CN109942649B - Indole glycoside compound and extraction and separation method and application thereof - Google Patents
Indole glycoside compound and extraction and separation method and application thereof Download PDFInfo
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Abstract
The invention discloses an indole glycoside compound and an extraction and separation method and application thereof, wherein the indole glycoside compound is obtained by separating rhizomes of plant Chiariella fringensis for the first time, and the molecular formula of the indole glycoside compound is C24H32N2O12(ii) a The structure is a group of chiral isomers. The invention adopts a special hydrophilic 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 extract and separate the indole glycoside compound from dried rhizome of Chinemys reevesii, and the compound can promote the uptake and transformation of glucose by cells, and can resist hepatitis, inflammation and gastric ulcer.
Description
Technical Field
The invention belongs to the field of medicines and health-care foods, and relates to an indole glycoside compound, and an extraction and separation method and application thereof.
Background
Along with the global industrialization and the modern life, the life style of people is greatly changed. Due to a plurality of factors such as improvement of living standard, change of dietary structure, increasingly tense life rhythm, and life style of little movement and sitting, the incidence rate of diabetes mellitus in the world is rapidly increased, and the diabetes mellitus becomes the third chronic disease which seriously threatens human health after tumor and cardiovascular disease. At present, the number of diabetic patients is more than 1.2 hundred million worldwide, and the patients in China are second in the world, so the research and development of diabetes medicines are not slow. The existing clinical medicines for treating diabetes have the defects of large toxic and side effects and the like, and natural botanical medicines are paid attention to by people due to the advantages of wide distribution, easily available materials, low price, small toxic and side effects and the like, so that the medicine has wide application prospect.
Pseudobulb and whole plant of Calanthe fimbriata franch (Calanthe) belonging to Calanthe of Orchidaceae (Orchidaceae), namely, the name of Reynaud's Ribes or Jiuzi's union, including provinces such as Shaanxi, Gansu, Yunnan, Sichuan, Tibet and Taiwan, and the distribution of the plant is also found in Xijin and Japan. It is often grown under mountain forests and on grass slopes with elevation of 1500-. 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
In order to overcome the defects of the prior art, the invention aims to provide an indole glycoside compound, an extraction and separation method and application thereof, the indole glycoside compound can be extracted and separated from calanthe fimbriata, and the compound has the functions of resisting hyperglycemia, hepatitis and inflammation and protecting gastric mucosa.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses an indole glycoside compound, the molecular formula of which is C24H32N2O12(ii) a The structural formula is shown as the following formula 1 and formula 2:
The invention also discloses an extraction and separation method of the indole glycoside compound, which comprises the following steps:
1) extracting dried rhizome of Stellaria schreberi under reflux for several times, mixing extractive solutions, and concentrating the extractive solution under reduced pressure to obtain total extract or concentrated solution;
2) suspending the total extract in water to obtain extract solution, sequentially extracting the extract solution or concentrated solution with petroleum ether, ethyl acetate and n-butanol for several times, separating out organic layer, and removing solvent under reduced pressure to obtain each 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), detecting the effluent, combining fractions with the eluent volume ratio of 7:3, and removing the solvent to obtain a first column passing 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 (20:80) - (100:0), combining fractions with the eluent volume ratio of 35:65, and removing the solvent to obtain a second column chromatography part;
5) loading the second 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 (5:95) - (100:0), combining fractions with the eluent volume ratio of 20:80, and removing the solvent to obtain a third column chromatography part;
6) and (4) loading the third column chromatography part on a high performance liquid chromatography separation column, and performing isocratic elution by using a mobile phase to obtain the indole glycoside compound.
Preferably, in the step 1), the reflux extraction times are 1-6 times, and each time lasts for 1-3 hours;
in the reflux extraction, methanol, water or ethanol with the volume fraction of 10-95% is used as an extracting agent, and the mass-volume ratio of dried rhizomes of the Chinemys fimbriata to the extracting agent is 1 kg: (1-5) L; when the extracting agent is methanol or ethanol with the volume fraction of 10-95%, recovering the solvent in the obtained extracting solution to obtain a total extract; and when the extracting agent is water, concentrating the volume of the extracting solution to 1/10-1/6 to obtain a concentrated solution.
