CN104592330B - A kind of kaempferol derivative isolated from Chinese scholar tree and its production and use - Google Patents

Abstract

The invention belongs to chemistry of forest product and trees active component technical field, and in particular to a kind of new kaempferol derivative is the O of Kaempferol 3 (4 " galloyl) α L rhamnopyranosyloxyhies glucosides [O of kaempferol 3 (4 " galloyl) α L rhamnopyranoside] and preparation method thereof and the application in antibacterial product is prepared.The present invention is with Chinese scholar tree (Sophorajapon1ca L.) root skin as raw material; its preparation technology specification is simple, and experiment proves that the O of Kaempferol 3 (4 " galloyl) growth of α L rhamnopyranosyloxyhies glucosides to bacterial strain has stronger inhibitory action.

Description

A kind of kaempferol derivative isolated from Chinese pagoda tree and its preparation method and application

technical field

The invention belongs to the technical field of forest product chemistry and tree active ingredients, and in particular relates to a new kaempferol derivative, namely kaempferol-3-O-(4″-galloyl) isolated from the root bark of Sophorajaponica L. -α-L-rhamnopyranoside [kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside] and its preparation method and use.

Background technique

Sophorajaponica L. is a deciduous tree of the genus Sophora L. in the Fabaceae subfamily of the Fabaceae family. Sophora japonica, etc., are widely distributed in most parts of our country. Chinese locust tree can reach 15-25m in height, has root nodules, and is suitable for moist and fertile soil (Yang Xin, et al., Northern Horticulture, 2011, (19): 175-178). Literature records show that the leaves, flowers, gum, fruit, bark and roots of Sophora japonica can be used as medicine (Zhao Kentian, et al., Plant Research, 1999, 19(3): 281-285). So far, researchers have extracted and isolated a variety of secondary metabolites from many parts of Sophora japonica, among which flavonoids are the main components (Zhang LB, eta1., Fitoterapia, 2013, 87:89-92).

In recent years, due to many reasons, drug-resistant strains have continued to increase, and the drug resistance of pathogenic bacteria in bacterial and fungal strains has continued to increase, resulting in a continuous increase in the mortality rate of clinical infections. At present, researchers in the food and pharmaceutical industries are actively working on finding safe, efficient, and broad-spectrum natural antibacterial agents. Plants are a natural treasure house of antibacterial active substances. Literature shows that more than 1,600 higher plants that have the ability to inhibit harmful organisms have been reported in the world, including 94 species that inhibit fungi, 11 species that inhibit bacteria, and 17 species that inhibit viruses. . It is also recorded that at least 1389 species of plants may be used as fungicides (Zhang Yingluo, et al., Journal of Southwest University for Nationalities (Natural Science Edition), 31(3):402-409). Therefore, searching for substances with antibacterial activity in natural components of plant secondary metabolism has become an important idea for the development of high-efficiency fungicides (Liu Haiyan, et al., Hebei Agricultural Sciences, 2005, 21(4): 254-257, 328).

Contents of the invention

The object of the present invention is to provide a kind of new kaempferol derivative namely kaempferol-3-O-(4 "-galloyl)-α-L-rhamnopyranoside [kaempferol-3-O-(4 "- galloyl)-α-L-rhamnopyranoside].

Another object of the present invention is to provide preparation of kaempferol-3-O- from the root bark of Sophora japonica L. (4″-galloyl)-α-L-rhamnopyranoside method.

The third object of the present invention is to provide kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside for its antibacterial activity and efficacy.

Technical scheme of the present invention is summarized as follows:

Kaempferol derivatives provided by the invention are compounds of the following structural formula:

The molecular formula of the compound of the present invention is C ₂₈ H ₂₄ O ₁₄ , its chemical name is kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside, and its corresponding English name is kaempferol-3 -O-(4″-galloyl)-α-L-rhamnopyranoside.

The method for preparing kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside from the root bark of Chinese pagoda tree comprises the following steps:

(1) Take the pulverized Sophora japonica root bark plant raw material, add acetone aqueous solution with a volume percentage concentration of 1% to 99% according to the mass ratio of 1:1 to 1:15, and extract at room temperature or heating or microwave or ultrasonic for 1 to 6 times, each time for 1 to 48 hours, filtered, and the filtrate was concentrated under reduced pressure to 1% to 20% of the original volume to obtain a crude extract;

(2) Add water with 1 to 6 times the mass of the crude extract, stir, add n-hexane with 1 to 6 times the mass of the crude extract, extract 1 to 6 times, separate, add the remaining water layer to 1 to 6 times the mass of the crude extract extracted 1 to 6 times with chloroform, separated, added ethyl acetate with 1 to 6 times the weight of the crude extract to the remaining water layer and extracted 1 to 6 times, separated the ethyl acetate layer and the final water layer, and separated the ethyl acetate layer Concentrate under reduced pressure to obtain ethyl acetate extract phase;

(3) The ethyl acetate extraction phase was prepared by at least one of silica gel column chromatography and gel column chromatography to obtain kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside.

