CN111808155B

CN111808155B - Ginseng coumarin compound and application thereof in medicine

Info

Publication number: CN111808155B
Application number: CN202010854436.2A
Authority: CN
Inventors: 杨丽; 何军伟; 刘荣华
Original assignee: Jiangxi University of Traditional Chinese Medicine
Current assignee: Jiangxi University of Traditional Chinese Medicine
Priority date: 2020-08-24
Filing date: 2020-08-24
Publication date: 2020-12-29
Anticipated expiration: 2040-08-24
Also published as: CN111808155A

Abstract

The invention relates to the technical field of development of medicaments for treating inflammatory diseases, in particular to application of coumarins in dendropanax japonicus in preparation of medicaments for treating rheumatoid arthritis or rheumatic arthritis, wherein the coumarins in dendropanax japonicus have one of the following chemical structural formulas:

the activity screening experiment proves that the three coumarin compounds in the dendropanax has obvious inhibiting effect on the release of nitric oxide from MH7A cells induced by tumor necrosis factor alpha (TNF-alpha), and the half inhibitory concentration (IC 50) values are 7.87 +/-0.7 mu M (formula I), 13.82 +/-1.1 mu M (formula II) and 39.88 +/-2.3 mu M (formula III) respectively. The invention provides a new medicine research direction for clinically developing the treatment of rheumatoid arthritis and rheumatoid arthritis.

Description

Ginseng coumarin compound and application thereof in medicine

Technical Field

The invention belongs to the field of medicines, and particularly relates to an application of coumarins in sappanwood in preparing a medicine for treating rheumatoid arthritis or rheumatic arthritis.

Background

Rheumatoid Arthritis (RA) is a common chronic, systemic autoimmune disease that can cause joint destruction, joint deformity, and even disability. At present, western medicines for treating RA are very expensive, and most of the medicines have a plurality of adverse reactions. Therefore, the search for drugs for treating RA from natural drugs, especially from the traditional significance, has a wide prospect.

MH7A cell is a human rheumatoid arthritis fibroblast-like synovial cell, and is closely related to rheumatoid arthritis and rheumatoid arthritis, and MH7A cell has an activity of releasing nitric oxide, which aggravates inflammation. Therefore, if it is found from natural products that MH7A cells can be inhibited from excessively releasing nitric oxide, the natural products can be used for treating rheumatoid arthritis and rheumatoid arthritis diseases.

Disclosure of Invention

Technical problem to be solved

In view of the defects and shortcomings of the prior art, based on the discovery that the coumarins in the dendropanax japonicas have the capability of inhibiting the activity of MH7A cells to release nitric oxide, the invention provides the application of the coumarins in the dendropanax japonicas in preparing the drugs for treating rheumatoid arthritis or rheumatic arthritis, and provides a new drug development approach for clinically developing the treatment of the rheumatoid arthritis and the rheumatic arthritis.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

the dendropanax japonicas coumarin compound has the following structural formula:

the compound of the formula I or the pharmaceutically acceptable salt or the hydrate of the salt has the activity of inhibiting tumor necrosis factor alpha (TNF-alpha) induced MH7A cells to release nitric oxide, and achieves the effects of relieving and treating rheumatoid arthritis and rheumatoid arthritis diseases.

The dendropanax japonicas coumarin compound and the application of the dendropanax japonicas coumarin compound and the derivatives thereof in preparing the medicine for treating rheumatoid arthritis or rheumatoid arthritis are disclosed, wherein the dendropanax japonicas coumarin compound has one of the following chemical structural formulas:

preferably, the derivatives of the coumarins of dendropanax japonicus refer to pharmaceutically acceptable salts or hydrates of the salts of the compounds with chemical structures of formula I, formula II and formula III.

Preferably, the dendropanax japonicas coumarin compound refers to a compound with a chemical structure shown in formula I, and the derivative refers to a pharmaceutically acceptable salt or a hydrate of the salt of the compound with the chemical structure shown in formula I.

Preferably, the compound with the chemical structure shown in formula I, formula II or formula III or the pharmaceutically acceptable salt or hydrate of the salt thereof has the efficacy of relieving and treating rheumatoid arthritis and rheumatoid arthritis diseases by inhibiting the activity of tumor necrosis factor alpha (TNF-alpha) induced MH7A cells to release nitric oxide.

