CN111518070A - Rotavirus-resistant compound in cassia wingnut, preparation method and application thereof - Google Patents

Abstract

The invention discloses an anti-rotavirus compound, a preparation method and application thereof. The structural formula of the compound is as follows:

(ii) a The preparation method comprises the steps of sample extraction, silica gel column chromatography, high performance liquid chromatography separation and gel column chromatography. The invention extracts a compound with novel structure and antiviral activity by using branches and leaves of Cassia alata as raw materials, and the compound has stronger anti-rotavirus activity, and anti-rotavirus experiments show that TC of the compound₅₀The value was 208.2 μ g/mL, IC₅₀The value is 7.62 mug/mL, and the therapeutic index TI is 27.3; the therapeutic index of the composition is far higher than that of the control ribavirin by 20.8; the compound has low toxicity and good antiviral activity, and can provide a high-efficiency and low-toxicity pilot compound for the research and development of anti-rotavirus medicaments. In addition, the first and second substrates are,the analysis of the rotavirus resisting activity of the compound provides a new compound skeleton type for screening antiviral drugs, and provides a new idea for the research and development of the antiviral drugs and mechanisms.

Description

A kind of compound, preparation method and application of anti-rotavirus in Cassia japonica

technical field

The invention belongs to the field of phytochemistry, and in particular relates to a compound against rotavirus in Cassia podae, a preparation method and an application thereof.

Background technique

Cassia alata L. is a species of the genus Cassia in the family Leguminosae. Native to tropical America, widely distributed in tropical regions of the world, in my country in Guangdong and southern Yunnan. The plant is colorful and has high ornamental value, and is often used as a landscaping tree species. At the same time, it is also an important medicinal plant with fungicidal, anti-inflammatory and antiviral properties, and can be used to treat skin diseases. It is one of the common raw materials for soaps, shampoos, and lotions. The saponins contained in its seeds can be used as anthelmintics to expel intestinal parasites; its leaf decoction is often used to treat high blood pressure, stomach problems, fever, asthma, snake bites, venereal diseases, etc. Finding high-efficiency and low-toxic antiviral active molecules from natural plants is also a research hotspot in natural product chemistry. Ginseng, rhubarb, chrysanthemum, etc. Yunnan is extremely rich in natural medicinal resources, and blends with ethnic diversity. Many characteristic medicines have been proven to have antiviral effects in long-term folk medicine. Therefore, the discovery of virus inhibitors from characteristic medicinal plant resources has a very broad prospect.

To sum up, in order to make full use of the abundant resources of Cassia japonica in Yunnan, and to find antiviral active molecules with high efficiency and low toxicity, the present invention studies the chemical constituents of Cassia japonica, and isolates a new skeleton type compound from it, The compound is a polymer of chromone and isocoumarin, and has obvious anti-rotavirus activity.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a compound against rotavirus in Cassia podae, the second object of the present invention is to provide a preparation method of the compound against rotavirus, and the third object of the present invention is to provide Application of anti-rotavirus compound in preparation of anti-rotavirus medicine.

The first object of the present invention is achieved in this way, a compound against rotavirus in Cassia serrata, the compound is a polymer of chromone and isocoumarin, named as Cassia spp. B, English name It is: alatain B, the molecular formula is C ₂₄ H ₂₀ O ₈ .

The structure is as follows:

Wherein, the a fragment of this compound is chromone, and the b fragment is isocoumarin.

