CN118440038A - Novel phloroglucinol compound and preparation method and application thereof - Google Patents
Novel phloroglucinol compound and preparation method and application thereof Download PDFInfo
- Publication number
- CN118440038A CN118440038A CN202410637050.4A CN202410637050A CN118440038A CN 118440038 A CN118440038 A CN 118440038A CN 202410637050 A CN202410637050 A CN 202410637050A CN 118440038 A CN118440038 A CN 118440038A
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- China
- Prior art keywords
- fraction
- hyperforidiol
- phloroglucinol
- compound
- petroleum ether
- Prior art date
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention belongs to the technical field of medicines, and particularly discloses a novel phloroglucinol compound and a preparation method and application thereof; research results show that the novel phloroglucinol compound has good anti-depression effect, wherein particularly remarkable is that the phloroglucinol compounds Hyperforidiol A, hyperforidiol B and Hyperforidiol C provided by the invention have remarkable protection effect on SH-SY 5Y cells induced by corticosterone, hyperforidiol A and Hyperforidiol C can remarkably reduce mouse tail suspension time, are obviously superior to positive medicaments fluoxetine and lu you tai, and can be used for developing medicaments for treating depression.
Description
Technical Field
The invention belongs to the technical field of medicines, and in particular relates to a novel phloroglucinol compound, a preparation method thereof, a pharmaceutical composition and application thereof in treating antidepressant medicines.
Background
The incidence of depression is now on the rise, statistically the second leading cause of loss of health life. Antidepressants remain an important therapeutic modality for the treatment of depression. As research is advanced, neuroprotective drugs are also of increasing interest because they can both improve psychological symptoms and protect nerve cells from damage, which makes the neuroprotective drug market have great potential.
In order to find novel medicines for treating depression, the inventor separates from the dry aerial parts of Hypericum perforatum to obtain a novel phloroglucinol compound Hyperforidiols A-D, and finds that Hyperforidiol A, hyperforidiol B and Hyperforidiol C have obvious antidepressant activity, are superior to positive medicines fluoxetine (Fluoxetine) and lupulone (Neurostan), and can be used for developing antidepressant medicines.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a novel phloroglucinol compound with 2- (2-hydroxypropyl) dihydrofuran substituted at 2,3 positions, and a preparation method, a pharmaceutical composition and application thereof.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the novel phloroglucinol compounds have structures shown in the following formulas (1), (2), (3) and/or (4):
（1）
（2）
（3）
（4）。
Wherein, including tautomers or pharmaceutically acceptable salts of the phloroglucinol compounds also fall within the scope of the present invention.
The invention also provides a preparation method of the phloroglucinol compound, wherein the phloroglucinol compound is obtained by extracting and separating Hypericum perforatum.
Further, the preparation method of the phloroglucinol compound comprises the following steps:
S1, taking the dried aerial parts of Hypericum perforatum, adding a solvent for reflux extraction, and concentrating after merging the extracting solutions to obtain extractum;
s2, adding the extractum into water for suspension, and extracting with petroleum ether to obtain an extract;
s3, performing gradient elution on the extract by using petroleum ether-ethyl acetate solution through silica gel column chromatography to obtain fraction E; when the silica gel thin layer chromatography is used for detection, the Rf value corresponding to the fraction E is 0.65-0.68;
s4, performing gradient elution on the fraction E by using petroleum ether-ethyl acetate solution through silica gel column chromatography to obtain fractions E3 and E7; when the silica gel thin layer chromatography is used for detection, the Rf value corresponding to the fraction E3 is 0.71-0.74, and the Rf value corresponding to the fraction E7 is 0.35-0.44;
S5, performing gradient elution on the fractions E7 and E3 by using a methanol-water solution through MCI column chromatography to obtain fractions E7A and E3F; when the silica gel thin layer chromatography is used for detection, the Rf value corresponding to the fraction E7A is 0.31-0.34, and the Rf value corresponding to the fraction E3F is 0.76-0.78;
s6, separating the phloroglucinol compound with the structure shown in the formula (1) and/or (2) from the fraction E7A by using an HPLC method;
The mobile phase used in the HPLC method is methanol-water solution.
The application takes methanol-water as mobile phase, adopts HPLC method, and uses C 18 chromatographic column to prepare phloroglucinol compound with structural formulas as shown in formulas (1) and (2) in fraction E7A.
S7, separating the phloroglucinol compound with the structure shown in the formula (3) and/or (4) from the fraction E3F by using an HPLC method;
the mobile phase used for the HPLC method was acetonitrile-water solution.
The application takes acetonitrile-water as a mobile phase, adopts an HPLC method, and utilizes a C 18 chromatographic column to prepare the phloroglucinol compound with the structural formula shown in the formula (3) and/or (4) in fraction E3F.
Further, in step S6, the volume ratio of methanol to water in the methanol-water solution is (70:30) - (80:20). Preferably, the volume ratio of methanol to water in the methanol-water solution is 75:25.
Further, in step S6, the fraction E7A is further separated according to the characteristic ultraviolet absorption (λ max =220, 280 nm) of phloroglucinol observed by HPLC analysis, including the retention time of the phloroglucinol compound represented by structural formula (1) being 38-42 min; the retention time of the phloroglucinol compound shown in the structural formula (2) is 43-48 min.
Further, in step S7, the volume ratio of acetonitrile to water in the acetonitrile-water solution is (55:45) - (65:35). Preferably, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 62:38.
Further, in step S7, the fraction E3F is further separated according to the characteristic ultraviolet absorption (λ max =220, 280 nm) of phloroglucinol observed by HPLC analysis, including the retention time of the phloroglucinol compound represented by structural formula (3) being 44-47 min; the retention time of the phloroglucinol compound shown in the structural formula (4) is 57-60 min.
In step S2, the extract is added into water with the mass of 8-15 times for suspension, and then petroleum ether is used for extraction.
The Rf value in the application is the Rf value of the fluorescent spot observed at scanning wavelengths 254 nm and 365 nm.
Further, in step S3, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution (i.e. corresponding to fraction E) when fraction E is eluted is (45:55) - (55:45) based on the total volume being 100; preferably 50:50.
Further, in step S4, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution (i.e., corresponding to fraction E7) when fraction E7 is eluted is (5:95) - (15:85); preferably 10:90.
Further, in step S4, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution (i.e., corresponding to fraction E3) when fraction E3 is eluted is (55:45) - (65:35); preferably, the volume ratio of petroleum ether to ethyl acetate is 60:40.
Further, in step S5, the volume ratio of methanol to water in the methanol-water solution (i.e., corresponding to fraction E7A) when fraction E7A is eluted is (65:35) - (73:27); preferably 70:30.
Further, in step S5, the volume ratio of methanol to water in the methanol-water solution (i.e., corresponding to fraction E3F) when fraction E3F is eluted is (85:15) - (95:5); preferably 90:10.
Further, in the step S1, the solvent is 88-98V% ethanol water solution; the solvent is added in an amount of 8-10 times that of herba Hyperici perforati, and the reflux extraction times are 1-3, and each time 1-3 h.
In yet another aspect, the invention provides pharmaceutical compositions comprising the novel phloroglucinol compounds described above.
Further, the pharmaceutical composition comprises a potentiating agent and a pharmaceutically acceptable carrier or excipient.
That is, pharmaceutical compositions containing the phloroglucinol compounds of the present invention as an active ingredient and conventional pharmaceutical excipients or adjuvants or carriers are also included in the present invention.
Further, the synergistic agent is one or more of the following substances:
Fluoxetine, paroxetine, fluvoxamine, sertraline, citalopram, escitalopram, venlafaxine, duloxetine, mirtazapine, bupropion, agomelatine, trazodone, reboxetine, imipramine, amitriptyline, chlorimipramine, doxepin, maprotiline, mollobemide, liver soothing capsules, san john's extract, flupentixol and melitracin.
