Rheum tanguticum extract as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and mainly relates to a rheum tanguticum extract as well as a preparation method and application thereof. The invention provides a rheum tanguticum extract which is obtained by performing ethanol microwave extraction on rheum tanguticum and then performing membrane filtration and macroporous resin separation. By adopting the extraction method disclosed by the invention, the extraction rate of total anthraquinone is high, and meanwhile, the prepared rheum tanguticum extract is safe and has a good anti-inflammatory effect.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a rhubarb tangut extract, a preparation method and an application thereof. Background Art

[0002] Rhubarb is one of the most important Chinese medicinal materials with wide application, and is also a commonly used medicine in traditional Chinese medicine. It has been recorded in Shennong's Herbal Classic, Treatise on Febrile and Miscellaneous Diseases, and Compendium of Materia Medica. Rhubarb, also known as General, Fire Ginseng, etc., mainly comes from the plants of the Liao family, Rheum palmatum, Rheum tanguticum, and medicinal rhubarb. Rhubarb is a perennial herb, and the medicinal part is the dried root and rhizome. It has many functions such as detoxification, clearing away heat and fire, promoting menstruation and removing blood stasis, cooling blood and stopping bleeding, and has always had huge market demand. Rheum tanguticum Maxim.ex Balf., Polygonaceae, Rheum genus, also known as chicken claw rhubarb, has strong cold resistance and grows mostly in mountain forest edges, shrubs, and grass slopes at 2000-4600 meters. It is one of the main sources of rhubarb.

[0003] Rhubarb tangutica contains a variety of chemical components such as anthraquinone, anthrone, tannin, diphenylethylene, flavonoids, polysaccharides, etc. Rhubarb tangutica has also been reported to have certain anti-inflammatory activity, but the specific anti-inflammatory active components of rhubarb tangutica are unclear, and the existing extraction methods have a low extraction rate for effective components. For example, the extraction rate of total anthraquinones in rhubarb tangutica by the existing technology is generally below 1.5%, and the extract obtained by further separation has poor anti-inflammatory effect. Therefore, it is particularly important to provide a preparation method for a rhubarb tangutica extract with a high extraction rate of anti-inflammatory components and good anti-inflammatory effect. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a Rheum tanguticum extract, a preparation method and application thereof.

[0005] The present invention provides a method for preparing a rhubarb tangut extract, comprising the following steps:

[0006] Tangut rhubarb is mixed with an ethanol solution with a volume fraction of 5 to 95%, and subjected to microwave extraction to obtain a crude ethanol extract;

[0007] The crude ethanol extract was dissolved in water, filtered through a membrane, and the filtrate was collected;

[0008] The filtrate is poured into a macroporous adsorption resin column and loaded with water, an ethanol solution with a volume fraction of 20 to 80%, and an ethanol solution with a volume fraction of 85 to 95% are used as eluents for elution in sequence, and then the ethanol eluate with a volume fraction of 85 to 95% is collected, and the ethanol is removed to obtain the rhubarb tangut extract.

[0009] Preferably, the solid-liquid ratio of the rhubarb tanguticus and the ethanol solution with a volume fraction of 5 to 95% is 1 g: (5 mL to 30 mL).

[0010] Preferably, the microwave extraction is performed at a temperature of 60 to 95° C. and a power of 200 W to 900 W for 1 to 4 times.

[0011] Preferably, during the microwave extraction, each extraction takes 10 to 90 minutes.

[0012] Preferably, the membrane filtration includes ceramic membrane filtration or organic membrane filtration.

[0013] Preferably, the particle size of the ceramic membrane is 0.1 μm to 0.5 μm.

[0014] Preferably, the molecular retention capacity of the organic membrane is 10,000 to 50,000 Da.

[0015] Preferably, the model of the macroporous adsorption resin column includes D101 or AB-8.

[0016] The present invention also provides a rheum tanguticum extract, which is prepared by the above-mentioned preparation method.

[0017] The present invention also provides the preparation method or application of the Rheum tanguticum extract in the preparation of anti-inflammatory products.

[0018] Beneficial effects of the present invention:

[0019] The invention provides a rhubarb tangut extract, which is obtained by extracting rhubarb tangut through ethanol microwave, and then filtering through a membrane and separating through a macroporous resin. The extraction method of the invention has a high extraction rate for total anthraquinones, and the prepared rhubarb tangut extract is safe and has a good anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the total ion current of the mass spectrometer detected by Rheum tanguticum extract.

