CN111808155A - Coumarin compounds of ginseng and their application in medicine - Google Patents

Abstract

The invention relates to the technical field of drug development for treating inflammatory diseases, in particular to the application of coumarin compounds in ginseng in the preparation of drugs for treating rheumatoid arthritis or rheumatoid arthritis, and ginseng coumarin compounds Has one of the chemical structural formulas shown below:

The activity screening experiment confirmed that the three coumarin compounds in the ginseng have a significant inhibitory effect on the release of nitric oxide from MH7A cells induced by tumor necrosis factor α (TNF-α), and the median inhibitory concentration (IC50) value were 7.87±0.7 μM (Formula I), 13.82±1.1 μM (Formula II), and 39.88±2.3 μM (Formula III), respectively. The invention provides a new pharmaceutical research direction for clinical development of rheumatoid arthritis and the treatment of rheumatoid arthritis.

Description

Coumarin compounds of ginseng and their application in medicine

technical field

The invention belongs to the field of medicine, and in particular relates to the application of coumarin compounds in ginseng in the preparation of medicines for treating rheumatoid arthritis or rheumatoid arthritis.

Background technique

Rheumatoid arthritis (RA) is a common chronic, systemic autoimmune disease that can cause joint destruction, joint deformity, and even disability. Western medicines currently used to treat RA are very expensive, and most of them have many adverse reactions. Therefore, there is a broad prospect to find drugs for RA treatment from natural medicines, especially traditional ones.

MH7A cells are human rheumatoid arthritis fibroblast-like synovial cells closely related to rheumatoid arthritis and rheumatoid arthritis, MH7A cells have the activity of releasing nitric oxide, which will aggravate inflammation. Therefore, if a natural product is found to inhibit the excessive release of nitric oxide from MH7A cells, the natural product could be useful in the treatment of rheumatoid arthritis and rheumatoid arthritis disease.

SUMMARY OF THE INVENTION

(1) Technical problems to be solved

In view of the above-mentioned shortcomings and deficiencies of the prior art, based on finding that the coumarin compounds in the ginseng have the ability to inhibit the activity of MH7A cells to release nitric oxide, the present invention provides the ginseng coumarin compounds in the preparation of the treatment of rheumatoid The application in arthritis or rheumatoid arthritis drugs provides a new drug development approach for the clinical development of rheumatoid arthritis and rheumatoid arthritis treatments.

(2) Technical solutions

In order to achieve the above-mentioned purpose, the main technical scheme adopted in the present invention includes:

Ginseng coumarins have the following structural formula:

The compound of formula I or a pharmaceutically acceptable salt or salt hydrate has the activity of inhibiting tumor necrosis factor α (TNF-α)-induced MH7A cells to release nitric oxide, so as to relieve and treat rheumatoid arthritis and rheumatoid arthritis disease.

Application of ginseng coumarins and derivatives thereof in the preparation of medicines for the treatment of rheumatoid arthritis or rheumatoid arthritis, said ginseng coumarins have one of the following chemical structural formulas:

Preferably, the derivatives of the ginseng coumarins refer to pharmaceutically acceptable salts or hydrates of the compounds of formula I, formula II, and formula III chemical structures.

Preferably, the ginseng-coumarin compound refers to a compound of the chemical structure of formula I, and the derivative refers to a pharmaceutically acceptable salt or hydrate of the salt of the compound of the chemical structure of formula I.

Preferably, the compound of formula I, formula II, formula III chemical structure or a pharmaceutically acceptable salt or hydrate of the salt can inhibit the release of nitric oxide from MH7A cells induced by tumor necrosis factor α (TNF-α). Active to achieve the effect of relieving and treating rheumatoid arthritis and rheumatoid arthritis diseases.

