A method for extracting and separating 7-deoxyloganin and 7beta-O-methylmorroniside from the pulp of the fruit of the plant of the genus of the plant of the genus of the plant of the genus of the plant of the genus of the plant of the genus of the plant of the genus of

Abstract

This invention discloses a method for extracting and separating 7-Deoxyloganin and 7β-O-methylmorroniside from the pulp of Cornus officinalis. The method involves using dried, mature Cornus officinalis pulp as raw material, extracting it with ethanol at room temperature, and then separating the extract using D101 macroporous adsorption column chromatography. Gradient elution is performed using water, 20% ethanol, 40% ethanol, 60% ethanol, and 95% ethanol as solvents. The 20% ethanol elution fraction (Fr.A) is selected for separation using silica gel column chromatography, eluted with dichloromethane and methanol to obtain nine fractions Fr1-Fr9. Further purification yields the pure compounds. This invention provides a method for extracting and separating 7-Deoxyloganin and 7β-O-methylmorroniside from dried, mature Cornus officinalis pulp, which is beneficial for better development and utilization of this medicinal plant and promotes its sustainable use. The extraction process is simple, easily yields the monomers of 7-Deoxyloganin and 7β-O-methylmorroniside, the solvent can be recovered, the extraction yield is large, and it is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction and separation technology, specifically relating to a method for effectively and rapidly extracting and separating compounds 7-Deoxyloganin and 7β-O-methylmorroniside from the dried mature pulp of Cornus officinalis Sieb. et Zucc. Background Technology

[0002] Cornus officinalis (Corni Fructus) is a plant belonging to the genus Cornus in the family Cornaceae. Cornus officinalis The dried, ripe pulp of *Sieb. et Zucc.*. The main components of *Corni Fructus* include flavonoids, saponins, alkaloids, steroids, and phenols, as well as a large amount of oil. *Corni Fructus* possesses various pharmacological effects, including anti-inflammatory, anti-diabetic, antioxidant, antitumor, antibacterial, neuroprotective, and hepatoprotective properties.

[0003] Currently, there are no reports on the extraction and separation of 7-Deoxyloganin and 7β-O-methylmorroniside from Cornus officinalis pulp. This invention, however, rapidly and effectively extracts and separates 7-Deoxyloganin and 7β-O-methylmorroniside from the ethanol extract of Cornus officinalis using macroporous resin column chromatography, silica gel column chromatography, gel column chromatography, and high-performance liquid chromatography. Summary of the Invention

[0004] The purpose of this invention is to overcome the deficiencies of the prior art and provide a method for effectively and rapidly extracting and separating the compounds 7-Deoxyloganin and 7β-O-methylmorroniside from the dried mature pulp of Cornus officinalis Sieb. et Zucc.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for extracting and isolating 7-Deoxyloganin and 7β-O-methylmorroniside from Cornus officinalis pulp, comprising the following steps: 1) Using dried mature fruit pulp of Cornus officinalis as raw material, the extract was extracted with ethanol, and the solvent was recovered under reduced pressure to obtain an extract paste. The extract paste was dissolved in methanol, mixed and chromatographically loaded onto a D101 macroporous adsorption column, and then the column was packed. Gradient elution was performed sequentially using water, 20% ethanol, 40% ethanol, 60% ethanol and 95% ethanol as solvents. The eluents were concentrated under reduced pressure to obtain the 20% ethanol eluent Fr.A, 40% ethanol eluent Fr.B, 60% ethanol eluent Fr.C and 95% ethanol eluent Fr.D. The 20% ethanol eluent Fr.A was selected for separation by silica gel column chromatography, and the fractions were detected and combined by silica gel thin-layer chromatography to obtain 9 fractions, which were labeled as Fr1-Fr9 according to the increasing polarity of the fractions. 2) The fraction Fr5 obtained in step 1) was separated by silica gel column chromatography and detected and combined by silica gel thin-layer chromatography to obtain two fractions, which were labeled as 5-1 to 5-2 in order of increasing polarity. Fraction 5-2 was separated by silica gel column chromatography under reduced pressure and detected and combined by silica gel thin-layer chromatography to obtain two fractions, which were labeled as 5-2-1 to 5-2-2 in order of increasing polarity. Fraction 5-2-2 was then separated by silica gel column chromatography to obtain crude compound 7-Deoxyloganin. 3) The fraction Fr6 obtained in step 1) was separated by silica gel column chromatography and detected by silica gel thin-layer chromatography to obtain two fractions. The fractions were labeled as 6-1 to 6-2 in order of increasing polarity. Fraction 6-1 was first separated by vacuum silica gel column chromatography to obtain fraction 6-1-1, and then purified by semi-preparative liquid chromatography to obtain pure compound 7β-O-methylmorroniside.

