Preparation process of morkotin a in wolfberry leaves

Abstract

This invention relates to a method for isolating and preparing the flavonoid compound Morkotin A from wolfberry leaves, which are both food and medicinal materials, belonging to the field of pharmaceutical technology. The method includes: (1) extracting wolfberry leaves by heating and reflux with an ethanol-water solution, and concentrating the extract; (2) enriching the concentrate with polyamide resin, removing impurities by water and 20% ethanol in sequence, and eluting with 70% ethanol to obtain a crude phenolic acid fraction; (3) removing impurities from the crude phenolic acid fraction with a strong acid cation exchange resin, and eluting with water to obtain a crude flavonoid fraction; (4) purifying the crude flavonoid fraction with dextran gel, eluting with 50% methanol, and collecting the Morkotin A fraction by TLC monitoring to obtain a crude product; (5) further refining the crude product with dextran gel, eluting with 50% methanol to obtain a Morkotin A monomer compound with a purity >98%. This process is low in cost, high in efficiency, simple and easy to implement, and can provide raw material guarantee for its bioactivity evaluation and product development.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a process for isolating and preparing the flavonoid compound Morkotin A from wolfberry leaves, which are both food and medicine. Background Technology

[0002] Morkotin A (also known as quercetin-3-O-rutinoside 7-O-glucoside, CAS No. 30311-61-6, molecular formula C) 33 H 40 O 21 Morkotin A (molecular weight 772.66) is a flavonoid compound. Previous studies have shown that Morkotin A possesses antioxidant and anti-colon cancer bioactivities, demonstrating promising potential for pharmaceutical applications. However, the China National Institutes for Food and Drug Control currently lacks a Morkotin A reference standard, and commercially available reference standards are expensive, severely hindering in-depth pharmacological research and drug development evaluation. Therefore, a low-cost, simple, and pilot-scale production method is urgently needed.

[0003] Goji berry leaves are a traditional medicinal and edible plant resource, rich in various active ingredients such as flavonoids, polysaccharides, and phenolic acids, with a high content of Morkotin A. Modern pharmacological studies have also shown that goji berry leaves have antioxidant and anti-inflammatory effects. Using goji berry leaves as raw material to prepare Morkotin A is not only abundant and inexpensive, but also aligns with the development direction of natural products, effectively meeting the needs for reference standard preparation, activity evaluation, and formulation development. Summary of the Invention

[0004] In view of this, the present invention provides a process for isolating and preparing Morkotin A from wolfberry leaves. This process involves reflux extraction with an ethanol-water solution to obtain a wolfberry leaf extract, followed by sequential purification using polyamide resin, cation exchange resin, and LH-20 dextran gel to obtain Morkotin A monomeric compound with a purity >98%. This process is low-cost, highly efficient, simple, and suitable for pilot-scale production, thus providing a raw material guarantee for the preparation of reference standards, bioactivity evaluation, and product development.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A process for isolating and preparing Morkotin A from wolfberry leaves, specifically including the following steps: (1) Ningxia wolfberry ( Lycium barbarum Using L.) leaves as raw material, a certain proportion of ethanol aqueous solution was added for soaking, heated and refluxed for extraction, filtered, the filtrates were combined and concentrated to obtain wolfberry leaf extract; (2) Dissolve the wolfberry leaf extract obtained in step (1) in a suitable solvent, load the sample onto a polyamide resin column, elute with water and 20% ethanol aqueous solution in sequence, discard the eluent; then elute with 70% ethanol aqueous solution, collect the eluent, concentrate, and obtain crude phenolic acid component; (3) Dissolve the crude phenolic acid component obtained in step (2) in a suitable solvent, load the sample onto a strong acid cation exchange resin column to remove impurities, elute with water, collect the eluent, concentrate, and obtain the crude flavonoid component. (4) Dissolve the crude flavonoid component described in step (3) in a suitable solvent, load it onto a dextran gel column, elute with 50% methanol aqueous solution, monitor the elution fraction with TLC, collect the Morkotin A fraction, concentrate it, and obtain crude Morkotin A (purity of about 90%). (5) Dissolve the crude Morkotin A obtained in step (4) in a suitable solvent, load it onto a dextran gel column again, elute with 50% methanol aqueous solution, collect the Morkotin A fraction, concentrate under reduced pressure, freeze dry, and obtain Morkotin A monomer compound with a purity greater than 98%.

[0006] Furthermore, the chemical structure of Morkotin A is shown in formula (1): Equation (1).

