High isoflavanoid compounds with tyrosinase inhibitory activity extracted from radix ophiopogonis and preparation method and application thereof

Abstract

The present invention relates to a high isoflavan compound with tyrosinase inhibitory activity extracted from the fibrous roots of Ophiopogon japonicus as shown in formula (I), and a preparation method and application thereof. The compound is a brand-new compound extracted and separated from the fibrous roots of Ophiopogon japonicus by methods such as alcohol extraction, macroporous resin column chromatography, silica gel column chromatography, Sephadex LH-20, ODS column chromatography, and its structure is identified by multiple spectral techniques such as NMR, HRESI, CD, and optical rotation. The chemical name is (3R)-7,2'-dihydroxy-3',4'-methylenedioxy high isoflavan. The compound of the present invention has strong tyrosinase inhibitory activity and is expected to become a candidate drug for treating skin diseases related to melanin biosynthesis, providing a new idea for the research and development of drugs, health products, and daily chemical products with whitening effects.

Description

Technical Field

[0001] The present invention relates to a new compound in a medicinal plant, belonging to the field of traditional Chinese medicine chemistry and pharmaceutical technology, and specifically relates to a new high-isoflavanoid compound extracted from ophiopogon japonicus root hairy roots, a preparation method thereof, and application in whitening products. Background Art

[0002] Ophiopogon japonicas (L. f) Ker-Gawl. is a dried root of Ophiopogon japonicas (L. f) Ker-Gawl., a plant of the genus Ophiopogon in the family Liliaceae. It is sweet, slightly bitter, slightly cold in nature, and enters the heart, lung, and stomach meridians. It has the effects of nourishing yin, promoting body fluid, moistening the lungs, and clearing the heart. It is used for dry cough due to lung dryness, tuberculosis due to yin deficiency, sore throat, thirst due to loss of body fluid, internal heat and thirst, restlessness and insomnia, and constipation due to dry intestines. At present, the main secondary metabolites isolated from this plant are steroidal saponins, homoisoflavonoids, polysaccharides, etc.

[0003] In traditional Chinese medicine, only the tuberous roots of Ophiopogon japonicus are used as medicine, while the fibrous roots are used as livestock feed or directly discarded. Existing studies have shown that the nutritional components and active substances of the fibrous roots of Ophiopogon japonicus are similar to those of the tuberous roots of Ophiopogon japonicus, and some are even higher than those of the tuberous roots. Therefore, its medicinal value may be similar to that of the tuberous roots of Ophiopogon japonicus, and it should have a good development and application prospect. The present invention takes the fibrous roots of Ophiopogon japonicus, a non-traditional medicinal part of Ophiopogon japonicus, as the research object, and discovers new active compounds therefrom, which is of great significance for fully tapping and utilizing Ophiopogon japonicus resources and promoting the sustainable, rapid and healthy development of the Ophiopogon japonicus industry. Summary of the invention

[0004] The purpose of the present invention is to provide a new high-isoflavanoid compound extracted from the fibrous roots of Ophiopogon japonicus, a preparation method thereof and application thereof in whitening products.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A new high-isoflavanoid compound, the structure of which is shown in formula (I):

[0007]

[0008] A method for preparing the new high-isoflavan compound comprises the following steps:

[0009] 1) After the dried Ophiopogon japonicus root is crushed, it is refluxed and extracted with 50% to 80% ethanol solution. The amount of extraction solvent is 8 to 15 times the mass of the raw material. The number of extractions is 2 to 4 times, each time for 1 to 3 hours. The extracts are combined, filtered, concentrated to a solid-liquid ratio of 1:5 to 1:15, and allowed to stand for 3 to 12 hours for use;

[0010] 2) The supernatant obtained in step 1) is subjected to a macroporous adsorption resin, and eluted with 2 to 10 column volumes of 50% to 70% ethanol and 90% ethanol, respectively, and the eluate is collected, and the 90% ethanol eluate is concentrated to dryness to obtain a crude extract containing the target compound, which is set aside;

[0011] 3) The crude extract obtained in step 2) was subjected to silica gel column chromatography, and gradient elution was performed using chloroform: methanol solvent system of 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1 as eluents, and the eluents were collected, and the eluents were combined by TLC spot plate detection to obtain 18 components Fr.1-18; Fr.10 was subjected to silica gel column chromatography, and gradient elution was performed using petroleum ether: ethyl acetate solvent system of 50:1, 20:1, 10:1, 5:1, and 1:1 as eluents, and the eluents were collected, and the eluents were combined by TLC spot plate detection to obtain 5 subcomponent segments Fr.10A-Fr.10E; Fr.10D was subjected to Sephadex LH-20 column chromatography, eluted with methanol, and 4 sample segments Fr.10D1-Fr.10D4 were separated; Fr.10D1 was subjected to ODS column chromatography, and eluted with 50% ethanol aqueous solution to obtain compound I.