The preparation method has the characteristics of high yield, low production cost and simple and standardized process.

Experiments confirm that kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside is effective against Staphylococcus aureus (Staphylococcus aureus) in Gram-positive bacteria and Gram-negative bacteria Klebsiella pneumonia (Klebsiella pneumonia) has a strong inhibitory effect (the corresponding minimum inhibitory concentration for the above two strains is 0.5 and 4.0 μg/ml), and its antibacterial effect is comparable to that of the positive control group neomycin The antibacterial effect of (neomycin) is quite (neomycin is respectively 0.5 and 2.0 μ g/ml to the minimum inhibitory concentration of Staphylococcus aureus and Klebsiella pneumoniae). This shows that kaempferol-3-O-(4″ -galloyl)-α-L-rhamnopyranoside can inhibit the growth of common pathogenic strains, and can be used as an efficient antibacterial agent in the fields of food, health care products, pharmaceuticals, cosmetics and the like.

detailed description

The present invention will be more readily and more fully understood by reference to the following examples, which are given to illustrate the invention and not to limit it in any way.

Example 1:

Preparation of Kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside

(1) Take the pulverized Sophora japonica root bark plant raw material, add acetone aqueous solution with a volume percentage concentration of 70% according to the mass ratio of 1:3, extract 3 times at normal temperature, each time for 36 hours, filter, and the filtrate is concentrated under reduced pressure to the original 10% of the volume to obtain the crude extract;

(2) Add water with 3 times the mass of the crude extract, stir, add 3 times the normal hexane of the mass of the crude extract to extract 3 times, separate, add the remaining water layer to extract 3 times with chloroform of 3 times the mass of the crude extract, separate, The remaining water layer was added to ethyl acetate with 3 times the weight of the crude extract to extract three times, the ethyl acetate layer and the final water layer were separated, and the ethyl acetate layer was concentrated under reduced pressure to obtain the ethyl acetate extract phase;

(3) The ethyl acetate extraction phase was prepared by silica gel column chromatography to obtain kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside.

The structural identification of the compound:

Fast atom bombardment mass spectrometry (FAB MS) shows that the m/z[M+Na] ⁺ of the compound is 607, and the m/z[M+H] ⁺ is 585, and its molecular formula is obtained by combining ¹ H NMR and ¹³ C NMR spectra is C ₂₈ H ₂₄ O ₁₄ .

Proton peaks [δ8.01 (2H, d, J ⁼ 8.5Hz, H-2′, 6′) and δ6.90 (2H, d, J=8.5Hz, H-3', 5')] shows that the ring B of the compound is a typical AA'BB' proton aromatic ring. Proton signals [δ6.20 (1H, d, J=2.3Hz, H-6) and δ6.39 (1H, d, J=2.3Hz, H-8)] indicated that the A ring was phloroglucinol type. And ¹³ C NMR peak data [δ159.4 (C-2), 134.8 (C-3), 179.4 (C-4)] revealed that the compound is a typical flavonol. Therefore, the aglycone of this compound was identified as kaempferol.

In the ¹ H NMR spectrum, it was shown that the compound contained a α-L-rhamnopyranoside [1 terminal hydrogen δ5.29 (1H, br s, H-1″), a methyl δ0.95 ( 3H, d, J=6.1Hz, H-6") and δ3.52～5.03 (4H, H-2", 3", 4", 5")].

Proton signal δ7.09 (2H, s, H-2"', 6"') combined with ¹³ C NMR data [δ121.9 (C-1"'), 110.1 (C-2"', 6"'), 146.4 (C-3"', 5"'), 139.9 (C-4"')] indicated that the compound contained a galloyl group.

The HMBC spectrum shows that the rhamnose end group hydrogen δ5.29 (1H, br s, H-1″) of the compound has a long-range correlation with the carbon signal δ134.8 (C-3) on kaempferol, and it is determined that α-L -Rhamnopyranoside is connected to the C-3 position of kaempferol. And the proton peak δ5.03 (1H, dd, J=10.3Hz, J=9.7Hz, H-4″) on the rhamnose is connected to the The carbon signal δ168.3 (C-7"') has a long-range correlation, which confirms that the 7"' position of galloyl group is connected with the 4" position of α-L-rhamnopyranoside.

In summary, the structure of the compound was determined to be kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside.

After retrieval, the compound is a new kaempferol derivative that has never been reported before. The compound is a white amorphous powder with a melting point of 191-193°C and an optical rotation of The compound has ultraviolet absorption at the wavelength of 254nm of an ultraviolet lamp, and is dark green in the spray color reaction with 1% ferric chloride ethanol solution (mass percentage). In the TLC test, when developing with a solvent system with a volume ratio of glacial acetic acid-water of 3:47, the R _f value of the compound was 0.49, while developing with a solvent system with a volume ratio of tert-butanol-glacial acetic acid-water of 3:1:1 When, the _Rf value of this compound is 0.73.