Further, the dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquid, medicinal granules, dripping pills or micropills.
The invention also provides application of the novel phloroglucinol compound in antidepressant drugs.
For convenience of description, the compounds having the formulas (1) to (4) are defined as the compounds Hyperforidiols A to D, respectively.
Compared with the prior art, the invention has the following advantages:
(1) The invention provides a novel phloroglucinol compound which is not reported at present. Experimental results show that the novel phloroglucinol compound Hyperforidiols A-D provided by the invention has good antidepressant effect; particularly, the compounds Hyperforidiol A, hyperforidiol B and Hyperforidiol C have remarkable protection effect on SH-SY5Y cells induced by corticosterone, hyperforidiol A and Hyperforidiol C can remarkably reduce the tail suspension immobility time of mice, are obviously superior to positive drugs fluoxetine and lupulone, and can be used for developing drugs for treating depression.
(2) The preparation method of the novel phloroglucinol compound has simple operation, good reproducibility and high extraction purity.
Drawings
FIG. 1 is a 1 H NMR spectrum (400 MHz, CDCl 3) of Hyperforidiol A prepared in example 1 of the present invention;
FIG. 2 is a 13 C NMR spectrum (100 MHz, CDCl 3) of Hyperforidiol A prepared in example 1 of the present invention;
FIG. 3 is a HMBC spectrum of Hyperforidiol A prepared in example 1 of the present invention;
FIG. 4 is a 1 H NMR spectrum (400 MHz, CDCl 3) of Hyperforidiol B prepared in example 1 of the present invention;
FIG. 5 is a 13 C NMR spectrum (100 MHz, CDCl 3) of Hyperforidiol B prepared in example 1 of the present invention;
FIG. 6 is a HMBC spectrum of Hyperforidiol B prepared in example 1 of the present invention;
FIG. 7 is a 1 H NMR spectrum (400 MHz, CDCl 3) of Hyperforidiol C prepared in example 2 of the present invention;
FIG. 8 is a 13 C NMR spectrum (100 MHz, CDCl 3) of Hyperforidiol C prepared in example 2 of the present invention;
FIG. 9 is a HMBC spectra of Hyperforidiol C prepared in example 2 of the present invention;
FIG. 10 is a 1 H NMR spectrum (400 MHz, CDCl 3) of Hyperforidiol D prepared in example 2 of the present invention;
FIG. 11 is a 13 C NMR spectrum (100 MHz, CDCl 3) of Hyperforidiol D prepared in example 2 of the present invention;
FIG. 12 is a HMBC spectrum of Hyperforidiol D prepared in example 2 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent.
It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In the examples, all means used are conventional in the art unless otherwise specified.
The terms "comprising," "including," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
Example 1
The embodiment discloses a preparation method of a novel phloroglucinol compound, which comprises the following steps:
S1, taking the aerial parts of Hypericum perforatum, adding a solvent for reflux extraction, combining the extracting solutions, and concentrating to obtain an extract; specifically, the solvent is 88-98V% ethanol water solution; the solvent is added in an amount of 8-10 times that of herba Hyperici perforati, and the reflux extraction times are 1-3, and each time 1-3 h.
Specific embodiments are as follows: weighing aerial parts of herba Hyperici perforati (208 kg), reflux-extracting with 95% ethanol water solution 10 times of herba Hyperici perforati as solvent for three times (each time 2.2 h), mixing extractive solutions, and concentrating to obtain extract (about 9.0. 9.0 kg).
S2, adding the extractum into water for suspension, and extracting with petroleum ether to obtain an extract;
In step S2, the extract is added into water with the mass of 8-15 times for suspension, and then petroleum ether is used for extraction.
The method comprises the following specific steps: the extract (9.0 kg) was suspended in 10 times by mass of water (90L), and then extracted 3 times with 1.5 times by volume of petroleum ether to obtain a petroleum ether extract (about 3100 g).
S3, performing gradient elution on the extract by using petroleum ether-ethyl acetate solution through silica gel column chromatography to obtain fraction E; when the silica gel thin layer chromatography is used for detection, the Rf value corresponding to the fraction E is 0.65-0.68; the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution (corresponding to fraction E) when fraction E is eluted is (45:55) - (55:45) based on the total volume of 100; preferably 50:50.
During gradient elution, fraction E can be eluted from petroleum ether-ethyl acetate solutions with volume ratios of (45:55) - (55:45), and fractions with Rf values of 0.65-0.68 are fraction E by silica gel thin layer chromatography. And wherein the volume ratio is 50:50 petroleum ether-ethyl acetate solution was most efficient to elute. The "preferred" principle is the same in the gradient elution process below.
The Rf value in the application is the Rf value of the fluorescent spot observed at scanning wavelengths 254 nm and 365 nm.
The method comprises the following steps: subjecting petroleum ether extract to silica gel column chromatography with volume ratio of 100: 0. 90: 10. 80: 20. 60: 40. 50: 50. 40: 60. 30: 70. 20: 80. 10:90 and 0:100, 1 fraction per 500mL volumes, to obtain a total of 56 fractions of 1-5, 6-10, 11-25, 26-29, 30-40, 41-44, 45-47, 48-51, 52-53, 54-56, respectively, using silica gel thin layer chromatography to examine, according to 254 nm and 365 nm, the fluorescence results of the Rf values of 0.81-0.83 (fraction 1-5), 0.78-0.80 (fraction 6-10), 0.75-0.77 (fraction 11-17), 0.72-0.74 (fraction 18-30), 0.65-0.68 (fraction 31-35), 0.64-0.63 (fraction 36-42), 0.57-0.62 (fraction 43-47), 0.52-0.54 (fraction 48-50), 0.46-0.47 (spot 51-53), 0.35-0.39 (fraction 54-56) are combined to obtain 10 fractions, respectively, and the J values of A, B, C, D, E, F, G, H, I and the J values are obtained sequentially; wherein, fraction E is obtained after combining fractions 31-35.
Fraction E was selected for further separation based on the characteristic uv absorption of phloroglucinol observed by HPLC analysis (λ max =220, 280 nm).
S4, performing gradient elution on the fraction E by using petroleum ether-ethyl acetate solution through silica gel column chromatography to obtain fraction E7; when the silica gel thin layer chromatography is used for detection, the Rf value corresponding to the fraction E7 is 0.35-0.44; the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution (corresponding to fraction E7) when fraction E7 is washed out is (5:95) - (15:85); preferably 10:90.
The specific operation steps comprise: subjecting fraction E to silica gel column chromatography at volume ratio of 100:0, 80: 20. 60: 40. 50: 50. 30: 70. 20: 80. 10: 90. 0:100 petroleum ether-ethyl acetate gradient elution to obtain 115 fractions of 1-14, 15-30, 31-38, 39-55, 56-77, 78-80, 81-100 and 101-115 in sequence, and then combining the fractions according to the observation of fluorescence spot Rf values of 0.79-0.85 (fraction 1-23), 0.75-0.78 (fraction 24-35), 0.71-0.74 (fraction 36-55), 0.63-0.69 (fraction 56-75), 0.50-0.62 (fraction 76-85), 0.45-0.49 (fraction 86-95), 0.35-0.44 (fraction 96-100) and 0.30-0.34 (fraction 101-115) at wavelengths of 254 nm and 365 nm to obtain fractions E1-E8; wherein fractions 96-100 are combined to form fraction E7.
S5, performing gradient elution on the fraction E7 by using a methanol-water solution through MCI column chromatography to obtain a fraction E7A; when the silica gel thin layer chromatography is used for detection, the Rf value corresponding to the fraction E7A is 0.31-0.34; eluting fraction E7A (i.e. corresponding to fraction E7A) to a volume ratio of methanol to water in the methanol-water solution of (65:35) - (73:27); preferably 70:30.