Preferably, the extraction and purification method of the ginseng coumarin compounds is:

S1: get the root of Araliaceae Radix Ginseng as raw material, add 8-12 times of quality, concentration 65-75% ethanol reflux extraction at least 3 times, at least 2h each time, the extract of extracting solution concentrated under reduced pressure;

S2: the extract is suspended with water, extracted with petroleum ether, ethyl acetate and n-butanol successively, and concentrated to dryness respectively, obtains the petroleum ether part, the ethyl acetate part and the n-butanol part successively; Get the n-butanol part extract, Silica gel column chromatography, eluted with a dichloromethane-methanol gradient to obtain 9 fractions;

S3: the 7th fraction is respectively subjected to octadecylsilane column chromatography, silica gel column chromatography, preparative HPLC column chromatography to obtain formula I compound ginseng coumarin A and formula II compound hapoperoside A; The fractions were respectively subjected to silica gel column chromatography and preparative HPLC column chromatography to obtain the compound of formula III, scopolactone.

The present invention further provides a medicament for treating rheumatoid arthritis or rheumatoid arthritis disease, the medicament contains the ethanol extract of the root of ginseng; the ethanol extract contains the chemical structures of formula I, formula II and formula III mixture of compounds.

The present invention further provides a medicament for treating rheumatoid arthritis or rheumatoid arthritis disease, the medicinal component of the medicament comprises: a coumarin compound derived from ginseng or a pharmaceutically acceptable salt or hydrate thereof ; Described ginseng coumarin compounds have one of the following chemical structural formulas:

The present invention further provides a medicament for treating rheumatoid arthritis or rheumatoid arthritis disease, the medicinal component of the medicament comprises the compound of formula I or a pharmaceutically acceptable salt or hydrate thereof.

(3) Beneficial effects

The present invention provides natural compounds, namely coumarin compounds and derivatives from ginseng, for preparing medicines for treating inflammatory diseases, especially rheumatoid arthritis and rheumatoid arthritis diseases, In order to replace the current expensive pure synthetic chemicals. Tree ginseng is rich in planting resources and easy to obtain raw materials, which helps to reduce the cost and price of drugs for such diseases, reduce the burden on patients, and reduce the adverse reactions of pure synthetic drugs.

After the activity screening experiment, it was confirmed that the three coumarin compounds of the ginseng were not toxic to cells at a concentration of 40 μmol/L, and had almost no effect on cell activity, but they had no effect on tumor necrosis factor α (TNF-α)-induced MH7A cells. Nitric oxide has a significant inhibitory effect, with the median inhibitory concentration (IC50) values of 7.87±0.7 μM (Formula I), 13.82±1.1 μM (Formula II) and 39.88±2.3 μM (Formula III), respectively.

The median inhibitory concentration (IC50) value of the compound of formula I is much smaller than that of the compounds of formula II and formula III. It can be seen that the compound of formula I has extremely high pharmacodynamics, and can significantly inhibit the release of nitric oxide from MH7A cells at a very small dose, so it can reduce the dose of drugs ingested by patients, and further Improve the speed of drug efficacy and reduce toxic and side effects.

Description of drawings

Fig. 1 is the hydrogen nuclear magnetic resonance spectrum ( ¹ H NMR) of the new compound ginseng coumarin A (structure of formula I).

Figure 2 is the carbon nuclear magnetic resonance spectrum ( ¹³ C NMR) of the new compound ginseng coumarin A (structure of formula I).

Fig. 3 is the nuclear magnetic resonance ¹ H- ¹ H COSY spectrum of the new compound ginseng coumarin A (structure of formula I).

Fig. 4 is the nuclear magnetic resonance HSQC spectrum of the new compound ginsengcoumarin A (structure of formula I) in the present invention.

Fig. 5 is the nuclear magnetic resonance HMBC spectrum of the new compound ginseng coumarin A (structure of formula I) in the present invention.

Figure 6 is the main HMBC (H→C) and COSY correlation of the new compound ginsengcoumarin A (structure of formula I) in the present invention.

Fig. 7 is the hydrogen nuclear magnetic resonance spectrum ( ¹ H NMR) of the compound hapoperoside A (structure of formula II) in the present invention.