Preferably, the method for extracting and purifying the dendropanax japonicas coumarin compound comprises the following steps:

s1, taking roots of sappanus japonicus of sappannaeus of Araliaceae as raw materials, adding 8-12 times of ethanol with the mass and the concentration of 65-75% for reflux extraction for at least 3 times, each time for at least 2h, and concentrating the extract under reduced pressure to obtain extract;

s2, suspending the extract with water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and respectively concentrating to dryness to obtain petroleum ether fraction, ethyl acetate fraction and n-butanol fraction; taking the n-butanol part extract, performing silica gel column chromatography, and performing gradient elution with dichloromethane-methanol to obtain 9 fractions;

s3, subjecting the 7 th fraction to octadecyl silane column chromatography, silica gel column chromatography and preparative HPLC column chromatography to obtain coumarins A of formula I and haploperoside A of formula II; and (4) respectively carrying out silica gel column chromatography and preparative HPLC column chromatography on the fraction to obtain the scopoletin compound shown in the formula III.

The invention also provides a medicament for treating rheumatoid arthritis or rheumatic arthritis diseases, which comprises the ethanol extract of the root of the dendropanax japonicus; the ethanol extract contains a mixture of compounds with chemical structures of formula I, formula II and formula III.

The invention also provides a medicament for treating rheumatoid arthritis or rheumatic arthritis diseases, which comprises the following medicinal components: coumarins from Panax quinquefolium or pharmaceutically acceptable salts or hydrates thereof; the dendropanax japonicas coumarin compound has one of the following chemical structural formulas:

the invention further provides a medicament for treating rheumatoid arthritis or rheumatic arthritis diseases, and the medicinal effect component of the medicament comprises a compound with the structure shown in the formula I or pharmaceutically acceptable salt or hydrate thereof.

(III) advantageous effects

The invention provides a natural compound, namely coumarin compound from dendropanax dentiger and derivatives thereof, which are used for preparing a medicament for treating inflammatory diseases, particularly rheumatoid arthritis and rheumatic arthritis, so as to replace the prior expensive pure synthetic chemical medicament. The planting resources of the sappanus japonicus are rich, the raw materials are easy to obtain, the cost and the price of the medicine for treating the diseases are reduced, the burden of patients is reduced, and the adverse reaction of the pure synthetic medicine is reduced.

The activity screening experiment proves that the three coumarin compounds of the dendropanax dentiger have no toxicity to cells and almost no influence on the activity of the cells at the concentration of 40 mu mol/L, but have obvious inhibition effect on the release of nitric oxide from MH7A cells induced by tumor necrosis factor alpha (TNF-alpha), and the half inhibition concentration (IC 50) values are 7.87 +/-0.7 mu M (formula I), 13.82 +/-1.1 mu M (formula II) and 39.88 +/-2.3 mu M (formula III), respectively.

Wherein the half maximal inhibitory concentration (IC 50) value of the compound having the structure of formula I is much smaller than that of the compounds having the structures of formula II and formula III. Therefore, the compound with the structure shown in the formula I has extremely high drug efficacy, and can play a significant role in inhibiting nitric oxide release of MH7A cells under the condition of small dosage, so that the dosage of the drug taken by a patient can be reduced, the drug efficacy speed is further improved, and the toxic and side effects are reduced.

Drawings

FIG. 1 shows NMR spectra of a novel compound, dendropanax coumarin A (structure of formula I) ((I))¹H NMR）。

FIG. 2 shows NMR spectrum of coumarin A (structure of formula I) of new compound (I)¹³C NMR）。

FIG. 3 shows NMR of coumarin A (structure of formula I) as a novel compound¹H-¹H COSY spectra.

FIG. 4 shows the NMR HSQC spectrum of coumarin A (structure of formula I) as a novel compound in the present invention.

FIG. 5 shows the NMR HMBC spectrum of the novel compound dendropanax coumarin A (structure of formula I).

FIG. 6 shows the correlation between HMBC (H → C) and COSY of coumarin A (structure of formula I) which is a novel compound in the present invention.

FIG. 7 shows a nuclear magnetic resonance hydrogen spectrum of a compound haloperoside A (structure of formula II) of the present invention: (¹H NMR）。

FIG. 8 shows the NMR spectrum of a compound haloperoside A (structure of formula II) of the present invention: (¹³C NMR）。

FIG. 9 shows the NMR spectrum of scopoletin (formula III) of the compound of the present invention: (¹H NMR）。

FIG. 10 shows the NMR spectrum of scopoletin (formula III) of the compound of the present invention: (¹³C NMR）。

Detailed Description

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.

