CN111690022B - Alkaloid separated from radix Caulophylli, and separation method and application thereof - Google Patents

Abstract

The invention discloses alkaloid separated from radix equiseti chinensis, a separation method and application thereof, and belongs to the technical field of biological medicines. The invention separates an alkaloid compound from the rhizome of Shaanxi ' seven-drug ' and Maya seven ' for the first time, and the molecular formula of the alkaloid compound is C₂₅H₃₈NO₁₃(ii) a The structure of the compound is an indole glycoside alkaloid compound. The invention adopts a special alkaline chromatographic column, can be well separated and purified, and has the characteristics of simple, convenient and effective method and high purity of the obtained compound. The invention can effectively extract and separate alkaloid compounds with anti-inflammatory and gastric mucosa protection effects from the dried rhizome of Shanxi ' Qiyao ' and Maya Qin '.

Description

Alkaloid separated from radix Caulophylli, and separation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and relates to alkaloid separated from radix equiseti, a separation method and application thereof.

Background

With the increase of modern life pressure and the influence of poor eating habits, the number of people suffering from gastritis, gastric ulcer and other stomach diseases is increasing, and the incidence rate of gastric ulcer is increasing year by year. At present, the medicines for treating gastric ulcer mainly comprise chemical medicines, and aiming at different causes of peptic ulcer, the medicines for treating gastric ulcer comprise antacid and histamine H₂Receptor blockers, proton pump inhibitors, gastrointestinal mucosa protectors, antioxidants, etc. However, these drugs have different side effects and disadvantages, such as large dose of antacid needed for multiple administrations; long-term application of Histamine H₂Receptor blocking drug therapy potential inducerIncrease of pH value in antrum of stomach and stress proliferation of gastric parietal cells; long-term use of proton pump inhibitors is also associated with osteoporosis, dementia, kidney damage, anemia, and iron and microbial B12 deficiencies. Although the triple therapy is applied to improve the cure rate of gastric ulcer and reduce the recurrence rate, the problems of overlong medication time, great side effect of the medicine, incomplete treatment and the like still exist. Therefore, the development of new anti-gastric ulcer drugs with high efficiency and low toxicity is necessary.

The traditional Chinese medicine has unique advantages in the research aspect of gastric ulcer resistance, has more clinical practical significance and development prospect, resists gastric ulcer, and has the advantages and characteristics of high cure rate, small side effect, low price and the like.

The 'seven-drug' in Shaanxi is mainly derived from plant drugs in Shaanxi, is an important component of national folk drugs, is a herbal medicine which is repeated and verified for tens of thousands of times in hundreds of years and gradually and definitely has exact curative effect in disease treatment practice, and is a precious folk cultural heritage. The Maya chinensis is one of seven medicines in Shanxi, is pseudobulb and whole grass of Calanthe fringensis (Calanthe Franch) which belongs to Calanthe of Orchidaceae (Orchidaceae), is mainly distributed in the southern mountain area and is usually grown under mountain forests with the altitude of 1500-3500 m and on grass slopes. It is light, slightly pungent and bitter, and cool in nature. Has little toxicity. It enters stomach, liver and lung meridians. Has the effects of clearing away heat and toxic materials, relieving swelling and pain, and removing blood stasis.

At present, the research on the whole calanthe plant at home and abroad mainly focuses on the aspects of ornamental value, cultivation technology, breeding method and the like, and the research on the effective components and the pharmacological activity of the calanthe plant is rarely reported.

Disclosure of Invention

The invention aims to provide alkaloid separated from radix equiseti immaturus, a separation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses an alkaloid separated from rhizoma gastrodiae, the molecular formula of the alkaloid is C₂₈H₃₅NO₁₃(ii) a The structural formula is as follows:

the invention also discloses application of the alkaloid separated from the root of nux vomica in preparing a medicament for protecting gastric mucosa.

The invention also discloses application of the alkaloid separated from the root of nux vomica in preparing anti-inflammatory drugs.

