Separation and purification method of two phenolic compounds in Polygonum capitatum Buch.-Ham. ex D. Don and its application

Abstract

The present invention relates to an extraction method and application of two new compounds, capitaone A and capitaone B, from Polygonum capitatum. The present invention uses modern spectroscopic techniques such as nuclear magnetic spectroscopy, high-resolution mass spectrometry, and the physical and chemical properties of the compounds to identify the structures of the isolated monomeric compounds and deduce the structures of the compounds. At the same time, the compounds have significant antioxidant activities and can be used as antioxidants in foods, cosmetics, beverages, etc., and can also be used to prepare drugs or health products with antioxidant activities and effects. This provides a material basis for further quality control and efficacy research of the Miao medicine Polygonum capitatum.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular, to two new compounds capitaone A and capitaone B in Polygonum capitatum Buch.-Ham. ex D.Don, and their preparation methods and antioxidant activities. Background Art

[0002] Biological oxidation refers to the oxidative decomposition process of chemical substances in living organisms. During the biological oxidation metabolism process in the human body, a large number of free radicals are generated. Research shows that if free radicals in the body are produced excessively and not treated and removed in time, it will lead to oxidative stress and imbalance of homeostasis, and may further cause some chronic diseases, such as diabetes, cardiovascular diseases, and cancer.

[0003] Polygonum capitatum comes from the whole herb of Polygonum capitatum Buch.-Ham. ex D.Don. Polygonum capitatum Research has found that Polygonum capitatum is rich in active small molecules, mainly including flavonoid compounds, lignan compounds, phenolic compounds, etc. The active small molecule compounds of Polygonum capitatum in Miao medicine have a wide range of pharmacological effects, such as antibacterial, anti-inflammatory, anticancer, and hypoglycemic effects. Therefore, Polygonum capitatum has broad research potential and can provide new development ideas for the development of clinical drugs.

[0004] In view of this, the present invention is particularly proposed. Summary of the Invention

[0005] One of the objectives of the present invention is to provide two compounds capitaone A and capitaone B, which are derived from the whole herb of Polygonum capitatum and have certain antioxidant activities.

[0006] Another objective of the present invention is to provide a preparation method for the above-mentioned compounds capitaone A and capitaone B.

[0007] The third objective of the present invention is to provide a drug whose preparation raw materials contain the above-mentioned compound capitaone A and / or compound capitaone B.

[0008] The present application can be implemented as follows:

[0009] In the first aspect, the present application provides two compounds capitaone A and capitaone B, wherein the chemical structural formula of compound capitaone A is:

[0010]

[0011] The chemical structural formula of compound capitaone B is:

[0012]

[0013] In a second aspect, the present application provides a method for preparing compound capitaone A and compound capitaone B as described in the foregoing embodiments, comprising the following steps:

[0014] The dried whole herb of Polygonum capitatum (30 kg) is crushed and soaked in an ethanol-aqueous solution with an ethanol content of 95 vt% for extraction for 7 days at a material-liquid ratio of 1:30; after the first filtration, the solid is soaked in an ethanol-aqueous solution with an ethanol content of 95 vt% for 7 days at a material-liquid ratio of 1:30; after the second filtration, the solid is soaked in an ethanol-aqueous solution with an ethanol content of 75 vt% for 7 days at a material-liquid ratio of 1:30, and the third filtration is carried out; the soaking solutions separated each time are combined; the filtrate is concentrated in vacuo to obtain an extract; the extract is extracted with five times the volume of ethyl acetate, and the extract is dispersed in distilled water and extracted 3 times; an ethyl acetate extract (350 g) is obtained, and then silica gel column chromatography is carried out. The conditions of silica gel column chromatography include: using petroleum ether-ethyl acetate as the eluent, and carrying out gradient elution in the order of the volume ratio of petroleum ether to ethyl acetate being 1:0, 50:1, 10:1, 8:1, 6:1, 4:1, 2:1, 2:1, and 0:1; the separation silica gel is 100-200 mesh. Finally, combining the TLC detection results, components A to J are obtained according to different polarities;

[0015] Component G is separated by silica gel column chromatography, using dichloromethane-methanol as the eluent, and carrying out gradient elution in the order of the volume ratio of dichloromethane to methanol being 20:1, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 0:1; combining the TLC detection results, fractions G1 to G3 are obtained; fraction G2 is passed through a reverse-phase chromatography column, using methanol-water as the eluent, and carrying out gradient elution in the order of the volume ratio of methane to water being 20:1, 40:1, 60:1, 80:1, 100:0; the concentrated solution is repeatedly passed through a Sephadex LH-20 column, eluted with methanol, the eluate containing the target compound is concentrated, and finally purified by semi-preparative high performance liquid chromatography to obtain new compound capitaone A (10 mg) and new compound capitaone B (58 mg).

