Method for extracting and detecting active substances of hemerocallis fulva and application of active substances

Abstract

The invention provides an extraction and detection method of a day lily active matter and application of the day lily active matter in anti-neuritis. Hemerocallis fulva contains various bioactive substances including flavone, polysaccharide and saponin, and the substances have potential application value in the fields of medicines and health care products. The invention provides a method for extracting the active components step by step, and rapid identification and detection are carried out by utilizing technologies such as fluorescence and ultraviolet-visible spectrophotometry. In addition, the invention also develops a detection method for evaluating the anti-neuritis effect of the active substances. Lipopolysaccharide (LPS) is used for stimulation to establish an inflammation model, day lily active substances are added, and the active substances are found to be capable of remarkably inhibiting generation and release of inflammatory factors and promoting growth and differentiation of neurons. Scientific basis is provided for developing new neuritis treatment methods, related drugs and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of extraction and identification of active ingredients of Chinese herbal medicine, and specifically relates to a preparation and rapid identification method of active substances of hemerocallis. BACKGROUND

[0002] Hemerocallis, also known as day lily, is also known as hemerocallis, forgetful grass, soothing herb, and golden needle herb in some places. It is a perennial herbaceous plant widely distributed in many provinces and cities in China. The roots and leaves of hemerocallis contain rich active substances and various secondary metabolites, such as flavonoids, polysaccharides, saponins, etc., which have antioxidant, anti-inflammatory, anticancer and other effects, and have wide application prospects in the fields of medicine, health care and cosmetics.

[0003] At present, the extraction methods of these active substances in hemerocallis have the problems of low extraction efficiency, complex operation, high cost, etc. For example, the prior art "Day lily flavone extractive and its preparation method and use" (CN201911050741) only discloses a technology for extracting a kind of active substance flavone from day lily, and "A total phenol extractive from hemerocallis leaf and its preparation method and application" (CN202111387924) discloses a preparation method for extracting active ingredients from the leaf parts of hemerocallis, and only discloses the extraction method of total phenol. The prior art does not disclose the step-by-step extraction and preparation method of hemerocallis bioactive components (BCHCB) flavone, polysaccharide and saponin. At the same time, the core steps of the prior art for extracting active ingredients are mainly filtration or centrifugation, and the preparation method is single and low in efficiency. Therefore, it is of great significance to develop a high-efficiency, simple and economical extraction method of hemerocallis bioactive components.

[0004] On the other hand, neuritis is a serious nervous system disease that can cause neuronal damage and dysfunction. If not treated in time, it may cause irreversible neuronal damage and disability. At present, although the technology has found that total flavones of bidens pilosa have the use in inhibiting neuritis, the efficacy is limited, therefore, for the development of special drugs for treating neuritis, the extraction and identification of hemerocallis bioactive components are of great research value and economic benefit. SUMMARY

[0005] The present application provides a step-by-step extraction and detection method of hemerocallis bioactive components and its use, specifically the step-by-step extraction and rapid detection of flavone, polysaccharide and saponin, and proposes its use in resisting neuritis.

[0006] A step-by-step extraction and preparation method of hemerocallis bioactive components, characterized by using different physicochemical properties of hemerocallis bioactive components to extract flavone, polysaccharide and saponin in turn, the specific steps are as follows:

[0007] (1) Steaming the fresh washed Hemerocallis dumortierii fresh vegetable and cooling to room temperature, drying, and making Hemerocallis dumortierii dry product coarse powder;

[0008] (2) Precisely taking the Hemerocallis dumortierii dry product coarse powder, soaking in a container with ethanol, and after ultrasonic treatment, transferring into a distillation flask;

[0009] (3) Refluxing the solution in the distillation flask in step (2) several times with water bath, filtering and recovering ethanol by heating, and stopping heating when the filtrate is concentrated to small volume and has no alcohol smell, thereby obtaining flavones; the filtrate and Hemerocallis dumortierii residue are reserved for later use;

[0010] (4) Refluxing the filtrate and Hemerocallis dumortierii residue in step (3) with ethanol;

[0011] (5) Soaking the residue after refluxing in step (4) with hot water, removing monosaccharides and oligosaccharides by suction filtration, repeating extraction once for the filtered material after suction filtration, combining the two filtrates, and removing macromolecular substances and impurities by microfiltration membrane;

[0012] (6) Taking the combined filtrate in step (5) and sequentially passing through ultrafiltration membranes with different molecular weight cut-offs for ultrafiltration, and repeatedly obtaining concentrated liquid retained by the ultrafiltration membrane and permeate liquid passing through the ultrafiltration membrane;

[0013] (7) Vacuum concentrating the concentrated liquid obtained in step (6) by using a concentration device to obtain crude polysaccharides;

[0014] (8) Concentrating the permeate liquid obtained in step (6) by rotary evaporation, vacuum concentration, etc. to small volume, and drying under reduced pressure to obtain crude saponins;

[0015] (9) Dissolving the crude saponins in deionized water, passing through an adsorption resin column, and eluting to obtain an eluate;

[0016] (10) Collecting the eluate in step (9) and concentrating to dryness to obtain saponins.

