CN112194648B - Novel phenanthrene compound separated from pholidota chinensis and preparation method and application thereof - Google Patents

Novel phenanthrene compound separated from pholidota chinensis and preparation method and application thereof Download PDF

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CN112194648B
CN112194648B CN202010750120.9A CN202010750120A CN112194648B CN 112194648 B CN112194648 B CN 112194648B CN 202010750120 A CN202010750120 A CN 202010750120A CN 112194648 B CN112194648 B CN 112194648B
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phenanthrene compound
methanol
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遆慧慧
赵昕
余茜
庄子熙
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Guangdong Pharmaceutical University
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    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
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Abstract

The invention discloses a novel phenanthrene compound separated from pholidota chinensis, wherein the molecular formula of the phenanthrene compound is C16H12O4The structural formula is as follows:
Figure DDA0002609730590000011
also discloses a preparation method of the novel phenanthrene compound and application of the novel phenanthrene compound in preparation of medicines with anti-staphylococcus aureus effect. The invention relates to a method for preparing 2-methoxy-3,7-dihydroxy-5H-phenanthro [4,5-bcd ] separated from pholidota chinensis]The pyran active substance is a novel phenanthrene compound, has the effect of resisting staphylococcus aureus, has wide application field and huge market potential, and has important significance for finding a novel antibacterial lead compound and promoting the development of antibacterial drugs.

