Method for extracting and purifying Calceolariside E and Paucifloside

Abstract

The invention discloses a method for extracting and purifying Calceolariside E and Paucifloside, and belongs to the technical field of separation and purification of plant extracts. The invention relates to a method for extracting and purifying Calceolarioside E and Paucifloside, which comprises the following steps of: mixing Fujian Jiangxi boea cavaleriei and a solvent, and performing ultrasonic extraction to obtain an extracting solution; separating the extracting solution by adopting a gel chromatographic column, and then separating the extracting solution by adopting a reversed-phase chromatographic column, so as to obtain an enriched solution of Calceolariside E and Paucifloside; the enrichment liquid is dried, then redissolved and dried again, and the Calceolariside E and the Paucifloside are obtained. According to the method disclosed by the invention, Calceolariside E and Paucifloside are separated from Jiangxi boea longissima for the first time, the speed of separating the compound is relatively high, and the purity of the obtained compound is relatively high.

Description

Technical Field

[0001] The present invention relates to the technical field of separation and purification of plant extracts, and particularly to a method for extracting and purifying Calceolarioside E and Paucifloside. Background Art

[0002] Calceolarioside E was first isolated from Calceolaria ascendens and has significant antioxidant effects and is a natural activator of NRF2. Paucifloside was first isolated from Lysionotus pauciflorus and also has strong antioxidant activity, and the SC value of its ability to scavenge DPPH free radicals 50 is 18.7 μM.

[0003] Didymocarpus heucherifolius W. T. Wang of the family Gesneriaceae is a perennial herb with a thick rhizome. It grows on the roadside of valleys, on the rocks by streams or under forests at an altitude of 530 m. The whole herb can be used as medicine. The whole herb is sweet and cool, and has the effects of detoxifying, detumescing and treating otitis media. Didymocarpus heucherifolius W. T. Wang contains a variety of active ingredients, mainly including terpenoids, flavonoids, phenols, fatty acids, chalcones, steroids, etc. These ingredients have wide application values in the medical field, such as functions of anti-urolithiasis, kidney protection, antibacterial, anti-cancer, anti-diabetes, cytotoxicity, wound healing and antioxidant. However, no method for extracting Calceolarioside E and Paucifloside from Didymocarpus heucherifolius W. T. Wang has been found. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for extracting and purifying Calceolarioside E and Paucifloside to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above purpose, the present invention provides the following solution:

[0006] One of the technical solutions of the present invention: A method for extracting and purifying Calceolarioside E and Paucifloside, comprising the following steps:

[0007] Mix Didymocarpus heucherifolius W. T. Wang and a solvent and perform ultrasonic extraction to obtain an extract;

[0008] Separate the extract by a gel chromatography column and then by a reversed-phase chromatography column to obtain an enriched solution of Calceolarioside E and Paucifloside;

[0009] Dry the enriched solution, redissolve it, and then dry it again to obtain the Calceolarioside E (molecular formula: C 28 H 34 O 15 ) and Paucifloside (molecular formula: C 33 H 42 O 19 ).

[0010] The chemical structural formulas of Calceolarioside E and Paucifloside are as follows:

[0011]

[0012] Furthermore, the solvent includes methanol or ethanol.

[0013] Furthermore, the mass / volume ratio of the Didymocarpus heucherifolius W. T. Wang and the solvent is 1 g:10 mL to 1 g:20 mL.

[0014] Furthermore, the gel chromatography includes an LH-20 gel column.

[0015] Furthermore, the eluent used for separation by the gel chromatography column includes methanol.

[0016] Furthermore, the reverse-phase chromatography column includes a C18 column.

[0017] Furthermore, the solvent used for redissolution includes methanol or ethanol.

[0018] Furthermore, the temperature of the ultrasonic extraction is 30 - 70 °C, the number of extractions is 2 - 6 times, and the time for each extraction is 1 - 5 h.