[0006] Specifically, in step 1), ethanol with a volume concentration of 75±5% is used for extraction at room temperature 2-4 times, each time for 2-4 days.

[0007] Furthermore, in step 1), when the 20% ethanol elution fraction Fr.A is separated by silica gel column chromatography, it is eluted sequentially with dichloromethane-methanol at volume ratios of 35:1, 25:1, 15:1, 10:1, 5:1, 3:1, and 1:1.

[0008] Specifically, in step 2), when component Fr5 is separated by silica gel column chromatography, it is eluted sequentially with ethyl acetate-methanol in volume ratios of 25:1, 20:1, 15:1, 10:1, 5:1, and 1:1.

[0009] Furthermore, in step 2), when component 5-2 is separated by a vacuum silica gel column, it is eluted sequentially with ethyl acetate-methanol in volume ratios of 25:1, 20:1, 15:1, 10:1, 5:1, and 1:1.

[0010] Furthermore, in step 2), during silica gel column chromatography separation, gradient elution is performed sequentially using dichloromethane-methanol at volume ratios of 8:1, 5:1, 3:1, and 1:1.

[0011] Specifically, in step 3), when component Fr6 is separated by silica gel column chromatography, it is eluted sequentially with dichloromethane-methanol at volume ratios of 100:1, 50:1, 25:1, 15:1, 5:1, and 1:1.

[0012] Furthermore, in step 3), when component 6-1 is separated by a vacuum silica gel column, isocratic elution is performed using dichloromethane-methanol at a volume ratio of 5:1; during semi-preparative liquid phase purification, methanol-water at a volume ratio of 37:63 is used as the eluent.

[0013] In a further preferred embodiment, in step 2), the crude 7-Deoxyloganin compound is dissolved in methanol, and semi-preparative liquid phase purification is performed using a methanol-water mixture with a volume ratio of 35:65 as the eluent to obtain pure 7-Deoxyloganin.