The specific operation comprises the following steps: the further separation of E7 was chosen according to the characteristic uv absorption of phloroglucinol observed by HPLC analysis (λ max =220, 280 nm), fraction E7 being chromatographed on MCI column using a volume ratio of 70: 30. 75: 25. 80: 20. 90: 10. 100:0, sequentially collecting 1-10, 11-17, 18-25, 21-30 and 31-35 total 35 fractions, detecting by silica gel thin layer chromatography, and combining 5 fractions (E7A-E7E respectively) according to Rf values of 0.31-0.34 (fraction 1-8), 0.47-0.49 (fraction 9-15), 0.55-0.62 (fraction 16-20), 0.64-0.69 (fraction 21-25) and 0.72-0.75 (fraction 26-35) of fluorescent spots observed at wavelengths 254-nm and 365-nm. Wherein fractions 1-8 are combined to form fraction E7A.
S6, separating the phloroglucinol compound with the structure shown in the formula (1) and/or (2) from the fraction E7A by using an HPLC method;
The mobile phase used in the HPLC method is methanol-water solution.
The application takes methanol-water as mobile phase, adopts HPLC method, and uses C 18 chromatographic column to prepare phloroglucinol compound with structural formulas as shown in formulas (1) and (2) in fraction E7A.
Wherein the volume ratio of methanol to water in the methanol-water solution is (70:30) - (80:20). Preferably, the volume ratio of methanol to water in the methanol-water solution is 75:25.
Further separation of fraction E7A according to the characteristic uv absorption of phloroglucinol (λ max =220, 280 nm) observed by HPLC analysis, including retention times of 38-42 min (purity 98%) for phloroglucinol compounds of formula (1); the retention time of the phloroglucinol compound with the structural formula shown in the formula (2) is 43-48 min (purity 99%).
Physical properties and detection data of the compound 1 produced in example 1 are as follows:
Colorless oily matter, and is easily dissolved in chloroform and methanol. The molecular weight is estimated to be 586 according to high-resolution mass spectrum, the molecular formula is determined to be C 35H54O7, and the unsaturation degree is calculated to be 9.
The 1H NMR (CDCl3, 400 MHz spectra shown in fig. 1 show that δ H 4.93.93 (1H, m), 4.99 (1H, t, j=7.0 Hz) suggests the presence of two double bond proton signals attached to methylene groups in the structure. In addition, δ H 4.87 (1H, m), 3.17 (1H, d, j=9.5 Hz) suggested two oxymethylene signals; 11 group methyl hydrogen signal [δH1.67 (3H, s),1.66 (3H, s),1.65 (3H, s),1.54 (3H, s),1.34 (3H, s),1.26 (3H, s),1.20 (3H, s),1.13 (3H, s),1.13 (3H, d,J= 6.6 Hz),1.13 (3H, d,J= 6.6 Hz),1.13 (3H, s)] .
The 13C NMR (CDCl3, 100 MHz spectra shown in fig. 2 show a total of 35 carbon signals, including 3 carbonyl carbon signals (δ C 209.2, 205.7, 190.7), 1 oxygen-olefin carbon signal δ C 171.6,5 olefin carbon signals (δ C 134.4, 133.7, 122.3, 120.8, 119.6), 1 quaternary carbon signal δ C 47.8,6 methylene carbon signals (δ C 39.6, 35.4, 29.4, 28.4, 27.6, 27.0), 2 methine carbon signals (δ C 42.5.5, 41.0), 10 methyl carbon signals (δ C 27.1, 26.9, 26.1, 26.0, 24.8, 24.0, 21.2, 21.2, 18.2, 18.1, 14.7). In addition, 2 tertiary carbon signals (δ C 94.3, 79.1) are included; 2 oxygen-linked quaternary carbon signals (delta C, 73.3, 71.4); quaternary carbon signals of 2 dicarbonyl groups (delta C 74.0.0, 64.0).
1H NMR、13 The signal assignment of C NMR is shown in Table 1.
Table 1 NMR data for Compound 1 delta (ppm)
As shown in the HMBC spectra of FIG. 3, H-27 (delta H, 4.87) was observed to correlate with C-26 (delta C94.3)、C-29 (δC, 8), H-26b (delta H, 3.00) correlated with C-27 (delta C94.3)、C-2 (δC171.6)、C-3 (δC, 120.8), demonstrating that the compound contains a 2- (2-hydroxypropyl) dihydrofuran substituent; the correlation between H-11 (delta H 2.48) and C-12 (delta C21.2)、C-10 (δC 209.2) indicates the presence of an isobutyryl group in the compound; correlation between H-15b (delta H 2.09) and C-8 (delta C47.8)、C-14 (δC14.7)、C-17 (δC27.6),H-17 (δH 3.17) and C-15 (delta C35.4)、C-16 (δC27.6)、C-18 (δC 73.3) and C-20 (delta C 24.0) indicates that 3, 4-dihydroxy-4-methylpentyl is attached at the C-8 position; in addition, the remote correlation between H-22 (δ H 4.93) and C-21 (δ C28.4)、C-7 (δC42.5)、C-24 (δC18.1),H-32 (δH 2.43) and C-31 (δ C29.4)、C-34 (δC18.2)、C-5 (δC 64.0) demonstrated that two 3-methyl-2-enebutyl groups were attached to carbon at positions 7 and 5, respectively.
Further comparison of this structure with the planar structure of Hypericumoxide B found a significant difference between H-27 (delta H 4.84.84) and Hypericumoxide B (delta H 4.61), combined with the difference in carbon spectra, speculates that compound 1 differs from the known compound Hypericumoxide B in the configuration of the carbon at C-27.
In summary, the structure of novel compound 1 (compound Hyperforidiol A) was determined as follows:
。
physical properties and detection data of the compound 2 prepared in example 1 are as follows:
A colorless oil; the molecular weight is estimated to be 586 according to high-resolution mass spectrum, the molecular formula is determined to be C 35H55O7, and the unsaturation degree is calculated to be 9.
As shown in figure 4 1H NMR (CDCl3, 400 MHz) spectrum δ H 4.91.91 (1H, t, j=6.9 Hz), 4.97 (1H, t, j=7.0 Hz) suggests the presence of two double bond proton signals attached to methylene groups in the structure. In addition, δ H 4.84 (1H, dd, j=11.6 Hz, 10.1 Hz), 3.15 (1H, d, j=9.7 Hz) was suggested as two methine signals; 11 group methyl hydrogen signal [δH1.66 (3H, s),1.65 (3H, s),1.63 (3H, s),1.54 (3H, s),1.33 (3H, s),1.26 (3H, s),1.20 (3H, s),1.18 (3H, s),1.10 (3H, s),1.14 (3H, d,J= 6.5 Hz),1.09 (3H, d,J= 6.4 Hz)] .
The 13C NMR (CDCl3, 100 MHz spectra shown in fig. 5 show a total of 35 carbon signals, including 3 carbonyl carbon signals (δ C 209.2, 205.6, 190.7), 1 oxygen olefin carbon signal δ C 171.8,5 olefin carbon signals (δ C 134.7, 133.7, 122.4, 120.7, 119.5), 1 quaternary carbon signal δ C 48.6,6 methylene carbon signals (δ C 40.0, 36.8, 29.5, 28.3, 28.0, 27.3), 2 methine carbon signals (δ C 43.6, 41.1), 10 methyl carbon signals (δ C 26.9, 26.4, 26.1, 26.0, 24.3, 23.7, 21.2, 21.2, 18.2, 18.1, 14.2). In addition, 2 tertiary carbon signals (delta C, 94.3, 79.6) are included; 2 oxygen-linked quaternary carbon signals (delta C, 73.2, 71.4); quaternary carbon signals of 2 dicarbonyl groups (delta C 73.3, 64.0).