Example 1: extraction and purification of three coumarin compounds in saprorrhiza

Taking 10.0 kg of sapiens root, heating and refluxing for 3 times with 100kg of 70% ethanol, 3 hours each time, and concentrating the extracting solution under reduced pressure to obtain 1.3 kg of extract. Suspending the extract with 1L water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol at equal volumes, and concentrating to dry respectively. Taking 216 g of n-butanol part extract, mixing with silica gel (weight ratio 1: 1), loading on silica gel column (column specification: 11 × 120 cm), and gradient eluting with dichloromethane-methanol (100: 0 → 0:100, v/v) to obtain 9 fractions Frs, C1-C9. (1 st to 9 th fractions)

Dichloromethane-methanol 5:1 elution fractions fr.c7 (7 th fraction 70.0 g) were subjected to octadecylsilane column chromatography (column size: 4.2 x 40 cm), methanol-water (5% → 30%) gradient elution, yielding 3 fractions fr.c 71-C73. Fraction C74 (4.92 g), stirred with silica gel (weight ratio 1: 1), applied to a silica gel column (column size: 6.4X 55 cm) and eluted with a gradient of dichloromethane-methanol (20: 1 → 10:1, v/v) to give 4 fractions Frs.C 741-C744. C742 (1.21 g) was subjected to preparative HPLC column chromatography (column size: 10 × 25 cm), and eluted with 20% acetonitrile to give 12.5 mg of the compound of the formula I (coumarina) and 31.7mg of the compound of the formula II (haloperoside A) according to the present invention.

The dichloromethane-methanol 30:1 eluted fractions Fr.C4 (4 th fraction 4.2 g), stirred with silica gel (weight ratio 1: 1), applied to a silica gel column (column size: 3.2 × 40 cm), and eluted with a dichloromethane-methanol (100: 0 → 0:100, v/v) gradient to give 8 fractions Frs.C 41-C48. C42 (256 mg) was subjected to preparative HPLC column chromatography (column format: 10 × 25 cm), and eluted with 35% methanol-water to give a structural compound of formula III (scopoletin) (100 mg).

Example 2: structure identification of three coumarin compounds in dendropanax dentiger

The compound with the structure shown in the formula I, the compound with the structure shown in the formula II and the compound with the structure shown in the formula III, which are separated in the example 1, are identified as three dendropanax coumarin compounds through high resolution mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance spectroscopy (1D NMR and 2D NMR). Wherein, the compound with the structure shown in the formula I is a novel coumarin derivative which is not reported in the literature, and the compound with the structure shown in the formula II and the compound with the structure shown in the formula III are known compounds (but the activity of treating rheumatoid arthritis and rheumatoid arthritis diseases is not reported).

Wherein, the compound with the structure shown in the formula I is light yellow powder. HR-ESI-MS gave the excimer peak M/z 531.17083 ([ M + H)]⁺Calculating the value: 531.17066) and determining that the molecular formula is C₂₃H₃₁O₁₄. The hydrogen spectrum of the compound with the structure of formula I is shown in figure 1, (DMSO-d ₆600 MHZ) has 2 methoxy hydrogen signals_H3.90 and 3.82 (each, 3H, s, 8/6-CH)₃) Triple alkene hydrogen proton signal_H7.97 (1H, d, J = 9.5 Hz, H-4), 7.11 (1H, s, H-5) and 6.39 (1H, d, J = 9.5 Hz, H-3).

The hydrogen (see figure 1) and carbon (table 1) spectra data (see figure 2) of a compound of formula I, which is a disaccharide-containing coumarin moiety comprising three olefinic hydrogensProton signal (_H7.97, 7.11 and 6.39), 8 aromatic carbon signals: (_C149.5, 144.4, 142.3, 141.5, 140.3, 114.8, and 105.4), 1 ester carbon signal: (_C159.8), 2 alkenylcarbons: (_C102.4 and 100.6) and alkene hydrogen signals: (_H5.07 and 4.39), and 1 glucose-based signal(s) ((s)_H 5.07 (1H, d, 7.2)，_C102.4 and 66.5) and 1 rhamnosyl signal: (_H 4.39，_C100.6 and 17.8).