Description

Novel phenanthrene compound separated from pholidota chinensis and preparation method and application thereof
Technical Field
The invention belongs to the technical field of pholidota chinensis, and particularly relates to a novel phenanthrene compound separated from pholidota chinensis, and a preparation method and application thereof.
Background
Shi Xian Tao is the whole herb of Shi Xian Tao Phlidota chinensis Lindl of Shi Xian peach of the genus Shi Xian in the family orchid, commonly known as Shi Shang Lian, Shi Ruo and Shi Chuan pan, and is recorded in the Bing Cao Yao Bian (the reserve of raw herb drugs). It is sweet in nature and cool in flavor, has the effects of nourishing yin, clearing heat, promoting diuresis and removing blood stasis, and is commonly used for treating hypertension, dizziness and headache caused by various reasons in folks.
The chemical components of the caryophyllus plant mainly comprise phenanthrene, bibenzyl, stilbene, triterpene, lignan, steroid, phenolic acid and other compounds, wherein the phenanthrene compound is used as the main component. The phenanthrene compound has good pharmacological activity, such as anti-inflammatory, antioxidant and antibacterial activity, and has wide development prospect.
The pholidota chinensis extract has been reported to have the effects of resisting tumor, bacteria, inflammation, oxidation and the like in documents, but the effective components are uncertain, so the action mechanism is not clear. Phenanthrene compounds are chemical frameworks with important medicinal values, and the frameworks have multiple active functions. Therefore, the research on the material basis of the pholidota chinensis, especially the phenanthrene monomers, determines the effective components, and has important scientific value. Not only can fully utilize Chinese medicine resources of the Chinese pholidota herb, but also is a good resource of a lead compound of an innovative medicine, can promote the health level of people, and creates great economic benefits and social benefits.
Disclosure of Invention
The invention aims to provide a novel phenanthrene compound separated from pholidota chinensis.
The invention also aims to provide a preparation method of the novel phenanthrene compound separated from the pholidota chinensis.
The invention finally aims to provide the application of the novel phenanthrene compound in preparing a medicament with an anti-staphylococcus aureus effect.
The first object of the present invention can be achieved by the following technical solutions: a novel phenanthrene compound separated from pholidota chinensis is characterized in that: the molecular formula of the phenanthrene compound is C16H12O4The structural formula is as follows:
Figure BDA0002609730570000021
the chemical name of the novel phenanthrene compound can be named as:
2-methoxy-3,7-dihydroxy-5H-phenanthro[4,5-bcd]pyran。
the second object of the present invention can be achieved by the following technical solutions: one preferable preparation method of the novel phenanthrene compound separated from the pholidota chinensis comprises the following steps:
(1) selecting whole herb of pholidota chinensis, and extracting with ethanol to obtain extract;
(2) loading the extract on normal phase silica gel column, gradient eluting with petroleum ether/acetone solution at volume ratio of 10:1, 7:1, 5:1, 3:1, 1:1 and 0:1, collecting the eluted part of petroleum ether/acetone solution at volume ratio of 0:1, concentrating until acetone is completely recovered;
(3) further loading the acetone eluate on a normal phase silica gel column, performing gradient elution by using chloroform/methanol with volume ratios of 30:1,10:1,5:1 and 1:1, collecting chloroform/methanol eluate parts with volume ratios of 5:1, concentrating, loading on a reverse phase silica gel chromatographic column, eluting with methanol/water with volume ratios of 5:5,6:4,7:3 and 8:2 respectively, collecting methanol/water eluate parts with volume ratios of 6:4, concentrating the eluate until methanol/water is completely recovered to obtain a concentrated solution;
(4) and (3) performing high performance liquid chromatography on the concentrated solution, eluting by using methanol/water with the volume ratio of 6:4, collecting the elution components, concentrating and drying to obtain the novel phenanthrene compound.
In the preparation method of the novel phenanthrene compound separated from the pholidota chinensis, the method comprises the following steps:
preferably, the whole herb of pholidota chinensis is selected in the step (2), and the ethanol is adopted for extraction, and the specific process of obtaining the extract is as follows: taking the dried whole herb part of the Chinese mesona herb, crushing, adding industrial ethanol with the volume percentage of 85% into the crushed whole herb part according to the mass-volume ratio of 1kg to 3-5L, extracting for three times, combining the three supernatants, concentrating in vacuum, and refrigerating at minus 80 ℃ for later use.
Preferably, the normal phase silica gel column in the steps (2) to (3) is a glass column with 4 times of column volume, the diameter of 3.5cm and the length of 40 cm.
Preferably, the drying in step (4) is freeze-drying.
The third object of the present invention can be achieved by the following technical solutions: the application of the novel phenanthrene compound in preparing a medicament with an anti-staphylococcus aureus effect.
The antibacterial experiments in the examples prove that the compound has the effect of resisting staphylococcus aureus and can be applied to the aspect of preparing medicines with the effect of resisting staphylococcus aureus.
The invention has the following advantages:
(1) the process of separating the novel phenanthrene compound from the pholidota chinensis adopts a mode of firstly performing gradient rough separation on a normal-phase silica gel column and then separating by combining reverse-phase column chromatography with high performance liquid chromatography, so that the high-efficiency separation of 2-methoxy-3,7-dihydroxy-5H-phenanthro [4,5-bcd ] pyran is realized, and the purity can reach more than 95%;
(2) the 2-methoxy-3,7-dihydroxy-5H-phenanthro [4,5-bcd ] pyran active substance prepared by the preparation method of the novel phenanthrene compound separated from the pholidota chinensis is a novel phenanthrene compound, has the effect of resisting staphylococcus aureus, is wide in application field and large in market potential, and has important significance for finding a novel antibacterial lead compound and promoting development of antibacterial drugs.
Drawings
FIG. 1 is a structural formula of the novel compound in example 2 of the present invention;
FIG. 2 is a key HMBC linkage diagram for the novel compound of example 2 of the present invention;
FIG. 3 is a hydrogen spectrum of the novel compound of example 2 of the present invention;
FIG. 4 is a carbon spectrum of the novel compound of example 2 of the present invention;
FIG. 5 is an HSQC spectrum of the novel compound of example 2 of the present invention;
FIG. 6 is a HMBC spectrum of the novel compound of example 2 of the present invention;
FIG. 7 is a ROESY spectrum of the novel compound of example 2 of the present invention;
FIG. 8 is a low resolution mass spectrum of the novel compound of example 2 of the present invention;