[0021] Figure 2 This is a microscopic observation of the inhibitory effect of Rheum tanguticum extract on CuSO4-induced inflammatory response.

[0022] Figure 3 This is a statistical analysis chart of the inhibitory effect of Rheum tanguticum extract on CuSO4-induced inflammatory response; ## indicates a very significant difference compared with the blank control group (Ctl) (P<0.01), *** indicates a very significant difference compared with the model group (Model) (P<0.001).

[0023] Figure 4The figures are the effects of Rheum tanguticum extract on the gene expression levels of inflammatory factors; A is the effect on the gene expression level of IL-1; B is the effect on the gene expression level of IL-2; C is the effect on the gene expression level of TNF-α; D is the effect on the gene expression level of NF-κB; ## indicates a very significant difference compared with the blank control group (Ctl) (P<0.01); * indicates a significant difference compared with the model group (Model) (P<0.05); ** indicates a very significant difference compared with the model group (Model) (P<0.01). DETAILED DESCRIPTION

[0024] The present invention provides a method for preparing a rhubarb tangut extract, comprising the following steps:

[0025] Tangut rhubarb is mixed with an ethanol solution with a volume fraction of 5 to 95%, and subjected to microwave extraction to obtain a crude ethanol extract;

[0026] The crude ethanol extract was dissolved in water, filtered through a membrane, and the filtrate was collected;

[0027] The filtrate is poured into a macroporous adsorption resin column and loaded with water, an ethanol solution with a volume fraction of 20 to 80%, and an ethanol solution with a volume fraction of 85 to 95% are used as eluents for elution in sequence, and then the ethanol eluate with a volume fraction of 85 to 95% is collected, and the ethanol is removed to obtain the rhubarb tangut extract.

[0028] The rhubarb used in the present invention is the dried root and rhizome of the plant Rheum tanguticum Maxim.exBalf. of the Rhizomeaceae family. The present invention has no special limitation on the source of the rhubarb, and conventional commercial products in the art can be used.

[0029] As an embodiment, Tanggut rhubarb is mixed with an ethanol solution with a volume fraction of 5 to 95%, and the volume fraction of the ethanol solution is preferably 40% to 85%, more preferably 70% to 80%, and further preferably 75%; the solid-liquid ratio of the Tanggut rhubarb mixed with the ethanol solution is preferably 1g: (5mL to 30mL), more preferably 1g: (15mL to 25mL), and further preferably 1g: 20mL. After mixing, microwave extraction is performed; the microwave extraction is preferably extracted 1 to 4 times at a temperature of 60 to 95°C with a power of 200W to 900W, and the temperature of the microwave extraction is more preferably 70 to 90°C, and further preferably 80°C; the power of the microwave extraction is more preferably 600 to 800W, and further preferably 700W; the number of extractions in one embodiment can be selected from 1, 2, 3, and 4 times. The time for each extraction is preferably 10 to 90min, more preferably 30min to 80min, and further preferably 40min. After the microwave extraction, the extract may be preferably concentrated to obtain a crude ethanol extract. The present invention does not specifically limit the concentration method, and conventional concentration methods in the art may be used. In one embodiment, reduced pressure concentration may be selected.

[0030] As an embodiment, the obtained crude ethanol extract is dissolved in water, filtered through a membrane, and the filtrate is collected; the membrane filtration preferably includes ceramic membrane filtration or organic membrane filtration; the particle size of the ceramic membrane is preferably 0.1μm to 0.5μm, and in one embodiment, 0.1μm, 0.2μm or 0.5μm can be selected; the molecular retention capacity of the organic membrane is preferably 10000 to 50000Da, and in one embodiment, 10000Da, 20000Da, 50000Da can be selected.

[0031] As an embodiment, the collected filtrate is poured into a macroporous adsorption resin column for sample loading, and the model of the macroporous adsorption resin preferably includes D101 or AB-8; after loading, water, ethanol solution with a volume fraction of 20-80%, and ethanol solution with a volume fraction of 85%-95% are used as eluents for elution in turn, and then the ethanol eluent with a volume fraction of 85%-95% is collected; in one embodiment, the ethanol solution with a volume fraction of 20-80% can be eluted by selecting one or more of 20%, 30%, 40%, 50%, 60%, 70% or 80% ethanol solution for gradient elution; the 85%-95% ethanol solution can be eluted by selecting one of 85%, 90% or 95% and collecting the corresponding ethanol eluent. The collected ethanol eluent is de-ethanoled to obtain a rhubarb extract. The present invention does not specifically limit the method for removing ethanol, and conventional methods in the art can be used.