[0019] The present invention discloses the following technical effects:

[0020] The present invention first separates Calceolarioside E and Paucifloside from Didymocarpus heucherifolius W. T. Wang, and the separation speed of the compounds is relatively fast, and the purity of the obtained compounds is relatively high, which has significant significance for the subsequent research and production of Calceolarioside E and Paucifloside in the pharmaceutical field. Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 is the secondary mass spectrometry spectrum of Calceolarioside E;

[0023] Figure 2 is the secondary mass spectrometry spectrum of Paucifloside;

[0024] Figure 3 is the HPLC detection result of Calceolarioside E;

[0025] Figure 4 is the HPLC detection result of Paucifloside. Detailed implementation manners

[0026] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0029] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0030] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0031] Instruments and materials adopted by the present invention:

[0032] Agilent 1260 high performance liquid chromatograph (including G1311B quaternary pump and G4212B DAD array diode detector, Agilent Technologies, Inc., USA)); Agilent 1220 high performance liquid chromatograph (Agilent Technologies, Inc., USA); KQ-300DE numerically controlled ultrasonic cleaner (Shanghai Yilin Scientific Instruments Co., Ltd.); AUW120D electronic analytical balance (Shimadzu Corporation, Japan); Mighty-10 ultrapure water machine (Shanghai Lidingshui Treatment Equipment Co., Ltd.); BZF-50 vacuum drying oven (Medical Equipment Factory of Shanghai Boxun Industry Co., Ltd.); Thin layer chromatography (Qingdao Ocean Chemical Factory); Didymocarpus heucherifolius W. T. Wang was collected from Simeng Town, Xupu County, Huaihua City, Hunan Province, 27°49′57.18″N, 110°31′4.02″E, collected in May 2023, and identified as Didymocarpus heucherifolius W. T. Wang by Professor Cai Wei of Hunan University of Medicine.

[0033] Example 1

[0034] A method for extracting and purifying Calceolarioside E and Paucifloside:

[0035] (1) Take 50.2189 g of Didymocarpus heucherifolius W. T. Wang leaves, slightly crush them with a pulverizer, pass through a No. 2 sieve (10 mesh), put them into a 2000 mL round-bottomed flask, add methanol according to the ratio of the amount of Didymocarpus heucherifolius W. T. Wang leaves to the solvent of 1 g:16 mL, and perform ultrasonic extraction 3 times at 50 °C for 1 h each time to obtain residue and filtrate. Combine the filtrate, and dry the filtrate under reduced pressure to obtain the Didymocarpus heucherifolius W. T. Wang leaf extract.

[0036] (2) Take 0.5145 g of the dried Didymocarpus heucherifolius W. T. Wang leaf extract, load it onto an LH-20 gel column with a column volume of 400 mL, elute with methanol as the eluent, collect the eluate, and determine the elution positions of Calceolarioside E and Paucifloside by thin layer chromatography (the developing agent is ethyl acetate, methanol and water with a volume ratio of 5:1:1, and the developing agent contains 0.1 vol.% formic acid based on the volume of the developing agent). Dry the eluate rich in Calceolarioside E and Paucifloside under reduced pressure to obtain the crude products of Calceolarioside E and Paucifloside.

[0037] (3) Using an Agilent 1220 high performance liquid chromatograph and a WondaSil C18-WR chromatographic column, analyze with an aqueous acetonitrile solution with a volume fraction of 12% for 42 min, collect the eluate of the chromatographic peaks where Calceolarioside E and Paucifloside are located, dry under reduced pressure, dissolve with methanol and filter, dry the filtrate to obtain pure Calceolarioside E and pure Paucifloside. The yields of Calceolarioside E and Paucifloside are 11.58 mg / g and 11.42 mg / g respectively.

[0038] Example 2

[0039] A method for extracting and purifying Calceolarioside E and Paucifloside:

[0040] (1) Take 30.4340 g of the roots of Didymocarpus heucherifolius, slightly crush them with a pulverizer, pass through a No. 2 sieve (10 mesh), put them into a 2000 mL round-bottomed flask, add methanol according to the ratio of the amount of Didymocarpus heucherifolius roots to the solvent of 1 g:16 mL, perform ultrasonic extraction 3 times at 50 °C for 1 h each time to obtain residues and filtrates, combine the filtrates, dry the filtrates under reduced pressure to obtain the extract of Didymocarpus heucherifolius roots.