FIG. 9 is a high resolution mass spectrum of the novel compound of example 2 of the present invention;
FIG. 10 is an infrared spectrum of the novel compound of example 2 of the present invention.
Detailed Description
The method of the present invention is further illustrated by the following examples. The following examples are for illustrative purposes only and are not to be construed as limiting the invention. The reagents or materials used in the examples, if not specifically mentioned, were commercially available, and the equipment used in the following examples, if not specifically mentioned, was conventional in the art.
Example 1
(1) Extracting the whole herb part of the pholidota chinensis with ethanol to obtain an ethanol extract;
the specific operation is as follows: taking the dry whole herb part of the Chinese pholidota herb, crushing, adding 85% industrial ethanol according to the mass-volume ratio of 1:3-5(15kg:45L), extracting for three times, combining the three supernatants, concentrating in vacuum, and refrigerating at-80 ℃ for later use;
(2) loading the ethanol extract on normal phase silica gel column, gradient eluting with petroleum ether/acetone solution (10:1, 7:1, 5:1, 3:1, 1:1, 0:1 (all acetone, volume ratio), respectively collecting eluates, and concentrating the 0:1 petroleum ether/acetone solution (acetone eluent) until acetone is completely recovered;
(3) further loading the acetone eluate on normal phase silica gel column, gradient eluting with chloroform/methanol (30:1,10:1,5:1,1:1, volume ratio), collecting chloroform/methanol (5:1) eluate, concentrating, loading on reverse phase silica gel column, eluting with methanol/water (5:5,6:4,7:3,8:2), collecting 6:4 methanol/water eluate, and concentrating the eluate until methanol/water is completely recovered to obtain concentrated solution;
(4) and (3) performing high performance liquid chromatography on the concentrated solution in the step (3), eluting with methanol/water (6:4, volume ratio), collecting the elution components in a concentrated manner, concentrating, and drying to obtain the novel phenanthrene compound.
The usage amount of the petroleum ether/acetone and acetone solution in the step (2) in the embodiment is 2L, the volume diameter of 4 times of the normal phase silica gel separation column is 3.5cm, and the length of the normal phase silica gel separation column is 40 cm.
The chloroform/methanol solution used in step (3) in this example was 2L, and the normal phase silica gel separation column was a glass column 4 times the column volume diameter of 3.5cm and 40cm long.
The drying in the step (4) is freeze drying.
Example 2
The structure identification process for the novel phenanthrene component in example 1 is as follows:
1. spectral data
2-methoxy-3,7-dihydroxy-5H-phenanthro[4,5-bcd]pyran, brown powder, ultraviolet spectrum (methanol) λ max (log ε) nm:238 (1.58); the infrared spectrum v max cm-1: v max 3450,3018,2976,2944,2904,2837,1627,1602,1529,1439,1388,1292,1152,1042,853;1h and13c NMR data, table 1; high resolution Mass Spectrometry HRESIMS (M/z 267.0663[ M-H ]]+(calcd for C16H11O4267.0736), low resolution mass spectrum M/z 267[ M-H ]]+。
2. Determination of structural formula
Compounds were prepared from HRESIMS (M/z 267.0663[ M-H ]]+(calcd for C16H11O4267.0736)) was deduced to be C16H12O4。
To it1H and13analysis of the C NMR data (Table 1) showed a total of 16 carbon atoms as a signal, 1 CH3Signal (delta)C56.0), 1 CH2Signal (delta)C64.3), 5 CH signals (δ)C106.6,102.1,103.9,123.9,126.7), 9 quaternary carbon signal (. delta.)C148.1,141.8,111.7,119.6,111.7,153.8,157.4,132.4,112.3). Two phenolic hydroxyl groups, four benzene ring hydrogen proton signals and one CH exist2(δ H5.57) and one methyl signal (δ H3.98). DeltaH6.58(1H, J ═ 2.2Hz) and δ H6.82 (1H, J ═ 2.2Hz) indicate the presence of a 1,3,4,5 substituted phenyl ring, δH7.29(1H, s) indicates the presence of a benzene ring with an aromatic hydrogen proton.
Table 1.1HNMR and13CNMR data (500MHz) (solvent deuterated Acetone)
Figure BDA0002609730570000051
The 4 structural fragments are connected by the following HMBC correlation formula to form a 9, 10-dihydrophenanthrene type phenanthrene compound skeleton: MeO H-12, H-1/C-2; h-11, H-1/C-3(HMBC conclusion). And the attachment position of MeO was further verified by HSQC and ROESY (MeO H-12/C-1). From these data, it is proposed that the planar structure of the compound is shown in fig. 1, specifically as follows:
Figure BDA0002609730570000061
key HMBC links for the new compounds are shown in fig. 2, specifically as follows:
Figure BDA0002609730570000062
the hydrogen spectra of the novel compounds are shown in FIG. 3.
The carbon spectrum of the new compound is shown in FIG. 4, with 16 carbon atoms present.
As can be seen from fig. 2 to 4, there are substantially no impurity peaks, and the purity of the obtained novel compound can reach 95% or more.
The HSQC spectrum of the novel compound is shown in FIG. 5, and the connection H-1/C-1, H-6/C-6, H-8/C-8, H-9/C-9, H-10/C-10 and H-11/C-11 exist.
HMBC spectra of the novel compounds are shown in FIG. 6, with 4 structural fragments linked to MeO H-12, H-1/C-2; h-11, H-1/C-3.
The ROESY spectrum of the novel compound is shown in FIG. 7, and the structural fragment is linked with MeO H-12/C-1.
Low resolution mass spectra of the novel compounds are shown in FIG. 8, with M/z 267[ M-H]+The molecular weight of the compound was deduced as 268.
High resolution mass spectra of the novel compounds are shown in FIG. 9, with M/z 267.0663[ M-H ]]+Further, the molecular weight of the compound 268 was deduced.
The infrared spectrum of the novel compound is shown in FIG. 10, with absorption 3400(OH),1529,1439,1388,1292,1152,1042,853 (aromatic ring).
Example 3
This example carries out the action of the novel phenanthrene compounds on staphylococcus aureus:
1. experimental materials and methods:
staphylococcus aureus (Aureous aureus) ATCC29213 was purchased from China General Microbiological Culture Collection Center (CGMCC). Adding a sample to be detected into a 96-well culture plate, and diluting the sample in a multiple ratio to make the final concentration of the sample be 200 mu M, 100 mu M and 50 mu M respectively; bacterial solution was added to each well to a final concentration of 5X 105CFU/mL; culturing at 37 deg.C for 24h, and measuring OD value at 625nm with microplate reader. The experiment is simultaneously provided with a culture medium blank control, a bacteria control and a penicillin G sodium positive drug control。
2. The experimental results are as follows:
as shown in Table 2, the results show that the treatment group of the novel compound (prepared in example 1) has good inhibitory effect on Staphylococcus aureus, and is concentration-dependent, i.e., the inhibitory rate gradually increases with the increase of the sample concentration.
TABLE 2 Activity of novel Compounds against Staphylococcus aureus
Figure BDA0002609730570000071
The above embodiments are only used for illustrating the present invention, and the scope of the present invention is not limited to the above embodiments. The object of the present invention can be achieved by those skilled in the art based on the above disclosure, and any improvements and modifications based on the concept of the present invention fall within the protection scope of the present invention, which is defined by the claims.

