CN114601828A - Ganoderma atrum extract and application thereof in preparing anti-inflammatory drugs - Google Patents

Ganoderma atrum extract and application thereof in preparing anti-inflammatory drugs Download PDF

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CN114601828A
CN114601828A CN202111554479.XA CN202111554479A CN114601828A CN 114601828 A CN114601828 A CN 114601828A CN 202111554479 A CN202111554479 A CN 202111554479A CN 114601828 A CN114601828 A CN 114601828A
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刘增根
方令豪
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Ruilin Beijing Biotechnology Co ltd
Northwest Institute of Plateau Biology of CAS
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Abstract

The invention belongs to the field of medicines, and particularly relates to a arenaria kansuensis extract and application thereof in preparing an anti-inflammatory drug. The extract can obviously reduce the content of NO and inflammatory factors (TNF-alpha, IL-1 beta and IL-6) generated by RAW264.7 cells or mice stimulated by Lipopolysaccharide (LPS), and has good anti-inflammatory effect. The components of the tricin, the compound salcolin B and the compound arenarine B which are separated from the extract of the arenaria kansuensis can obviously reduce the content of inflammatory factors (NO, TNF-alpha, IL-1 beta, IL-6 and PGE2), reduce the expression of COX-2 protein and prevent/treat various inflammatory symptoms. Therefore, the arenaria kansuensis extract provided by the invention has wide application prospect in preparation of safe and high-activity anti-inflammatory drugs.

Description

Arenaria kansuensis extract and application thereof in preparation of anti-inflammatory drugs
Technical Field
The invention belongs to the field of medicines, and particularly relates to a arenaria kansuensis extract and application thereof in preparing an anti-inflammatory drug.
Background
Inflammation is a common and very important pathological process, and is a defense response of living tissues with vascular systems to endogenous or exogenous stimuli. Generally, inflammation plays a positive role in the body, removes damage factors by phagocytosis of local pathogens and necrotic tissues, promotes healing of injured tissues, and is a natural physiological response. Once the balance between the body's anti-inflammatory capacity and the damage caused by inflammatory factors is lost, the inflammatory response is exacerbated. Long-term, acute inflammatory reactions can be involved in the pathological progression of many diseases, such as septic shock, arthritis, sepsis, diabetes, etc., and severe cases can initiate inflammatory storms, leading to failure of various organ functions. More and more research has shown that almost all diseases are associated with inflammation, especially chronic inflammation, the onset of which is the chronic inflammation for many metabolic diseases including cancer. The early inflammatory response involves a series of pathophysiological changes: such as the increase of capillary permeability of damaged tissues, the release of arachidonic acid metabolites and platelet activating factor, the release of pro-inflammatory factors (TNF-alpha, IL-6, IL-1, 5-hydroxytryptamine, histamine, PGE2), etc. These pathophysiological changes constitute the inflammatory response network, and NO plays a key regulatory role in the inflammatory cascade, particularly in the development of inflammatory responses and signaling. Therefore, it is necessary to find a drug effective for reducing the content of NO and inflammatory factors in the inflammatory tissue or cells
Anti-inflammatory drugs are drugs used to treat inflammatory reactions that occur after tissue damage. There are two main classes of anti-inflammatory drugs that are common today: one is steroidal anti-inflammatory drug such as hydrocortisone, prednisone, dexamethasone, etc., and the other is non-steroidal anti-inflammatory drug, i.e., antipyretic analgesic anti-inflammatory drug referred to in medical practice such as aspirin, acetaminophen, indomethacin, naproxen, naproxone, diclofenac, ibuprofen, nimesulide, rofecoxib, etc. However, after long-term administration of the medicines, organs such as liver, kidney, stomach and the like are easily damaged, and meanwhile, flora imbalance and certain vitamin deficiency are caused, so that secondary reactions such as bleeding and superinfection are caused; in addition, it is often accompanied by abnormal fever, nausea, vomiting, weakness of the whole body, and the like. Therefore, the development of natural therapeutic drugs with better curative effect, less adverse reaction and toxic and side effect and wide anti-inflammatory spectrum is urgently needed.
Gansu snow ganoderma (Arenaria kansuensis Maxim.) is a perennial cushion-shaped herbaceous plant of Anemoniaceae (Caryophyllaceae) No-Heart-vegetable (Arenaria L.) Sedum (Subgen. Eremogleastrum Williams), and mainly grows in gravel flow zone with the altitude of 4000-5000m and alpine meadow. The Tibetan medicine is named as the alzhonggabao, is prepared from whole herbs, is bitter and cold in nature and has the effects of clearing lung heat, relieving cough, reducing blood pressure, nourishing and the like, and is commonly used for treating diseases such as pneumonia, gonorrhea, scrofula, uterine diseases and the like. Meanwhile, the arenaria kansuensis is also a medicinal material with anti-inflammatory efficacy.