The invention also discloses an extraction and separation method of the alkaloid separated from the root of nux vomica, which comprises the following steps:

1) extracting dry rhizome of radix et rhizoma Rhei Palmatae with methanol or ethanol under reflux for several times, mixing extractive solutions, and concentrating under reduced pressure to obtain total extract;

or cold soaking in acid water at room temperature for several times, each for several hours, mixing the acid water solutions, and concentrating to obtain concentrated solution;

2) when a methanol or ethanol reflux extraction method is adopted in the step 1), suspending the obtained total extract in acid water, fully mixing, standing, filtering to remove insoluble substances, sequentially extracting the obtained filtrate with ethyl acetate and n-butanol for a plurality of times, and separating out an n-butanol extraction layer and an ethyl acetate extraction layer;

when the acid water cold leaching extraction method is adopted in the step 1), extracting the concentrated solution for a plurality of times by using ethyl acetate and n-butanol, and removing the organic solvent under reduced pressure to respectively obtain an ethyl acetate extraction layer and an n-butanol extraction layer;

3) loading the n-butanol extraction layer on a silica gel column, taking a chloroform-methanol system as eluent, carrying out gradient elution according to the volume ratio of (100:0) - (0:100), carrying out TLC detection on the effluent, combining fractions with the eluent volume ratio of 20:80, and removing the solvent to obtain a first column chromatography part;

4) loading the first column chromatography part on a reverse phase silica gel chromatographic column, taking a methanol-water system as eluent, performing gradient elution according to the volume ratio of (10:90) - (100:0), combining fractions with the eluent volume ratio of 25:75, and removing the solvent to obtain a second column chromatography part;

5) loading the second column chromatography part on a high performance liquid chromatography separation column, and performing isocratic elution with a mobile phase to obtain the target product alkaloid.

Preferably, in the step 1), the reflux extraction or the cold soaking extraction is performed for 1-5 times, and each time lasts for 1-3 hours.

Preferably, in the step 1), pure methanol or ethanol with the volume fraction of 10% -95% is adopted as an extracting agent in the reflux extraction, and a sulfuric acid aqueous solution or a hydrochloric acid aqueous solution with the concentration of 1% -5% is adopted as the extracting agent in the cold leaching extraction;

the mass volume ratio of the dry rhizome of the root of the manyleaf paris to the extracting agent is 1 kg: (1-8) L;

when the extractant is pure methanol or ethanol with the volume fraction of 10-95%, the solvent in the obtained extracting solution is recovered to obtain total extractum; and when the extracting agent is acid water, concentrating the volume of the combined acid water solution to 1/10-1/5 to obtain a concentrated solution.

Preferably, in the step 5), the flow rate of isocratic elution is 2-10 mL/min; the mobile phase is selected from a methanol-water system or an acetonitrile-water system, and the volume ratio of methanol to water in the methanol-water system is 20: 80; the volume ratio of acetonitrile to water in the acetonitrile-water system was 25: 75.

Compared with the prior art, the invention has the following beneficial effects:

the alkaloid compound is separated from the rhizome of Shaanxi ' seven-drug- ' Maya seven ' for the first time, and the molecular formula of the alkaloid compound is C₂₅H₃₈NO₁₃(ii) a The structure of the compound is an indole glycoside alkaloid.

The alkaloid compound has anti-inflammatory effect, and experiments prove that the alkaloid compound has good anti-inflammatory effect on RAW264.7 fine cells of mice, has the characteristics of high efficiency and low toxicity, and is expected to be developed into a new anti-inflammatory drug.

The alkaloid compound has the gastric mucosa protection effect, and experiments prove that the alkaloid compound has a certain protection effect on the gastric ulcer of a mouse induced by ethanol, has the characteristics of high efficiency and low toxicity, and is expected to be developed into a novel anti-gastric ulcer medicament.

The alkaloid compound obtained in the invention has good activity, but low content in plants, and large polarity, and is difficult to enrich and separate by using a conventional chromatographic method, so the alkaloid compound can be well separated and purified by using a special alkaline chromatographic column, and the alkaloid compound has the characteristics of simple, convenient and effective method and high purity of the obtained compound.