Preferably, in the step 2), the total extract is suspended in water, and the volume ratio of the total extract to the water is 1: 1-1: 4;
during extraction, petroleum ether and ethyl acetate are sequentially extracted, and n-butanol is further extracted; each extraction is equal volume extraction; each solvent is extracted for 1-6 times.
Preferably, after gradient elution in the step 3), collecting one flow portion every 300-800 mL; collecting one fraction per 100-300 mL after gradient elution in the step 4); collecting one flow portion per 100-300 mL after gradient elution in the step 5).
Preferably, in the step 6), the flow rate of isocratic elution is 3-5 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 25: 75; the volume ratio of acetonitrile to water in the acetonitrile-water system was 35: 65.
The invention also discloses application of the indole glycoside compound in preparation of antidiabetic drugs and/or health-care foods.
The invention also discloses application of the indole glycoside compound in preparation of anti-inflammatory drugs and/or health-care foods.
The invention also discloses application of the indole glycoside compound in preparation of anti-hepatitis drugs and/or health-care foods.
The invention also discloses application of the indole glycoside compounds in preparation of gastric mucosa protective medicines and/or health-care foods.
Compared with the prior art, the invention has the following beneficial effects:
the invention separates indole glycoside compound from the rhizome of the plant Chiariella fringensis for the first time, and the molecular formula of the indole glycoside compound is C24H32N2O12(ii) a The structure is a group of chiral isomers.
The indole glycoside compound obtained by the invention has good activity, but the content in plants is not high, the polarity is large, and the indole glycoside compound is difficult to enrich and separate by using a conventional chromatographic method. The invention adopts a special hydrophilic 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.
Experiments prove that the compound has a remarkable promoting effect at 3 mu M and has a concentration dependence relationship, and the compound has the characteristics of high efficiency and low toxicity, and is expected to be developed into a novel antidiabetic medicament or used for preparing a health-care food with the effect of preventing and treating diabetes.
The indole glycoside compound has anti-inflammatory effect, and experiments prove that the compound has good anti-inflammatory effect on RAW264.7 cells of mice, has the characteristics of high efficiency and low toxicity, and is expected to be developed into a new anti-inflammatory drug or used for preparing health-care food for preventing and treating inflammation.
The indole glycoside compound has the function of hepatitis resistance, tests prove that the indole glycoside compound has good inhibitory action on HBsAg and HBeAg generated by HepG2.2.15 cells, the compound has the characteristics of high efficiency and low toxicity, and the indole glycoside compound is expected to be developed into a new hepatitis resistance medicament or used for preparing health-care food for preventing and treating hepatitis.
The indole glycoside compound has the gastric mucosa protection effect, tests prove that the indole glycoside compound has certain resistance 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 or used for preparing health-care food for preventing and treating gastric ulcer.
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.
FIG. 8 is a drawing showing Compound 2 of the present invention1An H-NMR spectrum;
FIG. 9 shows Compound 2 of the present invention13A C-NMR spectrum;
FIG. 10 is a DEPT profile of Compound 2 of the present invention;
FIG. 11 is a drawing showing Compound 2 of the present invention1H-1H COSY map;
FIG. 12 is an HSQC spectrum of Compound 2 of the present invention;
FIG. 13 is an HMBC profile of compound 2 of the present invention;
FIG. 14 is a HR-ESI-MS spectrum of Compound 2 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 indole glycoside compounds from Chiariella fringensis, which comprises the following steps:
1) taking dried rhizomes of calanthe fringensis with a certain mass (kg), heating and refluxing for 1-6 times by using methanol with the volume being 1-5 times of the mass of the dried rhizomes of calanthe fringensis and ethanol with the volume fraction being 10-95% or water (L) near respective boiling points, wherein each time lasts for 1-3 hours, when an extracting agent is methanol or ethanol with the volume fraction being 10-95%, combining extracting solutions, decompressing, recovering and removing a solvent to obtain a total extract, and when the extracting agent is water, combining the extracting solutions, and concentrating the volume of the extracting solutions to one sixth to one tenth to obtain a concentrated solution;
2) suspending the total extract in water, wherein the volume ratio of the total extract to the water is 1: 1-1: 4 to obtain extract liquid, and sequentially extracting the extract liquid or the concentrated liquid with an equal volume of an organic solvent respectively, wherein the extract liquid or the concentrated liquid is subjected to equal volume extraction with petroleum ether during first extraction, an organic layer obtained after the previous extraction is separated out during each extraction, and the remaining aqueous layer is subjected to the next extraction with an equal volume of the organic solvent; extracting each solvent for 1-6 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 petroleum ether, ethyl acetate, n-butanol and the like, the extraction sequence is that the solvent with low polarity is used firstly, and then the organic solvent with high polarity is used, and the petroleum ether and the ethyl acetate can be omitted.