[0032] The present invention also provides a rheum tanguticum extract, which is prepared by the above-mentioned preparation method. The total anthraquinone content of the rheum tanguticum extract of the present invention is as high as 85%.

[0033] The present invention also provides the use of the preparation method or the Rheum tanguticum extract in the preparation of anti-inflammatory products. The Rheum tanguticum extract prepared by the preparation method of the present invention is safe and has good anti-inflammatory effect.

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] Example 1

[0038] (1) Add 20 times (1 g: 20 mL) of 75% ethanol solution to the rhubarb root, perform microwave extraction once at 80°C and 700 W power for 40 min, and collect the extract; concentrate the extract under reduced pressure of 0.05 MPa to obtain a crude ethanol extract;

[0039] (2) dissolving the crude ethanol extract in step (1) in water, filtering and removing impurities with an organic membrane with a molecular cutoff of 50,000 Da, and collecting the filtrate;

[0040] (3) pouring the filtrate in step (2) into a D101 macroporous adsorption resin column and loading the column, and eluting with 3 times the column volume of water, 3 times the column volume of 30% ethanol solution, 3 times the column volume of 60% ethanol solution, and 3 times the column volume of 90% ethanol solution as eluents, then collecting the eluent with a volume fraction of 90% ethanol solution, removing ethanol, and obtaining the tangutic rhubarb extract.

[0041] Example 2

[0042] (1) Add 30 times (1 g: 30 mL) of 95% ethanol solution to the rhubarb root, perform microwave extraction twice at 60°C and 900 W power for 90 min each time, and combine the extracts; concentrate the extracts under reduced pressure at 0.02 MPa to obtain a crude ethanol extract;

[0043] (2) dissolving the crude ethanol extract in step (1) in water, filtering and removing impurities with an organic membrane with a molecular cutoff of 10,000 Da, and collecting the filtrate;

[0044] (3) pouring the filtrate in step (2) into an AB-8 macroporous adsorption resin column and loading the sample, and eluting with 2 times the column volume of water, 3 times the column volume of 20% ethanol solution, 2 times the column volume of 50% ethanol solution, and 3 times the column volume of 85% ethanol solution as eluents, then collecting the eluent with a volume fraction of 85% ethanol solution, removing ethanol, and obtaining the tangutic rhubarb extract.

[0045] Example 3

[0046] (1) Add 5 times (1 g: 5 mL) of 5% ethanol solution to the rhubarb root, perform microwave extraction at 95°C and 200 W for 4 times, each time for 10 min, and combine the extracts; concentrate the extracts under reduced pressure at 0.07 MPa to obtain a crude ethanol extract;

[0047] (2) dissolving the crude ethanol extract in step (1) in water, filtering and removing impurities with an organic membrane with a molecular cutoff of 20,000 Da, and collecting the filtrate;

[0048] (3) pouring the filtrate in step (2) into a D101 macroporous adsorption resin column and loading the column, and eluting with 4 times the column volume of water, 4 times the column volume of 40% ethanol solution, 2 times the column volume of 80% ethanol solution, and 2 times the column volume of 95% ethanol solution as eluents, then collecting the eluent with a volume fraction of 95% ethanol solution, removing ethanol, and obtaining the tangutic rhubarb extract.

[0049] Example 4

[0050] (1) Add 20 times (1 g: 20 mL) of 75% ethanol solution to the rhubarb root, perform microwave extraction once at 80°C and 700 W power for 40 min, and collect the extract; concentrate the extract under reduced pressure of 0.05 MPa to obtain a crude ethanol extract;

[0051] (2) dissolving the crude ethanol extract in step (1) in water, filtering and removing impurities with a 0.2 μm ceramic membrane, and collecting the filtrate;

[0052] (3) pouring the filtrate in step (2) into a D101 macroporous adsorption resin column and loading the column, and eluting the column with 1 column volume of water, 2 column volumes of 70% ethanol solution, and 4 column volumes of 90% ethanol solution as eluents, and then collecting the eluent with a volume fraction of 90% ethanol solution, removing ethanol, and obtaining the rhubarb tanguticus extract.