Drawings

FIG. 1 is a drawing showing Compound 1 of the present invention¹An H-NMR spectrum;

FIG. 2 shows Compound 1 of the present invention¹³A C-NMR spectrum;

FIG. 3 is a DEPT profile of Compound 1 of the present invention;

FIG. 4 shows Compound 1 of the present invention¹H-¹H COSY map;

FIG. 5 is an HSQC spectrum of Compound 1 of the present invention;

FIG. 6 is an HMBC profile of compound 1 of the present invention;

FIG. 7 is a HR-ESI-MS spectrum of Compound 1 of the present invention.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

the invention discloses a method for extracting and separating alkaloid compounds from 'Maya gas', which comprises the following steps:

1) taking certain mass (kg) of dried rhizome of 'Maya chinensis', heating, refluxing or cold-soaking and extracting 1-5 times by using methanol with the volume being 1-5 times of the mass of the dried rhizome of 'Maya chinensis', ethanol with the volume fraction being 10-95% or acid water (L) near the respective boiling points for 1-3 hours each time, merging extracting solutions, recovering and removing a solvent under reduced pressure when an extracting agent is methanol or ethanol with the volume fraction being 10-95% to obtain a total extract, merging the extracting solutions and concentrating the volume of the extracting solutions to one fifth to one tenth when the extracting agent is the acid water to obtain a concentrated solution;

2) suspending the total extract in acid water, wherein the volume ratio of the total extract to the acid water is 1: 1-1: 4, filtering to remove insoluble substances to obtain acid water extract liquid, and sequentially extracting the extract liquid or the concentrated liquid with an equal volume of organic solvent respectively, wherein the extract liquid or the concentrated liquid is subjected to equal volume extraction with ethyl acetate during first extraction, an organic layer obtained after the previous extraction is separated out during each extraction, and the remaining acid water layer is subjected to the next extraction with an equal volume of organic solvent; extracting each solvent for 1-5 times, combining the extract liquor, and distilling under normal pressure or reduced pressure to remove the organic solvent to obtain each extraction layer and a water layer respectively. The organic solvent comprises ethyl acetate, n-butanol, etc., and the extraction order is that firstly the solvent with low polarity is used, and then the organic solvent with high polarity is used.

3) Taking the n-butanol extraction layer, and obtaining the alkaloid compound by adopting a separation method such as column chromatography purification.

The column chromatography comprises the following three stages:

the first stage is as follows: loading the n-butanol extraction layer on a silica gel column, taking a chloroform-methanol system as eluent, carrying out gradient elution according to the volume ratio (100:0) - (0:100), collecting one flow fraction every 300-800 mL, carrying out TLC detection on the effluent, combining the same flow fractions to obtain 20 flow fractions which are respectively named as MB1-MB20, evaporating the solvent at normal pressure or reduced pressure, and taking MB15 flow fractions, namely the flow fractions with the eluent volume ratio of 2:8, as first column passing fractions;

and a second stage: loading the first column-passing part on a reverse phase silica gel chromatographic column, taking a methanol-water system as an eluent, carrying out gradient elution according to the volume ratio of (10:90) - (100:0), collecting one flow part per 100-300 mL, carrying out TLC detection on the effluent, combining the same flow parts, removing the solvent under reduced pressure to obtain 16 flow parts, respectively named as MB15.1-FrB15.16, and taking the MB15.12 flow parts, namely the flow parts with the eluent volume ratio of 25:75, as a second column-passing part;

and a third stage: loading the second column passing part to high performance liquid chromatography separation column, and separating to obtain alkaloid compound.

Wherein the high performance liquid chromatography separation column is a COSMOSIL C18-PAQ column, and the high performance liquid chromatography separation is performed by using a differential refraction detector, using a methanol-water system or an acetonitrile-water system as a mobile phase and performing isocratic elution according to 3-5 mL/min. The volume ratio of methanol to water in the methanol-water system is 20: 80; the volume ratio of acetonitrile to water in the acetonitrile-water system is 25: 75.

the indole glycoside compound prepared by the method has the following structural formula:

in the research on chemical components and pharmacological activity of the radix equiseti, the alkaloid compound obtained by separation has an inhibitory effect on NO generated by RAW264.7 cells induced by LPS, and the compound has the characteristics of high efficiency and low toxicity, so that the compound is expected to be developed into a novel anti-inflammatory drug. Meanwhile, experiments prove that the compound has a certain protection effect on gastric ulcer of mice induced by ethanol, has the characteristics of high efficiency and low toxicity, and is expected to be developed into a novel anti-gastric ulcer medicament.