The second object of the present invention is achieved in this way. The preparation method of the compound against rotavirus includes sample extraction, silica gel column chromatography, high performance liquid chromatography separation and gel column chromatography, and specifically includes the following steps:

A. Extraction of samples: take the branches and leaves of Cassia japonica as raw materials, dry them in the sun, pulverize them to 30-50 mesh, soak and extract with the first solvent for 2-4 times, then combine the extracts, filter and concentrate to obtain branch and leaf extracts extract;

B, silica gel column chromatography: silica gel column chromatography is applied to the extract, with the volume ratio of chloroform and acetone being 10:0, 9:1, 8:2, 7:3, 6:4 and 5:5 successively A series of chloroform-acetone solutions were subjected to gradient elution, and a 20:1 chloroform-acetone eluent was subjected to gradient elution. After monitoring by TLC, the gradient eluents of each gradient were collected, concentrated and combined;

C, high pressure liquid chromatography separation: the sample obtained by 9:1 chloroform-acetone elution concentration in step B is separated and purified by high pressure liquid chromatography to obtain the crude product of the target;

D. Gel column purification: the crude product of the target product is dissolved in methanol, and pure methanol is used as the mobile phase, and further separation and purification is performed by gel column chromatography to obtain the pure product of the target product;

Wherein, the high-performance liquid chromatography separation and purification conditions are as follows: take the methanol aqueous solution with a volume concentration of 70 to 80% as the mobile phase, the flow rate is 15 to 25 mL/min, and the ZorbaxSB-C ₁₈ of 21.2 × 250 mm and 5 μm is used as the mobile phase. The reversed-phase preparative column is a stationary phase, and the detection wavelength of the UV detector is 340-365 nm, and each injection is 0.5-1.0 mL. The chromatographic peaks of 30-40 min are collected, and the crude product of the target substance is obtained after multiple accumulation and evaporation.

The third object of the present invention is achieved by the use of the anti-rotavirus compound in the preparation of an anti-rotavirus drug, the anti-rotavirus drug comprising the compound, viridian B.

The anti-rotavirus drug includes cassia B and/or cassia B pharmaceutically acceptable carrier or excipient.

The beneficial effects of the present invention are:

1. The present invention uses the branches and leaves of Cassia spp. as raw materials to extract a compound with novel structure and antiviral activity, which has strong anti-rotavirus activity. Anti-rotavirus experiments show that the compound Its TC ₅₀ value was 208.2 µg/mL, IC ₅₀ value was 7.62 µg/mL, and the therapeutic index TI was 27.3; its therapeutic index was much higher than the control ribavirin's therapeutic index of 20.8; in addition, the compound had low toxicity and good antiviral activity , which can provide high-efficiency and low-toxicity lead compounds for the development of anti-rotavirus drugs.

2. The preparation method of this compound is simple, and in addition, Cassia spp. is widely distributed in southern my country, with rapid growth, high biological yield, wide source of raw materials, and low cost; and the regeneration ability of branches and leaves is very strong, and it is sustainable as described in the present invention. The isolated preparation of the active compound provides the raw material so that the present compound can be produced and prepared on a sustainable scale.

3. The compound of the present invention is a polymer of chromone and isocoumarin, wherein the a fragment of the compound is chromone, and the b fragment is isocoumarin; the antiviral activity of this type of compound has never been reported in the open literature, Therefore, the analysis of the anti-rotavirus activity of this compound provides a new compound skeleton type for antiviral drug screening, and provides a new idea for the research and development of antiviral drugs and mechanisms.

Description of drawings

Fig. 1 Carbon nuclear magnetic resonance spectrum ( ¹³ C NMR) of the compounds of the present invention.

Figure 2 is the hydrogen nuclear magnetic resonance spectrum ( ¹ H NMR) of the compound of the present invention.

Figure 3 Key HMBC correlograms of compounds of the present invention.

Detailed ways

The present invention is further described below in conjunction with the examples, but the present invention is not limited in any way, and any transformation or replacement made based on the teachings of the present invention belongs to the protection scope of the present invention. Unless otherwise specified, the raw materials and equipment used in the present invention can be purchased from the market or are commonly used in the art. The methods in the examples, unless otherwise specified, are all conventional methods in the art.