[0041] (2) Take 0.5002 g of the dried extract of Didymocarpus heucherifolius roots, load it onto an LH-20 gel column with a column volume of 400 mL, elute with methanol as the eluent, collect the eluate, and determine the elution positions of Calceolarioside E and Paucifloside by thin layer chromatography (the developing agent is ethyl acetate, methanol and water with a volume ratio of 5:1:1, and the developing agent contains 0.1 vol.% formic acid based on the volume of the developing agent). Dry the eluate part enriched with Calceolarioside E and Paucifloside under reduced pressure to obtain the crude products of Calceolarioside E and Paucifloside.

[0042] (3) Using an Agilent 1220 high performance liquid chromatograph and a WondaSil C18-WR chromatographic column, analyze with an aqueous acetonitrile solution with a volume fraction of 12% for 42 min, collect the eluate of the chromatographic peaks where Calceolarioside E and Paucifloside are located, dry under reduced pressure, dissolve with methanol and filter, dry the filtrate to obtain pure Calceolarioside E and pure Paucifloside. The yields of Calceolarioside E and Paucifloside are 3.24 mg / g and 13.37 mg / g respectively.

[0043] The structural identification results of Calceolarioside E and Paucifloside prepared in Example 1 and Example 2 are as follows:

[0044] I. The mass spectrometry data of compound Calceolarioside E showed an ion of m / z 609.1823 for [M-H], indicating a molecular formula of C - H 28 H 34 O 15 .

[0045] The negative ion ESIMSMS spectrum showed further fragment ions at m / z 447.1518, m / z 163.0390, m / z 133.0283, and m / z 153.0547, indicating sequential fragmentation of the 3,4-dihydroxy phenethyl alcohol unit, pentose group, and hexose group. The fragment ions m / z 179.0340, m / z 161.0233, m / z 135.0440 showed the presence of the caffeoyl unit. The secondary mass spectrometry spectrum is shown in Figure 1 .

[0046] C 28 H 34 O 15 1 1H-NMR (700 MHz, MeOH-d4) δ: 7.60 (1H, d, J = 15.8 Hz, H-β”');

[0047] 7.05 (1H, s, H-2”'); 6.95 (1H, d, J = 8.2 Hz, H-6”'); 6.77 (1H, d, J = 8.2 Hz, H-5”'); 6.69 (1H, s, H-2); 6.66 (1H, d, J = 8.0 Hz, H-5); 6.56 (1H, d, J = 8.0 Hz, H-6); 6.28 (1H, d, J = 15.8 Hz, H-α”'); 5.34 (1H, s, H-1”); 4.38 (1H, d, J = 7.9 Hz, H-1'); 4.04 (1H, m, H-α); 3.89 (1H, s, H-2”); 3.40 - 3.77 (9H, m, Api / Glc-H); 2.80 (2H, m, H-β).

[0048] 1313C NMR (175 MHz, MeOH-d4) δ: 131.3 (C-1); 115.1 (C-2); 146.1 (C-3); 144.7 (C-4); 117.1 (C-5); 121.2 (C-6); 72.3 (C-α); 36.6 (C-β); 104.2 (C-1'); 75.8 (C-2'); 81.4 (C-3'); 70.5 (C-4'); 75.8 (C-5'); 62.3 (C-6'); 111.5 (C-1"); 78.2 (C-2"), 80.6 (C-3"); 75.2 (C-4"); 65.6 (C-5"), 127.6 (C-1'''); 114.9 (C-2'''); 146.9 (C-3'''); 149.8 (C-4'''); 116.3 (C-5'''); 123.1 (C-6'''); 116.5 (C'''-α'''); 147.7 (C-β'''); 168.3 (C=O).

[0049] II. The mass spectrometry data of the compound Paucifloside showed an ion of [M-H] at m / z 741.2247, indicating a molecular formula of C - H 33 H 42 O 19 .