The basic research on drug effect substances of the arenaria kansuensis is less reported, and the Chinese patent application 'CN 107913299A extraction and preparation method of arenaria kansuensis total flavonoids with high anti-pulmonary fibrosis activity' discloses a preparation method of the arenaria kansuensis extract aiming at the anti-pulmonary fibrosis activity, wherein active substances in the extract are mainly flavonoid compounds.
However, the research on the anti-inflammatory active ingredients of the ganoderma lucidum in vivo and in vitro is still blank at present. Thus, there is no extract of ganoderma lucidum in the prior art for the purpose of anti-inflammation. The method is very unfavorable for scientific and efficient utilization and application and popularization of the arenaria kansuensis medicinal material.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a snow ganoderma lucidum extract and application thereof in preparing an anti-inflammatory drug, aiming at: preparing extract of Arenaria plant with high content of antiinflammatory active ingredient, and using it in preparation of antiinflammatory medicine.
The invention provides application of a compound salcolin B and/or arenarine B in preparation of an anti-inflammatory drug.
The invention also provides a snow ganoderma lucidum extract, which contains the following components:
5.0-50.0 mg/g of lucernetin,
salcolin B 0.3～4.0mg/g，
arenarine B 1.5～18.0mg/g。
preferably, it is prepared by the following steps: separating the Arenaria kansuensis ethanol extract by column chromatography and silica gel column chromatography in sequence, and collecting thin layer chromatography detection specific shift value RfAnd (4) concentrating or drying the fraction of 0.2-0.8 to obtain the product.
Preferably, the ethanol extract of the Arenaria kansuensis maxim is an ethanol extract of Arenaria kansuensis maxim;
and/or, the column chromatography is macroporous adsorption resin column adsorption, and the eluent of the column chromatography is ethanol eluent with the volume fraction of 25-95%;
and/or the silica gel column chromatography adopts a mixed solvent of petroleum ether, ethyl acetate and methanol as an eluent;
and/or the developing solvent of the thin-layer chromatography is a mixed solvent of petroleum ether, ethyl acetate and methanol.
Preferably, the filler of the macroporous adsorbent resin column is selected from at least one of AB-8, DM301, D101, NKA-9 or HPD300, and the elution conditions of the column chromatography are as follows:
Figure BDA0003418146050000021
Figure BDA0003418146050000031
and/or in the mixed solvent, the volume ratio of petroleum ether, ethyl acetate and methanol is (10-5): (1-5): (0.1 to 1);
and/or the fraction is a thin-layer chromatography detection specific shift value RfA fraction of 0.3 and/or 0.6;
and/or the silica gel column filler of the silica gel column chromatography is 100-300 meshes of silica gel.
The invention also provides a preparation method of the arenaria kansuensis extract, which comprises the following steps:
step 1, preparing an ethanol extract of a snow ganoderma medicinal material;
step 2, sequentially carrying out column chromatography and silica gel column chromatography separation on the ethanol extract obtained in the step 1, and collecting a thin-layer chromatography detection specific shift value RfA fraction of 0.2 to 0.8;
and 3, concentrating or drying the fraction obtained in the step 2 to obtain the product.
Preferably, the Arenaria kansuensis maxim is Arenaria kansuensis maxim.A. of Gansu; the Arenaria kansuensis medicinal material is Arenaria kansuensis whole plant, and more preferably is powder obtained by crushing Arenaria kansuensis whole plant and sieving with a 40-250 mesh sieve;
and/or, in the step 1, the method for preparing the ethanol extract comprises the following steps: mixing the Arenaria kansuensis with 5-95% ethanol by volume according to the mass-volume ratio of 1: (6-90) extracting at g/mL; the extraction method is water bath extraction or ultrasonic extraction; the extraction temperature is 10-90 ℃; the extraction time is 1-60 hours;
and/or, in the step 1, the ethanol extract is further separated and purified, and the separation and purification steps are as follows: homogenizing the ethanol extract, centrifuging, separating supernatant, separating with membrane, and concentrating the filtrate to obtain purified ethanol extract.
Preferably, the separation and purification steps are as follows: grinding the ethanol extract with colloid mill, homogenizing, centrifuging to obtain supernatant, separating the supernatant with membrane to obtain filtrate, and concentrating to obtain extract of Arenaria plant. Further preferably, the grinding times are 2-6 times; the homogenizing pressure is 1-200 Mpa; the membrane used for membrane separation is a ceramic membrane with the aperture of 0.05-10 mu m, the flow speed of supernatant liquid during membrane separation is 0.1-10 t/h, the membrane separation temperature is 25-70 ℃, and the membrane separation pressure is 0.01-3 Mpa.