1H NMR、13 The signal assignment of C NMR is shown in Table 2.
Table 2 NMR data for Compound 2 delta (ppm)
| Position of | Chemical shift of hydrogen (coupling constant, hz) | Chemical shift of carbon | Position of | Chemical shift of hydrogen (coupling constant, hz) | Chemical shift of carbon |
| 1 | 73.3 | 19 | 1.18 (s) | 26.4 | |
| 2 | 171.8 | 20 | 1.10 (s) | 23.7 | |
| 3 | 120.7 | 21 | 1.49 (m); 1.25 (m) | 28.3 | |
| 4 | 190.7 | 22 | 4.91 (t, 6.9) | 122.4 | |
| 5 | 64.1 | 23 | 133.7 | ||
| 6 | 1.86 (dd, 13.8, 4.1); 1.36 (d, 13.1) | 40 | 24 | 1.66 (s) | 26 |
| 7 | 1.62 (m) | 43.6 | 25 | 1.54 (s) | 18.1 |
| 8 | 48.6 | 26 | 3.00 (dd, 14.9, 10.0); 2.84 (dd, 14.9, 11.8) | 27.3 | |
| 9 | 205.6 | 27 | 4.84 (dd, 11.6, 10.1) | 94.3 | |
| 10 | 209.2 | 28 | 71.4 | ||
| 11 | 2.51 (td, 13.8, 6.3) | 41.1 | 29 | 1.33 (s) | 26.9 |
| 12 | 1.09 (d, 6.4) | 21.2 | 30 | 1.26 (s) | 24.3 |
| 13 | 1.14 (d, 6.5) | 21.2 | 31 | 2.43 (m) | 29.5 |
| 14 | 1.20 (s) | 14.2 | 32 | 4.97 (t, 7.0) | 119.5 |
| 15 | 1.80 (dd, 13.6, 4.0); 1.68 (m) | 36.8 | 33 | 134.4 | |
| 16 | 2.07 (dd, 13.0, 4.6); 1.67 (m) | 28 | 34 | 1.65 (s) | 18.2 |
| 17 | 3.15 (d, 9.7) | 79.6 | 35 | 1.63 (s) | 26.1 |
| 18 | 73.2 |
In the HMBC spectra shown in FIG. 6, H-27 (delta H 4.87) was observed to correlate with C-28 (delta C71.4)、C-29 (δC24.3)、C-3 (δC 120.7.7), demonstrating that the compound contains a 2- (2-hydroxypropyl) dihydrofuran substituent; the correlation between H-11 (delta H 2.51) and C-12 (delta C21.2)、C-10 (δC 209.2) indicates the presence of an isobutyryl group in the compound; correlation between H-15b (delta H 1.80) and C-8 (delta C48.6)、C-14 (δC14.2),H-17 (δH 3.15) and C-15 (delta C36.8)、C-18 (δC 73.2) and C-20 (delta C 23.7.7) indicates that 3, 4-dihydroxy-4-methylpentyl is attached at the C-8 position; in addition, the remote correlation between H-22 (δ H 4.91) and C-21 (δC28.3)、C-7 (δC43.6)、C-24 (δC18.1)、C-25 (δC26.0),H-32 (δH4.97) and C-5 (δ C64.1)、C-31 (δC29.5)、C-34 (δC18.2)、C-35 (δC 36.1) demonstrated that two 3-methyl-2-enebutyl groups were attached to the carbon at positions 7 and 5, respectively.
By comparing nuclear magnetic data, the planar structures of the compound 2 and the compound 1 are the same, and further comparing the C-15 (delta C36.8）、C-7（δC43.6）、C-8（δC 48.6.6) and the compound 1 (delta C 35.4, 42.5, 47.8) are obviously different, and the configuration of the carbon at C-17 is presumed to be different between the compound 2 and the compound due to the difference of the combined carbon spectrums.
In summary, the structure of novel compound 2 (compound Hyperforidiol B) was determined as follows:
。
Example 2
The embodiment discloses a preparation method of a novel phloroglucinol compound, which comprises the following steps:
S1, taking the aerial parts of Hypericum perforatum, adding a solvent for reflux extraction, combining the extracting solutions, and concentrating to obtain an extract; specifically, the solvent is 88-98V% ethanol water solution; the solvent is added in an amount of 8-10 times that of herba Hyperici perforati, and the reflux extraction times are 1-3, and each time 1-3 h.
Specific embodiments are as follows: weighing aerial parts of herba Hyperici perforati (208 kg), reflux-extracting with 95% ethanol water solution 10 times of herba Hyperici perforati as solvent for three times (each time 2.2 h), mixing extractive solutions, and concentrating to obtain extract (about 9.0. 9.0 kg).
S2, adding the extractum into water for suspension, and extracting with petroleum ether to obtain an extract;
In step S2, the extract is added into water with the mass of 8-15 times for suspension, and then petroleum ether is used for extraction.
The method comprises the following specific steps: the extract (9.0 kg) was suspended in 10 times by mass of water (90L), and then extracted 3 times with 1.5 times by volume of petroleum ether to obtain a petroleum ether extract (about 3100 g).
S3, performing gradient elution on the extract by using petroleum ether-ethyl acetate solution through silica gel column chromatography to obtain fraction E; when the silica gel thin layer chromatography is used for detection, the Rf value corresponding to the fraction E is 0.65-0.68; the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution (corresponding to fraction E) when fraction E is eluted is (45:55) - (55:45) based on the total volume of 100; preferably 50:50.
The Rf value in the application is the Rf value of the fluorescent spot observed at scanning wavelengths 254 nm and 365 nm.
The method comprises the following steps: subjecting petroleum ether extract to silica gel column chromatography with volume ratio of 100: 0. 90: 10. 80: 20. 60: 40. 50: 50. 40: 60. 30: 70. 20: 80. 10:90 and 0:100, 1 fraction per 500mL volumes, to obtain a total of 56 fractions of 1-5, 6-10, 11-25, 26-29, 30-40, 41-44, 45-47, 48-51, 52-53, 54-56, respectively, using silica gel thin layer chromatography to examine, according to 254 nm and 365 nm, the fluorescence results of the Rf values of 0.81-0.83 (fraction 1-5), 0.78-0.80 (fraction 6-10), 0.75-0.77 (fraction 11-17), 0.72-0.74 (fraction 18-30), 0.65-0.68 (fraction 31-35), 0.64-0.63 (fraction 36-42), 0.57-0.62 (fraction 43-47), 0.52-0.54 (fraction 48-50), 0.46-0.47 (spot 51-53), 0.35-0.39 (fraction 54-56) are combined to obtain 10 fractions, respectively, and the J values of A, B, C, D, E, F, G, H, I and the J values are obtained sequentially; wherein, fraction E is obtained after combining fractions 31-35.
Fraction E was selected for further separation based on the characteristic uv absorption of phloroglucinol observed by HPLC analysis (λ max =220, 280 nm).
S4, performing gradient elution on the fraction E by using petroleum ether-ethyl acetate solution through silica gel column chromatography to obtain fraction E3; when the silica gel thin layer chromatography is used for detection, the Rf value corresponding to the fraction E3 is 0.71-0.74; the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution (corresponding to fraction E3) when fraction E3 is washed out is (55:45) - (65:35); preferably, the volume ratio of petroleum ether to ethyl acetate is 60:40.