[0016] In an alternative embodiment, the ethyl acetate extract obtained from the whole herb of Polygonum capitatum is obtained in the following manner: the whole herb of Polygonum capitatum is subjected to alcohol soaking extraction or alcohol reflux extraction, and the alcohol extract after extraction is subjected to ethyl acetate extraction.

[0017] Thirdly, the present application provides that the compound capitaone A and the compound capitaone B as described in the foregoing embodiments, or the ethanol extract or ethyl acetate extract in the preparation method of any one of the foregoing embodiments have antioxidant effects, and can be used to prepare antioxidants in foods, cosmetics, beverages, etc., and can also be used to prepare drugs or health products with antioxidant activity effects.

[0018] Fourthly, the present application provides two new compounds, and the raw materials for the preparation thereof contain at least one of the compound capitaone A and / or the compound capitaone B, pharmaceutically acceptable salts, esters, solvates, stereoisomers, tautomers and prodrugs as described in the foregoing embodiments, or contain the ethanol extract or ethyl acetate extract in the preparation method of any one of the foregoing embodiments.

[0019] In an alternative embodiment, the raw materials for the preparation of the drug further include a pharmaceutically acceptable carrier and / or excipient.

[0020] In an alternative embodiment, the raw materials for the preparation include at least one of a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, surfactant, adsorption carrier and lubricant;

[0021] In an alternative embodiment, the dosage forms of the drug include tablets, powders, pills, injections, capsules, films, suppositories, ointments or granules.

[0022] The beneficial effects of the present application include:

[0023] The compound capitaone A and the compound capitaone B provided by the present invention are new phenolic compounds first extracted from the whole herb of Polygonum capitatum. The present invention provides a method for separating and extracting the compound capitaone A and the compound capitaone B. The method is simple to operate and can prepare compounds with high yield and purity. In addition, the compound has the ability to scavenge DPPH free radicals and can be used as an antioxidant in foods, cosmetics, beverages, etc., and can also be used to prepare drugs or health products with antioxidant activity effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary

[0025] skilled in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0026] Figure 11H NMR spectrum of compound capitaone A in Example 2;

[0027] Figure 2 13C NMR spectrum of compound capitaone A in Example 2;

[0028] Figure 3 1H NMR spectrum of compound capitaone B in Example 2;

[0029] Figure 4 13C NMR spectrum of compound capitaone B in Example 2;

[0030] Figure 5 IC 50 results of the DPPH radical scavenging activities of compound capitaone A and compound capitaone B in Example 3;

[0031] Figure 6 Finished product effect diagram of the Polygonum capitatum hand cream ointment. Detailed implementation manners

[0032] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0033] The compounds capitaone A and capitaone B provided in the present application, their preparation methods and applications will be specifically described below.

[0034] The present application proposes a compound capitaone A and capitaone B, and the chemical structural formulas of the compounds are as follows:

[0035]

[0036] capitaoneA capitaone B

[0037] Both of the compounds capitaone A and capitaone B belong to phenolic compounds.

[0038] Example 1

[0039] Preparation of compound capitaone A and capitaone B

[0040] The preparation methods of the above-mentioned compounds capitaone A and capitaone B may include the following steps:

[0041] The dried whole plant of Polygonum capitatum Buch.-Ham. ex D. Don (30 kg) was pulverized and soaked in an ethanol-aqueous solution with an ethanol content of 95 vt% for 7 days at a solid-liquid ratio of 1:30; after the first filtration, the solid was soaked in an ethanol-aqueous solution with an ethanol content of 95 vt% for 7 days at a solid-liquid ratio of 1:30; after the second filtration, the solid was soaked in an ethanol-aqueous solution with an ethanol content of 75 vt% for 7 days at a solid-liquid ratio of 1:30, and then the third filtration was carried out; the soaking solutions separated each time were combined; the filtrate was concentrated in vacuo to obtain an extract; the extract was extracted with five times its volume of ethyl acetate, and the extract was dispersed in distilled water and extracted 3 times; an ethyl acetate extract (350 g) was obtained, and then silica gel column chromatography was carried out. The conditions for silica gel column chromatography included: using petroleum ether-ethyl acetate as the eluent, and performing gradient elution in the order of volume ratios of petroleum ether to ethyl acetate of 1:0, 50:1, 10:1, 8:1, 6:1, 4:1, 2:1, 2:1, and 0:1; the separation silica gel was 100-200 mesh. Finally, combining the results of TLC detection, components A to J were obtained according to different polarities;

[0042] Component G was separated by silica gel column chromatography using dichloromethane-methanol as the eluent, and gradient elution was carried out in the order of volume ratios of dichloromethane to methanol of 20:1, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, and 0:1; combining the results of TLC detection, fractions G1 to G3 were obtained; fraction G2 was passed through a reverse-phase chromatographic column using methanol-water as the eluent, and gradient elution was carried out in the order of volume ratios of methanol to water of 20:1, 40:1, 60:1, 80:1, and 100:0; the concentrated solution was repeatedly passed through a Sephadex LH-20 column, eluted with methanol, the eluate containing the target compound was concentrated, and finally purified by semi-preparative high performance liquid chromatography to obtain the new compound capitaone A (10 mg) and the new compound capitaone B (58 mg).

