CN111518070A

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

Info

Publication number: CN111518070A
Application number: CN202010513773.5A
Authority: CN
Inventors: 胡秋芬; 周敏; 杨光宇; 汪伟光; 董淼; 杨文武; 高茜; 刘欣; 李雪梅; 米其利; 李晶; 王晋; 黄海涛; 许�永
Original assignee: Yunnan Minzu University
Current assignee: Yunnan Minzu University
Priority date: 2020-06-08
Filing date: 2020-06-08
Publication date: 2020-08-11
Anticipated expiration: 2040-06-08
Also published as: CN111518070B

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

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

Technical Field

The invention belongs to the field of phytochemistry, and particularly relates to a rotavirus-resistant compound in cassia wingnut, a preparation method and application thereof.

Background

Cassia alata (A. heterophylla)Cassia alataL.) is a species of the genus Cassia of the family Leguminosae. The native America tropical region is widely distributed in the tropical regions all over the world and is distributed in the southern China, Guangdong and Yunnan. The plant has bright colorHas higher ornamental value and is commonly used as a species of landscaping trees. Meanwhile, it is also an important medicinal plant, has the functions of killing fungi, resisting inflammation and resisting viruses, can be used for treating skin diseases, and is one of common raw materials of soap, shampoo and lotion. Its seed contains saponin which can be used as anthelmintic for expelling intestinal parasites; the decoction of its leaves is often used to treat hypertension, gastropathy, fever, asthma, venomous snake bite, venereal disease, etc. The search for high-efficiency and low-toxicity antiviral active molecules from natural plants is also a research hotspot of the current natural product chemistry, and clinically, a plurality of medicinal plant resources are widely used for treating various viral infection diseases, such as isatis root, honeysuckle, liquorice, radix sophorae flavescentis, rheum officinale, chrysanthemum and the like. The natural medicine resources of Yunnan are very rich and are mutually blended with national diversity, and various characteristic medicines are proved to have antiviral efficacy in long-term folk medicine application, so that the prospect of finding the virus inhibitor from the characteristic medicinal plant resources is very wide.

In conclusion, in order to fully utilize the abundant cassia alata resources in Yunnan province and search for high-efficiency and low-toxicity antiviral active molecules, the invention researches the chemical components of the cassia alata and separates a compound with a new framework type from the cassia alata, wherein the compound is a polymer of chromone and isocoumarin and has obvious rotavirus resisting activity.

Disclosure of Invention

The invention aims to provide an anti-rotavirus compound in cassia wingless shell, a second aim of the invention is to provide a preparation method of the anti-rotavirus compound, and a third aim of the invention is to provide application of the anti-rotavirus compound in preparing anti-rotavirus medicaments.

The first object of the invention is achieved by a rotavirus-resistant compound in cassia wingless shell, which is a polymer of chromone and isocoumarin, named as cassia wingnut B, with the English name: alatain B of formula C₂₄H₂₀O₈。

The structural formula is as follows:

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

The second purpose of the invention is realized by that the preparation method of the rotavirus resisting compound comprises sample extraction, silica gel column chromatography, high performance liquid chromatography separation and gel column chromatography, and specifically comprises the following steps:

A. sample extraction: 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 mmμZorbaxSB-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.

The third object of the invention is achieved by the use of said anti-rotavirus compound in the preparation of an anti-rotavirus medicament comprising said compound, namely, wingnut B.

The anti-rotavirus medicament comprises a pharmaceutically acceptable carrier or excipient of a winged Cassia tora B and/or a winged Cassia tora B.

The invention has the beneficial effects that:

1. 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 experiments on anti-rotavirus 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; in addition, 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.

2. The preparation method of the compound is simple, and in addition, the cassia alata is widely released in south China, the growth is rapid, the biological yield is high, the raw material source is wide, and the cost is low; and the regeneration capacity of branches and leaves is very strong, which can continuously provide raw materials for the separation and preparation of the active compound, so that the compound can be continuously produced and prepared in a large scale.