3) Taking the n-butanol extraction layer, and obtaining the indole glycoside 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 an eluent, carrying out gradient elution according to the volume ratio (100:0) - (0:100), collecting one flow portion every 300-800 mL, carrying out TLC detection on the effluent liquid, combining the same flow portions to obtain 15 flow portions which are respectively named as FrB1-FrB15, evaporating the solvent at normal pressure or reduced pressure, and taking FrB12 flow portions, namely flow portions with the eluent volume ratio of 7:3 as a first column passing portion;
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 (20:80) - (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 12 flow parts which are respectively named as FrB12.1-FrB12.12, and taking FrB12.2 flow parts, namely the flow parts with the eluent volume ratio of 35:65, as a second column-passing part;
and a third stage: loading the second 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 (5:95) - (100:0), collecting one flow part per 100-300 mL, carrying out TLC detection on the effluent, combining the same flow parts, and removing the solvent under reduced pressure to obtain 16 flow parts which are respectively named as FrB12.2.1-FrB12.2.16, wherein the FrB12.2.13 flow parts, namely the flow parts with the eluent volume ratio of 20:80, are used as a third column passing part;
a fourth stage: and loading the third column passing part to a high performance liquid chromatography separation column, and separating to obtain the indole glycoside compound.
The high performance liquid chromatography separation is performed by using a differential refraction detector and 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 25: 75; the volume ratio of acetonitrile to water in the acetonitrile-water system is 35: 65.
the indole glycoside compound prepared by the method has the following structural formula:
the indole glycoside compound disclosed by the invention is applied to preparation of antidiabetic drugs and/or health-care food.
In the research on chemical components and pharmacological activity of the mirabilis rivularis, the invention discovers that the indole glycoside compound separated from the mirabilis rivularis has good effect of improving insulin sensitivity of fat cells, can remarkably promote glucose uptake of fat cells and convert the fat cells into triglyceride, and is expected to be developed into antidiabetic drugs and/or health-care foods for improving insulin resistance.
The application of the indole glycoside compound in the preparation of anti-inflammatory drugs and/or health-care food comprises the following steps: experiments prove that the compound has good anti-inflammatory effect on mouse RAW264.7 fine cells, has the characteristics of high efficiency and low toxicity, and is expected to be developed into a new anti-inflammatory drug or used for preparing health-care food for preventing and treating inflammation.
The application of the indole glycoside compound in the preparation of anti-hepatitis drugs and/or health-care food comprises the following steps: experiments prove that the compound has better inhibiting effect on HBsAg and HBeAg generated by HepG2.2.15 cells, has the characteristics of high efficiency and low toxicity, and is expected to be developed into a new anti-hepatitis medicament or used for preparing health-care food for preventing and treating hepatitis.
The application of the indole glycoside compound in the preparation of gastric mucosa protective drugs and/or health-care food comprises the following steps: experiments prove that the compound has a certain resistance effect on gastric ulcer of mice induced by ethanol, 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-gastric ulcer medicament or used for preparing health-care food for preventing and treating gastric ulcer.
Example 1
1. Extraction and separation of indole glycosides
1) Taking 10kg of dried rhizome of Chinemys reevesii, heating and refluxing with methanol with the volume 5 times of the mass of the dried rhizome of Chinemys reevesii for 4 times, each time for 3 hours, combining the extracting solutions, decompressing and recovering the solvent to obtain a total extract;
2) suspending the total extract in 4 times of water, isovolumetrically extracting 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, chloroform layer, ethyl acetate layer and n-butanol layer.
3) Taking 70g of n-butanol extraction layer, firstly performing gradient elution by silica gel column chromatography with chloroform/methanol according to a volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 500mL, and combining the same flow portions through TLC to obtain 15 flow portions (FrB1-FrB 15).