Table 1. ¹ H (400MHz) and ¹³ C NMR (100MHz) spectral data (CD ₃ OD) of kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside

Example 2:

Preparation of Kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside

(1) Take the pulverized Sophora japonica root bark plant raw material, add acetone aqueous solution with a volume percentage concentration of 60% according to the mass ratio of 1:4, microwave extraction 4 times, each time for 3 hours, filter, and the filtrate is concentrated under reduced pressure to the original 8% of the volume to obtain the crude extract;

(2) Add water with 2 times the mass of the crude extract, stir, add n-hexane with 2 times the mass of the crude extract to extract 4 times, separate, add the remaining water layer to chloroform with 2 times the mass of the crude extract, extract 4 times, separate, The remaining water layer was added to ethyl acetate twice the mass of the crude extract to extract 4 times, the ethyl acetate layer and the final water layer were separated, and the ethyl acetate layer was concentrated under reduced pressure to obtain the ethyl acetate extract phase;

(3) The ethyl acetate extract phase was prepared by gel column chromatography to obtain kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside.

Example 3:

Preparation of Kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside

(1) Take the pulverized Sophora japonica root bark plant raw material, add acetone aqueous solution with a volume percentage concentration of 80% according to the mass ratio of 1:2, ultrasonically extract 2 times, each time for 4 hours, filter, and the filtrate is concentrated under reduced pressure to the original 5% of the volume to obtain the crude extract;

(2) Add water with 4 times of the mass of the crude extract, stir, add 4 times of the mass of the crude extract with n-hexane to extract 2 times, separate, add the remaining water layer to chloroform with 4 times the mass of the crude extract, extract 2 times, separate, The remaining water layer was added to ethyl acetate with 4 times the weight of the crude extract to extract twice, the ethyl acetate layer and the final water layer were separated, and the ethyl acetate layer was concentrated under reduced pressure to obtain the ethyl acetate extract phase;

(3) The ethyl acetate extraction phase was prepared by gel column chromatography and silica gel column chromatography to obtain kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside.

Example 4:

Evaluation of the antibacterial activity of kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside by the minimum inhibitory concentration method

1. Experimental materials

1.1 Experimental strains: Staphylococcus aureus among Gram-positive bacteria and Klebsiella pneumoniae among Gram-negative bacteria were used as test strains in this experiment. The above two strains were provided by Microbiology Laboratory, School of Food Engineering, Tianjin University of Science and Technology.

1.2 Experimental equipment: constant temperature oscillator, constant temperature incubator, ultra-clean workbench, etc.

1.3 Drugs to be tested: the above-mentioned kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside has a purity of 99.9% after HPLC determination, and the selected positive control group is a broad-spectrum high-efficiency new Mycin.

2. Operation steps: The minimum inhibitory concentration method used in the test was carried out with reference to the steps described in the literature (Rabe T, van Staden J, Journal of Ethnopharmacology, 2000, 73, 171-174). Each group of experiments were carried out three times independently, and the average value was calculated.

The experimental results are shown in Table 2. The results showed that kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside had strong inhibitory activity against Staphylococcus aureus and Klebsiella pneumoniae, and its antibacterial The effect was equivalent to the antibacterial effect of neomycin in the positive control group.

Table 2. The minimum inhibitory concentration (μg/ml) of kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside and neomycin to experimental strains

Claims (1)

1. a kind of preparation technology of kaempferol derivative kaempferol-3-O-(4 "-galloyl)-α-L-rhamnopyranoside is characterized in that comprising the steps: (1) Take the pulverized Sophora japonica root bark plant raw material, add acetonitrile aqueous solution with a volume percentage concentration of 70% to 99% according to the mass ratio of 1:1 to 1:15, and extract at room temperature or heating or ultrasonic or microwave assisted extraction for 1 to 1. 6 times, each time for 1 to 48 hours, filter, and concentrate the filtrate under reduced pressure to 1% to 20% of the original volume to obtain a crude extract; (2) Add water with 1 to 6 times the mass of the crude extract, stir, add n-hexane with 1 to 6 times the mass of the crude extract, extract 1 to 6 times, separate, add the remaining water layer to 1 to 6 times the mass of the crude extract The petroleum ether is extracted 1 to 6 times, separated, and the petroleum ether layer is concentrated under reduced pressure to obtain the petroleum ether extract phase; (3) The petroleum ether extract phase was prepared by at least one of silica gel column chromatography and gel column chromatography to obtain kaempferol-3-O-(4″-galloyl)-α-L-rhamnopyranoside.