Preferably, in step 2, the column chromatography is performed by macroporous adsorbent resin column adsorption, the eluent of the column chromatography is ethanol eluent with a volume fraction of 25% -95%, the filler of the macroporous adsorbent resin column is at least one selected from AB-8, DM301, D101, NKA-9 or HPD300, and the elution conditions of the column chromatography are as follows:
Figure BDA0003418146050000041
preferably, in the column chromatography process, the sample loading liquid is a liquid obtained by mixing and dissolving the extractum ethanol extract of the arenaria kansuensis and 25-95% by volume of ethanol, and the mass ratio of the extractum ethanol extract of the arenaria kansuensis to 25-95% by volume of ethanol is 1: (2-20); the mass ratio of the extractum ethanol extract of the arenaria kansuensis to the macroporous resin filler is 1: (10-30);
and/or the silica gel column chromatography adopts a mixed solvent of petroleum ether, ethyl acetate and methanol as an eluent, the developing agent of the thin-layer chromatography is a mixed solvent of petroleum ether, ethyl acetate and methanol, and the volume ratio of the petroleum ether, the ethyl acetate and the methanol in the mixed solvent is (10-5): (1-5): (0.1 to 1); the silica gel column filler of the silica gel column chromatography is 100-300 meshes of silica gel.
Preferably, in the mixed solvent adopted by silica gel column chromatography, the volume ratio of petroleum ether, ethyl acetate and methanol is 3: 1: 0.2.
preferably, the thin layer chromatography detection conditions are as follows: the developing solvent is petroleum ether, ethyl acetate and methanol, and the weight ratio of the developing solvent to the solvent is 8: 2: 0.5 volume ratio, and the color developing agent is concentrated sulfuric acid ethanol solution with volume concentration of 10%.
The invention also provides application of the arenaria kansuensis extract in preparing anti-inflammatory drugs.
The invention also provides an anti-inflammatory pharmaceutical composition, which is characterized in that: the medicine is prepared by taking compound salcolin B, compound arenarine B or the extract of the arenaria kansuensis as active ingredients and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
The dosage form of the pharmaceutical composition is preferably granules, electuary, aerosol, tablets, nasal drops, sustained release agent, film agent or capsules.
In the invention, the ethanol with the volume fraction of 5-95% refers to an ethanol water solution with the volume fraction of 5-95%. "BV" refers to column volume.
The structural formula of the medicagenin is as follows:
Figure BDA0003418146050000051
the structural formula of the compound salcolin B is as follows:
Figure BDA0003418146050000052
the structural formula of the compound arenarine B is as follows:
Figure BDA0003418146050000053
the technical scheme of the invention has the following beneficial effects:
1. the extract and the separated active compound of the arenaria kansuensis maxim can obviously reduce the content of NO and inflammatory factors (TNF-alpha, IL-1 beta and IL-6) generated by RAW264.7 cells or mice stimulated by Lipopolysaccharide (LPS) and have good anti-inflammatory effect. Particularly, the flavone (medicacin and salcolin B) and beta-carboline alkaloid (arenarine B) in the extract of the snow ganoderma lucidum can obviously reduce the content of inflammatory factors (NO, TNF-alpha, IL-1 beta, IL-6 and PGE2), reduce the expression of COX-2 protein and prevent/treat various inflammatory symptoms. The arenaria kansuensis extract provided by the invention has wide application prospect in preparation of safe and high-activity anti-inflammatory drugs or health-care foods.
2. The preparation raw materials of the arenaria kansuensis extract are the arenaria kansuensis whole plants, the resource distribution is wide, the reserve capacity is large, the large-area planting can be realized, the quality is easy to control, and the production cost of the anti-inflammatory drug is effectively reduced.
3. In a preferred scheme, the preparation of the arenaria kansuensis extract adopts a colloid mill-high-pressure homogenization-membrane separation combined technology, so that the extraction rate and yield of active ingredients of medicinal materials can be effectively improved, the consumption of an extraction solvent in a production process is reduced, pectin, tannin, mucilage, bacteria, silt and some macromolecular substance impurities in an extracting solution can be effectively removed, the equipment investment and energy consumption are reduced, and the arenaria kansuensis extract is stable in property and simple in preparation process.
4. In a preferred scheme, the preparation of the arenaria kansuensis extract adopts macroporous resin enrichment and silica gel column chromatography separation processes, so that the purity of the anti-inflammatory active ingredients in the extract is improved (the content of total flavonoids is more than or equal to 70mg/g, the content of beta-carboline alkaloid is more than or equal to 10mg/g, and the figure 1), and the macroporous resin and the silica gel can be regenerated and recycled, so that the production cost can be effectively reduced. Furthermore, the content of the flavonoid component of the alfalfa element and the Salcolin B in the extract is 5.0-50.0 mg/g and 0.3-4.0 mg/g respectively, and the content of the beta-carboline alkaloid component of the Arenarine B is 1.5-18.0 mg/g.