The specific operation steps comprise: subjecting fraction E to silica gel column chromatography at volume ratio of 100:0, 80: 20. 60: 40. 50: 50. 30: 70. 20: 80. 10: 90. 0:100 petroleum ether-ethyl acetate gradient elution to obtain 115 fractions of 1-14, 15-35, 36-55, 56-59, 60-77, 78-80, 81-100 and 101-115 in sequence, and then combining the fractions according to the observation of fluorescence spot Rf values of 0.79-0.85 (fraction 1-23), 0.75-0.78 (fraction 24-35), 0.71-0.74 (fraction 36-55), 0.63-0.69 (fraction 56-75), 0.50-0.62 (fraction 76-85), 0.45-0.49 (fraction 86-95), 0.35-0.44 (fraction 96-100) and 0.30-0.34 (fraction 101-115) at wavelengths of 254 nm and 365 nm to obtain fractions E1-E8; wherein fractions 36-55 are combined to form fraction E3.
S5, performing gradient elution on the fraction E3 by using a methanol-water solution through MCI column chromatography to obtain a fraction E3F; when the silica gel thin layer chromatography is used for detection, the Rf value corresponding to the fraction E3F is 0.76-0.78; eluting fraction E3F (i.e. corresponding to fraction E3F) to a volume ratio of methanol to water in the methanol-water solution of (85:15) - (95:5); preferably 90:10.
The specific operation comprises the following steps: the E3 was selected for further separation according to the characteristic uv absorption of phloroglucinol observed by HPLC analysis (λ max =220, 280 nm), fraction E3 was subjected to MCI column chromatography using 60: 40. 65: 35. 70: 30. 75: 25. 80: 20. 90: 10. 100:0 are sequentially collected to obtain 1-5, 6-12, 13-17, 18-20, 21-28, 29-33 and 34-36 fractions, 36 fractions are combined by silica gel thin layer chromatography, and 7 fractions, namely E3A-E3G, are obtained according to the Rf values of fluorescent spots of 0.31-0.34 (fraction 1-3), 0.47-0.49 (fraction 4-11), 0.55-0.62 (fraction 12-18), 0.64-0.69 (fraction 19-24), 0.72-0.75 (fraction 25-28), 0.76-0.78 (fraction 29-33) and 0.80-0.85 (fraction 34-36) when the fluorescent spots are observed at wavelengths 254-nm and 365-nm respectively. Wherein fractions 29-33 are combined to form fraction E3F.
S6, separating the phloroglucinol compound with the structure shown in the formula (3) and/or (4) from the fraction E6D by using an HPLC method;
the mobile phase used for the HPLC method was acetonitrile-water solution.
The application uses acetonitrile-water as mobile phase, adopts HPLC method, and uses C 18 chromatographic column to prepare phloroglucinol compound with structural formulas as shown in formulas (3) and (4) in fraction E3F.
Wherein the volume ratio of acetonitrile to water in the acetonitrile-water solution is (55:45) - (65:35). Preferably, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 62:38.
Further separation of fraction E3F according to the characteristic uv absorption of phloroglucinol (λ max =220, 280 nm) observed by HPLC analysis, including retention times of 44-47 min (99% purity) for phloroglucinol compounds of formula (3); the retention time of the phloroglucinol compound with the structural formula shown in the formula (4) is 57-60 min (purity 99%).
Physical properties and detection data of the compound 3 prepared in example 2 are as follows:
A colorless oil; the molecular weight is estimated to be 586 according to high-resolution mass spectrum, the molecular formula is determined to be C 35H55O7, and the unsaturation degree is calculated to be 9.
In the 1H NMR (CDCl3, 400 MHz spectra shown in fig. 7, δ H 4.98 (1H, t, j=7.0 Hz), 4.94 (1H, t, j=8.5 Hz) suggests the presence of two double bond proton signals attached to methylene groups in the structure. In addition, δ H 4.79 (1H, t, j=10.5 Hz), 3.39 (1H, dd, j=9.7 Hz, 2.4 Hz) was suggested as two oxygen methine signals; 11 group methyl hydrogen signal [δH1.68 (3H, s),1.65 (3H, s),1.57 (3H, s),1.55 (3H, s),1.38 (3H, s),1.25 (3H, s),1.21 (3H, s),1.21 (3H, s),1.15 (3H, d,J= 5.0 Hz),1.14 (3H, d,J= 5.0 Hz),1.12 (3H, s)] .
35 Carbon signals are shown in total in the 13C NMR (CDCl3, 100 MHz spectra shown in fig. 8, including 3 carbonyl carbon signals (δ C 208.8, 208.2, 191.0), 1 oxygen-coupled olefin carbon signal δ C 172.1,5 olefin carbon signals (δ C 134.0.0, 131.9, 124.3, 122.0, 120.6), 1 quaternary carbon signal δ C 48.4,6 methylene carbon signals (δ C 40.6, 38.2, 32.9, 28.2, 26.8, 24.5), 2 methine carbon signals (δ C 42.4.4, 41.0), 10 methyl carbon signals (δ C 26.8, 26.0, 25.8, 25.7, 25.6, 24.5, 21.5, 21.2, 18.2, 17.9, 14.5). In addition, 2 tertiary carbon signals (δ C 94.4, 74.4) are included; 2 oxygen-linked quaternary carbon signals (delta C, 73.1, 71.1); quaternary carbon signals of 2 dicarbonyl groups (delta C 74.1, 63.3).
1H NMR、13 The signal assignment of C NMR is shown in Table 3.
Table 3 NMR data for Compound 3 delta (ppm)
In the HMBC spectra as shown in FIG. 9, H-27 (delta H 4.87) was observed to correlate with C-28 (delta C71.4)、C-29 (δC24.3)、C-2 (δC 120.7.7), demonstrating that the compound contains a 2- (2-hydroxypropyl) dihydrofuran substituent; the correlation between H-11 (delta H 2.51) and C-12 (delta C21.2)、C-10 (δC 209.2) indicates the presence of an isobutyryl group in the compound; the H-17 (delta H 3.15) and C-15 (delta C36.8)、C-16 (δC73.2) 、C-18 (δC 73.2) and C-19 (delta C 23.7) compounds have a 4-methyl-3-enamyl group; in addition, the remote correlation between H-22 (δ H 4.91) and C-21 (δC28.3)、C-7 (δC43.6)、C-24 (δC18.1)、C-25 (δC26.0),H-32 (δH4.97) and C-5 (δ C64.1)、C-31 (δC29.5)、C-35 (δC 36.1) demonstrated that 3-methyl-2-alkenobutyl and 2, 3-dihydroxy-3-methylbutyl are attached to carbons at positions 7 and 5, respectively.
By comparing nuclear magnetic data, the planar structures of the compound 4 and the compound 3 are the same, and further comparing the C-31 (delta C32.9）、C-32（δC 74.4.4) and the compound 1 (delta C 33.8, 75.1) are obvious differences, and the differences of the combined carbon spectrums are presumed that the configurations of the carbon at C-32 are different between the compound 2 and the compound.
In summary, the structure of novel compound 3 (compound Hyperforidiol C) was determined as follows:
。
physical properties and detection data of the compound 4 prepared in example 2 are as follows:
Colorless oily matter, and is easily dissolved in chloroform and methanol. The molecular weight is estimated to be 586 according to high-resolution mass spectrum, the molecular formula is determined to be C 35H54O7, and the unsaturation degree is calculated to be 9.
The 1H NMR (CDCl3, 400 MHz) spectra shown in fig. 10 show that δ H 4.98 (1H, t, j=7.0 Hz) and 4.92 (1H, t, j=7.9 Hz) suggest the presence of two double bond proton signals attached to methylene groups in the structure. In addition, δ H 4.80.80 (1H, t, j=10.5 Hz), 3.47 (1H, d, j=8.4 Hz) was suggested as two oxymethylene signals; 11 group methyl hydrogen signal [δH1.67 (3H, s),1.65 (3H, s),1.57 (3H, s),1.55 (3H, s),1.38 (3H, s),1.25 (3H, s),1.23 (3H, s),1.20 (3H, s),1.15 (3H, d,J= 7.3 Hz),1.13 (3H, d,J= 3.9 Hz),1.13 (3H, s)] .