4) Wherein the 12 th fraction FrB12 was isolated by reverse phase silica gel column chromatography with MeOH/H2And carrying out gradient elution on O according to the volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 200mL, and obtaining 12 flow portions (FrB12.1-FrB12.12) after identifying and combining the same flow portions by TLC.
5) Wherein the 2 nd fraction FrB12.2 is applied to a reverse phase silica gel column chromatography with MeOH/H2O is (5:95) - (100:0) in volume ratio (v/v)Gradient elution was performed, collecting one fraction per 200mL, and identifying by TLC detection and obtaining 16 fractions (FrB12.2.1-FrB12.2.16) after the same fraction.
6) FrB12.2.13 was then purified by semi-preparative high performance liquid chromatography on a Megres C18 column with a methanol-water solution (25:75, v/v) as the mobile phase at a flow rate of 3.0mL/min to give Compound 1 (t: (t))R30min) and compound 2 (t)R＝34min)。
The structure of the compound is identified by physicochemical constants and modern spectral technical means (HR-ESI-MS, 1D-NMR and 2D-NMR), and the compound 1 is:
(R)-5-(3-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-((((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)tetrah-ydro-2H-pyran-2-yl)oxy)-1H-indol-2-yl)pyrrolidin-2-one；
(S)-5-(3-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-((((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahy-dro-2H-pyran-2-yl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-1H-indol-2-yl)pyrrolidin-2-one。
2. structure identification of indole glycoside compounds
1) Structural characterization of novel Compound 2
Structural identification correlation maps for compound 2 are shown in FIGS. 8-14;
(S)-5-(3-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-((((2R,3R,4S,5S,6R)-3,4,5-tri-hydroxy-6-(hydroxymethyl)tetrahy-dro-2H-pyran-2-yl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-1H-indol-2-yl)pyrrolidin-2-one。
is a novel compound which is not reported in the literature. The structural formula is as follows:
the nuclear magnetic data are shown in table 1.
(2) Structural characterization of novel Compound 1
The structure identification related map of the compound 1 is shown in figures 1-7;
(R)-5-(3-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-((((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-1H-indol-2-yl)pyrrolidin-2-one。
is a novel compound which is not reported in the literature. Compound 1 is of the formula:
the nuclear magnetic data are shown in table 1.
TABLE 1 of Compounds 1 and 2 of the invention1H NMR and13c NMR data
3. Adipocyte triglyceride lipopexia experiment
The experimental method comprises the following steps:
3T3-L1 cells (5.0X 10)4cells/well) were seeded in 48-well plates, and 24 hours later, differentiation medium (DMEM (high glucose:4500mg/L, 1. mu.M dexamethasone, 0.5mM IBMX and 5. mu.g/mL insulin) for 3 days, the medium was then changed to a maintenance medium (DMEM containing 10% FBS (high glucose:4500mg/L and 5. mu.g/mL insulin), replacing fresh maintaining culture medium, culturing for 2 days, on day 8, the medium was aspirated, 200. mu.L of distilled water was added to each well, disrupted by sonication, and the TG content in the cell disruption solution was measured using a triglyceride liquid kit, the sample was dissolved in DMSO and added every time the medium was changed, wherein the final concentration of DMSO was 0.1%, Troglitazone was used as a positive control compound the values are expressed as the TG content increase from the control group MEAN ± SEM (n-4).<0.05,**p<0.01 (and pair)Compare to group).
The results are shown in Table 2.
TABLE 2 TG accumulation of 3T3-L1 adipocytes by Compound 1
The experimental results are as follows: the data in table 2 show that compounds 1 and 2 of the present invention have excellent effects of promoting accumulation of triglyceride in adipocytes, and both have significant promoting effects (p <0.01) at 3 μ M, and the promoting effects are enhanced with increasing concentration, i.e., have a concentration-dependent relationship.
The combination of the experiment and the experimental result shows that the indole glycoside compound has extremely strong effects of improving the sensitivity of fat cells to insulin and promoting the intake and transformation of glucose, so that the indole glycoside compound is expected to be developed into a new antidiabetic medicament; or used for preparing health food for preventing and treating diabetes.