A compound of the present invention against rotavirus in Cassia podae, the structural formula is as follows:

The preparation method of the anti-rotavirus compound of the present invention includes sample extraction, silica gel column chromatography, high performance liquid chromatography separation and gel column chromatography, and specifically includes the following steps:

A. Preparation of Cassia serrata branch and leaf extract extract: take the branches and leaves of Cassia serrata as raw material, dry and pulverize it to 30~50 meshes, soak and extract with the first solvent for 2~4 times, and then combine the extracts , filter and concentrate to obtain branch and leaf extract extract;

B, silica gel column chromatography: silica gel column chromatography is applied to the extract, with the volume ratio of chloroform and acetone being 10:0, 9:1, 8:2, 7:3, 6:4 and 5:5 successively A series of chloroform-acetone solutions were subjected to gradient elution, and a 20:1 chloroform-acetone eluent was subjected to gradient elution. After monitoring by TLC, the gradient eluents of each gradient were collected, concentrated and combined;

C, high pressure liquid chromatography separation: the sample obtained by 9:1 chloroform-acetone elution concentration in step B is separated and purified by high pressure liquid chromatography to obtain the crude product of the target;

D. Gel column purification: the crude product of the target product is dissolved in methanol, and pure methanol is used as the mobile phase, and further separation and purification is performed by gel column chromatography to obtain the pure product of the target product;

Wherein, the high-performance liquid chromatography separation and purification conditions are as follows: take the methanol aqueous solution with a volume concentration of 70 to 80% as the mobile phase, the flow rate is 15 to 25 mL/min, and the Zorbax SB-C of 21.2 × 250 mm and 5 μm is used as the mobile phase. ₁₈ The reversed-phase preparative column is the stationary phase, and the detection wavelength of the UV detector is 340-365 nm. Each injection is 0.5-1.0 mL, and the chromatographic peaks of 30-40 min are collected, and the crude product of the target substance is obtained by evaporation after multiple accumulation.

The organic solvent is an aqueous solution of 60%-100% ethanol, methanol or acetone.

In the step A, 1.5 to 4 times the sample mass parts of the solvent is used for extraction each time, and the extraction time is 12 h to 72 h.

In the step B, the extract is dissolved with acetone, ethanol or methanol of 1 to 3 times the weight of the extract, and then used 1 to 1.6 times the weight of the extract of 60 to 120 mesh before being subjected to silica gel column chromatography. The samples were loaded after the silica gel was mixed.

In the step B, the column packing silica gel is 160-300 mesh, and the weight of the silica gel used is 2-5 times the weight of the extract.

The application of the anti-rotavirus compound of the present invention in the preparation of anti-rotavirus medicine.

The anti-rotavirus drug includes the compound.

The anti-rotavirus drug comprises the compound and/or a pharmaceutically acceptable carrier or excipient for the compound.

The present invention will be further described below in conjunction with the examples.

Example 1

In this example, the branches and leaves of Cassia serrata were collected in Dehong, Yunnan.

The branches and leaves of Cassia serrata were sun-dried and crushed to 30 mesh, and 3.0 kg was sampled and extracted with 70% acetone aqueous solution for 3 times, each extraction was 24 hours, the extracts were combined, filtered, and concentrated under reduced pressure to obtain 150 g of extract.

Dissolve the extract with 180 g of methanol, then add 200 g of 80-mesh thick silica gel to mix the sample, pack the column with 1.0 kg of 160-mesh silica gel, and load the column after mixing the sample; the volume ratios are 10:0, 9:1, and 8:1. A series of chloroform-acetone solutions of 2, 7:3, 6:4 and 5:5 were subjected to gradient elution, and the eluent obtained by elution with a 9:1 volume ratio of chloroform-acetone solution was collected and concentrated to obtain 16.8 gram concentrate. The concentrate used 78% methanol aqueous solution as the mobile phase, Zorbax SB-C ₁₈ (21.2 × 250 mm, 5 μm ) preparative column as the stationary phase, the mobile phase flow rate was 20 mL/min, and the detection wavelength of the UV detector was 357 nm. , inject 1.0 mL each time, collect chromatographic peaks with a residence time of 36.8 min, and evaporate to dryness after accumulating for many times to obtain the crude cassia B of the present invention; the crude cassia B is treated with pure methanol Dissolve, use pure methanol as mobile phase, and purify by Sephadex LH-20 gel column chromatography to obtain pure cassia B.