[0053] Example 5

[0054] (1) Add 10 times (1 g: 10 mL) of 50% ethanol solution to the rhubarb root, perform microwave extraction at 70°C and 800 W for 3 times, each time for 70 min, and combine the extracts; concentrate the extracts under reduced pressure at 0.04 MPa to obtain a crude ethanol extract;

[0055] (2) dissolving the crude ethanol extract in step (1) in water, filtering and removing impurities with a 0.5 μm ceramic membrane, and collecting the filtrate;

[0056] (3) pouring the filtrate in step (2) into a D101 macroporous adsorption resin column and loading the column with 3 column volumes of water, 1 column volume of 30% ethanol solution, and 2 column volumes of 85% ethanol solution as eluents, and then collecting the eluent with a volume fraction of 85% ethanol solution, removing ethanol, and obtaining the rhubarb tanguticus extract.

[0057] Example 6

[0058] (1) Add 15 times (1 g: 15 mL) of 65% ethanol solution to the rhubarb root, perform microwave extraction twice at 90°C and 400 W power for 30 min each time, and combine the extracts; concentrate the extracts under reduced pressure at 0.06 MPa to obtain a crude ethanol extract;

[0059] (2) dissolving the crude ethanol extract in step (1) in water, filtering and removing impurities with a 0.1 μm ceramic membrane, and collecting the filtrate;

[0060] (3) pouring the filtrate in step (2) into an AB-8 macroporous adsorption resin column and loading the column with 2 column volumes of water, 2 column volumes of 60% ethanol solution, and 1 column volume of 95% ethanol solution as eluents, and then collecting the eluent with a volume fraction of 95% ethanol solution, removing ethanol, and obtaining the rhubarb tanguticus extract.

[0061] Comparative Example 1

[0062] (1) Add 20 times (1 g: 20 mL) of 75% ethanol solution to the rhubarb root, perform microwave extraction once at 80°C and 700 W power for 40 min, and collect the extract; concentrate the extract under reduced pressure of 0.05 MPa to obtain a crude ethanol extract;

[0063] (2) dissolving the crude ethanol extract in step (1) in water, filtering and removing impurities with an organic membrane with a molecular cutoff of 50,000 Da, and collecting the filtrate;

[0064] (3) pouring the filtrate in step (2) into a D101 macroporous adsorption resin column and loading the column with 2 times the column volume of water and 3 times the column volume of 60% ethanol solution as eluents, then collecting the eluent with a volume fraction of 60% ethanol solution, removing ethanol, and obtaining the Rheum tanguticum extract.

[0065] Comparative Example 2

[0066] (1) Add 20 times (1 g: 20 mL) of 75% ethanol solution to the rhubarb root, perform microwave extraction once at 80°C and 700 W power for 40 min, and combine the extracts; concentrate the extracts under reduced pressure at 0.05 MPa to obtain a crude ethanol extract;

[0067] (2) dissolving the crude ethanol extract in step (1) in water, filtering and removing impurities with an organic membrane with a molecular cutoff of 50,000 Da, and collecting the filtrate;

[0068] (3) pouring the filtrate in step (2) into a D101 macroporous adsorption resin column and loading the column with 3 column volumes of water and 1 column volume of 30% ethanol solution as eluents, then collecting the eluent with a volume fraction of 30% ethanol solution, removing ethanol, and obtaining the Rheum tanguticum extract.

[0069] Test Example 1

[0070] (1) Investigation of microwave power

[0071] 10 g of dried Rheum tanguticum medicinal material was accurately weighed and placed in a round-bottom flask. 200 mL of 75% ethanol solution was added and microwave extraction was performed once for 40 min. The extraction temperature was 50° C. The power of the microwave extraction was shown in Table 1. The content of total anthraquinone in the extracts obtained under different microwave powers was determined, and the extraction rate of total anthraquinone was calculated according to the following formula.

[0072] E=(C 提取物 ×m 提取物 / m 原料 )×100%

[0073] E is the extraction rate, C is 提取物 is the total anthraquinone content of the extract, m 提取物 is the mass of the extract, m 原料 For the quality of raw materials.