3. The compound is a polymer of chromone and isocoumarin, wherein a segment of the compound is chromone, and b segment is isocoumarin; the antiviral activity of the compound is never reported in the published literature, so that 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.

Drawings

FIG. 1 NMR spectra of compounds of the invention: (¹³C NMR）。

FIG. 2 shows the NMR spectra of the compounds of the present invention: (¹H NMR）。

FIG. 3 key HMBC correlation scheme for compounds of the present invention.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to be limiting in any way, and any modifications or alterations based on the teachings of the present invention are intended to fall within the scope of the present invention. The raw materials and equipment used in the invention can be obtained from the market or are commonly used in the field if not specified, and the methods in the examples are conventional in the field if not specified.

The invention relates to a rotavirus-resistant compound in cassia wingnut, which has the following structural formula:

the preparation method of the rotavirus resisting compound comprises the steps of sample extraction, silica gel column chromatography, high performance liquid chromatography separation and gel column chromatography, and specifically comprises 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;

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 m₁₈The 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, and the color is collected for 30-40 minAnd (5) performing spectrum peak, accumulating for multiple times, and evaporating to dryness to obtain a crude target product.

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

In the step A, 1.5-4 times of the mass of the solvent is used for extraction every time, and the extraction time is 12-72 hours.

And in the step B, before the extract is subjected to silica gel column chromatography, dissolving the extract by using 1-3 times of acetone, ethanol or methanol by weight, and mixing the sample by using 1-1.6 times of 60-120-mesh silica gel by weight and then loading the sample.

And in the step B, the silica gel filled in the column is 160-300 meshes, and the weight of the silica gel is 2-5 times that of the extract.

The invention relates to application of a compound for resisting rotavirus in preparing a medicament for resisting rotavirus.

The anti-rotavirus medicament comprises the compound.

The anti-rotavirus medicament comprises the compound and/or a carrier or excipient which is acceptable in the pharmacy of the compound.

The present invention is further illustrated by the following examples.

Example 1

In the embodiment, branches and leaves of the cassia tora are collected from Dehong in Yunnan.

Drying branches and leaves of Cassia alata L.in the sun, pulverizing to 30 mesh, sampling 3.0 kg, extracting with 70% acetone water solution for 3 times, each time for 24 hr, mixing extractive solutions, filtering, and concentrating under reduced pressure to obtain 150g extract.

Dissolving the extract with 180g of methanol, adding 200 g of 80-mesh crude silica gel, mixing, loading into a column with 1.0 kg of 160-mesh silica gel, mixing, and loading into the column; gradient eluting with a series of chloroform-acetone solutions of volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4 and 5:5 in sequence, collecting the eluate eluted with chloroform-acetone solution of volume ratio of 9:1 and concentrating to obtain 16.8 g of concentrate. The concentrate is prepared by using 78% methanol aqueous solution as mobile phase, Zorbax SB-C₁₈(21.2 × 250 mm, 5μm) the preparation column is a stationary phase, the flow rate of the mobile phase is 20 mL/min, the detection wavelength of the ultraviolet detector is 357 nm, 1.0mL of sample is injected each time, and the collection and retention time is 36.8 minThe chromatographic peak is evaporated to dryness after being accumulated for a plurality of times, and the crude product of the winged Cassia obtusifolia L extract B is obtained; and dissolving the crude product of the Cassia alata essence B by using pure methanol, taking the pure methanol as a mobile phase, and purifying by using Sephadex LH-20 gel column chromatography to obtain the pure product of the Cassia alata essence B.

Example 2

In the embodiment, branches and leaves of the cassia tora are collected from Yunnan Xishuangbanna.