4. In vitro anti-inflammatory assay
The experimental method comprises the following steps:
RAW264.7 cells were seeded in a 96-well plate, cultured for 24h, and LPS (lipopolysaccharide) was added. Then, different groups were added with different concentrations of samples ( inventive compounds 1 and 2, indomethacin as positive drug) and the blank group was added with an equal volume of vehicle. 50 μ L of cell culture broth mixed with an equal volume of Griess reagent I was added to the above 96-well plate followed by Griess reagent II. OD value of each group of samples under 546nm wavelength is measured by a microplate reader, NO generation inhibition rate is calculated by the following formula, and half Inhibition Concentration (IC) of the tested samples is calculated by SPSS software50Value, μ M).
NO inhibition (%) [ 1- (sample group/blank group) ] × 100%.
The experimental results are as follows:
TABLE 3 inhibitory Effect of Compounds 1 and 2 of the present invention on NO production by LPS-stimulated cells RAW264.7
And (4) analyzing results: the data in Table 3 show that the compounds 1 and 2 in the invention have good in vitro anti-inflammatory effect, have good inhibition effect on NO production of mouse RAW264.7 cells stimulated by LPS (lipopolysaccharide), and half inhibition concentration IC50The concentration of the indometacin is 25.6 +/-1.3 mu M and 30.8 +/-3.0 mu M respectively, which are better than that of the positive control medicament indometacin at 39.8 +/-3.1 mu M. The indole glycoside compounds 1 and 2 in the invention have better anti-inflammatory effect.
5. Gastric mucosa protection experiment
Healthy Kunming male mice with the body weight of about 20-25 g are randomly divided into 9 groups, and each group comprises 8 mice, namely a blank control group, a model control group, a positive control group, a compound 1 low, medium and high dose group and a compound 2 low, medium and high dose group. The low, medium and high dose groups of compounds 1 and 2 were each gavaged with 20,40 and 80mg/kg, the positive control group with omeprazole 20mg/kg, and the blank and model groups with an equal volume of saline. After 1h of administration, the stomach was perfused with 0.1ml of 70% ethanol, and the blank group was given an equal amount of physiological saline. The mice are killed by a cervical vertebra dislocation method for 1h, the stomach is taken out quickly after laparotomy, then the mice are fixed by 10% of formaldehyde for 10-15 min, the mice are cut along the greater curvature, washed by physiological saline, flattened and dried by suction. The ulcer area of the mouse was precisely calculated using a vernier caliper: calculation of ulcer index and inhibition rate was performed according to Guth's criteria.
Ulcer inhibition rate (mean model group UI-mean experimental group UI)/mean model group UI × 100%
The experimental results are as follows:
TABLE 4 inhibitory Effect of Compounds 1 and 2 of the present invention on ethanol-induced gastric ulcer in mice
And (4) analyzing results: the data in Table 4 show that the compounds 1 and 2 in the invention have certain gastric ulcer resisting effect and certain resistance effect on the gastric ulcer of mice induced by ethanol, wherein the gastric ulcer resisting effect of the compound 1 is better than that of the compound 2, and the indole glycoside compounds 1 and 2 in the invention have certain gastric ulcer resisting effect.
6. In vitro anti-hepatitis experiments
HepG2.2.15 cells were seeded in 96-well plates, and after 24h of culture samples ( inventive compounds 1 and 2, positive drug lamivudine) of different concentrations were added to each group, and an equal volume of vehicle was added to the blank group. Culturing for 6 days, collecting cell culture supernatant, uniformly detecting HBsAg and HBeAg in the supernatant by ELISA method, after color reaction, reading absorbance value at 490nm by enzyme labeling instrument, and calculating 50% drug inhibition concentration (IC 50).
TABLE 5 inhibitory Effect of Compounds 1 and 2 of the present invention on the production of HBsAg and HBeAg in HepG2.2.15 cells
And (4) analyzing results: the data in Table 5 show that the compounds 1 and 2 in the invention have better in-vitro anti-hepatitis effect, have better inhibition effect on HBsAg and HBeAg generated by HepG2.2.15 cells, and show that the indole glycoside compounds 1 and 2 in the invention have better anti-hepatitis effect.
