cordata extract obtained in Step S1 with distilled water, and extract it with a first solvent, with the mass ratio of the extract to the first solvent being 1:10-30; concentrate the residue after extraction under reduced pressure to obtain Miltaria cordata extraction residue; Step S3, dissolve the Miltaria cordata extraction residue obtained in Step S2 in ethyl acetate, with the solid-liquid ratio of the residue to ethyl acetate being 1:10-20 kg / L; stir to dissolve thoroughly, stand for 30-100 min, pour the supernatant into another container, and repeat the stirring and standing operations for 3-5 times; finally, concentrate the supernatant under reduced pressure to dryness; Step S4, macroporous resin chromatography separation: use D101 as the packing material, dissolve the dry substance obtained in Step S3 in ethanol with a volume concentration of 10-20%, with the solid-liquid ratio of the dry substance to ethanol being 10-5:1 g / mL; load the sample, use ethanol with a volume concentration of 20-100% as the mobile phase, collect the 40% ethanol eluent, and concentrate the eluent to a specific gravity of 2.0-3.0 to obtain Miltaria cordata loading solution; Step S5, medium-high pressure liquid chromatography separation: connect the first chromatographic column and the second chromatographic column in series, use MCI as the packing material for the first chromatographic column and C18 as the packing material for the second chromatographic column, equilibrate the columns with acetonitrile or methanol with a volume concentration of 5-30%, and set the wavelength of the ultraviolet detector to 200-400 nm; after the baseline is stable, load the loading solution in Step S4 into the pre-packed column, use acetonitrile or methanol with a volume concentration of 30-70% as the mobile phase, collect the eluent according to the UV detection, set the detection wavelength of the UV detector to 250-300 nm, collect the eluent flowing out, and concentrate, filter and dry the eluent to obtain high-purity 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O-galloyl)-glucopyranoside; The solvent in Step S1 is ethanol or methanol with a volume concentration of 95%; The first solvent in Step S2 is petroleum ether; The pressure in the medium-high pressure liquid chromatography separation in Step S5 is less than 0.8 MPa.

2. The process for the extraction of 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O- gallolyl)-glucopyranoside from Murraya koenigii according to claim 1, characterized in that, The ultrasonic extraction conditions in Step S1 are as follows: the solid-liquid ratio of the powder to the solvent is 1:5-10 kg / L, the extraction temperature is 40-80°C, the ultrasonic power is 300-600 W, the extraction time is 30-90 min, and the extraction times are 1-4.

3. The process for the extraction of 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O- gallolyl)-glucopyranoside from Murraya koenigii according to claim 1, characterized in that, The ultrasonic extraction conditions in Step S1 are as follows: the solid-liquid ratio of the powder to the solvent is 1:5-7 kg / L, the extraction temperature is 40-60°C, the ultrasonic power is 400-600 W, the extraction time is 30-60 min, and the extraction times are 2-4.

4. The process for the extraction of 2,4,6-trimethoxyphenol-1-O-β-D-(6'-O- gallolyl)-glucopyranoside from Murraya koenigii according to claim 1, characterized in that, The particle size of the packing material of the chromatographic column in Step S4 is 0.3-1.25 mm, the column length is 900-1000 mm, and the column inner diameter is 40-60 mm.

5. The process for the isolation of 2,4,6-trimethoxyphenol-l-O-β-D-(6'-O- gallolyl)-glucopyranoside from the plant Murraya koenigii according to claim 1, characterized in that, In the first chromatographic column in step S5, the particle size of the filler is 10-100 μm, the column length is 310-620 mm, and the column inner diameter is 15-100 mm; in the second chromatographic column, the particle size of the filler is 5-50 μm, the column length is 310-620 mm, and the column inner diameter is 15-100 mm.

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