[0007] Furthermore, in step (2), the loading flow rate of the polyamide resin is 1.2 to 1.8 BV / h; the water elution volume is 3 BV and the elution flow rate is 2 BV / h; the elution volume of the 20% ethanol aqueous solution is 2 BV and the elution flow rate is 2 BV / h; and the elution volume of the 70% ethanol aqueous solution is 4 BV and the elution flow rate is 1.5 BV / h.

[0008] Furthermore, in step (3), the strong acid cation exchange resin is type 001×7; the loading flow rate is 1.2 to 1.8 BV / h; the elution volume of purified water is 5 BV, and the elution flow rate is 2 BV / h.

[0009] Furthermore, in step (4), the dextran gel was Sephadex LH-20, the loading flow rate was 0.5 BV / h, and the elution flow rate was 0.3 BV / h.

[0010] Furthermore, after concentration in step (5), the product is freeze-dried to obtain Morkotin A monomer compound, which is a pale yellow powder with a purity >98% as determined by HPLC.

[0011] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a process for separating and preparing Morkotin A from wolfberry leaves, and for the first time separates and prepares high-purity Morkotin A from wolfberry leaves. The process established by the invention is simple, and only uses the preparation and purification process of polyamide resin enrichment → cation exchange resin impurity removal → dextran gel purification to obtain Morkotin A with a purity >98%. It does not require expensive instruments such as liquid chromatography, is simple to operate, has high preparation efficiency and low cost, and is suitable for industrial production, providing raw material guarantee for the preparation of reference standards, bioactivity evaluation and product development. Attached Figure Description

[0012] Figure 1 The attached figure is a flowchart of the process for separating and preparing Morkotin A from wolfberry leaves.

[0013] Figure 2 The attached figure shows the HPLC chromatogram of wolfberry leaf extract.

[0014] Figure 3 The attached figure shows the HPLC chromatogram of the crude phenolic acid component obtained by enriching wolfberry leaf extract with polyamide resin.

[0015] Figure 4 The attached figure shows the HPLC chromatogram of the crude flavonoid sample obtained after removing impurities from the crude phenolic acid sample using a cation exchange resin.

[0016] Figure 5 The attached figure shows the HPLC chromatogram of Morkotin A crude flavonoid after purification by Sephadex LH-20 gel electrophoresis.

[0017] Figure 6 The attached figure shows the HPLC chromatogram of crude Morkotin A after purification with Sephadex LH-20 gel permeation.

[0018] Figure 7 The attached figure shows the LC-MS primary and secondary mass spectra of Morkotin A. Figure 7 A is a primary mass spectrum in positive ion mode. Figure 7 B is the primary mass spectrum in negative ion mode. Figure 7 C is a second-order mass spectrum in positive ion mode. Figure 7 D is a secondary mass spectrum in negative ion mode.

[0019] Figure 8 The attached figure shows the ¹H NMR and ¹³C NMR spectra of Morkotin A. Figure 8 A is the ¹H-NMR spectrum. Figure 8 B is the ¹³C-NMR spectrum. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] All reagents used in this invention are commercially available analytical grade; experimental methods not mentioned are conventional experimental methods and will not be described in detail here. Example 1

[0022] Take clean and dried Ningxia wolfberries ( Lycium barbarum Lycium barbarum (L.) leaves were crushed and passed through a 40-mesh sieve. 2.0 kg of the raw powder was added to 16 L of 60% ethanol aqueous solution and soaked for 0.5 hours. The mixture was then refluxed twice, 1 hour each time. The filtrates were combined and concentrated under reduced pressure to obtain 510 g of Lycium barbarum leaf extract. The extract was suspended in water and loaded onto an 80-100 mesh polyamide resin column with a bed volume of 20 L (Φ150 mm × H1130 mm) at a flow rate of 1.2 BV / h. Elution was performed sequentially with 3 BV of purified water and 2 BV of 20% ethanol aqueous solution (2 BV / h flow rate), discarding the eluent. Elution was then performed with 4 BV of 70% ethanol aqueous solution (1.5 BV / h flow rate), collecting the eluent and concentrating under reduced pressure to obtain 110 g of crude phenolic acid fraction. The crude phenolic acid fraction was suspended in water and loaded onto a 001×7 type strong acid cation exchange resin column with a bed volume of 30 L (Φ150 mm × H1700 mm) and a loading flow rate of 1.2 BV / h. Elution was performed with 5 BV of purified water, and the eluent was collected and concentrated under reduced pressure to obtain 72 g of crude flavonoids. The crude flavonoids were dissolved in 50% methanol (minimum volume) and loaded onto a Sephadex LH-20 gel column with a bed volume of 15 L (Φ100 mm × H1900 mm) and a loading flow rate of 0.5 BV / h. Isocratic elution was performed with 50% methanol aqueous solution (0.3 BV / h flow rate). The Morkotin A main peak fraction was monitored and collected by TLC in real time, yielding 2.18 g of crude Morkotin A with a purity of approximately 90%. The above gel purification steps were repeated once, the main peak fractions were combined, concentrated under reduced pressure, and freeze-dried to obtain 2.12 g of pure Morkotin A with a purity >98%. The flowchart for the isolation and preparation of Morkotin A from wolfberry leaves is shown below. Figure 1 As shown.