The 13C NMR (CDCl3, 100 MHz spectra shown in fig. 11 show a total of 35 carbon signals, including 3 carbonyl carbon signals (δ C 208.2, 206.7, 192.7), 1 oxygen-olefin carbon signal δ C 172.6,5 olefin carbon signals (δ C 134.0, 131.9, 124.3, 122.1, 120.5), 1 quaternary carbon signal δ C 48.4,6 methylene carbon signals (δ C 41.2, 38.3, 33.8, 28.1, 26.7, 24.5), 2 methine carbon signals (δ C 42.3.3, 41.2), 10 methyl carbon signals (δ C 26.9, 26.0, 25.8, 25.6, 25.6, 24.3, 21.4, 21.2, 18.2, 17.9, 14.4). In addition, 2 tertiary carbon signals (δ C 94.4, 75.1) are included; 2 oxygen-linked quaternary carbon signals (delta C, 73.2, 71.2); quaternary carbon signals of 2 dicarbonyl groups (delta C 74.2.2, 63.3).
1H NMR、13 The signal assignment of C NMR is shown in Table 4.
Table 4 NMR data for Compound 4 delta (ppm)
| Position of | Chemical shift of hydrogen (coupling constant, hz) | Chemical shift of carbon | Position of | Chemical shift of hydrogen (coupling constant, hz) | Chemical shift of carbon |
| 1 | 75.1 | 19 | 1.55 (s) | 18.2 | |
| 2 | 172.6 | 20 | 1.65 (s) | 25.8 | |
| 3 | 120.5 | 21 | 2.07 (m); 1.70 (m) | 28.1 | |
| 4 | 192.7 | 22 | 4.92 (t, 7.9) | 122.1 | |
| 5 | 63.3 | 23 | 134.0 | ||
| 6 | 1.91 (dd, 13.5, 4.0);1.40 (m) | 41.2 | 24 | 1.67 (s) | 26.0 |
| 7 | 1.68 (m) | 42.3 | 25 | 1.57 (s) | 17.9 |
| 8 | 48.4 | 26 | 3.01 (d, 11.0) | 26.7 | |
| 9 | 208.2 | 27 | 4.80 (t, 10.5) | 94.4 | |
| 10 | 206.7 | 28 | 71.2 | ||
| 11 | 2.49 (dt, 13.0, 6.5) | 41.2 | 29 | 1.38 (s) | 26.9 |
| 12 | 1.13 (d, 3.9) | 21.4 | 30 | 1.25 (s) | 25.6 |
| 13 | 1.15 (d, 7.3) | 21.2 | 31 | 2.06 (m);1.99 (m) | 33.8 |
| 14 | 1.13 (s) | 14.4 | 32 | 3.47 (d, 8.4) | 74.2 |
| 15 | 1.64 (m) | 38.3 | 33 | 73.2 | |
| 16 | 1.97 (m) | 24.3 | 34 | 1.23 (s) | 25.6 |
| 17 | 4.98 (t, 7.0) | 124.3 | 35 | 1.20 (s) | 24.5 |
| 18 | 131.9 |
In the HMBC spectra as shown in FIG. 12, it can be observed that H-27 (delta H 4.80) correlates with C-3 (delta C120.5)、C-29 (δC 26.9.9) and H-26 (delta H 3.01) correlates with C-27 (δC94.4)、C-28 (δC71.2)、C-2 (δC172.6)、C-3 (δC120.5) 、C-4 (δC192.7), demonstrating that the compound contains a 2- (2-hydroxypropyl) dihydrofuran substituent; the correlation between H-11 (delta H 2.49) and C-12 (delta C21.2)、C-10 (δC 208.2) indicates the presence of an isobutyryl group in the compound; in addition, the remote correlation between H-22 (δ H 4.92) and C-24 (δ C18.2)、C-25 (δC 26.0.0) demonstrated a 3-methyl-2-alkenylbutyl group in the structure.
Further comparison of this structure with the planar structure of Uralione K found a significant difference between H-27 (delta H 4.80) and Uralione K (delta H 4.67), combined with the difference in carbon spectra, speculates that compound 3 is different from the known compound Uralione K in the configuration of the carbon at C-27. In addition, the chemical shift of compound 3 at C-32 and the like is different, and it is suggested that the configuration of C-32 carbon of the compound is different from that of the known compound.
In summary, the structure of novel compound 4 (compound Hyperforidiol D) was determined as follows:
。
Comparative example 1
Comparative example 1 of the present invention provides a process for the preparation of novel phloroglucinol compounds, which is similar to example 1, except that in step S5, fraction E7 is subjected to MCI column chromatography using a methanol-water solution having a methanol to water volume ratio of less than 65:35 or higher than 73:27 in methanol-water. The results show that compounds 1 and 2 could not be prepared.
Comparative example 2
Comparative example 2 of the present invention provides a process for the preparation of phloroglucinol, which is similar to example 1, except that in step S4, fraction E is subjected to silica gel column chromatography using a volume ratio of less than 55:45, or above 65:35 petroleum ether-ethyl acetate gradient elution. The results show that compounds 3 and 4 cannot be prepared.
In order to better understand the essence of the present invention, the novel application of the above phloroglucinol compounds in the pharmaceutical field will be described below in conjunction with pharmacological tests and results.
Test examples
This test example discloses experiments on the antidepressant activity of the above compounds Hyperforidiol A, hyperforidiol B, hyperforidiol C, hyperforidiol D.
(1) Experimental materials and instruments
Animal C57BL/6JNifdc mice, male, 20+ -2 g, purchased from Peking Vitrelli laboratory animal technologies Co., ltd;
human neuroblastoma SH-SY5Y was purchased from basic research institute of Chinese medical science sciences;
DMEM medium, PBS buffer, fetal Bovine Serum (FBS) and trypsin were all purchased from Gibco, usa;
fluoxetine (Shanghai derived leaf Biotechnology Co., ltd.);
luyou Tai (Weima Shu Peibo, germany pharmaceutical Co.);
CCK-8 cell activity detection kit (Elabscience company);
corticosterone, DMSO (Sigma, usa);
96 well plates (Corning corporation, usa);
carbon dioxide cell incubator (Thermo FISHER SCIENTIFIC company, usa);
full wavelength microplate reader (Thermo FISHER SCIENTIFIC, usa);
BIOFUGE STRATOS centrifuge (Thermo FISHER SCIENTIFIC, usa);
IX73 inverted Electron microscope (Olympus Corp., japan);
Pipettes (Eppendorf, germany);
a corticosterone ELISA kit (Elabscience company);
electronic balance (Sartorius company, germany);
Tissue refiner: DY89-I (Ningbo Xinzhi Bio Inc.);
Related consumables such as ultra-clean bench, centrifuging tube, straw.
(2) Method of
1) Establishment and evaluation of corticosterone injury SH-SY5Y cell model (corticosterone-induced SH-SY5Y is a common cell model for evaluation of antidepressant activity)
Taking out the labeled SH-SY5Y cell freezing tube in the liquid nitrogen, immediately putting the tube into a water bath kettle at 37 ℃ and completing quick thawing within 1 min as much as possible; after alcohol sterilization is carried out on the freezing tube, the freezing solution in the tube is transferred into a 15 mL sterile centrifuge tube, and a corresponding culture medium is added to mix evenly, and then the supernatant is removed by centrifugation; the above steps were repeated once for washing, and then 10 mL culture solution was added to mix them uniformly to resuspend the cells, which were then transferred to 10 mL dishes and incubated in a constant temperature incubator at 37℃with 5% CO 2.