[0074] Table 1 Effect of different extraction powers on total anthraquinone extraction rate

[0075] Power(W) 200 300 400 500 600 700 800 900 Extraction rate (%) 1.57 1.98 2.45 2.88 3.02 3.87 3.34 3.12

[0076] The results are shown in Table 1. The results show that the total anthraquinones in Rheum tanguticum can be effectively extracted in the range of 200W to 900W. Among them, the total anthraquinone extraction rate in the range of 600W to 900W can reach more than 3%.

[0077] (2) Investigation of extraction time

[0078] 10 g of dried Rheum tanguticum was accurately weighed and placed in a round-bottom flask. 200 mL of 75% ethanol solution was added. The extraction temperature was 60 ° C. and the microwave power was 700 W. The extraction was performed once. The content of total anthraquinone in the extract obtained at different extraction times was determined and the extraction rate was calculated. The results are shown in Table 2.

[0079] Table 2 Effect of different extraction time on total anthraquinone extraction rate

[0080] Time (min) 10 20 30 40 50 60 70 80 90 Extraction rate (%) 1.23 1.72 2.27 3.67 3.58 3.22 2.83 2.27 1.98

[0081] The results showed that the total anthraquinones in Rheum tanguticum could be effectively extracted within the range of 10min to 90min, and the total anthraquinone extraction rate within the range of 40min to 60min could reach more than 3%.

[0082] (3) Investigation of liquid-to-solid ratio

[0083] 10 g of dried Rheum tanguticum was accurately weighed and placed in a round-bottom flask. The microwave power was 700 w and extracted once for 40 min. When the ethanol concentration was 75%, the content of total anthraquinone in the extract obtained under different liquid-to-solid ratios was determined and the extraction rate was calculated. The results are shown in Table 3.

[0084] Table 3 Effect of different liquid-to-solid ratios on total anthraquinone extraction rate

[0085] Material-liquid ratio (W) 1:5 1:10 1:15 1:20 1:25 1:30 Extraction rate (%) 1.09 1.98 2.45 3.62 3.09 2.67

[0086] The results showed that the total anthraquinones in Rheum tanguticum could be effectively extracted in the range of 1:5 to 1:30, and the total anthraquinone extraction rate in the range of 1:20 to 1:25 could reach more than 3%.

[0087] (4) Temperature investigation

[0088] 10 g of dried Rheum tanguticum was accurately weighed and placed in a round-bottom flask. 200 mL of 75% ethanol solution was added. The microwave power was 700 w and the extraction was performed once for 40 min. The content of total anthraquinone in the extract obtained at different temperatures was determined and the extraction rate was calculated. The results are shown in Table 4.

[0089] Table 4 Effect of different temperatures on the extraction rate of total anthraquinone

[0090] Temperature(℃) 60 70 80 90 95 Extraction rate (%) 2.23 3.45 3.86 3.62 3.21

[0091] The results showed that the total anthraquinones in Rheum tanguticum could be effectively extracted within the range of 60-95°C, and the total anthraquinone extraction rate within the range of 70-95°C could reach more than 3%.

[0092] (5) Investigation of extraction methods

[0093] 10 g of dried Rheum tanguticum was accurately weighed and extracted by heating reflux, ultrasonic extraction and microwave extraction respectively. The specific extraction parameters are shown in Table 5. The content of total anthraquinone in the extracts obtained by different extraction methods was determined and the extraction rate was calculated.

[0094] Table 5 Effect of different extraction methods on the extraction rate of total anthraquinone

[0095]

[0096] The results showed that the total anthraquinone extraction rate obtained by microwave extraction was higher than that by heating reflux extraction and ultrasonic extraction.

[0097] (6) Investigation of purification methods

[0098] The crude ethanol extract obtained in step (1) of Example 1 was dissolved in water, purified by the treatment methods listed in Table 6, and the purified liquid was collected to determine the total anthraquinone content. The enrichment effect of different purification methods on total anthraquinone was investigated.

[0099] Table 6 Effect of different purification methods on total anthraquinone content

[0100]

[0101] The results showed that both ceramic membrane and organic membrane filtration could effectively enrich total anthraquinones, and the enrichment effect was significantly better than that of centrifugation.