[0050] The negative ion ESI-MS / MS spectrum showed further fragment ions at m / z 447.1518, m / z 163.0390, m / z 133.0283 and m / z 153.0547, indicating sequential fragmentation of the 3,4-dihydroxyphenethyl alcohol unit, the pentose group and the hexose group. The fragment ions m / z 579.1929, m / z 447.1518 indicated fragmentation of the hexose unit, and the fragment ions m / z 179.0340, m / z 161.0233, m / z 135.0440 showed the presence of the caffeoyl unit. The secondary mass spectrometry spectrum is shown in Figure 2 .

[0051] C 33 H 42 O 19 1 1H-NMR (700 MHz, MeOH-d4) δ: 7.60 (1H, d, J = 15.8 Hz, H-β''');

[0052] 7.05 (1H, s, H-2”'); 6.95 (1H, d, J = 8.2 Hz, H-6””); 6.78 (1H, d, J = 8.2 Hz, H-5””); 6.69 (1H, s, H-2); 6.67 (1H, d, J = 8.0 Hz, H-5); 6.57 (1H, d, J = 8.0 Hz, H-6); 6.28 (1H, d, J = 15.8 Hz, H-α”'); 5.34 (1H, s, H-1”); 4.90 (1H, s, H-1”'); 4.38 (1H, d, J = 7.9 Hz, H-1'); 4.00 (1H, m, H-α); 3.89 (1H, s, H-2”); 3.86 (1H, s, H-2”'); 3.40 - 3.77 (15H, m, Api / Glc-H); 2.80 (2H, m, H-β).

[0053] 13 13C NMR (175 MHz, MeOH-d4) δ: 131.3 (C-1); 115.1 (C-2); 146.1 (C-3); 144.7 (C-4); 116.3 (C-5); 121.3 (C-6); 72.4 (C-α); 36.6 (C-β); 104.2 (C-1'); 75.7 (C-2'); 81.3 (C-3'); 70.8 (C-4'); 74.6 (C-5'); 68.4 (C-6'); 111.5 (C-1”); 78.2 (C-2”), 80.6 (C-3”); 75.2 (C-4”); 65.5 (C-5”); 111.0 (C-1”'); 78.0 (C-2”'), 80.6 (C-3”'); 75.1 (C-4”'); 65.5 (C-5”'); 127.6 (C-1””); 114.9 (C-2””); 146.9 (C-3””); 149.8 (C-4””); 116.5 (C-5””); 123.2 (C-6””); 117.1 (C”'-α””); 147.8 (C-β””); 168.2 (C=O).

[0054] The chemical structures of Calceolarioside E and Paucifloside are as follows:

[0055]

[0056] Effect Example 1

[0057] Purity determination:

[0058] (1) Thin layer chromatography (TLC) method

[0059] The prepared Calceolarioside E, Paucifloside and reference substances were spotted on a silica gel plate respectively, and silica gel thin-layer chromatography was carried out using a mixed solution of ethyl acetate, methanol and water with a volume ratio of 5:1:1 (the developing agent contained 0.1 vol.% formic acid based on the volume of the developing agent) as the developing agent. Sulfuric acid-ethanol (v:v, 1:9) was used as the color-developing agent, heated, and the samples were observed under a daylight lamp.

[0060] When detected by the above developing system method, the samples showed single spots and no impurity spots.

[0061] (2) High Performance Liquid Chromatography (HPLC) method

[0062] Chromatographic column: Agilent Poroshell 120 EC-C18 column (150 nm × 4.6 mm, 4 μm);

[0063] Mobile phase: 0.5 vol.% formic acid water-acetonitrile solution; the gradient elution program is shown in Table 1, and it was filtered through a 0.45 μm microporous filter membrane and ultrasonically degassed before use;

[0064] Flow rate: 1 mL / min;

[0065] Detection wavelength: 210 nm;

[0066] Column temperature: 25 °C.

[0067] Table 1 Mobile phase elution program

[0068] Time (min) Acetonitrile 0.1 vol.% formic acid in water 0.00 10.0 90.0 3.00 13.0 87.0 25.00 13.0 87.0 40.00 15.0 85.0 45.00 95.0 5.0 50.00 95.0 5.0 50.10 10.0 90.0 55.00 10.0 90.0

[0069] The HPLC detection results of Calceolarioside E are shown in Figure 3 ; the HPLC detection results of Paucifloside are shown in Figure 4 .