Figure 8 is the carbon nuclear magnetic resonance spectrum ( ¹³ C NMR) of the compound hapoperoside A (structure of formula II) in the present invention.

Figure 9 is the hydrogen nuclear magnetic resonance spectrum ( ¹ H NMR) of the compound scopolactone (structure of formula III) in the present invention.

Figure 10 is the carbon nuclear magnetic resonance spectrum ( ¹³ C NMR) of the compound scopolactone (structure of formula III) in the present invention.

Detailed ways

In order to better explain the present invention and facilitate understanding, the present invention will be described in detail below with reference to the accompanying drawings and through specific embodiments.

Example 1: Extraction and purification of three coumarin compounds in ginseng roots

Take 10.0 kg of ginseng roots, use 100 kg of 70% ethanol, heat and reflux for 3 times for 3 hours each time, and concentrate the extract under reduced pressure to obtain 1.3 kg of extract. The extract was suspended in 1 L of water, extracted with equal volumes of petroleum ether, ethyl acetate and n-butanol in turn, and concentrated to dryness respectively. Take 216 g of n-butanol extract, mix the sample with silica gel (weight ratio 1:1), put it on a silica gel column (column size: 11*120cm), and dichloromethane-methanol (100:0→0:100, v /v) Gradient elution to obtain 9 fractions Frs. C1-C9. (1st to 9th streams)

Dichloromethane-methanol 5:1 elution fraction Fr.C7 (70.0 g of the seventh fraction) was subjected to octadecylsilane column chromatography (column size: 4.2*40 cm), methanol-water (5%→30 %) gradient elution to obtain 3 fractions Frs.C71-C73. Fraction C74 (4.92g) was mixed with silica gel (weight ratio 1:1), applied to a silica gel column (column size: 6.4*55 cm), mixed with dichloromethane-methanol (20:1→10:1, v/ v) Gradient elution to obtain 4 fractions Frs. C741-C744. C742 (1.21 g) was subjected to preparative HPLC column chromatography (column size: 10*25 cm) and eluted with 20% acetonitrile to obtain 12.5 mg of the compound of the formula I of the present invention (Ginsengcoumarin A) and the compound of the formula II of the present invention. Compound (hapoperoside A) 31.7 mg.

Dichloromethane-methanol 30:1 elution fraction Fr.C4 (4.2 g of the fourth fraction) was mixed with silica gel (weight ratio 1:1), applied to a silica gel column (column size: 3.2*40 cm), Elution was carried out with a gradient of dichloromethane-methanol (100:0→0:100, v/v) to obtain 8 fractions of Frs. C41-C48. C42 (256 mg) was subjected to preparative HPLC column chromatography (column size: 10*25 cm) and eluted with 35% methanol-water to obtain the compound of formula III (scopolactone) (100 mg).

Example 2: Structural identification of three coumarin compounds in ginseng

The compound of formula I, the compound of formula II, and the compound of formula III isolated in Example 1 were identified as three kinds of Panax Radix by high-resolution mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance spectroscopy (1D NMR and 2D NMR). leguminous compounds. Among them, the compound of formula I is a new coumarin derivative that has not been reported in the literature, the compound of formula II and the compound of formula III are known compounds (but the activity of treating rheumatoid arthritis and rheumatoid arthritis diseases is not yet known). see report).