5. The preparation method of the arenaria kansuensis extract is simple in production process, easy in control of technical parameters and wide in application range, and can be used for industrial large-scale production to form a biological medicine product industrial chain.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is an HPLC chromatogram of the extract of Ganoderma snowflake of example 1, wherein the names of the identified compounds are Tricin (Tricin), Salcolin B and Arenarine B;
FIG. 2 shows the effect of the compounds Tricin, Salcolin B and Arenarine B in the extract of Ganoderma sinense on the viability of RAW264.7 macrophages (A) and on the content of NO in macrophages stimulated by LPS (B). Wherein: compared to the blank control, # P < 0.01; p <0.01, P <0.05 compared to LPS model group;
FIG. 3 shows the effect of compounds Tricin, Salcolin B and Arenarine B in extracts of Ganoderma sinense on the levels of IL-1 β (A), IL-6(B), TNF- α (C) and PGE2(D) inflammatory factors in LPS-stimulated RAW264.7 macrophages. Wherein: compared to blank control, # P < 0.01; p <0.01, P <0.05 compared to LPS model group.
FIG. 4 Effect of compounds Tricin (A), Salcolin B (B) and Arenarine B (C) in the extract of Ganoderma sinense on the expression of COX-2 protein levels in RAW264.7 macrophages stimulated by LPS. Wherein: compared to the blank control, # P < 0.01; p <0.01, P <0.05 compared to LPS model group.
Detailed Description
The raw materials, consumables and equipment used in the invention are all known products, and are obtained by purchasing products sold in the market.
Example 1 preparation of Tibetan drug Gansu snow Ganoderma extract
The method comprises the steps of manually selecting, cleaning and removing impurities from the whole grass of Gansu Xue ganoderma, drying the grass in the shade, crushing the grass and sieving the crushed grass with a 40-mesh sieve to obtain Gansu Xue ganoderma medicinal material powder;
the Arenaria kansuensis medicinal material powder and 80% of ethanol in volume fraction are added in an amount of 1: heating in water bath at 70 deg.C for extraction at a ratio of 20(g/mL) to obtain extractive solution 24 hr;
thirdly, grinding the extracting solution for 6 times by using a colloid mill, homogenizing under the pressure of 1Mpa, and performing centrifugal separation to obtain a supernatant;
fourthly, separating and removing impurities from the supernatant through a ceramic membrane with the aperture of 2 microns at the flow speed of 0.5t/h at the temperature of 65 ℃ and the pressure of 1Mpa to obtain filtrate, and concentrating the filtrate under reduced pressure to obtain a ganoderma lucidum extract-like extract;
fifthly, mixing the arenaria kansuensis extractum extract with 75% by volume of ethanol according to a ratio of 1: 10, adsorbing by AB-8 macroporous adsorption resin, eluting by 6BV pure water to remove sugar, eluting by 25 percent, 45 percent, 75 percent and 95 percent ethanol step gradient elution of 4BV, 2BV, 4BV and 2BV respectively, collecting 25 percent and 75 percent ethanol eluent, mixing, and concentrating under reduced pressure to obtain extractum to obtain crude extract of the arenaria kansuensis extract. Wherein the mass ratio of the arenaria kansuensis extract to the AB-8 macroporous resin is 1: 8 (g/g);
sixthly, separating the crude product of the snow ganoderma lucidum extract by silica gel column chromatography, eluting by using a petroleum ether/ethyl acetate/methanol mixed solvent with the volume of 3 times of the column volume, collecting fractions according to 200mL of each fraction, and detecting and collecting a specific displacement value (R) by using thin-layer chromatographyf) The fractions of 0.3 and 0.6 were concentrated and dried under reduced pressure to obtain yellow powdered extract of Arenaria kansuensis with yield of 0.54% based on the whole plant of Arenaria kansuensis. Wherein, the mixed solvent of petroleum ether/ethyl acetate/methanol refers to that the mixed solvent of petroleum ether, ethyl acetate and methanol is prepared according to the ratio of 6: 1: a solvent obtained by mixing at a volume ratio of 0.2 (mL/mL/mL); the mass ratio of the crude extract of the arenaria kansuensis to the silica gel filler is 1: 15 (g/g); the size of the silica gel column is 50mm multiplied by 80 mm; the specification of the silica gel is 100-200 meshes; conditions for thin layer chromatography detection: the developing solvent is petroleum ether/ethyl acetate/methanol according to the weight ratio of 8: 2: a mixed solvent mixed by a volume ratio (mL/mL/mL) of 0.5, wherein the color developing agent is concentrated sulfuric acid ethanol solution with the volume concentration of 10%; the vacuum concentration and drying conditions refer to that the vacuum degree is 0.06MPa and the temperature is 50 ℃.
In this example, the chromatogram obtained after HPLC detection of the prepared ganoderma lucidum extract is shown in fig. 1A, and the chromatogram of the reference products of Tricin (Tricin), Salcolin B and Arenarine B is shown in fig. 1B.
HPLC chromatographic conditions: german VDSP pher C18-AQ column (4.6mm × 250mm,5 μm), isocratic-gradient mixed elution with 0.2% formic acid (a) and acetonitrile (B) as mobile phase: 0-30min, 14% B; 30-50min, 14-19% B; 50-80min, 19% B; 80-100min, 19-27% B; 100-165min, 27% B. The sample injection amount is 20 mu L, the flow rate is 1.0mL/min, the column temperature is 35 ℃, and the detection wavelength is 360 nm.