Drying branches and leaves of Cassia alata L.and pulverizing to 50 mesh, sampling 3.5 kg, extracting with 95% ethanol for 3 times, each time for 48 hr, mixing extractive solutions, filtering, and concentrating under reduced pressure to obtain 140 g extract. Dissolving the extract with 280g of methanol, mixing with 150g of 120-mesh crude silica gel, loading into a column with 0.9 kg of 200-mesh silica gel, and loading into the column after mixing; gradient eluting with a series of chloroform-acetone solutions of 10:0, 9:1, 8:2, 7:3, 6:4 and 5:5 in sequence, collecting the eluate eluted with chloroform-acetone solution of 9:1 in volume ratio and concentrating to obtain 26.4 g concentrate. Using 70% methanol aqueous solution as mobile phase, Zorbax SB-C₁₈(21.2 × 250 mm, 5μm) taking a preparation column as a stationary phase, taking a mobile phase at the flow rate of 25mL/min, taking a sample of 1.0mL each time at the detection wavelength of 357 nm by using an ultraviolet detector, collecting chromatographic peaks with the retention time of 36.8 min, accumulating for multiple times, and evaporating to dryness to obtain a crude product of the pterocarya heterophylla cassia element B. Dissolving the crude product of the Cassia alata extract B in a methanol solution, taking the methanol solution as a mobile phase, and carrying out SephadexLH-20 gel column chromatography separation to obtain the pure product of the Cassia alata extract B.

Test example 1

The compound is a yellow colloidal compound, and the anti-rotavirus compound separated in the embodiment 1-2 is subjected to structural identification through spectral techniques such as MS, HRMS, 1H NMR, 1H and 13C NMR, HMBC, DEPT and the like, and specific data and analysis are as follows:

compound spectral data: ultraviolet spectrum (methanol as solvent)

210 (4.26), 258 (3.68), 292(3.72) and 357 (3.47) nm. Infrared spectrum (Potassium bromide tablet)

3418、2943、1722、1657、1606、1548、1432、1359、1146、1050、978、826 cm^–1. ESIMS peakm/z459 [M+Na]⁺；HRESIMSm/z459.1050[M+Na]⁺(calculated 459.1056, C₂₄H₂₀O₈Na)；¹H and¹³c NMR spectrum (FIGS. 1 and 2), data are shown in Table 1.

The compound has characteristic absorption of hydroxyl, carbonyl and aromatic ring in ultraviolet spectrum and infrared spectrum. High resolution mass spectrometry (HRESIMS) gives the peak of the excimer ionm/z459.1050 [M+Na]⁺(calculated 459.1056). Bonding of¹H and¹³the CNMR spectrum gives a molecular formula C₂₄H₂₀O₈The unsaturation degree was 15.

Preparation of obtusin A of winged pod¹The H NMR spectrum (fig. 1) shows some characteristic signals including 1 methyl group, 1 methoxy group, three methylene groups, two double-bonded unimodal signals, one 1,2,3, 5-tetra-substituted benzene ring signal and one 1,2,3, 4-tetra-substituted benzene ring signal. It is composed of₁₃The C and DEPT NMR spectra (FIG. 2) show that the 24 carbon signals include 2 methyl carbons (including an oxygen-containing methyl group), 3 methylene groups (including a1 oxygen-containing carbon signal), and 6 ene-carbon methine ethyl 13 quaternary carbon atom signals (including 3 carbonyl groups and 5 oxygen-containing carbon signals). Wherein 3 carbonyl groups and 18 alkene carbons occupy 12 unsaturations, suggesting that the compound is a compound of 1 tetracyclic system. In summary, it was preliminarily determined that Castorasin A is a highly aromatized heterodimer (see FIG. 3) consisting essentially of a C₁₃The chromone parent nucleus (segment 1 a) and a bicyclic aromatic hydrocarbon segment form (segment 1 b), and the plane structure of the chromone parent nucleus is determined by 1D and 2D nuclear magnetic resonance comprehensive analysis: chromone core (rings A and B) passage characteristics in fragment 1a¹H and¹³the C signal is determined. The fragment is produced by the reaction of H-3: (_H6.28, s) and C-2/C-4/C-10, H-6 (C: (B)_H6.56, d,J= 1.8 Hz) and C-8/C-10 and H-8 (_H6.67, d,J= 1.8 Hz) was determined in relation to HMBC at C-6/C-10. In addition, a carbonyl moiety (three carbon units of C-11 to C-13) and a hydroxyl group (7-OH) are attached at the C-5 and C-7 positions of the chromonic moiety, respectively, the inference being based on H₂-11 (_H4.15, s) and C-6/C-10, and 7-OH: (_H10.70, s) and C-6/C-7/C-8 were confirmed. These features are very similar to the known compound 5-acetoxy-7-hydroxy-2-methyihromone, except that the C-14 methyl group in the A ring was originally changed to the methylene group in 1a, presumably via H-3 and C-14, and H ₂14 and C-2, C-3, C-9 (⁴ J _CH) It was confirmed that the 1a fragment was presumably linked to the 1b fragment through C-14.