[0012] The macroporous adsorption resin in step 2) is one of HPD-100, D-101 and AB-8.

[0013] The structures of the new compounds are 1 H-NMR, 13 The products were identified by C-NMR, DEPT, HSQC, COSY, HMBC, HR-ESI-MS, CD and optical rotation spectroscopy.

[0014] The embodiment of the present invention provides the inhibitory effect of the novel high-isoflavanoid compound on the activity of tyrosinase.

[0015] The new compound described in the present invention has no literature reports on its structure, preparation method and pharmacological activity. High isoflavanoid compounds are first discovered from plants of the genus Ophiopogonis. Tyrosinase activity inhibition experiments have proven that the new high isoflavanoid compounds have a strong inhibitory effect on tyrosinase, and can be used as whitening active molecules or as lead compounds, providing new ideas for the development of drugs, health products and daily chemicals with whitening effects. In addition, the preparation of the new compound described in the present invention comes from the non-traditional medicinal part of Ophiopogon japonicus, the fibrous root of Ophiopogon japonicus, which is of great significance for the comprehensive utilization of Ophiopogon japonicus medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the compound of the present invention;

[0017] Figure 2The carbon nuclear magnetic resonance spectrum of the compound of the present invention;

[0018] Figure 3 is the HRESI spectrum of the compound of the present invention;

[0019] Figure 4 is the CD diagram of the compound of the present invention;

[0020] Figure 5 The compounds of the present invention 1 H- 1 H COSY and main HMBC signal correlation diagram;

[0021] Figure 6 is the structural formula of the compound of the present invention. DETAILED DESCRIPTION

[0022] The present invention will be further described in detail below by way of examples, and those skilled in the art can refer to the content of the present invention and appropriately improve the relevant parameters according to the specific actual equipment. The protection scope of the present invention is not limited to the content, and the methods in the embodiments are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0023] Example 1: Extraction and separation of compounds

[0024] 1) Take 15 kg of dried Ophiopogon japonicus root, crush it, put it in a multifunctional extraction tank, add 70% ethanol 10 times the mass of the raw material, reflux extraction, add 10 times the amount of 70% ethanol each time to extract for 2 h, extract 3 times in total, filter the extract, combine the filtrate and place it in a double-effect concentrator (60-70 ℃) to concentrate it to a solid-liquid ratio of 1:10, let it stand for 8 h, and set aside;

[0025] 2) The supernatant obtained in step 1) was separated using HPD-100 macroporous adsorption resin, and eluted with 5 column volumes of 70% ethanol and 90% ethanol in sequence, the eluate was collected, and the 90% ethanol eluate was concentrated to dryness to obtain 150 g of a crude extract containing the target compound, which was set aside;

[0026] 3) The crude extract obtained in step 2) was ultrasonically dissolved with an appropriate amount of ethanol, mixed with 100-200 mesh silica gel at a 2-fold mass ratio, evaporated to dryness, and dry-packed with 200-300 mesh silica gel at a 50-fold mass ratio, and gradient eluted with chloroform: methanol solvent system of 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, 0:1 as eluent, and the eluent was collected, and the eluent was combined after TLC spot plate detection to obtain 18 components Fr.1-18; Fr.10 was subjected to silica gel column chromatography, and gradient eluted with petroleum ether: ethyl acetate solvent system of 50:1, 20:1, 10:1, 5:1, 1:1 as eluent, and the eluent was collected, and the eluent was combined after TLC spot plate detection to obtain 5 subcomponent segments Fr.10A- Fr.10E and Fr.10D were separated by Sephadex LH-20 column chromatography and eluted with methanol to obtain four sample segments Fr.10D1-Fr.10D4; Fr.10D1 was separated by ODS column chromatography and eluted with 50% ethanol aqueous solution to obtain compound Ⅰ (16.7 mg).