[0023] High-performance liquid chromatography (HPLC) was used to observe the changes in chromatographic peaks at different purification stages. Chromatographic conditions: Agilent 1260 II HPLC system, COSMOSIL® C18 column (250 mm × 4.6 mm, 5 μm); mobile phase A was 0.3% phosphoric acid solution, mobile phase B was methanol; flow rate was 1 mL / min, column temperature was 40℃, detection wavelength was 320 nm, and injection volume was 10 μL.

[0024] HPLC chromatogram of wolfberry leaf extract is shown below. Figure 2 As shown, the chromatogram shows a large number of impurity peaks, high baseline noise, and a low response to the target peak, indicating that the Lycium barbarum leaf extract is complex and contains a large number of impurities. The target component, Morkotin A, is present in a high proportion in the crude extract, but further enrichment and purification are needed. The HPLC chromatogram of the crude phenolic acid fraction obtained after polyamide resin enrichment of the Lycium barbarum leaf extract is shown below. Figure 3 As shown. With Figure 2 In comparison, the impurity peaks were significantly reduced, the baseline tended to be stable, and the target peak response was significantly enhanced, indicating that the polyamide resin can effectively remove a large number of impurities and play a good enrichment role for the target component Morkotin A. The HPLC chromatogram of the crude flavonoid component obtained after impurity removal by cation exchange resin for the crude phenolic acid component is shown below. Figure 4 As shown. With Figure 3 Compared to the previous method, most polar impurities were removed, resulting in a cleaner chromatographic background and a further increase in the proportion of the target peak. This indicates that cation exchange resin can effectively remove charged impurities and further improve the purity of the target component. The HPLC chromatogram of Morkotin A crude flavonoid fraction purified by Sephadex LH-20 gel electrophoresis is shown below. Figure 5 As shown. With Figure 4 In comparison, structurally similar impurity peaks largely disappeared, while the proportion of the target peak significantly increased, indicating that gel purification can effectively separate structurally similar impurities and further purify the target component. The HPLC chromatogram of crude Morkotin A after gel purification with Sephadex LH-20 is shown below. Figure 6 As shown in the figure, the chromatogram shows a single, symmetrical main peak with a stable baseline and no obvious impurity peaks, indicating that the two gel purification processes can basically remove residual impurities and obtain the purified target component. The resulting product has a high purity, reaching over 98%.

[0025] The structure of the obtained products was identified by LC-MS, ¹H-NMR, and ¹³C-NMR spectroscopy. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was methanol. A Waters Symmetry® C18 column (2.1 mm × 100 mm, 3.5 µm) was used, the column temperature was maintained at 30 °C, the flow rate was set at 0.2 ml / min, the injection volume was 1 μl, and gradient elution was used.

[0026] The HPLC-MS primary and secondary mass spectra of the obtained product are shown in the figure. Figure 7 As shown ( Figure 7 A is a primary mass spectrum in positive ion mode. Figure 7 B is the primary mass spectrum in negative ion mode. Figure 7 C is a second-order mass spectrum in positive ion mode. Figure 7 D is the second-order mass spectrum in negative ion mode. In positive ion mode, the quasi-molecular ion peak is m / z 773.2178 [M+H]⁺, and in negative ion mode, the quasi-molecular ion peak is m / z 771.1983 [M−H]⁻, both of which are consistent with the molecular ion mass of Morkotin A. The ¹H-NMR and ¹³C-NMR spectra of the obtained product are shown below. Figure 8 As shown, Figure 8 A is the ¹H-NMR spectrum. Figure 8 B is a ¹³C-NMR spectrum, which is consistent with the proton and carbon spectra of Morkotin A structure. Example 2