[0014] As a preferred technical solution, the present invention provides a method for extracting and separating 7-Deoxyloganin and 7β-O-methylmorroniside from Cornus officinalis, specifically including the following steps: 1) Using dried mature fruit pulp of Cornus officinalis as raw material, it was extracted three times at room temperature with 75% ethanol for three days each time. The extracts were combined and the solvent was recovered under reduced pressure to obtain an extract paste. The extract paste was dissolved in methanol, mixed and chromatographically loaded onto a D101 macroporous adsorption column. The column was packed and eluted sequentially with water, 20% ethanol, 40% ethanol, 60% ethanol and 95% ethanol as solvents. The eluents were concentrated under reduced pressure to obtain the 20% ethanol eluent (Fr.A), 40% ethanol eluent (Fr.B), 60% ethanol eluent (Fr.C) and 95% ethanol eluent (Fr.D). The fraction eluted with 20% ethanol (Fr.A) was separated by silica gel column chromatography. The silica gel column was eluted sequentially with dichloromethane-methanol (35:1, 25:1, 15:1, 10:1, 5:1, 3:1, 1:1, volume ratio). The fractions were detected and combined by silica gel thin-layer chromatography, resulting in 9 fractions, which were labeled Fr1-Fr9 according to their polarity from smallest to largest. 2) The fraction Fr5 obtained in step 1) was separated by silica gel column chromatography with gradient elution of ethyl acetate-methanol (25:1, 20:1, 15:1, 10:1, 5:1, 1:1, v / v). The fractions were then detected and combined by silica gel thin-layer chromatography, yielding two fractions, labeled 5-1 to 5-2 according to their increasing polarity. Fraction 5-2 was separated by vacuum silica gel column chromatography with gradient elution of ethyl acetate-methanol (25:1, 20:1, 15:1, 10:1, 5:1, 1:1, v / v). The fractions were then detected and combined by silica gel thin-layer chromatography, yielding two fractions, labeled 5-2-1 to 5-2-2 according to their increasing polarity. Fraction 5-2-2 was further separated by silica gel column chromatography with gradient elution of dichloromethane-methanol (8:1, 5:1, 3:1, 1:1, v / v), yielding crude compound 7-Deoxyloganin. The crude compound 7-Deoxyloganin was dissolved in methanol and purified by semi-preparative liquid chromatography (eluent: methanol-water 35:65, volume ratio) to obtain pure compound 7-Deoxyloganin. 3) The fraction Fr6 obtained in step 1) was separated by silica gel column chromatography with gradient elution using dichloromethane-methanol (100:1, 50:1, 25:1, 15:1, 5:1, 1:1, v / v). The fractions were then detected and combined by silica gel thin-layer chromatography, yielding two fractions, which were labeled as 6-1 to 6-2 according to their polarity from smallest to largest. Fraction 6-1 was first separated by vacuum silica gel column chromatography with isocratic elution using dichloromethane-methanol (5:1, v / v) to obtain fraction 6-1-1, which was then purified by semi-preparative liquid chromatography (eluent: methanol-water 37:63, v / v) to obtain pure compound 7β-O-methylmorroniside.

[0015] The 7-Deoxyloganin described in this invention has the molecular formula: C 17 H 26 O9, molecular weight: 374.4, its structural formula is shown below.

[0016]

[0017] The 7β-O-methylmorroniside described in this invention has the molecular formula: C 18 H 28 O 11 Its molecular weight is 420.41, and its structural formula is shown below.

[0018]

[0019] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1) To date, there have been no reports of extracting and isolating compounds 7-Deoxyloganin and 7β-O-methylmorroniside from the plant Cornus officinalis. This invention provides a simple process for extracting and isolating such compounds from Cornus officinalis, which is beneficial for better development and utilization of this medicinal plant.

[0020] 2) The 7-Deoxyloganin and 7β-O-methylmorroniside extracted and separated by this invention have a wide range of biological activities. This invention provides a method for extracting and separating the compounds 7-Deoxyloganin and 7β-O-methylmorroniside, which facilitates in-depth research on them.

[0021] 3) The separation materials used in this invention include reversed-phase chromatography, silica gel chromatography, and gel column chromatography. These materials are insoluble in water and any solvent, non-toxic and odorless, chemically stable, and exhibit good selectivity for organic compounds. They are unaffected by the presence of inorganic salts and strong ionic low-molecular-weight compounds, and are easily eluted and regenerated, making them suitable for multiple uses. The preparative high-performance liquid chromatography (HPLC) used in the purification process is currently the most popular new separation method, saving time and effort and significantly improving sample yield. Detailed Implementation

[0022] To make the technical objectives, technical solutions, and beneficial effects of the present invention clearer, the technical solutions of the present invention will be further described below with reference to specific embodiments. However, the embodiments described are intended to explain the present invention and should not be construed as limiting the present invention. In the following examples, all raw materials used are common commercially available products that can be directly purchased, or can be prepared using conventional techniques in the art. Where specific techniques or conditions are not specified in the examples, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions.

[0023] In this embodiment, the Cornus officinalis used as raw material was collected from Luanchuan County, Luoyang City, Henan Province; the concentrations of ethanol and methanol are both volume concentrations.