As can be seen from FIG. 1, the extracts prepared in this example contained Tricin (Tricin), Salcolin B and Arenarine B. According to the calculation of the chromatographic peak area, the contents of the three compounds of Tricin (Tricin), Salcolin B and Arenarine B are 43.69, 2.84 and 13.72mg/g in sequence.
Example 2 preparation of extract of Kansu Katsumadai Ganoderma
The method comprises the steps of manually selecting, cleaning and removing impurities from the whole grass of Gansu Xue ganoderma, drying the grass in the shade, crushing the grass and sieving the crushed grass with a 250-mesh sieve to obtain Gansu Xue ganoderma medicinal material powder;
the Arenaria kansuensis medicinal material powder and ethanol with the volume fraction of 60% are added in an amount of 1: carrying out water bath ultrasonic extraction at the temperature of 50 ℃ according to the feed-liquid ratio of 6(g/mL), and obtaining an extracting solution after 1 h;
thirdly, grinding the extracting solution for 2 times by using a colloid mill, homogenizing under the pressure of 200Mpa, and performing centrifugal separation to obtain a supernatant;
fourthly, separating and removing impurities from the supernatant through a ceramic membrane with the aperture of 10 mu m at the flow speed of 0.1t/h at the temperature of 25 ℃ and the pressure of 0.01Mpa to obtain filtrate, and concentrating under reduced pressure to obtain the arenaria kansuensis extract;
fifthly, mixing the arenaria officinalis extract-like extract with 60% ethanol by volume according to a ratio of 1: 20, adsorbing by a DM301 macroporous adsorption resin, eluting by 6BV pure water to remove sugar, performing gradient elution by 25%, 45%, 75% and 95% ethanol step by step with 6BV, 2BV, 6BV and 2BV, collecting 25% and 75% ethanol eluent, mixing, and concentrating under reduced pressure to obtain extractum to obtain a crude product of the arenaria kansuensis extract. Wherein the mass ratio of the arenaria kansuensis extract to the AB-8 macroporous resin is 1: 10 (g/g);
sixthly, separating the crude product of the snow ganoderma lucidum extract by silica gel column chromatography, eluting by using a mixed solvent of petroleum ether, ethyl acetate and methanol with the volume of 6 times of the column volume, collecting fractions according to 500mL of each fraction, and detecting and collecting a specific displacement value (R) by using thin-layer chromatographyf) The fractions of 0.2 and 0.8 were concentrated and dried under reduced pressure to obtain yellow powdered extract of Arenaria kansuensis with yield of 0.46% based on the whole plant of Arenaria kansuensis. Wherein, the mixed solvent of petroleum ether/ethyl acetate/methanol refers to that the mixed solvent of petroleum ether, ethyl acetate and methanol is prepared according to the ratio of 5: 5: 1 (mL/mL/mL) in a volume ratio of 1; the mass ratio of the crude product extracted from the arenaria kansuensis to the silica gel filler is 1: 30 (g/g); the size of the silica gel column is 100mm multiplied by 200 mm; the specification of the silica gel is 100-200 meshes; conditions for thin layer chromatography detection: the developing solvent is petroleum ether/ethyl acetate/methanol according to the weight ratio of 8: 2:a mixed solvent mixed by a volume ratio (mL/mL/mL) of 0.5, wherein the color developing agent is concentrated sulfuric acid ethanol solution with the volume concentration of 10%; the vacuum concentration and drying conditions refer to that the vacuum degree is 0.07MPa and the temperature is 70 ℃.