[0070] Determined according to the above chromatographic conditions, after deducting the solvent interference peak, the normalization method was used to calculate the content, and the peak area percentages of Calceolarioside E and Paucifloside were greater than 90.0% at each wavelength.

[0071] (3) UHPLC-QE Orbitrap MS (High-resolution mass spectrometry)

[0072] Chromatographic separation was carried out using Thermo Scientific Hypersil GOLD TM aQ (100 mm × 2.1 mm, 1.9 μm) at a column temperature of 40 °C and a flow rate of 0.3 mL / min.

[0073] Mobile phase: Water containing 0.1 vol.% formic acid as eluent A and acetonitrile as eluent B.

[0074] The flow rate was set as a linear gradient: 0 - 5 min, A was 95 - 80%; 5 - 20 min, A was 80 - 70%; 20 - 30 min, A was 70 - 5%; 30 - 35 min, A was 5 - 5%; 35 - 35.1 min, A was 5 - 95%; 35.1 - 40 min, A was 95 - 95%.

[0075] The sample injection volume was 2 μL.

[0076] MS analysis was performed using electrospray ionization (ESI) in positive and negative ionization modes.

[0077] The key parameters were as follows: spray voltage, 3.5 kV (+); spray voltage, 3.2 kV (-); sheath gas flow rate, 35 Arb; auxiliary gas flow rate, 10 Arb; capillary temperature, 320 °C; heater temperature, 350 °C; S-lens RF level, 60.

[0078] The MS spectra were recorded in Full MS-ddMS 2 mode; the scanning range was m / z 100 - 1500. The stepped normalized collision energies were 20%, 40% and 60%.

[0079] The UHPLC-QE Orbitrap MS detection results of Calceolarioside E are shown in Figure 1 ; the UHPLC-QE Orbitrap MS detection results of Paucifloside are shown in Figure 2 . The experimental results showed that the obtained products could be confirmed as Calceolarioside E and Paucifloside under this method.

[0080] Compared with the yields of calceolarioside E and paucifloside of 0.18 mg / g and 0.010 mg / g in the current research by Nicoletti M. et al., the yields in the present invention (the yields of calceolarioside E and paucifloside are 11.58 mg / g and 11.42 mg / g) have been significantly improved. At the same time, changing the extraction solvent (methanol or ethanol), extraction method (the temperature of ultrasonic extraction is 30 - 70 °C, the number of extractions is 2 - 6 times, and the extraction time for each time is 1 - 5 h), the chromatographic column used (LH-20, C18 column), and the reagent used for redissolution (methanol or ethanol) will all affect the yields and purities of calceolarioside E and paucifloside. That is, if the extraction method is different from that of the present invention, the yields and purities of calceolarioside E and paucifloside will decrease.

[0081] The embodiments described above are only for describing the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for extracting and purifying Calceolarioside E and Paucifloside, characterized in that: The following steps are involved: Mixing the spatholobiaceae with a solvent and then ultrasonically extracting the mixture to obtain an extract; The extract is separated by a gel chromatography column and then by a reverse phase chromatography column to obtain an enriched solution of Calceolarioside E and Paucifloside; The enriched solution is dried, redissolved, and dried again to obtain the Calceolarioside E and Paucifloside.

2. The method according to claim 1, characterized in that The solvent includes methanol or ethanol.

3. The method according to claim 1, characterized in that The mass / volume ratio of the Fujian-Ganzhou Longicorn Moss and the solvent is 1g:10mL to 1g:20mL.

4. The method according to claim 1, characterized in that: The gel chromatography column comprises an LH-20 gel column.

5. The method according to claim 1, characterized in that The eluent used in the separation by gel chromatography column includes methanol.

6. The method according to claim 1, characterized in that The reverse phase chromatography column comprises a C18 column.

7. The method according to claim 1, characterized in that The solvent used for the re-dissolution includes methanol or ethanol.

8. The method according to claim 1, characterized in that The temperature of the ultrasonic extraction is 30-70° C., the number of extractions is 2-6 times, and the time of each extraction is 1-5 hours.

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