When the cell density reaches 70% -80%, passaging the cells; the old culture medium is firstly sucked, PBS is added for washing for 2 times, trypsin containing EDTA is added into a culture dish and placed in a culture box at 37 ℃ for 3 min, then the culture medium is added for stopping digestion, 800 rpm is centrifuged for 5 min, after the digestion liquid is sucked, the culture medium containing serum is added again and cells are repeatedly blown off to form cell suspension, and finally the cell suspension is transferred into a new culture dish according to the number of the cells, so that the cell passage is completed.
SH-SY5Y cells were plated in 96-well plates at 1X 10 5 cells/well, 6 wells per group were plated, and cultured at 80. Mu.L/well for 24h, fluoxetine, lulutidine, hypericumoxide B, uralione K, hyperforin and tetrahydrohyperforin were dissolved and diluted to 10. Mu.M, and compounds Hyperforidiol A, hyperforidiol B, hyperforidiol C and Hyperforidiol D were dissolved and diluted to 5. Mu.M, 10. Mu.M and 10. Mu.L/well for 1h, and then 10. Mu.L of corticosterone at 25. Mu.M was added, and cultured at 37℃for 48 h, and then CCK-8. Mu.L and 1h were added, and absorbance at 450 nm was measured by using a microplate reader, and cell viability was calculated.
Cell viability (%) = [ (a control-a sample)/(a control-a blank) ]x100%.
2) Mouse tail suspension experiment (gold index of antidepressant drug evaluation)
The feed is adaptively fed for 3 days before experiments, maintains 12 h circadian rhythms, and is free to ingest drinking water at room temperature (23+/-2). After 3 days of adaptive feeding, C57BL/6J mice (21.+ -. 2 g) were divided into 11 groups, 12 groups each, by mass balance and randomization. The blank group, the positive drug fluoxetine group (10 mg/kg), the Luyoutai group (78 mg/kg), the Hypericumoxide B group (2.5 mg/kg), the Uralione K group (2.5 mg/kg), the hyperforin group (2.5 mg/kg), the tetrahydrohyperforin group (2.5 mg/kg) and the experimental group Hyperforidiol A group (1.25 mg/kg, 2.5 mg/kg, 5 mg/kg), the Hyperforidiol C group (1.25 mg/kg, 2.5 mg/kg, 5 mg/kg) respectively. Each group of mice was given an intraperitoneally injected drug, except for the blank group, which was given the same dose of physiological saline. After the administration of 0.5 h on the 4 th day, a tail suspension experiment is carried out, namely, the mice are fixed on a tail suspension box at a position which is 2 to cm away from the tail tip by using a medical adhesive tape, so that the mice are in an inverted suspension state, the head is about 10 to cm away from the ground, animal vision is separated by using plates on two sides of the suspension, 6 min is observed in total, and after the mice are adapted to 2min, the accumulated immobility time in 4 min is recorded. The judgment standard is that the animal stops struggling, and the body is in a vertical and inverted state and is still.
(3) Experimental results
The neuroprotective activity test results (Table 5) show that various novel phloroglucinol compounds disclosed by the application have good antidepressant effect, and particularly, compared with a model group, hyperforidiol A, hyperforidiol B and Hyperforidiol C with low and high doses (5 mu M and 10 mu M) have remarkable protective effect on the injury of SH-SY5Y nerve cells induced by corticosterone. The neuroprotective activity of low doses Hyperforidiol A, hyperforidiol B and Hyperforidiol C is significantly better than that of the positive drugs, namely lulutidine (the first international antidepressant natural plant drug, 10. Mu.M), hypericumoxide B (10. Mu.M), uralione K (10. Mu.M), hyperforin (10. Mu.M) and tetrahydrohyperforin (10. Mu.M); the neuroprotective activity of high doses Hyperforidiol A, hyperforidiol B and Hyperforidiol C was significantly better than that of the positive drug fluoxetine (first line chemical for clinical treatment of depression, 10 μm).
Neuroprotection of compounds of Table 5 against corticosterone-induced SH-SY5Y cell injury±s, n=5）
| Group of | Concentration (mu M) | Cell viability (%) |
| Blank space | 0 | 100.00±7.89 |
| Model | 0 | 52.49±2.10#### |
| Fluoxetine | 10 | 65.79±2.33** |
| Luyoutai (Chinese character) | 10 | 60.03±3.33* |
| Hypericumoxide B | 10 | 50.79±3.71 |
| Uralione K | 10 | 53.79±3.71 |
| Hyperforin | 10 | 55.83±2.71 |
| Tetrahydrohyperforin | 10 | 58.03±3.33* |
| Hyperforidiol A | 10 | 88.10±8.61**** |
| Hyperforidiol A | 5 | 71.29±3.42** |
| Hyperforidiol B | 10 | 72.30±6.08*** |
| Hyperforidiol B | 5 | 65.66±2.44* |
| Hyperforidiol C | 10 | 67.07±4.71** |
| Hyperforidiol C | 5 | 61.29±3.50* |
| Hyperforidiol D | 10 | 59.91±4.39 |
| Hyperforidiol D | 5 | 58.56±4.01 |
Note that: comparison to the blank: #### P is less than 0.0001; *P＜0.05,**P＜0.01,***P＜0.001,**** P < 0.0001 compared to model group
In the mouse tail suspension experiment (Table 6), compared with the blank group, the low and high doses (1.25 mg/kg, 5 mg/kg) of Hyperforidiol A and Hyperforidiol C significantly shorten the immobility time of the mouse tail suspension, and the effect of the mouse tail suspension experiment is superior to that of a positive drug fluoxetine (first-line chemical for clinically treating depression, 5 mg/kg) and is significantly superior to that of a positive drug, namely, lulutidine (first antidepressant natural plant drug in International, 78 mg/kg), hypericumoxide B (5 mg/kg), uralione K (5 mg/kg), hyperforin (5 mg/kg) and tetrahydrohyperforin (5 mg/kg).
TABLE 6 results of mice tail-suspension experiments±s, n=12）
| Group of | Dosage (mg/kg) | Tail suspension immobility time(s) |
| Blank space | 0 | 129.25±23.18 |
| Fluoxetine | 5 | 96.32±12.61** |
| Luyoutai (Chinese character) | 78 | 100.12±26.13* |
| Hypericumoxide B | 5 | 125.23±23.16 |
| Uralione K | 5 | 120.13±23.27 |
| Hyperforin | 5 | 113.58±13.57 |
| Tetrahydrohyperforin | 5 | 121.31±15.82 |
| Hyperforidiol A | 1.25 | 92.94±12.32** |
| Hyperforidiol A | 5 | 74.22±14.68**** |
| Hyperforidiol C | 1.25 | 96.41±19.50** |
| Hyperforidiol C | 5 | 92.46±11.79** |
Note that: comparison to the blank: *P＜0.05,**P＜0.01,**** P < 0.0001
The results of the open field experiments in mice (Table 7) showed that there was no significant difference in the horizontal scores of Hyperforidiol A, hyperforidiol C at low, medium and high doses (1.25 mg/kg, 5 mg/kg) compared to the vertical scores and the blank, and false positive results of the compounds due to neuronal excitation could be excluded.
TABLE 7 open field experimental results±s, n=12）
| Group of | Dosage (mg/kg) | Number of horizontal exercises | Number of vertical movements |
| Blank space | 0 | 102.70±28.75 | 25.18±7.22 |
| Fluoxetine | 5 | 101.30±13.38 | 23.00±5.32 |
| Hyperforidiol A | 1.25 | 99.30±22.28 | 25.20±7.00 |
| Hyperforidiol A | 5 | 93.00±24.43 | 20.33±6.71 |
| Hyperforidiol C | 1.25 | 103.20±20.58 | 23.90±4.01 |
| Hyperforidiol C | 5 | 88.70±29.89 | 20.36±8.02 |
In conclusion, the novel phloroglucinol compound Hyperforidiols A-D has good antidepressant effect; particularly, hyperforidiol A, hyperforidiol B and Hyperforidiol C show remarkable antidepressant activity, are superior to first-line antidepressant medicaments of fluoxetine and lupulone, and can be used as medicaments for treating depression.