[0102] (7) Investigation of separation methods

[0103] The filtrate collected in step (2) of Example 1 was poured into D101, AB-8 and HPD-450 macroporous adsorption resin columns for loading, and then 3 column volumes of water, 2 column volumes of 30% ethanol solution, 2 column volumes of 60% ethanol solution and 2 column volumes of 90% ethanol solution were used as eluents for elution, and then 30%, 60% and 90% ethanol eluents were collected respectively. The model of the macroporous adsorption resin column and the ethanol concentration of the collected eluents are shown in Table 7. The content of total anthraquinone in each eluent was determined.

[0104] Table 7 Effects of different types of macroporous resin columns and different elution concentrations of ethanol solutions on the total anthraquinone content in the eluent

[0105]

[0106] Test Example 2

[0107] The Rheum tanguticum extract obtained in Example 1 was used as a sample to measure the chemical components contained therein.

[0108] (1) Sample pre-experimental treatment method

[0109] Weigh 0.2 g of the sample, add 15 ml of methanol, ultrasonicate for 15 min, centrifuge at 12,000 rpm for 20 min, and take the supernatant for testing.

[0110] (2) Instruments and Equipment

[0111] UPLC-QE system: AcquityTM ultra high performance liquid chromatograph (Waters, USA), TripleTOF 5600+ time-of-flight mass spectrometer equipped with electrospray ion source (AB SCIEX, USA); Eppendorf minispan centrifuge (Eppendorf, Germany).

[0112] (3) Test conditions

[0113] Chromatographic conditions: the chromatographic column is Waters ACQUITY UPLC HSS T3 (150mm×2.1mm id, 1.8μm); 0.1% formic acid solution is used as mobile phase A, 0.1% formic acid acetonitrile is used as mobile phase B, linear gradient elution, 0-10min 5-15% acetonitrile, 10-30min 15-25% acetonitrile, 30-40min 25-40% acetonitrile, 40-45min40-70% acetonitrile, 45-50min70-80% acetonitrile, 50-60min 80-95% acetonitrile, detection wavelength: 254nm, 280nm, 320nm, 360nm, molecular weight 150-1500; injection volume: 2μL.

[0114] Mass spectrometry conditions: UPLC-Triple-TOF 5600+ time-of-flight liquid chromatography-mass spectrometer: negative ion scanning mode; scanning range: m / z 100-1500; nebulizer gas (GS1): 55psi; nebulizer gas (GS2): 55psi; curtain gas (CUR): 35psi; ion source temperature (TEM): 600℃ (positive); ion source voltage (IS): 5500V (positive); primary scan: declustering voltage (DP): 100V; focusing voltage (CE): 10V; secondary scan: use TOF MS~Product Ion~IDA mode to collect mass spectrometry data, CID energies are -20, -40 and -60V, and before injection, use CDS pump to perform mass axis correction to make the mass axis error less than 2ppm.

[0115] (4) Test results and data analysis

[0116] Table 8 Test results of chemical components of Rheum tanguticum extract

[0117]

[0118] The test results are shown in Table 8 and Figure 1The results show that the main compounds in the Tangut rhubarb extract prepared by the present invention include: rhubarb-1-O-β-D-glucoside, cassia ketone-8-O-β-D-pyranoglucoside, chrysophanol-1-O-β-D-pyranoglucoside, chrysophanol-8-O-β-D-pyranoglucoside, rhubarb-8-O-β-D-pyranoglucoside, rhein-8-O-β-D-pyranoglucoside, rhein-8-O-β-D-pyranoglucoside, 4-(3-Oxobutyl)phenyl-6-O-[-3-(4-hydroxyphenyl)prop-2-enoyl]-2-O-(3,4,5-trihydrox ybenzoyl)-β-D-glucopyranoside, aloe-rhubarb, rhein, 4′-hydroxyphenyl-2-butanone cinnamoyl galloyl glycoside, Rhein-ethylation, rhubarb.

[0119] Test Example 3

[0120] 1. Animals, reagents, and instruments

[0121] Healthy macrophage fluorescent transgenic zebrafish (Tg:lyz-EGFP) was provided by the zebrafish drug screening platform of the Institute of Biology, Shandong Academy of Sciences.

[0122] Sample solution: The Rheum tanguticum extracts prepared in Example 1 (sample 161), Comparative Example 1 (sample 162) and Comparative Example 2 (sample 164) were used as samples, and DMSO was added to prepare a solution; the samples and the prepared solutions were stored in a -20°C refrigerator.