Example 2

In this example, the branches and leaves of Cassia serrata were collected in Xishuangbanna, Yunnan.

The branches and leaves of Cassia serrata were sun-dried and crushed to 50 mesh, 3.5 kg were sampled, extracted with 95% ethanol for 3 times, each extraction was 48 hours, the extracts were combined, filtered, and concentrated under reduced pressure to obtain 140 g of extract. After the extract was dissolved in 280g methanol, the sample was mixed with 150g of 120-mesh coarse silica gel, and the column was packed with 0.9 kg of 200-mesh silica gel. A series of chloroform-acetone solutions of 7:3, 6:4 and 5:5 were subjected to gradient elution, and the eluent obtained by elution with a 9:1 volume ratio of chloroform-acetone solution was collected and concentrated to obtain 26.4 g of concentrated thing. A 70% methanol aqueous solution was used as the mobile phase, a Zorbax SB-C ₁₈ (21.2 × 250 mm, 5 μm ) preparative column was used as the stationary phase, the flow rate of the mobile phase was 25 ml/min, and the detection wavelength of the UV detector was 357 nm. Inject 1.0 mL of sample, collect the chromatographic peaks with a residence time of 36.8 min, accumulate for many times and evaporate to dryness to obtain the crude product of Cassia B. The crude cassia B is dissolved in methanol solution, and then the methanol solution is used as the mobile phase to separate by SephadexLH-20 gel column chromatography to obtain the pure cassia B.

Test Example 1

This compound is a yellow colloidal compound. The structure of the anti-rotavirus compound isolated in Example 1-2 was identified by spectroscopic techniques such as MS, HRMS, 1H NMR, 1H and 13C NMR, HMBC and DEPT. The specific data and analyse as below:

210 (4.26)、258 (3.68)、292(3.72)、357 (3.47) nm。红外光谱(溴化钾压片)

3418、2943、1722、1657、1606、1548、1432、1359、1146、1050、978、826 cm^–1。ESIMS峰m/z 459 [M+Na]⁺；HRESIMS m/z 459.1050[M+Na]⁺ (计算值459.1056，C₂₄H₂₀O₈Na)；¹H和¹³C NMR谱(图1和图2)，数据见表1。Compound spectral data: UV spectrum (solvent is methanol)

210 (4.26), 258 (3.68), 292 (3.72), 357 (3.47) nm. Infrared Spectroscopy (Potassium Bromide Tablets)

3418, 2943, 1722, 1657, 1606, 1548, 1432, 1359, 1146, 1050, 978, 826 cm ^–1 . ESIMS peak m/z 459 [M+Na] ⁺ ; HRESIMS m/z 459.1050 [ _M +Na] ⁺ (calcd _459.1056 for _C24H20O8Na ); ¹ H and ¹³ C NMR spectra (Fig. 1 and Fig. 1 2), see Table 1 for the data.

The ultraviolet spectrum and infrared spectrum of this compound show the characteristic absorption of hydroxyl, carbonyl and aromatic ring. High resolution mass spectrometry (HRESIMS) gave a quasi-molecular ion peak m/z 459.1050 [M+Na] ⁺ (calcd. 459.1056). Combining the ¹ H and ¹³ C NMR spectra gave a molecular formula of C ₂₄ H ₂₀ O ₈ with an unsaturation of 15.