[0017] A detection method of Hemerocallis dumortierii active substances, characterized by realizing rapid detection of flavone compounds, polysaccharides and saponins by fluorescence and ultraviolet-visible spectrophotometry. Specifically, the method comprises the following steps:

[0018] Step one, rapid detection of flavone compounds

[0019] 1.1 Establishing a standard curve by ultraviolet-visible spectrophotometry to quantitatively detect Hemerocallis dumortierii total flavones;

[0020] 1.2 Qualitatively detecting flavones by fluorescence: dropping Hemerocallis dumortierii extract on filter paper and observing under ultraviolet light, and observing bright yellow-green fluorescence, which indicates that the extract contains C3-hydroxyl flavones, i.e. flavone alcohols.

[0021] Step two, rapid detection of polysaccharide

[0022] The standard curve equation is established by ultraviolet-visible spectrophotometry to quantitatively detect the peony polysaccharide;

[0023] Step three, rapid detection of saponin

[0024] The standard curve equation is established by ultraviolet-visible spectrophotometry to quantitatively detect the peony saponin.

[0025] The step one of the rapid detection of flavonoids is characterized by detecting the rutin standard solution by a spectrophotometer to establish a regression equation standard curve.

[0026] The step two of the rapid detection of polysaccharide is characterized by utilizing the sulfuric acid-phenol method standard curve equation to determine the polysaccharide content.

[0027] The step three of the rapid detection of saponin is characterized by utilizing the vanillin glacial acetic acid solution colorimetric method to determine the total saponin content.

[0028] The application further provides a use of peony active substances, which is characterized by detecting and analyzing the effects of the active substances on anti-neuroinflammation to obtain the use of the flavonoids, polysaccharides and saponins in resisting neuroinflammation; a cell inflammation model is established by stimulating BV2 microglial cells with lipopolysaccharides (LPS), and the peony active substances are added to compare the cell activity, inflammatory factor secretion and gene expression with those of a control group, so as to detect and evaluate the anti-neuroinflammation effects of the peony active substances in vitro and in vivo.

[0029] The use of peony active substances is characterized by that the anti-neuroinflammation effects of the peony active substances in vitro and in vivo are detected and proved in the following manner: in the first stage, the active substances in the peony are detected for anti-neuroinflammation in vitro.

[0030] The BV2 microglial cells are cultured for 12 hours after being contacted with LPS. The peony active substances are used to protect the cells before being stimulated by LPS. The BV2 microglial cells are divided into four groups: a normal control group, a model group (stimulated by LPS), a BCHCB group and a BCHCB combined with LPS treatment group.

[0031] 1.1 MTT method is used to detect the cell survival rate;

[0032] 1.2 Griess method is used to detect the NO (nitric oxide) release;

[0033] 1.3 ELISA method is used to detect the contents of inflammatory factors and anti-inflammatory factors;

[0034] 1.4 RT-qPCR method to detect the mRNA expression levels of inflammatory factors and anti-inflammatory factors.

[0035] The BV2 microglial cells in step 1.1 are treated according to the aforementioned grouping. The absorbance (A) value is measured at 490 nm by a microplate reader. Cell survival rate (%) = (experimental group A490nm / cell control group A490nm) x 100%.

[0036] In step 1.2, the BV2 microglial cells are collected according to the aforementioned grouping. The absorbance (A) value of each group is measured at 540 nm. The wavelength set may vary depending on different markers or substrates.

[0037] In step 1.3, the cell culture medium is collected according to the aforementioned grouping. The absorbance (A) value of each group is measured at 405 nm by a microplate reader.

[0038] Step 1.4 is characterized by the RT-qPCR primer sequences in Table 4 and the reaction conditions in the examples.

[0039] Based on the experimental data, it is evaluated whether BCHCB has a protective effect on LPS-induced BV2 microglial cell damage.

[0040] The second stage: in vivo detection of active substances in Hemerocallis middendorffii Regel against neuroinflammation;

[0041] 2.1 Brain tissue pathological structure examination to evaluate the damage degree of hippocampal structure in brain tissue;

[0042] 2.2 Detection of NO content in brain tissue;

[0043] 2.3 ELISA method to detect the content of inflammatory factors in brain tissue;

[0044] 2.4 Western blot detection of inflammation-related protein expression.

[0045] The second stage in vivo experiment is characterized by detecting the anti-neuroinflammatory effect of BCHCB through animal experiment method; the experimental animals are grouped, dosed, modeled and sampled according to appropriate conditions; the animal grouping is similar to the in vivo detection method.