Application example 1
The application example of the invention discloses a capsule taking Hyperforidiol A as a raw material medicine, which comprises the following components:
Hyperforidiol A18.0 mg
Starch 6.0 g
Sodium metabisulfite 0.2 g
Magnesium stearate 0.2 g
Proper amount of absolute ethyl alcohol
Making into 100 granules.
The preparation process comprises the following steps:
mixing Hyperforidiol A with starch and sodium metabisulfite, adding absolute ethanol to obtain soft material, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and making into capsule.
Application example 2
The application example of the invention discloses a granule taking a compound Hyperforidiol B as a raw material medicine, which comprises the following components:
Hyperforidiol B35.0 mg
Starch 6.0 g
Sodium bisulfite 0.2 g
Magnesium stearate 0.2 g
Proper amount of absolute ethyl alcohol
Making into 100 bags.
The preparation process comprises the following steps:
Mixing Hyperforidiol B with starch and sodium bisulphite, adding absolute ethanol to obtain soft material, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and bagging.
Application example 3
The application example of the invention discloses an oral liquid taking a compound Hyperforidiol C as a raw material medicine, which comprises the following components:
Hyperforidiol C 25.0 mg
Sucrose 3.0 g
Sodium bisulfite 0.2 g
Methyl parahydroxybenzoate 0.2 g
Sodium bicarbonate 0.1 g
Injection water 1000 mL
100 Pieces of the Chinese herbal medicine are prepared.
The preparation process comprises the following steps:
mixing the above components, preparing into oral liquid by conventional method, and packaging.
Application example 4
The application example of the invention discloses an injection taking a compound Hyperforidiol B as a raw material medicine respectively, which comprises the following components:
Hyperforidiol B45.0 mg
vitamin C0.2 g
Sodium chloride 6.0 g
Sodium bicarbonate 0.1 g
Injection water 1000 mL
100 Pieces of the Chinese herbal medicine are prepared.
The preparation process comprises the following steps:
After the components are evenly mixed, 100 pieces can be obtained by adopting the conventional preparation method of injection.
Application example 5
The application example of the invention discloses a tablet taking a compound Hyperforidiol B and fluoxetine as raw material medicines, which comprises the following components:
Hyperforidiol B20.0 mg
Fluoxetine 0.5g
Hydroxypropyl methylcellulose 18 g
Talc powder 0.4g
Lactose 0.2 g
Magnesium stearate 0.2 g
Proper amount of absolute ethyl alcohol
Making into 100 pieces.
The preparation process comprises the following steps:
Mixing Hyperforidiol B, fluoxetine, hydroxypropyl methylcellulose, talcum powder, lactose and magnesium stearate, adding absolute ethyl alcohol to prepare a soft material, sieving with a 24-mesh sieve, granulating, drying, adding magnesium stearate, mixing uniformly, and tabletting.
Application example 6
The application example of the invention discloses a capsule taking a compound Hyperforidiol A and sertraline as raw material medicines, which comprises the following components:
Hyperforidiol A 18.0 mg
sertraline 2.0 g
Starch 6.0 g
Sodium metabisulfite 0.2 g
Magnesium stearate 0.2 g
Proper amount of absolute ethyl alcohol
Making into 100 granules.
The preparation process comprises the following steps:
Mixing Hyperforidiol A, sertraline, starch and sodium metabisulfite, adding absolute ethanol to obtain soft material, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and making into capsule.
Application example 7
The application example of the invention discloses an injection taking a compound Hyperforidiol C and escitalopram as raw material medicines, which comprises the following components:
Hyperforidiol C30.0 mg
Escitalopram 2.0 g
Vitamin C0.2 g
Sodium chloride 6.0 g
Sodium bicarbonate 0.1 g
Injection water 1000 mL
100 Pieces of the Chinese herbal medicine are prepared.
The preparation process comprises the following steps:
After the components are evenly mixed, 100 pieces can be obtained by adopting the conventional preparation method of injection.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention.
It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A novel phloroglucinol compound characterized by having a structure represented by formula (1), formula (2), formula (3) and/or formula (4):
（1）
（2）
（3）
（4）。
2. A process for the preparation of the novel phloroglucinol compound of claim 1, wherein the phloroglucinol compound is isolated from the upper part of Hypericum perforatum.
3. A process according to claim 2, characterized in that it comprises the following steps:
s1, taking the aerial parts of Hypericum perforatum, adding a solvent for reflux extraction, combining the extracting solutions, and concentrating to obtain an extract;
s2, adding the extractum into water for suspension, and extracting with petroleum ether to obtain an extract;
s3, performing gradient elution on the extract by using petroleum ether-ethyl acetate solution through silica gel column chromatography to obtain fraction E; when the silica gel thin layer chromatography is used for detection, the Rf value corresponding to the fraction E is 0.65-0.68;
s4, performing gradient elution on the fraction E by using petroleum ether-ethyl acetate solution through silica gel column chromatography to obtain fractions E3 and E7; when the silica gel thin layer chromatography is used for detection, the Rf value corresponding to the fraction E3 is 0.71-0.74, and the Rf value corresponding to the fraction E7 is 0.35-0.44;
S5, performing gradient elution on the fractions E7 and E3 by using a methanol-water solution through MCI column chromatography to obtain fractions E7A and E3F respectively; when the silica gel thin layer chromatography is used for detection, the Rf value corresponding to the fraction E7A is 0.31-0.34, and the Rf value corresponding to the fraction E3F is 0.76-0.78;
s6, separating the phloroglucinol compound with the structure shown in the formula (1) and/or (2) from the fraction E7A by using an HPLC method;
The mobile phase used in the HPLC method is methanol-water solution.
S7, separating the phloroglucinol compound with the structure shown in the formula (3) and/or (4) from the fraction E3F by using an HPLC method;
the mobile phase used for the HPLC method was acetonitrile-water solution.
4. The method according to claim 3, wherein in step S6, the volume ratio of methanol to water in the methanol-water solution is (70:30) - (80:20).
5. The process according to claim 3, wherein in step S6, the fraction E7A is further separated according to the characteristic ultraviolet absorption of phloroglucinol observed by HPLC analysis, and the retention time of the phloroglucinol compound represented by the structural formula (1) is 38-42 min;
The retention time of the phloroglucinol compound shown in the structural formula (2) is 43-48 min.
6. The method according to claim 3, wherein in step S7, the volume ratio of acetonitrile to water in the acetonitrile-water solution is (55:45) - (65:35).
7. The process according to claim 3, wherein in step S7, the fraction E3F is further separated according to the characteristic ultraviolet absorption of phloroglucinol observed by HPLC analysis, and the retention time of the phloroglucinol compound represented by the structural formula (3) is 44-47 min;
The retention time of the phloroglucinol compound shown in the structural formula (4) is 57-60 min.
8. The method according to claim 3, wherein in step S2, the extract is suspended in 8-15 times by mass of water and extracted with petroleum ether.
9. The process according to claim 3, wherein in step S3, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution in fraction E is washed out based on 100 total volumes is (45:55) - (55:45).
10. The process according to claim 3, wherein in step S3, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution in fraction E is 50, based on the total volume of 100: 50.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410637050.4A CN118440038A (en) | 2024-05-22 | 2024-05-22 | Novel phloroglucinol compound and preparation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202410637050.4A CN118440038A (en) | 2024-05-22 | 2024-05-22 | Novel phloroglucinol compound and preparation method and application thereof |
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