Example 3 preparation of Tibetan drug Gansu snow Ganoderma extract
The method comprises the steps of manually selecting, cleaning and removing impurities from the whole grass of Gansu xuelian ganoderma, drying the grass in the shade indoors, crushing the grass, and sieving the crushed grass with a 60-mesh sieve to obtain Gansu xuelian ganoderma medicinal material powder;
the preparation method comprises the following steps of mixing the arenaria kansuensis medicinal material powder with 5% of ethanol by volume percentage according to 1: heating and extracting in water bath at 90 deg.C for 60 hr to obtain extractive solution with a material-to-liquid ratio of 90 (g/mL);
thirdly, grinding the extracting solution by a colloid mill for 4 times, homogenizing under the pressure of 100Mpa, and performing centrifugal separation to obtain a supernatant;
fourthly, separating and removing impurities from the supernatant through a ceramic membrane with the aperture of 1 mu m at the flow speed of 0.5t/h at the temperature of 50 ℃ and the pressure of 0.05Mpa to obtain filtrate, and concentrating under reduced pressure to obtain the arenaria kansuensis extract;
fifthly, mixing the arenaria kansuensis extractum extract with 20% ethanol by volume according to a ratio of 1: 15, adsorbing by D101 macroporous adsorption resin, eluting by 6BV pure water to remove sugar, eluting by 25%, 45%, 75% and 95% ethanol step by 5BV, 2BV, 5BV and 2BV, collecting 25% and 75% ethanol eluent, mixing, and concentrating under reduced pressure to obtain extractum to obtain crude product of the extract of the arenaria kansuensis. Wherein the mass ratio of the arenaria kansuensis extract to the D101 macroporous resin is 1: 10 (g/g);
sixthly, separating the crude product of the snow ganoderma lucidum extract by silica gel column chromatography, eluting by using a mixed solvent of petroleum ether, ethyl acetate and methanol with the volume of 6 times of the column volume, collecting fractions according to 300mL of each fraction, and detecting and collecting a specific displacement value (R) by using thin-layer chromatographyf) The fractions of 0.3 and 0.7 were concentrated and dried under reduced pressure to obtain yellow powdered extract of Arenaria kansuensis with yield of 0.60% based on the whole plant of Arenaria kansuensis. Wherein, the mixed solvent of petroleum ether/ethyl acetate/methanol refers to that the mixed solvent of petroleum ether, ethyl acetate and methanol is prepared according to the ratio of 7: 2: mixing at a volume ratio of 0.4 (mL/mL/mL)The solvent thus obtained; the mass ratio of the crude product extracted from the arenaria kansuensis to the silica gel filler is 1: 10 (g/g); the size of the silica gel column is 150mm multiplied by 200 mm; the specification of the silica gel is 100-200 meshes; conditions for thin layer chromatography detection: the developing solvent is petroleum ether/ethyl acetate/methanol according to the weight ratio of 8: 2: a mixed solvent mixed by a volume ratio (mL/mL/mL) of 0.5, wherein the color developing agent is concentrated sulfuric acid ethanol solution with the volume concentration of 10%; the vacuum concentration and drying conditions refer to that the vacuum degree is 0.08MPa and the temperature is 60 ℃.
Control example 1 preparation method of control Tibetan drug Gansu snow Ganoderma extract
(1) Manually picking, cleaning and removing impurities from the whole plant of Gansu xue ganoderma, drying in the shade, crushing, and sieving with a 60-mesh sieve to obtain the medicinal powder of Xuezhi ganoderma;
(2) mixing the snow ganoderma medicinal material powder with 75% of ethanol by volume percent according to the proportion of 1: heating in water bath at 70 deg.C for extraction at a ratio of 25(g/mL) to obtain Arenaria kansuensis extract after 3 hr;
(3) concentrating the Arenaria plant extract under reduced pressure, and drying to obtain Arenaria plant extract; wherein, the vacuum concentration and drying conditions refer to that the vacuum degree is 0.07MPa and the temperature is 55 ℃.
The beneficial effects of the present invention are demonstrated by the following experimental examples.
Experimental example 1 anti-inflammatory Effect test of the extract of Ganoderma sinense of the invention
1. Experimental methods
(1) Cell viability and inflammatory factor detection:
RAW264.7 macrophage (1 × 10) with good growth state4Per well) were cultured in 96-well plates. Wherein, each well is added with 1mL DMEM medium containing 10% fetal calf serum and placed in 5% CO2The culture was carried out in an incubator (37 ℃) for 4 hours. The cells were then randomly grouped into blank (cells + medium), LPS (1. mu.g/mL LPS + cells), experimental (different mass concentrations of extract of either example or control case of Arenaria kansuensis + 1. mu.g/mL LPS + cells), 3 replicates per group. In the examples or the control example, the Ganoderma atrum extract pretreatment RAW264.7 macrophage mass concentration is 100 (high dose group), 50 (middle dose group) and 10 (low dose group) mug/mL respectively, LPS stimulation cells with the final mass concentration of 1 mug/mL are added, and after 24h, the supernatant is obtainedThe expression of the inflammatory factors TNF-alpha, IL-1 beta and IL-6 is detected by an ELISA kit. In addition, the toxicity of the extract of Ganoderma lucidum of examples or control on RAW264.7 macrophages was measured by MTT method, and the OD value (570nm) in each well of 96-well plate was measured by microplate reader, and the cell viability was ═ ODAdministration of drugs-ODBlank space)/(ODControl-ODBlank space)×100％
(2) The Griess method is used for detecting the NO content of the cells:
preparation of Single cell suspensions (5X 10)4mL), placed in 96-well plates (100. mu.L/well) and cultured for 12 h. After grouping treatment of cells, 50 mu L/hole of cell supernatant is taken to operate according to the instruction of the NO detection kit, the OD value is measured at 545nm, and the content of NO secreted by the cells is calculated according to a standard curve.
(3) Evaluation of the anti-inflammatory activity in vitro of the compounds tricin, salcolin B and arenarine B:
when the monomeric compounds of tricin, salcolin B and arenarine B in the extract of the arenaria kansuensis of the examples or the control examples are evaluated for the anti-inflammatory activity of the cells, the experimental procedures are the same except that the administration dosage in the experimental group is different and the compound is respectively used for replacing the extract of the arenaria kansuensis. In addition, PGE2 content was measured according to the instructions of its Elisa kit, and COX-2 protein level was measured according to the protocol of the western blot experiment.