Example 2
1) Taking 10kg of dried rhizomes of calanthe fringensis, heating and refluxing the 10kg of dried rhizomes of calanthe fringensis for 5 times by using 95 percent ethanol with the volume amount being 3 times of the mass of the dried rhizomes of calanthe fringensis 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 water, isovolumetrically extracting with petroleum ether for 4 times, isovolumetrically extracting with chloroform, ethyl acetate and n-butanol for 4 times, and removing solvent from the extraction layer under reduced pressure to obtain petroleum ether layer, chloroform layer, ethyl acetate layer and n-butanol layer.
3) Taking 70g of n-butanol extraction layer, firstly performing gradient elution by silica gel column chromatography with chloroform/methanol according to a volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 400mL, and combining the same flow portions through TLC to obtain 15 flow portions (FrB1-FrB 15).
4) Wherein the 12 th fraction FrB12 was isolated by reverse phase silica gel column chromatography with MeOH/H2And carrying out gradient elution on O according to the volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 300mL, and obtaining 12 flow portions (FrB12.1-FrB12.12) after identifying and combining the same flow portions by TLC.
5) Wherein the 2 nd fraction FrB12.2 is applied to a reverse phase silica gel column chromatography with MeOH/H2O was subjected to gradient elution at a volume ratio (v/v) of (5:95) to (100:0), and one aliquot was collected per 200mL, followed by TLC detection to identify 16 aliquots (FrB12.2.1-FrB12.2.16).
6) FrB12.2.13 was then purified by semi-preparative high performance liquid chromatography on a Megres C18 column with a methanol-water solution (25:75, v/v) as the mobile phase at a flow rate of 3.0mL/min to give Compound 1 (t: (t))R30min) and compound 2 (t)R＝34min)。
Example 3
1) Taking 10kg of dried rhizomes of calanthe fringensis, heating and refluxing for 3 times by using water with the volume of 8 times of the mass of the dried rhizomes of calanthe fringensis 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 1 time of water, isovolumetrically extracting with ethyl acetate for 4 times, isovolumetrically extracting with 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) Taking 70g of n-butanol extraction layer, firstly performing gradient elution by silica gel column chromatography with chloroform/methanol according to a volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 600mL, and combining the same flow portions through TLC detection to obtain 15 flow portions (FrB1-FrB 15).
4) Wherein the 12 th fraction FrB12 was isolated by reverse phase silica gel column chromatography with MeOH/H2And carrying out gradient elution on O according to the volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 200mL, and obtaining 12 flow portions (FrB12.1-FrB12.12) after identifying and combining the same flow portions by TLC.
5) Wherein the 2 nd fraction FrB12.2 is applied to a reverse phase silica gel column chromatography with MeOH/H2Performing gradient elution on O according to the volume ratio (v/v) of (5:95) - (100:0), and collecting every 200mLOne fraction, identified by TLC detection and the same fraction yielded 16 fractions (FrB12.2.1-FrB12.2.16).
6) FrB12.2.13 was then purified by semi-preparative HPLC using a Megres C18 column with a mobile phase of acetonitrile-water (35:65, v/v) at a flow rate of 3.0mL/min to give Compound 1 (t: (t))R28min) and compound 2 (t)R＝31min)。
Example 4
1) Taking 10kg of dried rhizomes of calanthe fringensis, heating and refluxing for extraction for 4 times by using 10% ethanol with the volume of 4 times of the mass of the dried rhizomes of calanthe fringensis 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 4 times of water, isovolumetrically extracting with ethyl acetate for 3 times, isovolumetrically extracting with n-butanol for 3 times, and removing solvent from the extraction layer under reduced pressure to obtain 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 according to a volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 500mL, and combining the same flow portions through TLC to obtain 15 flow portions (FrB1-FrB 15).
4) Wherein the 12 th fraction FrB12 was isolated by reverse phase silica gel column chromatography with MeOH/H2And carrying out gradient elution on O according to the volume ratio (v/v) of 100: 0-0: 100, collecting one flow portion per 300mL, and obtaining 12 flow portions (FrB12.1-FrB12.12) after identifying and combining the same flow portions by TLC.
5) Wherein the 2 nd fraction FrB12.2 is applied to a reverse phase silica gel column chromatography with MeOH/H2O was subjected to gradient elution at a volume ratio (v/v) of (5:95) to (100:0), and one aliquot was collected per 200mL, followed by TLC detection to identify 16 aliquots (FrB12.2.1-FrB12.2.16).