[0024] Room temperature refers to 25±5℃. Example 1

[0025] A method for extracting and separating 7-Deoxyloganin and 7β-O-methylmorroniside from Cornus officinalis specifically includes the following steps: (1) Using 20 kg of dried mature Cornus officinalis pulp as raw material, it was extracted three times with 75% ethanol at room temperature for three days each time. The extracts were combined and the solvent was recovered under reduced pressure to obtain an extract paste. The extract paste was dissolved in methanol and separated by D101 macroporous adsorption column chromatography. Gradient elution was performed with water, 20% ethanol, 40% ethanol, 60% ethanol and 95% ethanol as solvents. The eluents were concentrated under reduced pressure and weighed to obtain the 20% ethanol eluent (Fr.A), 40% ethanol eluent (Fr.B), 60% ethanol eluent (Fr.C) and 95% ethanol eluent (Fr.D). The fraction eluted with 20% ethanol (Fr.A) was separated by silica gel column chromatography. The silica gel column was sequentially eluted with dichloromethane-methanol (35:1, 25:1, 15:1, 10:1, 5:1, 3:1, 1:1, volume ratio). The fractions were detected and combined by silica gel thin-layer chromatography, resulting in 9 fractions, which were labeled Fr1-Fr9 in ascending order of polarity.

[0026] (2) The fraction Fr5 obtained in step 1) was separated by silica gel column chromatography with gradient elution of ethyl acetate-methanol (25:1, 20:1, 15:1, 10:1, 5:1, 1:1, volume ratio), and detected and combined by silica gel thin-layer chromatography to obtain two fractions, which were labeled as 5-1 to 5-2 in order of increasing polarity. Fraction 5-2 was first separated by silica gel column chromatography under reduced pressure with gradient elution of ethyl acetate-methanol (25:1, 20:1, 15:1, 10:1, 5:1, 1:1, volume ratio), and detected and combined by silica gel thin-layer chromatography to obtain two fractions, which were labeled as 5-2-1 to 5-2-2 in order of increasing polarity. Fraction 5-2-2 was then separated by silica gel column chromatography with gradient elution of dichloromethane-methanol (8:1, 5:1, 3:1, 1:1, volume ratio) to obtain crude compound 7-Deoxyloganin. The crude compound 7-Deoxyloganin was dissolved in methanol and purified by semi-preparative liquid chromatography (C18 reversed-phase column, eluent, methanol-water 35:65, volume ratio) to obtain pure 7-Deoxyloganin.

[0027] (3) The fraction Fr6 obtained in step 1) was separated by silica gel column chromatography and eluted with dichloromethane-methanol (100:1, 50:1, 25:1, 15:1, 5:1, 1:1, v / v). The fractions were then detected and combined by silica gel thin-layer chromatography to obtain two fractions, which were labeled as 6-1 to 6-2 in order of increasing polarity. Fraction 6-1 was first separated by vacuum silica gel column chromatography and eluted isocratically with dichloromethane-methanol (5:1, v / v) to obtain fraction 6-1-1. Then it was purified by semi-preparative liquid chromatography (C18 reversed-phase column, eluent, methanol-water 37:63, v / v) to obtain pure compound 7β-O-methylmorroniside.

[0028] The obtained compound 7-Deoxyloganin is a light brown oil with a yield of 0.00050% and a purity ≥96%. Compound 7β-O-methylmorroniside is a pale yellow crystal with a yield of 0.00040% and a purity ≥98%. The structures of the obtained compounds were identified using various spectroscopic techniques, as detailed below.

[0029] Instruments and materials: Ultraviolet light was measured using a UV-210A ultraviolet spectrometer; 1 H, 13 C-NMR spectra were measured using a Brukeram-500 MHz nuclear magnetic resonance spectrometer. The experimental results are as follows.