The remaining 11 carbon signals (including 1 methoxy, 1 oxygen containing methylene, 3 ene carbon methine and 6 quaternary carbon signals) are assigned to 1C 9 bicyclic structure containing 1 methoxy and one hydroxymethyl group (fragment 1 b). Further by key HMBC signals H-4 'and C-1' ((R))⁴ J _CH) C-3', C-4a ', C-5', C-8a ' and C-9', 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 that the C9 mother nucleus is an isocoumarin skeleton. In addition, the substituents 1 methoxy and one hydroxymethyl are attached at the C-8 'and C-3' positions, respectively, as determined by the corresponding HMBC. Finally, fragments 1a and 1b pass through the critical H₂The correlation of-14 with C-4a '/C-5'/C-6 'HMBC demonstrates that it is linked by C-14 and C-5', and thus the structure of the compound Cassia alathin B has been determined, which is known under the English name alataiin B.

Test example 2 detection of anti-rotavirus Activity of Compound of the present invention

The method comprises the following steps: the compound prepared in the embodiment 1-2 is taken as a sample to carry out an anti-rotavirus activity test, an in vitro cell test method is adopted, namely, after the sample and the virus act on MA104 cells simultaneously, the protective effect of the sample on cell death caused by virus infection is detected by an Alarmablue method, so that the activity effect of the sample on the rotavirus is measured.

(a) Drug cytotoxicity assays

After MA104 cells are cultured in a 96-well cell culture plate to form a monolayer, adding sample solutions with different concentrations, continuously culturing for 3 days, replacing the culture solution containing Alamarblue, continuously culturing for 2-3 hours, detecting the fluorescence value at 530/590nm of the Alamarblue, detecting the toxicity of the sample to the MA104 cells, and calculating the concentration of half number of cytotoxic cells (TC)₅₀）。

(b) Detection of anti-rotavirus effect of drug

After MA104 cells are cultured in a 96-well cell culture plate to form a monolayer, a virus solution of 100TCID50 and a drug solution with gradient concentration not more than 20% of cytotoxicity are simultaneously added to the MA104 cells, after the cells are continuously cultured for 4-6 days, a culture solution containing Alamarblue is replaced to continuously culture for 2-3 hours, the fluorescence value at 530/590nm of the cells is detected, and the median Inhibitory Concentration (IC) is calculated₅₀）。

(c) Root TC₅₀/ IC₅₀Calculating the therapeutic index of the compound

The results show that TC of the compounds of the invention₅₀Value of 208.2µg/mL、IC₅₀A value of 7.62µg/mL, the therapeutic index TI is 28.8; its therapeutic index exceeds that of control ribavirin (TC)₅₀Value 258.4, IC₅₀A value of 9.5) is 27.3, which shows that the compound has good anti-rotavirus activity.

In conclusion, the compound has good application prospect in preparing anti-rotavirus medicaments; the compound has simple structure and better activity, and can be used as a leading compound for research and development of anti-rotavirus medicaments for research and development of anti-rotavirus medicinal preparations.

Claims

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:

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.