[0043] Example 2

[0044] Identification of compounds capitaone A and capitaone B

[0045] The hydrogen spectrum and carbon spectrum of compound capitaone A are as Figure 1 and Figure 2 shown, and the hydrogen spectrum and carbon spectrum of compound capitaone B are as Figure 3 and Figure 4 shown, and the specific data are shown in Table 1:

[0046] Table 1: NMR data of compounds 1-2( 1H: 600 MHz, 13 C: 150 MHz, DMSO- d 6 )

[0047]

[0048]

[0049] Structure of compound capitaone A (1)

[0050]

[0051]

[0052] Structure of compound capitaone B (2)

[0053] Example 3

[0054] In vitro antioxidant activities of Polygonum capitatum extract, compound capitaone A and compound capitaone B

[0055] ① Experimental materials and methods.

[0056] Accurately weigh the DPPH material and add it to an ethanol solution to make its concentration 0.1 mmol / L. Then add the solution of compound capitaone A (100 μL, 40 μg / mL) and the DPPH solution (100 μL) (A i ) to a 96-well plate and incubate at room temperature for 30 min. Accurately weigh the DPPH material and add it to an ethanol solution to make its concentration 0.1 mmol / L. Then add the solution of compound capitaone B (100 μL, 40 μg / mL) and the DPPH solution (100 μL) (A i ) to a 96-well plate and incubate at room temperature for 30 min. Subsequently, use Thermo Scientific Varioskan LUX (Berthold, VL0L00D0, USA) to measure the activity, and the absorbance is 570 nm. Use vitamin C (40 μg / mL) as the positive control.

[0057] The calculation formula for the free radical scavenging effect is:

[0058] Free radical scavenging rate = [1−(A i −A j ) / A0] × 100%

[0059] (The clearance rate of 100 μL ethanol and 100 μL DPPH solution is A0, and the clearance rate of 100 μL ethanol and 100 μL compound capitaone A is A j1 , and the clearance rate of 100 μL ethanol and 100 μL compound capitaone A is A j2 )

[0060] ②. Experimental results

[0061] Both compound capitaone A and compound capitaone B have obvious scavenging activities on DPPH free radicals, and the IC 50 of compound capitaone A: 9.68 μg / mL, and the IC 50 of compound capitaone B: 3.90 μg / mL, indicating that both compound capitone A and compound capitone B have good antioxidant effects in vitro.

[0062] Example 4

[0063] Preparation of Polygonum capitatum Hand Cream Using Compound capitaone A and capitaone B

[0064] First, in a clean workshop, weigh the following into a beaker: 0.2 g of xanthan gum, 0.5 g of carbomer, 1 g of sodium alginate, and 500 g of water. Heat and stir evenly at 85 °C - 90 °C until there is no white solid;

[0065] Second step, add the following to the above beaker: 5 g of glycerol, 2 g of propylene glycol, and 0.5 g of triethanolamine, stir evenly, stop heating, and cool to below 45 °C;

[0066] Third step, add 10 mg of compound capitaone A and compound capitaone B, 5 mg of potassium sorbate, 30 mg of squalane, 5 mg of niacinamide, and 50 mg of rose hydrosol, stir evenly, emulsify with an emulsifier to obtain a preliminary ointment product, and let it stand for 2 - 3 days;

[0067] Fourth step, after irradiating and sterilizing the preliminary ointment product, can it to form a finished Polygonum capitatum ointment, which is used as a hand cream for skin care. The prepared Polygonum capitatum hand cream is as Figure 6 shown.

[0068] Test Example 1 Evaluation Test on the Antioxidant Ability of the Polygonum capitatum Hand Cream Ointment Prepared from Compound capitaone A and Compound capitaone B

[0069] Take an appropriate amount of the hand cream ointment prepared in Example 4 (0.1 mg / mL), use vitamin C at the same concentration as the positive control, and use a DPPH free radical scavenging ability detection kit to measure the antioxidant ability of the Polygonum capitatum Buch.-Ham. ex D. Don ointment.