[0030] 1) Compound 1: 7-Deoxyloganin, light brown oil, m / z: 374.4, molecular formula: C 17 H 26 O9. 1H-NMR (CD3OD, 500 MHz) δH: 7.49 (1H ,s, H-3), 5.63 (1H, d, J = 2.9 Hz, H-1), 4.68 (1H, d, J = 7.9 Hz, H-1′), 3.90 (1H, dd, J = 11.9, 2.1 Hz, H-6′a), 3.70(3H, s, 11-OCH3), 3.66 (1H, dd, J = 11.9, 6.1 Hz, H-6′b), 3.37 (1H, t, J =9.0 Hz, H-3′), 3.30 – 3.25 (2H, m, H-4′,5′), 3.20 (1H, dd, J = 9.2, 8.0 Hz,H-2′), 2.34 (1H, dd, J = 6.7, 3.5 Hz, H-9), 2.09 (1H, m, H-8), 1.15 (3H, d, J= 7.4 Hz, H-10) 13 C-NMR (CD3OD, 126 MHz) δC: 77.4 (C-1), 201.0 (C-2), 117.5 (C-3), 161.7 (C-4), 129.2 (C-5), 124.8 (C-6), 159.4 (C-7), 113.5 (C-8), 147.8(C-9), 121.8 (C-10), 38.2 (C-11), 67.1 (C-12), 17.3 (C-13), 33.1 (C-14), 16.1(C-15).

[0031] Tests: Using TLC, no fluorescence was observed at UV 254nm; with sulfuric acid developer, a dark brown color was observed after 5 minutes at 110℃.

[0032] 2) Compound 2: 7β-O-methylmorroniside, pale yellow crystals, m / z: 420.41, molecular formula: C 18 H 28 O 11 . 1H-NMR (CD3OD, 500 MHz) δH: 7.51 (1H, s, H-3), 5.89 (1H, d, J = 9.2Hz, H-1), 4.80 (1H, d, J = 7.9 Hz, H-1′), 4.75 (1H, d, J = 3.7 Hz, H-7), 4.30(1H, qd, J = 6.9, 2.4 Hz, H-8), 3.90 (1H, dd, J = 12.2, 2.0 Hz, H-6′b), 3.69(3H, s, 11-OCH3), 3.66 (1H, d, J = 5.9 Hz, H-6′a), 3.40 (1H, t, J = 8.7 Hz,H-3′), 3.36 (3H, s, 7-OCH3), 3.32 – 3.27 (2H, m, H-4′,5′), 3.23 (1H, t, J =8.7 Hz, H-2′), 3.05 (1H, dt, J = 12.9, 4.7 Hz, H-5), 1.93 (1H, dd, J = 13.9, 4.7 Hz, H-6b), 1.85 – 1.79 (1H, m, H-9), 1.52 (1H, td, J = 13.4, 3.9 Hz, H-6a), 1.35 (3H, d, J = 7.0 Hz, 10-CH3). 13 C-NMR (CD3OD, 126 MHz) δC: 95.6 (C-1), 154.5 (C-3), 111.6 (C-4), 28 (C-5), 33.7 (C-6), 99.5 (C-7), 66.3 (C-8), 40.3 (C-9), 19.6 (C-10), 168.7 (C-11), 51.7 (11-OCH3), 55 (7-OCH3), 100.1 (C-1′), 75 (C-2′), 77.9 (C-3′), 71.6 (C-4′), 78.5 (C-5′), 62.8 (C-6′).

[0033] Tests: Using TLC, no fluorescence was observed at UV 254nm; with sulfuric acid developer, a dark purple color was observed after 5 minutes at 110℃.

[0034] In summary, the method for extracting and separating compounds 7-Deoxyloganin and 7β-O-methylmorroniside from the mature pulp of dried Cornus officinalis described in this invention is beneficial for better development and utilization of Cornus officinalis as a medicinal plant and promotes the sustainable use of the plant. The extraction process of this invention is simple, easily yields the monomers of 7-Deoxyloganin and 7β-O-methylmorroniside, the solvent can be recycled, the extraction yield is large, and it can be industrialized.