[0027] Take clean and dried Ningxia wolfberries ( Lycium barbarumLycium barbarum (L.) leaves were crushed and passed through a 40-mesh sieve. 2.0 kg of the raw powder was added to 20 L of 70% ethanol aqueous solution and soaked for 1 hour. Extraction was performed by heating and reflux, repeated three times for 1 hour each time. The extract was filtered while hot, and the filtrates were combined. The filtrate was concentrated under reduced pressure until no alcohol odor remained, yielding 532 g of Lycium barbarum leaf extract. An 80-100 mesh polyamide resin column with a bed volume of 20 L (Φ150 mm × H1130 mm) was used. The Lycium barbarum leaf extract was suspended in deionized water and loaded at a flow rate of 1.5 BV / h. Impurities were removed by elution with 3 BV purified water, 2 BV 20% ethanol (2 BV / h flow rate), and then 4 BV 70% ethanol (1.5 BV / h flow rate). The eluent was collected and concentrated under reduced pressure to obtain 130 g of crude phenolic acid fraction. A 001×7 strong acid cation exchange resin was used with a column bed volume of 30 L (Φ150 mm × H1700 mm). The crude phenolic acid fraction was suspended in water and loaded at a flow rate of 1.5 BV / h. Elution was performed with 5 BV purified water (2 BV / h flow rate), and the eluent was collected and concentrated under reduced pressure to obtain 80 g of crude flavonoids. Sephadex LH-20 gel was packed with a column bed volume of 15 L (Φ100 mm × H1900 mm). The crude flavonoids fraction was dissolved in a minimum volume of 50% methanol and loaded at a flow rate of 0.5 BV / h. Isocratic elution was performed with 50% methanol aqueous solution (0.3 BV / h flow rate). The Morkotin A main peak fraction was monitored and collected in real time by TLC, concentrated, and 2.35 g of crude Morkotin A with a purity of approximately 90% was obtained. Repeat the above gel purification steps once, combine the main peak fractions, concentrate under reduced pressure, and freeze-dry to obtain 2.3g of Morkotin A monomer, with a purity >98% as determined by HPLC. Example 3

[0028] Take dried Ningxia wolfberries ( Lycium barbarumLycium barbarum leaf extract (2.0 kg) was soaked in 24 L of 80% ethanol aqueous solution for 1 hour, then extracted by reflux twice, 1 hour each time. The extract was filtered while hot, and the filtrates were combined. The filtrate was concentrated under reduced pressure until no alcohol odor remained, yielding 500 g of Lycium barbarum leaf extract. An 80-100 mesh polyamide resin column with a bed volume of 20 L (Φ150 mm × H1130 mm) was used. The Lycium barbarum leaf extract was suspended in water and loaded at a flow rate of 1.8 BV / h. The extract was eluted sequentially with 3 BV purified water, 2 BV 20% ethanol (2 BV / h flow rate), and then with 4 BV 70% ethanol (1.5 BV / h flow rate). The eluent was collected and concentrated under reduced pressure to obtain 105 g of crude phenolic acid fraction. A 001×7 strong acid cation exchange resin was used with a column bed volume of 30 L (Φ150 mm × H1700 mm). The crude phenolic acid fraction was suspended in water and loaded at a flow rate of 1.8 BV / h. Elution was performed with 5 BV purified water (2 BV / h flow rate), and the eluent was collected and concentrated under reduced pressure to obtain 70 g of crude flavonoid fraction. Sephadex LH-20 gel was packed with a column bed volume of 15 L (Φ100 mm × H1900 mm). The crude flavonoid fraction was dissolved in a minimum volume of 50% methanol and loaded at a flow rate of 0.5 BV / h. Isocratic elution was performed with a 50% methanol aqueous solution (0.3 BV / h flow rate). The Morkotin A main peak fraction was monitored and collected in real-time by TLC, concentrated, and yielded 2.12 g of crude Morkotin A with a purity of approximately 90%. The gel purification steps were repeated once, the main peak fractions were combined, concentrated under reduced pressure, and freeze-dried to obtain 2.08 g of Morkotin A monomer, with a purity of >98% as determined by HPLC. Example 4