Claims (4)

1. A preparation method of phenanthrene compounds separated from pholidota chinensis is characterized by comprising the following steps:
(1) selecting whole herb of pholidota chinensis, and extracting with ethanol to obtain extract;
(2) loading the extract on normal phase silica gel column, gradient eluting with petroleum ether/acetone solution at volume ratio of 10:1, 7:1, 5:1, 3:1, 1:1 and 0:1, collecting the eluted part of petroleum ether/acetone solution at volume ratio of 0:1, concentrating until acetone is completely recovered;
(3) further loading the acetone eluate on a normal phase silica gel column, performing gradient elution by using chloroform/methanol with volume ratios of 30:1,10:1,5:1 and 1:1, collecting chloroform/methanol eluate parts with volume ratios of 5:1, concentrating, loading on a reverse phase silica gel chromatographic column, eluting with methanol/water with volume ratios of 5:5,6:4,7:3 and 8:2 respectively, collecting methanol/water eluate parts with volume ratios of 6:4, concentrating the eluate until methanol/water is completely recovered to obtain a concentrated solution;
(4) performing high performance liquid chromatography on the concentrated solution, eluting with methanol/water with volume ratio of 6:4, collecting eluate, concentrating, and drying to obtain phenanthrene compound;
the molecular formula of the phenanthrene compound is C16H12O4The structural formula is as follows:
Figure DEST_PATH_IMAGE002
2. the method for producing a phenanthrene compound isolated from pholidota chinensis as claimed in claim 1, wherein: the whole herb of Chinese pholidota herb is selected in the step (2), and the extract is obtained by ethanol extraction, and the specific process of the extract is as follows: taking the dried whole herb part of the Chinese mesona herb, crushing, adding industrial ethanol with the volume percentage of 85% into the crushed whole herb part according to the mass-volume ratio of 1kg to 3-5L, extracting for three times, combining the three supernatants, concentrating in vacuum, and refrigerating at-80 ℃ for later use.
3. The method for producing a phenanthrene compound isolated from pholidota chinensis as claimed in claim 1, wherein: in the step (2) to the step (3), the normal phase silica gel column is a glass column which is 4 times of the column volume, has the diameter of 3.5cm and the length of 40 cm.
4. The method for producing a phenanthrene compound isolated from pholidota chinensis as claimed in claim 1, wherein: the drying in the step (4) is freeze drying.
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