First, preparation example

Example 1

An extraction and separation method of alkaloid compounds comprises the following steps:

1) taking 10kg of dry rhizomes of the root of the manyleaf paris, heating and refluxing the dry rhizomes of the root of the manyleaf paris for 4 times by using methanol with the volume of 5 times of the mass of the dry rhizomes of the root of the manyleaf paris, extracting for 3 hours each time, combining extracting solutions, decompressing and recovering a solvent to obtain a total extract;

2) suspending the total extract in 4 times of 2% sulfuric acid aqueous solution, mixing thoroughly, standing, filtering to remove insoluble substances, isovolumetrically extracting the filtrate with ethyl acetate for 4 times, isovolumetrically extracting with n-butanol for 4 times, and removing organic solvent from the extraction layer under reduced pressure to obtain ethyl acetate layer and n-butanol layer respectively.

3) Collecting 100g of n-butanol extraction layer, performing gradient elution with chloroform/methanol at volume ratio (v/v) of 100: 0-0: 100 by silica gel column chromatography, collecting one fraction per 400mL, and combining the same fractions by TLC to obtain 20 fractions (MB1-MB 20).

4) Wherein the 15 th fraction MB15 is separated by reverse phase silica gel column chromatography, and is eluted by MeOH/H2O in a gradient of 100: 0-0: 100 in a volume ratio (v/v), one fraction is collected per 300mL, and 16 fractions (MB15.1-MB15.16) are obtained after TLC identification and combination of the same fractions.

5) Wherein the 12 th fraction MB15.12 was purified by semi-preparative high performance liquid chromatography on a COSMOSIL C18-PAQ column with a methanol-water solution (20:80, v/v) as the mobile phase at a flow rate of 4.0mL/min to give compound 1.

The structure of the compound is determined by physical and chemical constants and modern spectral technical means (HR-ESI-MS, 1D-NMR and 2D-NMR).

2. Structural identification of alkaloid compounds

Compound 1: yellow oil, readily soluble in methanol. According to HR-ESI-MS spectrum, the peak of the quasi-molecular ion is M/z 594.21678[ M + H ]]+ (calcd for 594.21812) determined to be C28H35NO13 with 12 unsaturations. In that¹H-NMR (FIG. 1) and¹³in C-NMR (fig. 2), proton signals δ H7.72 (1H, d, J ═ 7.1Hz), δ H6.96 (1H, td, J ═ 7.1,0.9Hz), δ H7.00(1H, td, J ═ 7.0,1.2Hz), δ H7.18 (1H, d, J ═ 7.5Hz) and the carbon signal δ C134.5,134.7,128.8,122.7,122.0,119.7,118.6,111.9 indicated that the molecule contained a disubstituted indole structure, the proton signal δ H6.87 (1H, s), δ H6.72 (1H, d, J ═ 8.2Hz), δ H6.71 (1H, d, J ═ 8.2Hz) and the carbon signal δ C148.9,145.8,132.8,122.3,116.1,113.6 indicated that the molecule contained a trisubstituted benzene ring, δ H3.79 (3H, s) indicated that the benzene ring contained a methoxy substituent, as H-7 ' is related to C-2 ' and C-6 ' in the HMBC spectrum (figure 6), a methylene substituent on a benzene ring is a benzyl, the third substituent is hydroxyl group through carbon spectrum chemical shift, and the connection position of the benzyl and indole ring is determined because H-7' is related to both C-2 and C-3. In HMBC spectra, correlation of H-5 'with C-3' indicates that the C-3 'position is methoxy, and correlation of H-6' with C-4 'indicates that the substituent at the C-4' position is hydroxy. In that¹H-NMR and¹³in C-NMR, terminal proton signals δ H4.68 (1H, D, J ═ 7.6Hz), δ H4.30 (1H, D, J ═ 7.7Hz) and carbon signals δ C107.4, 104.9 indicated that the molecule contained 2 β -configuration sugar units, and after acid hydrolysis of this compound, all were D-form glucose as determined by GC analysis. And because in the HMBC (FIG. 6) spectrum, H-1 ' is related to C-6 ' and H-1 ' is related to C-3, the position of glucose is determined, and the structure of the compound is finally determined as (2S,3R,4S,5S,6R) -2- ((2- (4-hydroxy-3-methylbenzyl) -1H-indole-3-yl) oxy) -6- (((2R,3R,4S,5S,6R) -3,4,5-trihydroxy-6- (hydroxymethy) tetrahydro-2H-pyran-2-yl) oxy) methyl) tetrahydro-2H-pyran-3,4,5-triol and is named calanthan H, which is a new compound not reported in the literature. The structural formula is as follows:

the nuclear magnetic data are shown in table 1, and the related maps are shown in attached figures 1-7;

TABLE 1 of the compounds of the invention¹H NMR and¹³c NMR data

No.	δH(ppm)	δC(ppm)
			2		128.8
3		134.5
			4	7.72(1H,d,J＝7.1Hz)	118.6
5	6.96(1H,m)	119.7
			6	7.00(1H,m)	122.0
7	7.18(1H,d,J＝7.5Hz)	111.9
			8		134.7
9		122.7
			1′		132.8
2′	6.87(1H,s)	113.6
			3′		148.9
4′		145.8
			5′	6.71(1H,d,J＝8.2Hz)	116.1
6′	6.72(1H,d,J＝8.2Hz)	122.3
			7′	4.18(1H,d,J＝15.7Hz)，4.06(1H,d,J＝15.8Hz)	31.3
3′-OCH3	3.79(3H,s)	56.5
			1″	4.69(1H,d,J＝7.9Hz)	107.4
2″	3.52(1H,m)	75.3
			3″	3.42(1H,m)	78.0
4″	3.54(1H,m)	71.4
			5″	3.35(1H,m)	77.0
6″	3.80(1H,m),4.07(1H,m)	70.0
			1″′	4.29(1H,d,J＝7.7Hz)	104.9
2″′	3.21(1H,m)	75.1
			3″′	3.30(1H,m)	77.9
4″′	3.28(1H,m)	71.6
			5″′	3.23(1H,m)	77.9
6″′	3.65(1H,dd,J＝11.9,5.4Hz),3.83(1H,dd,J＝12.0,2.2Hz)	62.8

Example 2

1) Taking 10kg of dry rhizome of the root of the Chinese paris, heating and refluxing the dry rhizome of the root of the Chinese paris for 5 times by using 95 percent ethanol with the volume amount being 3 times of the mass of the dry rhizome of the root of the Chinese paris for 2 hours each time, combining extracting solutions, and recovering a solvent under reduced pressure to obtain a total extract;

2) suspending the total extract in 3 times of 2% hydrochloric acid aqueous solution, shaking thoroughly, mixing, standing for 12 hr, filtering to remove precipitate, isovolumetrically extracting the filtrate with petroleum ether for 4 times, isovolumetrically extracting with ethyl acetate and n-butanol for 4 times, and removing solvent from the extraction layer under reduced pressure to obtain petroleum ether layer, ethyl acetate layer and n-butanol layer respectively.

3) Taking 70g of n-butanol extraction layer, firstly performing gradient elution by silica gel column chromatography with chloroform/methanol at a volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 400mL, and combining the same flow portions through TLC detection to obtain 20 flow portions (MB1-FrB 20).

4) The 15 th fraction MB15 was isolated by reverse phase silica gel column chromatography on MeOH/H₂Performing gradient elution on O according to the volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 300mL, and obtaining 16 flow portions (MB15.1-MB15.16) after identifying and combining the same flow portions by TLC.

5) Wherein the 12 th fraction MB15.12 was subjected to semi-preparative high performance liquid chromatography using a COSMOSIL C18-PAQ column with a methanol-water solution (20:80, v/v) as the mobile phase at a flow rate of 3.0mL/min to give Compound 1.