Wherein, the compound of formula I is light yellow powder. HR-ESI-MS gave a quasi-molecular ion peak m/z 531.17083 ([M+H] ⁺ , calculated: 531.17066), which was determined to be C ₂₃ H ₃₁ O ₁₄ . The hydrogen spectrum of the compound of formula I is shown in Figure 1, (DMSO- d ₆ , 600MHZ) has two methoxy hydrogen signals δ _H 3.90 and 3.82 (each, 3H, s, 8/6-CH ₃ ), Three alkene hydrogen proton signals δ _H 7.97 (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 spectrum (see Fig. 1) and carbon spectrum data (Table 1) of the compound of formula I show (see Fig. 2) that the compound is a disaccharide-containing coumarin-like component, including three alkene hydrogen proton signals (δ _H 7.97, 7.11 and 6.39), 8 aromatic carbon signals (δ _C 149.5, 144.4, 142.3, 141.5, 140.3, 114.8, 114.8 and 105.4), 1 ester carbon signal (δ _C 159.8), 2 alkenyl carbons ( δ _C 102.4 and 100.6) and alkene hydrogen signals (δ _H 5.07 and 4.39), and 1 glucosyl signal (δ _H 5.07(1H, d, 7.2), δ _C 102.4 and 66.5) and 1 rhamnosyl signal (δ _H 4.39, δ _C 100.6 and 17.8).

The hydrogen spectrum and carbon spectrum data of the compound of formula I are very similar to the known compound hapoperoside A (the compound of formula II), except that the compound of formula I has one more methoxyl signal than the compound of formula II (δ _H 3.90 (3H ). , s), δ _C 61.3, 8-OCH ₃ ). The HMBC spectrum showed that 8-OCH ₃ was related to C-8, that is, the extra methoxy group was attached to the C-8 position (Figures 5 and 6). In addition, H-1' ( δ _H 5.07) was correlated with C-7 ( δ _C 148.8), and H-1" ( δ _H 4.39) was correlated with C-6' ( δ _C 66.5), indicating that β-D- Glucose group and α-L-rhamnosyl group are attached at C-7 and C-6' positions, respectively. Further through 1H-1H COSY (see Figure 3), HSQC (see Figure 4) and HMBC (see Figure 5) maps The structure of the compound of formula I can be determined, named as ginseng coumarin A, the structure is as follows:

Table 1 Hydrogen spectrum (600MHZ) and carbon spectrum (150MHZ) data of compound 1

Among them, the compound of formula II is a known compound, and its structure is determined to be hapoperoside A by comparing with the NMR data in the literature (see Figures 7 and 8 for the H and C nuclear magnetic resonance spectra of the compound of formula II, respectively). Its molecular formula is determined to be C ₂₂ H ₂₈ O ₁₃ , and it is a pale yellow powder. ¹ H NMR (600 MHZ, DMSO- d ₆ ): δ (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 glycosyl ), 3.81 (3H, s, 6-OCH ₃ ), 5.39 (1H, s, H-1"), 1.06 (3H, d, J = 5.8 Hz, H-6"). ¹³ C NMR (150 MHZ, DMSO- d ₆ ): δ (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-OCH ₃ ).

Among them, the compound of formula III (the H NMR spectrum and C NMR spectrum of the compound of formula III are shown in Figures 9 and 10, respectively) are also known compounds. By comparing with the NMR data in the literature, it is determined that its structure is scopolamine. ester. C ₁₀ H ₈ O ₄ , colorless powder. ¹ H NMR (600 MHZ, DMSO- d ₆ ): δ (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 ). ¹³ C NMR (150 MHZ, DMSO- d ₆ ): δ (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-OCH ₃ ).

Example 3: Cytotoxicity evaluation experiment of coumarin compounds in ginseng to MH7A

Cell culture: MH7A cells (Guangdong Genio Biotechnology Co., Ltd.) were subcultured for 5-10 generations, and the culture conditions were containing penicillin (final concentration of 100 U/mL), streptomycin (final concentration of 100 μg/mL), 10 % FBS in high glucose DMEM medium, when the cells are confluent to 90%, discard the old medium, wash the cells twice with 2 ml PBS, add 2 mL of 0.25% (w/v) Trypsin-0.53 after discarding the PBS The digestion solution was mixed with mM EDTA and observed under a microscope for about 30 s. When the cells became round, 2 ml of complete medium was quickly added to terminate the digestion, and the cells were collected by gently pipetting. Centrifuge at 800 rpm, 4 °C for 5 min, discard the supernatant, resuspend the cells in complete medium, culture in separate flasks, and change the medium every other day.