The hydrogen and carbon spectra data of the compound of the structure I are very similar to those of the known compound haploperoside A (compound of the structure II), except that the compound of the structure I has 1 more methoxyl signal than the compound of the structure II: (_H 3.90 (3H, s)，_C 61.3, 8-OCH₃). HMBC spectral display, 8-OCH₃There is a correlation with C-8, i.e., the extra methoxy group is attached at the C-8 position (FIGS. 5 and 6). Furthermore, H-1' ((II))_H5.07) and C-7 (_C148.8) related, H-1' (_H4.39) and C-6' ((II)_C66.5) shows that beta-D-glucosyl and alpha-L-rhamnosyl are linked at the C-7 and C-6' positions, respectively. The structure of the compound of formula I, named as conginoumarin A, can be further determined by 1H-1H COSY (see figure 3), HSQC (see figure 4) and HMBC (see figure 5) mapping as follows:

TABLE 1 Hydrogen (600 MHZ) and carbon (150 MHZ) spectra data for Compound 1

Wherein, the compound with the structure of formula II is a known compound, and the structure is haploperoside A by comparing with NMR data in the literature (the nuclear magnetic resonance hydrogen spectrum and the nuclear magnetic resonance carbon spectrum of the compound with the structure of formula II are respectively shown in figures 7 and 8). Determining the molecular formula as C₂₂H₂₈O₁₃A pale yellow powder.¹H NMR（600 MHZ，DMSO-d ₆）：（ppm）：6.35 (1H, d, J = 9.5 Hz, H-3)、7.97 (1H, d, J = 9.5 Hz, H-4)、7.29 (1H, s, H-5)、7.15 (1H, s, H-8)、5.07 (1H, d, J= 6.0 Hz, H-1'), 3.12-4.68 (10H, hydrogen on the glycosyl), 3.81 (3H, s, 6-OCH)₃）、5.39 (1H, s, H-1")、1.06 (3H, d, J = 5.8 Hz, H-6")。¹³C NMR（150 MHZ，DMSO-d ₆）：（ppm）：161.0 (C-2)、113.3 (C-3)、144.4 (C-4)、109.7 (C-5)、146.1 (C-6)、148.8 (C-7)、103.2 (C-8)、149.9 (C-9)、112.4 (C-10)、100.5 (Glu-1')、73.1 (Glu-2')、76.7 (Glu-3')、69.7 (Glu-4')、75.5 (Glu-5')、66.0 (Glu-6')、99.8 (Rha-1")、70.4 (Rha-2")、70.7 (Rha-3")、71.8 (Rha-4")、68.4 (Rha-5")、17.9 (Rha-6")、56.0 (6-OCH₃)。

Wherein, the compound with the structure shown in formula III (the hydrogen spectrum and the carbon spectrum of the compound with the structure shown in formula III are respectively shown in figures 9 and 10) is also a known compound, and the structure is scopoletin through comparison with NMR data in the literature. C₁₀H₈O₄A colorless powder.¹H NMR（600 MHZ，DMSO-d ₆）：（ppm）：6.20 (1H, d, J = 9.6 Hz, H-3)、7.90 (1H, d, J = 9.6 Hz, H-4)、7.20 (1H, s, H-5)、6.77 (1H, s, H-8)、3.81（3H, s, 6-OCH₃）。¹³C NMR（150 MHZ，DMSO-d ₆）：（ppm）：160.7 (C-2)、111.4 (C-3)、144.5 (C-4)、110.3 (C-4a)、109.5 (C-5)、145.4 (C-6)、151.6 (C-7)、102.7 (C-8)、149.6 (C-9)、55.9 (6-OCH₃)。

Example 3: cytotoxicity evaluation experiment of coumarin compounds in dendropanax dentiger on MH7A

Cell culture: MH7A cells (Guangdong Jiniu Euro Biotechnology company) were subcultured for 5-10 passages in high-sugar DMEM medium containing penicillin (final concentration 100U/mL), streptomycin (final concentration 100. mu.g/mL), and 10% FBS, when the cells fused to 90%, the old medium was discarded, the cells were washed with 2 mL of PBS for 2 times, after discarding PBS, 2 mL of 0.25% (w/v) Trypsin-0.53 mM EDTA mixed digest was added, the mixture was observed under a microscope for about 30 s, when the cells became round, 2 mL of complete medium was rapidly added to terminate the digestion, and the cells were collected by gently pipetting. Centrifuging at 800 rpm and 4 deg.C for 5 min, discarding supernatant, suspending cells with complete culture medium, culturing in bottles, and changing the culture medium every other day.