The ¹ H NMR spectrum of Cassia A (Figure 1) showed some characteristic signals, including 1 methyl group, 1 methoxy group, three methylene groups, two double bond singlet signals, one 1, 2,3,5-tetrasubstituted benzene ring signal and one 1,2,3,4-tetrasubstituted benzene ring signal. Its ₁₃ C and DEPT NMR spectra (Fig. 2) showed 24 carbon signals including 2 methyl carbons (including an oxygenated methyl group), 3 methylene groups (including an oxygenated carbon signal), 6 13 quaternary carbon atom signals (including 3 carbonyl and 5 oxygen-containing carbon signals) of alkenylcarbmethineethyl. Among them, 3 carbonyl groups and 18 alkene carbons occupy 12 unsaturations, suggesting that the compound is a compound of a tetracyclic system. In summary, it can be preliminarily judged that cassiacin A is a highly aromatized heterodimer (see Figure 3), which is mainly composed of a C ₁₃ chromone nucleus (fragment 1a) and a bicyclic ring The aromatic hydrocarbon fragment constitutes (fragment 1b), and its planar structure was determined by comprehensive 1D and 2D NMR analysis: the chromone nucleus (rings A and B) in fragment 1a was determined by the characteristic ¹ H and ¹³ C signals. The fragment passes through H-3 ( δ _H 6.28, s) and C-2/C-4/C-10, H-6 ( δ _H 6.56, d, J = 1.8 Hz) with C-8/C-10 and The HMBC correlation of H-8 ( δ _H 6.67, d, J = 1.8 Hz) with C-6/C-10 was determined. In addition, a carbonyl fragment (three carbon units from C-11 to C-13) and a hydroxyl group (7-OH) are attached at the C-5 and C-7 positions of the chromone fragment, respectively, which is inferred by H ₂ -11 ( δ _H 4.15, s) with C-6/C-10, and 7-OH ( δ _H 10.70, s) with C-6/C-7/C-8 were confirmed. These features are very similar to the known compound 5-acetonyl-7-hydroxy-2-methylchromone, except that the original C-14 methyl group in the A ring has been changed to a methylene group in 1a, which is presumed to be caused by H- 3 was confirmed with C-14, as well as H2-14 and C- ₂ , C-3, _{C-9 (4JCH} ⁾ , and it was speculated that the 1a fragment was linked to the 1b fragment through C-14.

The remaining 11 carbon signals (including 1 methoxy group, 1 oxygen-containing methylene group, 3 alkenyl carbonmethine groups, and 6 quaternary carbon signals) were assigned to 1 containing 1 methoxy group and one hydroxyl group. C9 bicyclic structure of methyl (fragment 1b). Further related to C-1' ( ^4JCH ), _C -3', C-4a', C-5', C-8a' and C-9' through the key HMBC signal H-4', H -7 ' is related to C-5', C-6', C-8' and C-8a', and H-8' is related to C-4a', C-6', C-7' and C-8a' to determine the The C9 parent nucleus is an isocoumarin skeleton. In addition, the substituents 1 methoxy and one hydroxymethyl were attached at the C-8' and C-3' positions, respectively, as determined by the corresponding HMBC. Finally, fragments 1a and 1b correlate with C-4a'/C-5'/C-6' HMBC via the key _H2-14 , confirming that they are linked via C-14 and C-5', so far, the compound The structure of Mingsu B has been determined, and its English name is alatain B.

Test Example 2 Detection of anti-rotavirus activity of the compounds of the present invention

Methods: The compounds prepared in Examples 1-2 were taken as samples for anti-rotavirus activity test, and the in vitro cell test method was used, that is, after the samples and the virus acted on MA104 cells at the same time, the alarmablue method was used to detect the effect of the samples on cell death caused by virus infection. protection, to determine the activity of the sample against rotavirus.

(a) Cytotoxicity assay of drugs

MA104 cells were cultured in a 96-well cell culture plate to form a monolayer, and sample solutions of different concentrations were added. After culturing for 3 days, the culture solution containing Alamarblue was replaced. After culturing for 2 to 3 hours, the fluorescence values at 530/590 nm were detected. , so as to detect the toxicity of the sample to MA104 cells, and calculate the half cytotoxic concentration (TC ₅₀ ).

(b) Detection of drug anti-rotavirus effect

After MA104 cells were cultured in a 96-well cell culture plate to form a monolayer, 100 TCID50 of virus solution and a gradient concentration of drug solution with no more than 20% cytotoxicity were added to MA104 cells at the same time. After continuing to culture for 4-6 days, the culture containing Alamarblue was replaced After incubation for 2-3 hours, the fluorescence value at 530/590nm was detected, and the median inhibitory concentration (IC ₅₀ ) was calculated.