[0027] Example 2: Extraction and separation of compounds

[0028] 1) Take 15 kg of dried Ophiopogon japonicus root, crush it, put it in a multifunctional extraction tank, add 80% ethanol in an amount of 8 times the mass of the raw material, reflux extract, add 8 times the amount of 80% ethanol each time to extract for 3 hours, extract 4 times in total, filter the extract, combine the filtrate, and place it in a double-effect concentrator (60-70 ℃) to concentrate it to a solid-liquid ratio of 1:5, let it stand for 3 hours, and set aside;

[0029] 2) The supernatant obtained in step 1) was separated with AB-8 macroporous adsorption resin, and eluted with 2 column volumes of 60% ethanol and 90% ethanol in sequence, the eluate was collected, and the 90% ethanol eluate was concentrated to dryness to obtain 155 g of a crude extract containing the target compound, which was set aside;

[0030] 3) Same as step 3) in Example 1.

[0031] Example 3: Extraction and separation of compounds

[0032] 1) Take 15 kg of dried Ophiopogon japonicus root, crush it, put it in a multifunctional extraction tank, add 50% ethanol 15 times the mass of the raw material, reflux extraction, add 15 times the amount of 50% ethanol each time to extract for 1 hour, extract twice in total, filter the extract, combine the filtrate and place it in a double-effect concentrator (60-70 ℃) to concentrate it to a solid-liquid ratio of 1:15, let it stand for 12 hours, and set aside;

[0033] 2) The supernatant obtained in step 1) was separated with D-101 macroporous adsorption resin, and eluted with 10 column volumes of 50% ethanol and 90% ethanol in sequence, the eluate was collected, and the 90% ethanol eluate was concentrated to dryness to obtain 146 g of a crude extract containing the target compound, which was set aside;

[0034] 3) Same as step 3) in Example 1.

[0035] Example 4: Structural Identification of New Compounds

[0036] Compound I, a pale yellow amorphous powder, showed a deprotonated ion with a mass-to-charge ratio of 299.0920 (calculated as C 17 H 15 O5, 299.0920), corresponding to the molecular formula C 17 H 16 O5, unsaturation is 10. 1 The H-NMR spectrum showed a 1,3,4-trisubstituted benzene ring [δ H 6.75 (d, J = 8.0 Hz, H-5), 6.26 (dd, J = 8.0, 2.5 Hz, H-6) and 6.16 (d, J = 2.5 Hz, H-8)] and a 1,2,3,4-tetrasubstituted benzene ring [δ H 6.30 (d, J = 8.0 Hz, H-5') and 6.50 (d, J = 8.0 Hz, H-6')] proton signals, 13 The C-NMR spectrum shows 12 sp2 hybridized carbon atoms (δ C 101.0 ~157.4) signals (see Table 1). In addition, an sp3 methine (δ C 34.4) and four sp3 methylene groups, including one dioxygen substituted (δ C 102.1) methylene. These data indicate that compound I is a homoisoflavanoid compound, similar to the compound 7-hydroxy-3',4'-methylenedioxy homoisoflavanoid reported in the literature, except that it has an additional hydroxyl group. In the HMBC two-dimensional spectrum, H-5, H-6 and C-7 (δ C 157.4), H-9, H-6' and C-2' (δ C 140.3), -O-CH2-O (δ H 5.85) and C-3' (δ C 148.5), C-4' (δ C136.0) have related absorption signals, and there is also a 1 H- 1 H COSY correlation, this information indicates that there are two hydroxyl groups located at C-7 and C-2', and a methylenedioxy substituent located at C-3' and C-4'. Therefore, the planar structure of compound I was determined to be 7,2'-dihydroxy-3',4'-methylenedioxyhomoisoflavane. By comparing the specific rotation and CD data of compound I with the data of the reported compound 7,4'-dihydroxy-6-methoxyhomoisoflavane, the absolute configuration of compound I can be inferred. Compound I and 7,4'-dihydroxy-6-methoxyhomoisoflavane have similar specific rotations (compound I is [α] 25 D +69; 7,4'-dihydroxy-6-methoxyhomoisoflavane is [α] 25 D +58) and the Cotton effect in the CD spectrum (positive Cotton effect at 207 and 285 nm, negative Cotton effect at 239 nm) indicate that the absolute configuration of compound I is 3R. Therefore, compound I was confirmed to be (3R)-7,2'-dihydroxy-3',4'-methylenedioxyhomoisoflavane. Its structural formula with atomic numbering is:

[0037]

[0038] Table 1 Compound I of the present invention 1 H-NMR and 13 C-NMR data and their assignment (CD3OD)

[0039]

[0040] Example 5: Tyrosinase activity inhibition experiment

[0041] According to the method described in the literature, we used spectrophotometry to determine the inhibition of tyrosinase activity. Briefly, the solution of the new compound described in the present invention (20 μM) was mixed with L-DOPA (1.25 mM) and then diluted with 970 μL of 0.05 mM sodium phosphate buffer (PBS, pH 6.8) in a test tube. The reaction was initiated by adding mushroom tyrosinase (25 U / mL) and then incubated at room temperature for 5 minutes. The inhibition rate (%) of the compound described in the present invention on tyrosinase was calculated by measuring the absorbance of each well at 490 nm, and the concentration of the compound when the tyrosinase activity inhibition rate reached 50% was taken as the IC 50 The results are shown in Table 2. Vitamin C was used as a positive control. The inhibition percentage of tyrosinase was calculated according to the following formula:

[0042] Tyrosinase activity inhibition rate (%) = (1 – sample well OD 490 nm / OD of experimental control well 490 nm )×100

[0043] Table 2 Inhibitory effect of compound I of the present invention on tyrosinase

[0044]

[0045] As can be seen from Table 2, the novel high isoflavanoid compound of the present invention has a significant inhibitory effect on tyrosinase, which is significantly better than the positive drug vitamin C, and can be used as a potential tyrosinase inhibitor and whitening active agent for preventing and treating skin pigmentation such as skin spots, freckles, and age spots. It is expected to become a candidate drug for treating skin diseases related to melanin biosynthesis, and provides a new idea for the research and development of drugs, health products, and daily chemical products with whitening effects.

Claims

1. A high isoflavanoid compound extracted from the fibrous roots of Ophiopogon japonicus, characterized in that: The structure is shown in formula (I):

2. The method for preparing high isoflavanoid compounds extracted from the fibrous roots of Ophiopogon japonicus according to claim 1, characterized in that: The following steps are involved: 1) After the dried radix ophiopogonis is crushed, it is refluxed and extracted with a 50% to 80% ethanol solution, the amount of the extraction solvent is 8 to 15 times the mass of the raw material, the number of extractions is 2 to 4 times, each time for 1 to 3 hours, the extracts are combined, the extracts are filtered, concentrated to a solid-liquid ratio of 1:5 to 1:15, and allowed to stand for 3 to 12 hours for use; 2) The supernatant obtained in step 1) is subjected to a macroporous adsorption resin, and eluted with 2 to 10 column volumes of 50% to 70% ethanol and 90% ethanol, respectively, and the eluate is collected, and the 90% ethanol eluate is concentrated to dryness to obtain a crude extract containing the target compound, which is set aside; 3) The crude extract obtained in step 2) was subjected to silica gel column chromatography, and gradient elution was performed using chloroform: methanol solvent system of 100:1, 50:1, 20:1, 10:1, 5:1, 1:1, and 0:1 as eluents, and the eluents were collected and combined for TLC spot plate detection to obtain 18 components Fr.1-18; Fr.10 was subjected to silica gel column chromatography, and petroleum ether: ethyl acetate solvent system of 50:1, 20:1, 10:1, 5:1, 1:1 were used as eluents for gradient elution, and the eluents were collected and combined for TLC spot plate detection to obtain 5 subcomponent segments Fr.10A-Fr.10E; Fr.10D was separated by Sephadex LH-20 column chromatography and eluted with methanol to obtain 4 sample segments Fr.10D1-Fr.10D4; Fr.10D1 was separated by ODS column chromatography and eluted with 50% ethanol aqueous solution to obtain the compound of formula (I).

3. The method for preparing high isoflavanoid compounds extracted from the fibrous roots of Ophiopogon japonicus according to claim 2, characterized in that: The macroporous adsorption resin in the step 2) is one of HPD-100, D-101 and AB-8.

4. Use of the high isoflavanoid compounds extracted from the fibrous roots of Ophiopogon japonicus according to claim 1 in the preparation of whitening products.

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