Cell viability assay: MH7A cell density was adjusted to 5X 10⁴one/mL, inoculated in a 96-well plate at 100. mu.L/well in 5% CO at 37 ℃₂After 24 hours of incubation in the incubator, the stock culture was aspirated and washed 2 times with PBS. The experiment was performed in a blank (DMEM-supplemented high-sugar medium) and drug groups (final concentration 40. mu. mol/L) at 100. mu.L per well and 3 duplicate wells per concentration. After further culturing for 24 h, the cell viability was determined by the CCK8 method. The result shows that the sapiens coumarin A (the compound shown in the formula I), the haloperoside A and the scopoletin do not show obvious cytotoxicity under the concentration of 40 mu mol/L, and the cell activities are respectively 90.1 +/-4.6, 97.6 +/-4.0 and 94.4 +/-3.8%; meanwhile, the cell viability of the blank group was 100. + -. 7.3%. Therefore, the three coumarin compounds of the dendropanax dentiger have no influence on the activity at the concentration of 40 mu mol/L, have no toxicity on cells and have cell safety.

Example 4: inhibition experiment of sapiens japonicas coumarin compound on tumor necrosis factor alpha induced MH7A cell release nitric oxide

The standard working solution or cell supernatant was added to the wells in sequence, 50. mu.l per well. Then, 50. mu.l of each well was added to each well at room temperature Griess Reagent I. Finally, 50. mu.l per well of a room temperature Griess Reagent II was added to each well. Immediately, the OD value of each well was measured at a wavelength of 450nm by a microplate reader, and NO inhibition (%) = (model group OD value-OD value after administration)/(model group OD value-control group OD value).

The results showed that the half inhibitory concentration (IC 50) values of the activity of saproperoside A (compound of formula I), haloperoside A (compound of formula II), and scopoletin (compound of formula III) on the release of nitric oxide from MH7A cells were 7.87 + -0.7 μ M (formula I), 13.82 + -1.1 μ M (formula II), and 39.88 + -2.3 μ M (formula III), respectively.

Therefore, the three coumarin compounds in the dendropanax dentiger have obvious inhibition effect on the activity of the MH7A cells for releasing nitric oxide, and can be used for treating rheumatoid arthritis and rheumatic arthritis diseases. Particularly significant among them is the aegilops tauschii coumarin A (compound of formula I), the half inhibitory concentration (IC 50) value is only 7.87 +/-0.7, and the aegilops tauschii coumarin A is 1/2 or 1/5 of the compound of formula II and the compound of formula III IC50 under the same conditions. Therefore, the saprophytarin A (compound shown in the formula I) can show extremely strong inhibition capability of MH7A cells to release nitric oxide at extremely low concentration, and the pharmacological activity of the saproperoside A (compound shown in the formula II) and scopoletin (compound shown in the formula III) is remarkably superior to that of haloperoside A (compound shown in the formula II) and scopoletin (compound shown in the formula III). The dendropanax japonicus coumarin A (compound shown in formula I) is used as a main drug effect component for preparing the drug for treating rheumatoid arthritis and rheumatoid arthritis diseases, so that the drug dosage of a patient can be reduced, the drug effect speed is further improved, and the toxic and side effects are reduced.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The dendropanax japonicas coumarin compound has the following structural formula:

2. the application of the dendropanax japonicas coumarin compound and the pharmaceutically acceptable salt thereof in preparing the medicine for treating rheumatoid arthritis or rheumatic arthritis is characterized in that the dendropanax japonicas coumarin compound has one of the following chemical structural formulas:

3. the use according to claim 2, wherein the compound of formula I, formula II or pharmaceutically acceptable salt thereof has the effect of alleviating and treating rheumatoid arthritis and rheumatoid arthritis by inhibiting the tumor necrosis factor α -induced nitric oxide releasing activity of MH7A cells.

4. The medicine for treating rheumatoid arthritis or rheumatic arthritis diseases is characterized by comprising an ethanol extract of the root of dendropanax dentiger, wherein the ethanol extract contains a mixture of compounds with chemical structures shown in formula I and formula II; the chemical structural formulas of the formula I and the formula II are as follows:

5. a medicament for treating rheumatoid arthritis or rheumatic arthritis diseases, which is characterized in that the effective components of the medicament comprise: coumarins from Panax quinquefolium or pharmaceutically acceptable salts thereof; the dendropanax japonicas coumarin compound has one of the following chemical structural formulas:

6. a medicament for treating rheumatoid arthritis or rheumatoid arthritis diseases, which comprises the compound of claim 1 or a pharmaceutically acceptable salt thereof as a pharmaceutically effective ingredient.