Claims

1. A method for extracting and separating 7-Deoxyloganin and 7β-O-methylmorroniside from the pulp of Cornus officinalis, characterized in that, Includes the following steps: 1) Using dried mature fruit pulp of Cornus officinalis as raw material, the extract was extracted with ethanol, and the solvent was recovered under reduced pressure to obtain an extract paste. The extract paste was dissolved in methanol, mixed and chromatographically loaded onto a D101 macroporous adsorption column, and then the column was packed. Gradient elution was performed sequentially using water, 20% ethanol, 40% ethanol, 60% ethanol and 95% ethanol as solvents. The eluents were concentrated under reduced pressure to obtain the 20% ethanol eluent Fr.A, 40% ethanol eluent Fr.B, 60% ethanol eluent Fr.C and 95% ethanol eluent Fr.D. The 20% ethanol eluent Fr.A was selected for separation by silica gel column chromatography, and the fractions were detected and combined by silica gel thin-layer chromatography to obtain 9 fractions, which were labeled as Fr1-Fr9 according to the increasing polarity of the fractions. 2) The Fr5 component obtained in step 1) was separated by silica gel column chromatography and detected and combined by silica gel thin-layer chromatography to obtain two components, which were labeled as 5-1 to 5-2 in order of increasing polarity. Component 5-2 was separated by vacuum silica gel column chromatography and detected by silica gel thin-layer chromatography to obtain two components, which were labeled as 5-2-1 to 5-2-2 in order of increasing polarity. Component 5-2-2 was then separated by silica gel column chromatography to obtain crude compound 7-Deoxyloganin. 3) The fraction Fr6 obtained in step 1) was separated by silica gel column chromatography and detected by silica gel thin-layer chromatography to obtain two fractions. The fractions were labeled as 6-1 to 6-2 in order of increasing polarity. Fraction 6-1 was first separated by vacuum silica gel column chromatography to obtain fraction 6-1-1, and then purified by semi-preparative liquid chromatography to obtain pure compound 7β-O-methylmorroniside.

2. The method as described in claim 1, characterized in that, In step 1), use ethanol with a volume concentration of 75±5% for extraction 2-4 times at room temperature, each time for 2-4 days.

3. The method as described in claim 1, characterized in that, In step 1), when separating the 20% ethanol elution fraction Fr.A using silica gel column chromatography, gradient elution was performed sequentially with dichloromethane-methanol at volume ratios of 35:1, 25:1, 15:1, 10:1, 5:1, 3:1, and 1:

1.

4. The method as described in claim 1, characterized in that, In step 2), when component Fr5 is separated by silica gel column chromatography, it is eluted sequentially with ethyl acetate-methanol in volume ratios of 25:1, 20:1, 15:1, 10:1, 5:1, and 1:

1.

5. The method as described in claim 1, characterized in that, In step 2), when component 5-2 is separated by a vacuum silica gel column, it is eluted sequentially with ethyl acetate-methanol in volume ratios of 25:1, 20:1, 15:1, 10:1, 5:1, and 1:

1.

6. The method as described in claim 1, characterized in that, In step 2), during silica gel column chromatography, gradient elution is performed sequentially using dichloromethane-methanol at volume ratios of 8:1, 5:1, 3:1, and 1:

1.

7. The method as described in claim 1, characterized in that, In step 3), when component Fr6 is separated by silica gel column chromatography, it is eluted sequentially with dichloromethane-methanol at volume ratios of 100:1, 50:1, 25:1, 15:1, 5:1, and 1:

1.

8. The method as described in claim 1, characterized in that, In step 3), when component 6-1 is separated by a vacuum silica gel column, isocratic elution is performed using dichloromethane-methanol at a volume ratio of 5:1; during semi-preparative liquid chromatography purification, methanol-water at a volume ratio of 37:63 is used as the eluent.

9. The method as described in claim 1, characterized in that, In step 2), the crude 7-Deoxyloganin compound was dissolved in methanol, and semi-preparative liquid phase purification was performed using a methanol-water mixture with a volume ratio of 35:65 as the eluent to obtain pure 7-Deoxyloganin.

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