[0123] Instruments: SZX16 fluorescence microscope and DP2-BSW image acquisition system (Olympus, Japan); X51 inverted microscope (Olympus, Japan); Forma 3111 water-jacketed CO2 incubator (Forma, USA); zebrafish breeding equipment (Beijing Aisheng Technology Co., Ltd.).

[0124] 2. Screening of safe doses

[0125] Samples (161, 162, 164) were prepared into samples with a series of concentrations (3.125, 6.25, 12.5, 25, 50, 100, 200, 400 μg / mL).

[0126] Female and male zebrafish were raised separately, with alternating lighting for 14h / darkness for 10h, and were fed with artificial granular feed and newly hatched Artemia nauplii at regular intervals. When collecting eggs, healthy and sexually mature zebrafish were placed in a mating tank at a ratio of 1:2 or 2:2 between female and male, and fertilized eggs were obtained at 10-11 am the next day. After disinfection and washing, the fertilized eggs were transferred into zebrafish embryo culture water (containing 5.0mM NaCl, 0.17mM KCl, 0.4mM CaCl2, 0.16mM MgSO4) and cultured at 28°C with controlled light.

[0127] At 3dpf (days post fertilization) of fertilized eggs, normal zebrafish embryos were selected under a stereomicroscope and transferred into 12-well culture plates, 15 per well, and two replicate wells per group each time. A blank control group and a sample group were set up. The sample groups were added with 2.0mL of samples with concentrations of 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400μg / mL, respectively. The blank control group was only added with fish water of the same volume as the sample group without adding drugs. The 12-well plate was then placed in a light incubator (28°C) to allow the embryos to continue to develop, and the survival and deformity of the zebrafish were observed after 24h of incubation.

[0128] The experimental results are shown in Table 9, which show that: 1) At high concentrations of 12.5, 25, 50, 100, 200, and 400 μg / mL, all zebrafish fry died within 3 hours after treatment with 161, and no death occurred after treatment with 6.25 μg / mL and 3.125 μg / mL of 161 for 24 hours; 2) At high concentrations of 25, 50, 100, 200, and 400 μg / mL, all zebrafish fry died within 3 hours after treatment with 162, and no death occurred after treatment with 6.25 μg / mL and 3.125 μg / mL of 162 for 24 hours; 3) At high concentrations of 50, 100, 200, and 400 μg / mL, all zebrafish fry died within 3 hours after treatment with 164, and no death occurred after treatment with 12.5, 6.25 μg / mL, and 3.125 μg / mL of 164 for 24 hours.

[0129] Therefore, the working concentrations of samples for anti-inflammatory activity detection were set at 1.25, 2.5, and 5 μg / mL.

[0130] Table 9 Mortality of zebrafish at different concentrations

[0131]

[0132]

[0133] 3. Anti-inflammatory Experiment

[0134] Concentration of samples to be tested (μg / mL): 1.25, 2.5, 5.0 μg / mL.

[0135] At 3 dpf (days post fertilization) of fertilized eggs, normal AB zebrafish embryos were selected under a stereomicroscope and transferred into 12-well culture plates, 15 per well, with two replicate wells per group each time. A blank control group (corresponding to Figure 2 and Figure 3 Ctl in), model group (corresponding to Figure 2 and Figure 3 Model in), positive drug group (corresponding to Figure 2 and Figure 3 Indo in), sample group (corresponding to Figure 2 and Figure 3 161, 162, 164 concentration groups). 2mL of 40μM indomethacin (Indometacin, Indo) was added to the positive drug group, and 2mL of 1.25, 2.5, and 5μg / mL samples (161, 162, 164) were added to the sample group. No drug was added to the blank control group and the model group, and only the same volume of fish water as the sample group was added. Then the 12-well plate was placed in a light incubator (28°C) to allow the embryos to continue to develop. After incubation for 2h, except for the blank control group, they were treated with 20μM CuSO4 for 1h. Then the macrophage inflammatory response was observed under a fluorescence microscope, and photos were taken. The macrophages around the zebrafish lateral line neuromasts were counted, and then statistical analysis was performed using Imagepro-plus software. The statistical results of the anti-inflammatory effects of each group are shown in Table 10.

[0136] Table 10 Statistical table of anti-inflammatory effects of each group

[0137]

[0138]

[0139] Note: ## indicates that there is a very significant difference compared with the blank control group (Ctl) (P<0.01), *** indicates that there is a very significant difference compared with the model group (Model) (P<0.001).