Medicaginin (tricin), salcolin B and arenarine B were isolated from the extract of Arenaria plant of example 1 by the following method:
the extract was subjected to silica gel column chromatography using ethyl acetate: methanol: elution was performed with a water (10: 1: 0.1, 8: 2: 0.5, 7: 3: 1) system and combined according to TLC detection to give 4 fractions, of which 2 and 3 fractions were subjected to HPLC (70% acetonitrile containing 0.2% formic acid as mobile phase at a flow rate of 15mL/min) with a Xamide preparative column to give compounds salcolin B, tricin and arenarine B, respectively.
(4) Determination of NO and inflammatory factor content in mouse serum:
the anti-inflammatory activity of the extract of Ganoderma lucidum of the examples or the control examples was evaluated by LPS-stimulated modeling. The experimental procedure was as follows: the blank group was not treated with any drug, and the experimental group was gavaged with 100 (high dose group), 50 (medium dose group), 10 (low dose group) mg/kg of the extract of the snow ganoderma lucidum of the examples or the control example, respectively, and 24 hours later, was intraperitoneally stimulated with LPS (10. mu.g/kg) together with the model group for 2 hours. Among them, the number of experimental mice per group was 7. Taking eyeball and blood, centrifuging and taking supernatant, and measuring the content of inflammatory factors (TNF-alpha, IL-1 beta and IL-6) according to the operation flow of an ELISA kit. And simultaneously, measuring the content of NO in serum according to the NO detection kit instruction.
2. Results of the experiment
The results of the experiments are shown in tables 1-3 and FIGS. 2-4.
TABLE 1 Effect of extract of Ganodermataceae on LPS stimulation of NO, TNF-alpha, IL-1 beta and IL-6 production by RAW264.7 cells (n ═ 3)
Figure BDA0003418146050000111
Note: compared to the blank control, # P < 0.01; p <0.01, P <0.05 compared to LPS model group; compared with the control arenaria kansuensis extract under the same dosage, the P is less than 0.05.
TABLE 2 influence of extract of Arenaria plant on the levels of NO and inflammatory factors (TNF-. alpha., IL-1. beta. and IL-6) in the serum of LPS-stimulated mice results (n. 7)
Figure BDA0003418146050000112
Figure BDA0003418146050000121
Note: compared to blank control, # P < 0.01; p <0.01, P <0.05 compared to LPS model group; p <0.05 compared to the control ganoderma lucidum extract at the same dose.
TABLE 3 Effect of treatment with Arenaria plant extract on RAW264.7 cell growth
Figure BDA0003418146050000122
Figure BDA0003418146050000131
As can be seen, compared with the blank control group, the content of each inflammatory factor in the LPS model group is obviously increased, which indicates that the molding is successful; compared with an LPS model group, the arenaria kansuensis extract obtained in the embodiment 1-3 can effectively reduce the content of NO, TNF-alpha, IL-1 beta and IL-6 generated by LPS stimulation RAW264.7 macrophage and mice, and shows good anti-inflammatory activity; especially the medium and high dose groups were able to significantly reduce the expression levels of NO and inflammatory factors in macrophages and mice. Compared with the control arenaria kansuensis extract, the arenaria kansuensis extract obtained by extraction in the embodiments 1-3 of the invention has obviously improved anti-inflammatory activity (P <0.05) under the same dosage. Meanwhile, treatment with the extract of arenaria kansuensis has no significant effect on the normal growth of RAW264.7 macrophages (table 3).
In addition, 3 monomeric compounds, which were isolated from the extract of Arenaria plant of example 1, were tricin (tricin), salcolin B and arenarine B, respectively, the first two compounds belonging to flavonoids and the third to the β -carboline alkaloid fraction. By evaluating the anti-inflammatory activity of the cells of these 3 compounds, it was found that: (1) compared with the blank group, the content of NO and various inflammatory factors is obviously increased, which indicates that the molding is successful; (2) none of the 3 compounds (200, 100, 50, 25, 10, 5 μ M) was cytotoxic (fig. 2A); (4) compared with the model group, 5-200 μ M of lucernin, 25-200 μ M of salcolin B and 50-200 μ M of arenarine B can significantly reduce the secretion of NO in the cells (FIG. 2B); (5) compared with the model group, the medicanin, the salcolin B and the arenarine B with different concentrations can reduce the content of inflammatory factors in cells, and particularly can obviously reduce the content of IL-1 beta in the cells (figure 3); (6) both 10-100 μ M of medicacin and salcolin B significantly reduced COX-2 protein expression compared to the model group (FIG. 4). Therefore, the tricin, salcolin B and arenarine B compounds separated from the extract of the Arenaria kansuensis of example 1 can reduce the expression of NO and inflammatory factors (TNF-alpha, IL-1 beta and IL-6) in cells induced by LPS and show stronger anti-inflammatory bioactivity, and the tricin and salcolin B can obviously reduce the content of PGE2 and lower the level of COX-2 protein so as to achieve the effect of eliminating inflammation.