Example 3

1) Taking 10kg of dry rhizomes of the root of the Chinese paris, cold-soaking and extracting for 3 times at room temperature by using 2 percent of hydrochloric acid aqueous solution with the volume amount being 8 times of the mass of the dry rhizomes of the root of the Chinese paris for 8 hours each time, combining the extracting solutions, and concentrating under reduced pressure to one fifth of the original volume to obtain concentrated acid aqueous solution;

2) extracting the acid water concentrated solution with equal volume of ethyl acetate for 4 times, then extracting with equal volume of n-butanol for 4 times, and removing solvent from the extraction layer under reduced pressure to obtain ethyl acetate layer and n-butanol layer respectively.

3) Collecting 80g of n-butanol extraction layer, performing gradient elution with chloroform/methanol at volume ratio (v/v) of 100: 0-0: 100 by silica gel column chromatography, collecting one fraction per 500mL, and combining the same fractions by TLC to obtain 20 fractions (MB1-MB 20).

4) The 15 th fraction MB15 was isolated by reverse phase silica gel column chromatography on MeOH/H₂Performing gradient elution on O according to the volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 300mL, and obtaining 16 flow portions (MB15.1-MB15.16) after identifying and combining the same flow portions by TLC.

5) Of these, the 12 th fraction, MB15.12, was purified by semi-preparative HPLC using a COSMOSIL C18-PAQ column with a mobile phase of acetonitrile-water solution (25:75, v/v) at a flow rate of 4.0mL/min to give Compound 1.

Second, Activity test

1. In vitro anti-inflammatory assays on Compound 1 prepared in the examples

1) Experimental methods

RAW264.7 cells were seeded in 96-well plates and Lipopolysaccharide (LPS) was added after 24h of culture. Samples (the compound of the invention and the indomethacin as a positive drug) with different concentrations are respectively added, equal volume of DMSO is added into a blank group, after 48 hours of culture, 50 mu L of cell culture supernatant is mixed with equal volume of Griess reagent I, and then Griess reagent II is added for reaction. The samples of each group are measured by a microplate readerOD value at 546nm, NO production inhibition rate was calculated using the following formula, and half Inhibitory Concentration (IC) of the test sample was calculated using SPSS software₅₀Value, μ M).

NO inhibition (%) [ 1- (sample group OD value/blank group OD value) ] × 100%.

2) Results of the experiment

The results of the experiment are shown in table 2:

TABLE 2 inhibition of NO production by LPS stimulated cells RAW264.7 by the compounds of the present invention

And (4) analyzing results: the data in Table 2 show that the compound 1 in the invention has good in-vitro anti-inflammatory effect, has good inhibition effect on NO production of mouse RAW264.7 cells stimulated by LPS, and has half inhibitory concentration IC₅₀Is 41.3 +/-3.8 mu M, and is equivalent to 37.9 +/-2.1 mu M of the positive control drug indometacin. The indole glycoside alkaloid compound 1 has good anti-inflammatory effect.

2. Gastric mucosa protection experiment

1) Experimental methods

Male Kunming mice weighing 20-25 g were randomly divided into 5 groups of 8 mice each, i.e., normal group, model group, omeprazole group, low dose group (10mg/kg), medium dose group (20mg/kg) and high dose group (40mg/kg) of Compound 1. Omeprazole 20mg/kg is administered for the positive control group by intragastric administration, and the same volume of physiological saline is administered for the blank group and the model group. After 1h of administration, 100. mu.L of 70% ethanol was gavaged, and the blank group was given an equal amount of physiological saline. 1h, removing the neck, killing the mouse, performing laparotomy, quickly taking out the stomach, cutting along the greater curvature, flushing with physiological saline, flattening, draining, fixing with 10% formaldehyde for 10-15 min, precisely measuring the ulcer area of the mouse by using a vernier caliper, and calculating the ulcer index and the inhibition rate.

The ulcer inhibition rate is (mean value of ulcer index of model group-mean value of ulcer index of experiment group)/mean value of ulcer index of model group x 100%

2) Results of the experiment

The results of the experiment are shown in table 3:

TABLE 3 inhibitory Effect of the Compounds of the present invention on gastric ulcer in mice induced by ethanol

And (4) analyzing results: the data in table 3 show that the compound of the present invention has a certain anti-gastric ulcer effect and a certain protective effect on gastric ulcer of mice induced by ethanol.