[0140] The results are as follows Figure 2 , Figure 3As shown in Table 10, 20 μM CuSO4 can significantly promote the migration of zebrafish neutrophils to zebrafish lateral line neuromast cells, and 40 μM indomethacin can significantly inhibit the inflammatory response induced by CuSO4; the results after sample treatment show that: at 1.25, 2.5, and 5 μg / mL doses, samples 162 and 164 have no inhibitory effect on the inflammatory response induced by CuSO4, and there is no statistical difference; at 1.25 and 2.5 μg / mL doses, sample 161 has no inhibitory effect on the inflammatory response induced by CuSO4, and at 5 μg / mL doses, sample 161 has an inhibitory effect on the inflammatory response induced by CuSO4, and there is a statistical difference. At the same time, the results show that sample 161 can dye the zebrafish body yellow. This shows that 5 μg / mL sample 161 has a certain anti-inflammatory effect.

[0141] 4. Molecular Detection

[0142] After treating AB zebrafish embryos by the method described in step 3 of this embodiment, the total RNA of zebrafish embryos of the blank control group (labeled as Ctl), the model group (labeled as Model), the positive drug group (indomethacin, labeled as Resv) and the 161 sample group (labeled as Sample) was extracted using a kit and reversely transcribed into cDNA. The gene expression levels of IL1, IL2, TNF-α and NF-κB in each group were detected by fluorescent real-time quantitative PCR amplification. β-actin was used as an internal reference during the analysis of the results. The primer sequences of the internal reference target genes are shown in Table 11.

[0143] Table 11 Primer sequences of target genes involved in molecular detection

[0144]

[0145] The results are as follows Figure 4 As shown: Compared with the blank control group, IL1, IL2, TNF-α and NF-κB in the model group were significantly upregulated, indicating that inflammation occurred, and the expression of genes in the 5μg / mL sample group was significantly downregulated, indicating that 5μg / mL of 161 can significantly downregulate the expression of inflammatory factor genes and effectively inhibit the inflammatory response.

[0146] The above results indicate that sample 161 (Rheum tanguticum extract prepared in Example 1 of the present invention) has anti-inflammatory effects, and its anti-inflammatory molecular mechanism is to achieve anti-inflammatory effects by reducing the expression of pro-inflammatory factors IL1, IL2, TNF-α and NF-κB.

[0147] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a rhubarb extract, characterized in that: The following steps are involved: Tangut rhubarb is mixed with an ethanol solution with a volume fraction of 5 to 95%, and subjected to microwave extraction to obtain a crude ethanol extract; The crude ethanol extract was dissolved in water, filtered through a membrane, and the filtrate was collected; The filtrate is poured into a macroporous adsorption resin column and loaded with water, an ethanol solution with a volume fraction of 20 to 80%, and an ethanol solution with a volume fraction of 85 to 95% are used as eluents for elution in sequence, and then the ethanol eluate with a volume fraction of 85 to 95% is collected, and the ethanol is removed to obtain the rhubarb tangut extract.

2. The preparation method according to claim 1, characterized in that: The material-liquid ratio of the Tangut rhubarb mixed with the ethanol solution with a volume fraction of 5-95% is 1g: (5mL-30mL).

3. The preparation method according to claim 1, characterized in that: The microwave extraction is performed at a temperature of 60 to 95° C. and a power of 200 W to 900 W for 1 to 4 times.

4. The preparation method according to claim 3, characterized in that: During the microwave extraction, each extraction lasts 10 to 90 minutes.

5. The preparation method according to claim 1, characterized in that: The membrane filtration includes ceramic membrane filtration or organic membrane filtration.

6. The preparation method according to claim 5, characterized in that: The particle size of the ceramic membrane is 0.1 μm to 0.5 μm.

7. The preparation method according to claim 5, characterized in that: The molecular retention capacity of the organic membrane is 10000-50000Da.

8. The preparation method according to claim 1, characterized in that: The models of the macroporous adsorption resin column include D101 or AB-8.

9. A rhubarb extract, characterized in that: The Rheum tanguticum extract is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the preparation method according to any one of claims 1 to 8 or the Rheum tanguticum extract according to claim 9 in the preparation of anti-inflammatory products.

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