In conclusion, the invention provides a arenaria kansuensis extract and application thereof in preparing anti-inflammatory drugs. The extract can remarkably reduce the expression level of NO and inflammatory factor in macrophage and mouse stimulated by LPS, has good anti-inflammatory effect, and can prevent/treat pneumonia, hepatitis, gynecological inflammation diseases, etc. The arenaria kansuensis extract provided by the invention has wide application prospect in preparation of safe and high-activity anti-inflammatory drugs.

Claims (10)

1. The application of the compound salcolin B and/or arenarine B in preparing anti-inflammatory drugs.
2. A Arenaria kansuensis extract is characterized in that: it comprises the following components:
5.0-50.0 mg/g of lucernetin,
salcolin B 0.3～4.0mg/g，
arenarine B 1.5～18.0mg/g。
3. the Arenaria kansuensis extract of claim 2, wherein: the preparation method comprises the following steps: separating the Arenaria kansuensis ethanol extract by column chromatography and silica gel column chromatography in sequence, and collecting thin layer chromatography detection specific shift value RfAnd (4) concentrating or drying the fraction of 0.2-0.8 to obtain the product.
4. The Arenaria kansuensis extract of claim 3, wherein: the ethanol extract of the Arenaria kansuensis maxim is an ethanol extract of Arenaria kansuensis maxim;
and/or, the column chromatography is macroporous adsorption resin column adsorption, and the eluent of the column chromatography is ethanol eluent with the volume fraction of 25-95%;
and/or, the silica gel column chromatography adopts a mixed solvent of petroleum ether, ethyl acetate and methanol as an eluent;
and/or the developing solvent of the thin-layer chromatography is a mixed solvent of petroleum ether, ethyl acetate and methanol.
5. The Arenaria kansuensis extract of claim 4, wherein: the filler of the macroporous adsorption resin column is selected from at least one of AB-8, DM301, D101, NKA-9 or HPD300, and the elution conditions of the column chromatography are as follows:
Figure FDA0003418146040000011
and/or in the mixed solvent, the volume ratio of petroleum ether, ethyl acetate and methanol is (10-5): (1-5): (0.1 to 1);
and/or the fraction is a thin-layer chromatography detection specific shift value RfA fraction of 0.3 and/or 0.6;
and/or the silica gel column filler of the silica gel column chromatography is 100-300 meshes of silica gel.
6. The method for preparing the Arenaria plant extract as claimed in any of claims 2-5, comprising the steps of:
step 1, preparing an ethanol extract of a Arenaria kansuensis medicinal material;
step 2, sequentially carrying out column chromatography and silica gel column chromatography separation on the ethanol extract obtained in the step 1, and collecting a thin-layer chromatography detection specific shift value RfA fraction of 0.2 to 0.8;
and 3, concentrating or drying the fraction obtained in the step 2 to obtain the product.
7. The method of claim 6, wherein: the Arenaria kansuensis maxim is a Gansu Arenaria kansuensis maxim;
and/or, in the step 1, the method for preparing the ethanol extract comprises the following steps: mixing the Arenaria kansuensis with 5-95% ethanol by volume according to the mass-volume ratio of 1: (6-90) extracting at g/mL; the extraction method is water bath extraction or ultrasonic extraction; the extraction temperature is 10-90 ℃; the extraction time is 1-60 hours;
and/or, in the step 1, the ethanol extract is further separated and purified, and the separation and purification steps are as follows: homogenizing the ethanol extract, centrifuging, separating supernatant, performing membrane separation, and concentrating the filtrate to obtain purified ethanol extract.
8. The method of claim 6, wherein: in the step 2, the column chromatography is macroporous adsorption resin column adsorption, the eluent of the column chromatography is ethanol eluent with the volume fraction of 25-95%, the filler of the macroporous adsorption resin column is at least one selected from AB-8, DM301, D101, NKA-9 or HPD300, and the elution condition of the column chromatography is as follows:
Figure FDA0003418146040000021
and/or the silica gel column chromatography adopts a mixed solvent of petroleum ether, ethyl acetate and methanol as an eluent, the developing agent of the thin-layer chromatography is a mixed solvent of petroleum ether, ethyl acetate and methanol, and the volume ratio of the petroleum ether, the ethyl acetate and the methanol in the mixed solvent is (10-5): (1-5): (0.1 to 1); the silica gel column filler of the silica gel column chromatography is 100-300 meshes of silica gel.
9. Use of the extract of Arenaria plant according to any one of claims 2-5 for the preparation of an anti-inflammatory agent.
10. An anti-inflammatory pharmaceutical composition characterized by: the medicine is prepared by taking compound salcolin B, compound arenarine B or the extract of the arenaria kansuensis as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
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