In conclusion, the alkaloid compounds disclosed by the invention are extracted and separated from dried roots and stems of Shanxi 'Qiyao' and 'Maya Qi', and experimental researches show that the compounds have a good inhibition effect on NO generated by RAW264.7 cells stimulated by LPS, namely the compounds have a good in vitro anti-inflammatory effect and are expected to be developed into anti-inflammatory medicaments. The alkaloid compound has a certain protection effect on the gastric ulcer of a mouse induced by ethanol, and has the characteristics of high efficiency and low toxicity, so that the alkaloid compound is expected to be developed into a novel anti-gastric ulcer medicament.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Claims (6)

1. An alkaloid separated from radix Asteris Lasiocladi, wherein the alkaloid has a molecular formula of C₂₈H₃₅NO₁₃(ii) a The structural formula is as follows:

2. use of the alkaloid isolated from Aequoria dichotoma as defined in claim 1 in the preparation of a medicament for protecting gastric mucosa.

3. Use of the alkaloid isolated from Aequoria dichotoma according to claim 1 in the preparation of anti-inflammatory agent.

4. The method for extracting and separating alkaloid from rhizoma et radix Parthenocissi Tricuspidatae as claimed in claim 1, comprising the steps of:

1) extracting dry rhizome of radix et rhizoma Rhei Palmatae with methanol or ethanol under reflux for several times, mixing extractive solutions, and concentrating under reduced pressure to obtain total extract;

or cold soaking in acid water at room temperature for several times, each for several hours, mixing the acid water solutions, and concentrating to obtain concentrated solution;

2) when a methanol or ethanol reflux extraction method is adopted in the step 1), suspending the obtained total extract in acid water, fully mixing, standing, filtering to remove insoluble substances, sequentially extracting the obtained filtrate with ethyl acetate and n-butanol for a plurality of times, and separating out an n-butanol extraction layer and an ethyl acetate extraction layer;

when the acid water cold leaching extraction method is adopted in the step 1), extracting the concentrated solution for a plurality of times by using ethyl acetate and n-butanol, and removing the organic solvent under reduced pressure to respectively obtain an ethyl acetate extraction layer and an n-butanol extraction layer;

3) loading the n-butanol extraction layer on a silica gel column, taking a chloroform-methanol system as eluent, carrying out gradient elution according to the volume ratio of (100:0) - (0:100), carrying out TLC detection on the effluent, combining fractions with the eluent volume ratio of 20:80, and removing the solvent to obtain a first column chromatography part;

4) loading the first column chromatography part on a reverse phase silica gel chromatographic column, taking a methanol-water system as eluent, performing gradient elution according to the volume ratio of (10:90) - (100:0), combining fractions with the eluent volume ratio of 25:75, and removing the solvent to obtain a second column chromatography part;

5) loading the second column chromatography part on a high performance liquid chromatography separation column, and performing isocratic elution by using a mobile phase to obtain a target product alkaloid;

the flow rate of the isocratic elution is 2-10 mL/min; the mobile phase is selected from a methanol-water system or an acetonitrile-water system, and the volume ratio of methanol to water in the methanol-water system is 20: 80; the volume ratio of acetonitrile to water in the acetonitrile-water system was 25: 75.

5. The method for extracting and separating alkaloids from rhizoma et radix Parthenocissi Tricuspidatae as claimed in claim 4, wherein in step 1), the reflux extraction or cold soaking extraction is performed 1-5 times for 1-3 h each time.

6. The method for extracting and separating alkaloids from rhizoma et radix Veratri as claimed in claim 4, wherein in step 1), pure methanol or ethanol with volume fraction of 10% -95% is used as extractant in reflux extraction, and sulfuric acid aqueous solution or hydrochloric acid aqueous solution with concentration of 1% -5% is used as extractant in cold leaching extraction;

the mass volume ratio of the dry rhizome of the root of the manyleaf paris to the extracting agent is 1 kg: (1-8) L;

when the extractant is pure methanol or ethanol with the volume fraction of 10-95%, the solvent in the obtained extracting solution is recovered to obtain total extractum; and when the extracting agent is acid water, concentrating the volume of the combined acid water solution to 1/10-1/5 to obtain a concentrated solution.

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