(c) Calculation of the therapeutic index of the compound based on _TC50 / _IC50

The results showed that the TC ₅₀ value of the compound of the present invention was _208.2 μg/mL, the IC ₅₀ value was 7.62 μ g /mL, and the therapeutic index TI was 28.8 _; ), the therapeutic index was 27.3, indicating that the compound had good anti-rotavirus activity.

To sum up, the compound of the present invention has a good application prospect in the preparation of anti-rotavirus drugs; its structure is simple and the activity is good, and it can be used as a leading compound for the development of anti-rotavirus drugs for anti-rotavirus drugs Development of formulations.

Claims (9)

1. A rotavirus-resistant compound in Cassia alata is characterized in that the structural formula is as follows:

。

2. the preparation method of the rotavirus resisting compound as claimed in claim 1, which comprises the steps of sample extraction, silica gel column chromatography, high performance liquid chromatography separation and gel column chromatography, and is characterized by comprising the following steps:

A. preparing extract of branches and leaves of Cassia alata: taking branches and leaves of Cassia alata as raw materials, drying the branches and leaves of Cassia alata in the sun, crushing the raw materials to 30-50 meshes, soaking and extracting the raw materials for 2-4 times by using a first solvent, combining extracting solutions, filtering and concentrating to obtain a branch and leaf extract;

B. silica gel column chromatography: performing silica gel column chromatography on the extract, performing gradient elution by using a series of chloroform-acetone solutions with chloroform-acetone volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4 and 5:5 in sequence, performing gradient elution by using chloroform-acetone eluent with 20:1, monitoring by TLC, collecting gradient eluent of each gradient, concentrating and combining;

C. high-pressure liquid chromatography separation: separating and purifying the sample obtained by eluting and concentrating the chloroform-acetone of 9:1 in the step B by high performance liquid chromatography to obtain a crude product of the target substance;

D. purifying by using a gel column: dissolving the crude target product with methanol, taking pure methanol as a mobile phase, and further separating and purifying by gel column chromatography to obtain a pure target product;

wherein the high performance liquid chromatography separation and purification conditions are that methanol water solution with volume concentration of 70-80% is used as mobile phase, the flow rate is 15-25 mL/min, and the flow rate is 21.2 × 250 mm, 5 mmμZorbax SB-C of m₁₈And the reversed-phase preparation column is a stationary phase, the detection wavelength of an ultraviolet detector is 340-365 nm, 0.5-1.0 mL of sample is fed each time, chromatographic peaks of 30-40 min are collected, and a crude target product is obtained by evaporation after multiple accumulation.

3. The method of claim 2, wherein the organic solvent is an aqueous solution of 60% to 100% ethanol, methanol or acetone.

4. The method for preparing the anti-rotavirus compound according to claim 2, wherein in the step A, the extraction is performed by using 1.5-4 times of the solvent by mass of the sample every time, and the extraction time is 12-72 hours.

5. The method for preparing the anti-rotavirus compound according to claim 2, wherein in the step B, the extract is dissolved by using 1-3 times of acetone, ethanol or methanol of the weight of the extract before the extract is subjected to silica gel column chromatography, and then the sample is mixed by using 1-1.6 times of 60-120 meshes of silica gel of the weight of the extract and then loaded.

6. The method for preparing an anti-rotavirus compound according to claim 2, wherein in the step B, the silica gel packed in the column is 160-300 meshes, and the weight of the silica gel is 2-5 times of the weight of the extract.

7. Use of an anti-rotavirus compound according to any one of claims 1 to 6 in the manufacture of an anti-rotavirus medicament.

8. The use according to claim 7 wherein the anti-rotavirus medicament comprises the anti-rotavirus compound.

9. The use according to claim 7, wherein the anti-rotavirus medicament comprises the anti-rotavirus compound and/or a pharmaceutically acceptable carrier or excipient of the compound.

Images