6) FrB12.2.13 was then purified by semi-preparative high performance liquid chromatography on a Megres C18 column with a methanol-water solution (25:75, v/v) as the mobile phase at a flow rate of 3.0mL/min to give Compound 1 (t: (t))R30min) and compound 2 (t)R＝34min)。
The invention discloses an indole glycoside compound and application thereof. The indole glycoside compounds are extracted and separated from dried rhizomes of calanthe fringensis, and experimental research shows that the compounds have a promoting effect on accumulation of triglyceride of 3T3-L1 fat cells, namely promoting effect on glucose uptake of the cells, and the compounds are extracted and separated from wild plants, have the characteristics of high efficiency and low toxicity, and are expected to be developed into medicines and/or health-care foods with anti-diabetic effect. Secondly, the indole glycoside compounds have good inhibition effect on NO production of mouse RAW264.7 cells stimulated by LPS, namely, the compounds have good in-vitro anti-inflammatory effect, and are expected to be developed into medicaments and/or health-care foods with anti-inflammatory effect. Moreover, the indole glycoside compounds have good inhibitory action on HBsAg and HBeAg generated by HepG2.2.15 cells, namely, the compounds have good in-vitro anti-hepatitis action and are expected to be developed into medicaments and/or health-care foods with anti-hepatitis action. In addition, the indole glycoside compound has a certain resistance effect on gastric ulcer of mice induced by ethanol, and the compound has the characteristics of high efficiency and low toxicity, so that the indole glycoside compound is expected to be developed into a novel anti-gastric ulcer medicament or used for preparing health-care food for preventing and treating gastric ulcer.
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. The indole glycoside compound is characterized in that the molecular formula of the indole glycoside compound is C24H32N2O12(ii) a The structural formula is shown as the following formula 1 and formula 2:
formula 1 and formula 2 are each a chiral isomer.
2. The method for extracting and separating an indole glycoside compound according to claim 1, comprising the steps of:
1) taking 10kg of dried rhizomes of calanthe fringensis, heating and refluxing for 4 times by using methanol with the volume 5 times of the mass of the dried rhizomes of calanthe fringensis for 3 hours each time, combining extracting solutions, and concentrating the extracting solution under reduced pressure to obtain a total extract;
2) suspending the total extract in 4 times of water to obtain extract solution, isovolumetrically extracting the extract solution with petroleum ether for 4 times, isovolumetrically extracting with ethyl acetate and n-butanol for 4 times, and removing solvent under reduced pressure to obtain petroleum ether layer, chloroform layer, ethyl acetate layer and n-butanol layer;
3) loading 70g of 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), collecting one flow component per 500mL, carrying out TLC detection on the effluent, and combining the same flow components to obtain 15 flow components, namely FrB1-FrB 15;
4) loading the 12 th flow fraction FrB12 to a reversed phase silica gel chromatographic column, taking a methanol-water system as an eluent, performing gradient elution according to the volume ratio of 100: 0-0: 100, collecting one flow fraction per 200mL, performing TLC detection on the effluent, and combining the same flow fractions to obtain 12 flow fractions, namely FrB12.1-FrB12.12;
5) loading the 2 nd flow part FrB12.2 onto 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 (5:95) - (100:0), collecting one flow part per 200mL, carrying out TLC detection on the effluent, and combining the same flow parts to obtain 16 flow parts, namely FrB12.2.1-FrB12.2.16;
6) loading FrB12.2.13 fraction to semi-preparative high performance liquid chromatography column using Megres C18 column, the mobile phase is methanol-water solution with a weight ratio of 25:75 volume ratio, flow rate of 3.0mL/min, tRWhen the reaction time is 30min, compound 1, t is obtainedRCompound 2 was obtained when 34 min.
3. The use of the indole glycoside compound of claim 1 in the preparation of an antidiabetic agent.
4. The use of the indole glycosides of claim 1 in the preparation of anti-hepatitis medicaments.
5. The use of the indole glycosides of claim 1 in the preparation of a medicament for protecting the gastric mucosa.
6. The use of the indole glycosides of claim 1 in the preparation of anti-inflammatory agents.
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| CN111690022B (en) * | 2020-05-22 | 2021-09-07 | 西安交通大学 | Alkaloid separated from radix Caulophylli, and separation method and application thereof |
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