[0029] Take dried Ningxia wolfberries ( Lycium barbarumLycium barbarum leaf extract (2.0 kg) was soaked in 20 L of 80% ethanol aqueous solution for 0.5 hours, then extracted by reflux twice for 3 hours each time. The extract was filtered while hot, and the filtrates were combined and concentrated under reduced pressure until no alcohol odor was detected, yielding 526 g of Lycium barbarum leaf extract. An 80-100 mesh polyamide resin column with a bed volume of 20 L (Φ150 mm × H1130 mm) was used. The Lycium barbarum leaf extract was suspended in water and loaded at a flow rate of 1.2 BV / h. The extract was eluted sequentially with 3 BV purified water, 2 BV 20% ethanol (2 BV / h flow rate), and then 4 BV 70% ethanol (1.5 BV / h flow rate). The eluent was collected and concentrated under reduced pressure to obtain 125 g of crude phenolic acid fraction. A 001×7 strong acid cation exchange resin was used with a column bed volume of 30 L (Φ150 mm × H1700 mm). The crude phenolic acid fraction was suspended in water and loaded at a flow rate of 1.2 BV / h. Elution was performed with 5 BV purified water (2 BV / h flow rate), and the eluent was collected and concentrated under reduced pressure to obtain 76 g of crude flavonoids. Sephadex LH-20 gel was packed with a column bed volume of 15 L (Φ100 mm × H1900 mm). The crude flavonoids were dissolved in a minimum volume of 50% methanol and loaded at a flow rate of 0.5 BV / h. Isocratic elution was performed with a 50% methanol aqueous solution (0.3 BV / h flow rate). The Morkotin A main peak fraction was monitored and collected in real time by TLC, concentrated, and yielded 2.30 g of crude Morkotin A with a purity of approximately 90%. The gel purification steps were repeated once, the main peak fractions were combined, concentrated under reduced pressure, and freeze-dried to obtain 2.22 g of Morkotin A monomer, with a purity of >98% as determined by HPLC.

Claims

1. A process for isolating and preparing Morkotin A from wolfberry leaves, characterized in that... Includes the following steps: (1) Ningxia wolfberry ( Lycium barbarum Using L.) leaves as raw material, a certain proportion of ethanol-water solution was added for soaking, heated and refluxed for extraction, filtered, the filtrates were combined and concentrated to obtain wolfberry leaf extract; (2) Dissolve the wolfberry leaf extract obtained in step (1) in a suitable solvent, load the sample onto a polyamide resin column, elute with water and 20% ethanol aqueous solution in sequence, discard the eluent; then elute with 70% ethanol aqueous solution, collect the eluent, concentrate, and obtain crude phenolic acid component; (3) Dissolve the crude phenolic acid component obtained in step (2) in a suitable solvent, load the sample onto a strong acid cation exchange resin column to remove impurities, elute with water, collect the eluent, concentrate, and obtain the crude flavonoid component. (4) Dissolve the crude flavonoid component described in step (3) in a suitable solvent, load it onto a dextran gel column, elute with 50% methanol aqueous solution, monitor the elution fraction with TLC, collect the Morkotin A fraction, concentrate it, and obtain crude Morkotin A (purity of about 90%). (5) Dissolve the crude Morkotin A obtained in step (4) in a suitable solvent, load it onto a dextran gel column again, elute with 50% methanol aqueous solution, collect the Morkotin A fraction, concentrate under reduced pressure, freeze dry, and obtain Morkotin A monomer compound with a purity greater than 98%.

2. The process for separating and preparing Morkotin A from wolfberry leaves according to claim 1, characterized in that, The Morkotin A, also known as quercetin-3-O-rutin-7-O-glucoside, has the structure shown in formula (1): Equation (1).

3. The process for separating and preparing Morkotin A from wolfberry leaves according to claim 1, characterized in that, In step (1), the volume concentration of the ethanol aqueous solution is 60% to 80%, and the extraction is carried out by reflux 2 to 3 times, each time for 1 to 3 hours.

4. The process for separating and preparing Morkotin A from wolfberry leaves according to claim 1, characterized in that, In step (2), the loading flow rate of the polyamide resin is 1.2 to 1.8 BV / h; the water elution volume is 3 BV and the elution flow rate is 2 BV / h; the elution volume of the 20% ethanol aqueous solution is 2 BV and the elution flow rate is 2 BV / h; and the elution volume of the 70% ethanol aqueous solution is 4 BV and the elution flow rate is 1.5 BV / h.

5. The process for separating and preparing Morkotin A from wolfberry leaves according to claim 1, characterized in that, In step (3), the strong acid cation exchange resin is type 001×7; the loading flow rate is 1.2 to 1.8 BV / h; the elution volume of purified water is 5 BV, and the elution flow rate is 2 BV / h.

6. The process for separating and preparing Morkotin A from wolfberry leaves according to claim 1, characterized in that, In step (4), the dextran gel is Sephadex LH-20, the loading flow rate is 0.5 BV / h, and the elution flow rate is 0.3 BV / h.

7. The process for separating and preparing Morkotin A from wolfberry leaves according to claim 1, characterized in that, After concentration in step (5), the product is freeze-dried to obtain Morkotin A monomer compound. Morkotin A is a pale yellow powder with a purity >98% as determined by HPLC.

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