Cell viability assay: adjust the density of MH7A cells to 5×10 ⁴ cells/mL, inoculate 100 μL per well in a 96-well culture plate, and incubate at 37°C and 5% CO ₂ for 24 h in an incubator. The culture medium was washed twice with PBS. The experiment set blank group (add DMEM high glucose culture medium), each drug group (final concentration of 40 μmol/L), 100 μL per well, and 3 replicate wells for each concentration. After culturing for 24 h, the cell viability was detected by CCK8 method. The results showed that ginseng coumarin A (compound of formula I), hapoperoside A and scopolactone did not show obvious cytotoxicity at the concentration of 40 μmol/L, and the cell viability was 90.1±4.6, 97.6±4.0 and 90.1±4.6, respectively. 94.4±3.8%; meanwhile, the cell viability of blank group was 100±7.3%. It can be seen that the three coumarin compounds of ginseng have no effect on the activity at a concentration of 40 μmol/L, and have no toxicity to cells, and have cell safety.

Example 4: Inhibition experiment of ginseng coumarin compounds on tumor necrosis factor α-induced MH7A cells to release nitric oxide

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

The results showed that the median inhibitory concentration (IC50) values of ginseng coumarin A (compound of formula I), hapoperoside A (compound of formula II), and scopolactone (compound of formula III) on nitric oxide release activity of MH7A cells were respectively were 7.87±0.7 μM (Formula I), 13.82±1.1 μM (Formula II), and 39.88±2.3 μM (Formula III).

It can be seen that the three coumarin compounds in the ginseng have obvious inhibitory effects on the activity of MH7A cells to release nitric oxide, and can be used for the treatment of rheumatoid arthritis and rheumatoid arthritis diseases. Among them, ginsengcoumarin A (compound of formula I) is particularly notable, and its median inhibitory concentration (IC50) value is only 7.87±0.7, which is 1/2 or 1 of the IC50 of compound of formula II and compound of formula III under the same conditions. /5. This shows that ginseng coumarin A (compound of formula I) can exhibit a strong inhibitory ability of MH7A cells to release nitric oxide at a very low concentration, and its pharmacological activity is significantly better than that of hapoperoside A (compound of formula II) and scopolactone (a compound of formula III). Using ginseng coumarin A (compound of formula I) as the main medicinal component for preparing medicines for treating rheumatoid arthritis and rheumatoid arthritis diseases can reduce the dosage of medicines taken by patients, and further improve the speed of medicines. Reduce toxic side effects.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features thereof can be equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention. scope.

Claims (9)

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

。

2. the application of the dendropanax japonicas coumarin compound and the derivatives thereof in preparing the drugs 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 as claimed in claim 2, wherein the derivatives of coumarins of dendropanax japonicas refer to pharmaceutically acceptable salts or hydrates of salts of compounds having chemical structures of formula I, formula II and formula III.

4. The use as claimed in claim 2, wherein the coumarins of dendropanax japonicas refers to compounds of formula I, and the derivatives refer to pharmaceutically acceptable salts or hydrates of the salts of the compounds of formula I.

5. The use according to claim 2, wherein the compound of formula I, formula II, formula III or the pharmaceutically acceptable salt or hydrate of the 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.

6. The application of claim 2, wherein 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.

7. The medicine for treating rheumatoid arthritis or rheumatic arthritis diseases is characterized by comprising an ethanol extract of the roots of panax ginseng, wherein the ethanol extract contains a mixture of compounds with chemical structures shown in formula I, formula II and formula III.

8. 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 or hydrates thereof; the dendropanax japonicas coumarin compound has one of the following chemical structural formulas:

。

9. a medicament for the treatment of rheumatoid arthritis or rheumatoid arthritis disease, wherein the pharmaceutically effective component of the medicament comprises a compound having the structure of formula I as claimed in claim 1 or a pharmaceutically acceptable salt or hydrate of the salt thereof.

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