[0070] The antioxidant evaluation results of the Polygonum capitatum Buch.-Ham. ex D. Don ointment are shown in Table 1:

[0071]

[0072] As can be seen from Table 1, the hand cream ointment of Polygonum capitatum Buch.-Ham. ex D. Don (capitaone A and capitaone B) has good DPPH free radical scavenging ability. Compared with the DPPH scavenging rate of vitamin C at 0.1 mg / mL, its DPPH scavenging rate is 15% higher, indicating that the hand cream ointment of Polygonum capitatum Buch.-Ham. ex D. Don (capitaone A and capitaone B) has good antioxidant effects.

[0073] In summary, both the compound capitaone A and the compound capitaone B provided by the present invention are new phenolic compounds first extracted from the whole herb of Polygonum capitatum Buch.-Ham. ex D. Don. The present invention provides a separation and extraction method for the compound capitaone A and the compound capitaone B. This method is simple to operate and can prepare compounds with high yields and purities. In addition, this compound has the ability to scavenge DPPH free radicals and can be used to prepare antioxidants for foods, cosmetics, beverages, etc., and can be used to prepare drugs or health products with antioxidant activity effects. A typical example is that the compounds capitaone A and capitaone B in the Miao medicine Polygonum capitatum Buch.-Ham. ex D. Don are used as the main raw materials, and a cream agent is prepared by adding appropriate excipients. It has strong antioxidant ability and good skin care effects. Therefore, it can be externally applied to the skin to prepare a hand cream or other types of skin care products.

[0074] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Two new compounds, capitaone A and capitaone B, in Polygonum capitatum Buch.-Ham. ex D. Don, have the following structural formulas: 。 2. The preparation method of the compound capitaone A and the compound capitaone B according to claim 1, characterized in that, It includes the following steps: Step 1) After making the whole herb of Polygonum capitatum Buch.-Ham. ex D. Don into dry powder, soak and extract it in an ethanol-aqueous solution with an ethanol content of 95 v% for 7 days according to a material-liquid ratio of 1:30; after the first filtration, soak the solid in an ethanol-aqueous solution with an ethanol content of 95 v% for 7 days again according to a material-liquid ratio of 1:30; after the second filtration, soak the solid in an ethanol-aqueous solution with an ethanol content of 75 v% for 7 days according to a material-liquid ratio of 1:30, and then conduct the third filtration; combine the soaking solutions separated each time; Step 2) Concentrate the soaking solution in Step 1) in vacuo to obtain an extract; extract the extract with five times its volume of ethyl acetate, disperse the extract in distilled water, and extract 3 times; Step 3) Perform silica gel column chromatography on the ethyl acetate extract in Step 2). The conditions for silica gel column chromatography include: using petroleum ether-ethyl acetate as the eluent, and performing gradient elution according to the volume ratios of petroleum ether to ethyl acetate of 1:0, 50:1, 10:1, 8:1, 6:1, 4:1, 2:1, 2:1, and 0:1 in sequence; the separation silica gel is 100 - 200 mesh; finally, according to the TLC detection results, obtain components A to J according to different polarities; Step 4) Perform silica gel column chromatography separation on component G in Step 3), using dichloromethane-methanol as the eluent, and performing gradient elution according to the volume ratios of dichloromethane to methanol of 20:1, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, and 0:1 in sequence; according to the TLC detection results, obtain fractions G1 to G3; Step 5) Pass fraction G2 in Step 4) through a reverse-phase chromatographic column, using methanol-water as the eluent, and performing gradient elution according to the volume ratios of methanol to water of 20:1, 40:1, 60:1, 80:1, and 100:0 in sequence; repeatedly pass the concentrated solution through a Sephadex LH-20 gel chromatographic column, elute with methanol, concentrate the eluate containing the target compound, and finally purify it by semi-preparative high-performance liquid chromatography to obtain the new compound capitaone A and the new compound capitaone B.

3. The preparation method according to claim 2, wherein: During the purification process of the Sephadex LH-20 gel chromatographic column in Step 5), perform isocratic elution with methanol as the eluent, with a flow rate of 1 drop of eluent corresponding to every 4 - 5 s, and the volume of the eluent is 1.5 - 2.5 L.

4. The preparation method according to claim 2, characterized in that: The conditions for the semi-preparative high-performance liquid chromatography in Step 5) include: the chromatographic column is a YMC Pack ODS-A column, the specification of the chromatographic column is 10 mm × 250 mm, and the particle size of the packing in the chromatographic column is 5 μm; the eluent is methanol and water with a volume ratio of 25 - 45:75 - 55, the detection wavelength is 254 - 365 nm, and the flow rate is 1.8 - 2.2 mL / min.

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