[0046] In particular, BCHCB is divided into high, medium and low concentration groups; the experimental operations in steps 2.1, 2.2 and 2.3 are carried out at low temperature; the frozen brain tissue is washed with pre-cooled buffer, which can be PBS (0.01M, pH=7.4), and is thoroughly ground and homogenized in an ice bath; by comprehensively evaluating the in vivo detection results of this stage, it is judged whether an inflammatory reaction has occurred, and then it is evaluated whether BCHCB can inhibit neuroinflammatory reaction.

[0047] Step 2.1 is characterized in that brain tissue HE staining can be performed, and the morphology of nerve cells in the brain tissue is observed under a light microscope.

[0048] Step 2.2 is characterized in that an enzyme marker 405 nm is detected in each group (A). The wavelength is set according to different markers or substrates.

[0049] Step 2.3 is characterized in that the content of inflammatory factors in the brain tissue homogenate of each group of experimental animals is directly detected.

[0050] Step 2.4 is characterized in that the brain tissue block is ground in RIPA lysis buffer on ice for 30 min, the lysis buffer is centrifuged at 12000 rpm at 4 DEG C for 5 min, and the protein concentration of each group is determined by BCA method.

[0051] The effect of BCHCB on significantly inhibiting the production and release of inflammatory factors is evaluated.

[0052] The present application has the following advantages:

[0053] 1. The step-by-step extraction process disclosed in the present application can efficiently utilize various active ingredients of the daylily plant.

[0054] 2. The step-by-step extraction process disclosed in the present application has high extraction efficiency, energy saving, and high extraction efficiency.

[0055] 3. The detection technology of the step-by-step extraction process disclosed in the present application is simple and easy to operate, and the technical and equipment requirements are low.

[0056] 4. Similarly, the present application not only helps to better and efficiently utilize the natural resource of daylily, but also provides a reference for the extraction and detection of active substances of other plants in the daylily family.

[0057] 5. The use of the active substances of daylily in the treatment of neuroinflammation provides a new idea and experimental basis for the development of new neuroinflammation treatment methods. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 Standard curve for measuring total flavonoids in daylily by aluminum nitrate method.

[0059] Figure 2 Standard curve for measuring glucose in daylily by sulfuric acid-phenol method.

[0060] Figure 3 Vanillin glacial acetic acid solution colorimetric method for determining total saponin content.

[0061] Figure 4 UV absorption spectrum of daylily polysaccharide.

[0062] Figure 5 The infrared absorption spectrum of the Hemerocallis citrina Baroni polysaccharide.

[0063] Figure 6 MTT method was used to detect the effect of different doses of BCHCB on the survival rate of LBP inflammatory model cells.

[0064] Figure 7 The effect of BCHCB on the release of NO in BV2 microglial cells induced by LPS. b P<0.01 vs control group; c P<0.01 vs LPS group.

[0065] Figure 8 The effect of BCHCB on the release of TNF-α, IL-1β, IL-6 and IL-10 in BV2 microglial cells induced by LPS.

[0066] Figure 9 The effect of BCHCB on the expression level of TNF-α, IL-1β, IL-6 and IL-10 mRNA in BV2 microglial cells induced by LPS.

[0067] Figure 10 A process diagram of a Hemerocallis citrina Baroni flower active substance preparation and rapid detection method and anti-neuroinflammatory use. DETAILED DESCRIPTION

[0068] In the following, the present application will demonstrate the beneficial effects of the present application through examples. Those skilled in the art will know that these examples are exemplary and not limiting. These examples will not limit the scope of the present application in any way. The experimental operations described in the following examples are routine operations unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0069] Example 1

[0070] Extraction method of active substance

[0071] Different physicochemical properties of Hemerocallis citrina Baroni active substance are utilized to extract flavonoids, polysaccharides and saponins in sequence.

[0072] 1.1 Extraction and content detection of flavonoids

[0073] Freshly washed Hemerocallis citrina Baroni was steamed for 5-6 min, cooled to room temperature, dried, and made into coarse powder. 25 g of Hemerocallis citrina Baroni dry coarse powder was precisely weighed, 500 mL of 70% ethanol was added in a stoppered conical flask, and ultrasonic extraction was performed at 60 Hz for 40 min. The extraction was transferred into a distillation flask, and water bath reflux was performed several times. Filtration was performed (filtrate was reserved for subsequent polysaccharide and saponin extraction), and heating was performed under normal pressure. When the extraction concentrate was 20 mL and had no alcohol smell, heating was stopped, and flavonoids were obtained. The filtrate and the reserved residue were used for subsequent polysaccharide and saponin extraction.

[0074] UV-visible spectrophotometry was used to determine the total flavonoids in the herb of Hemerocallis citrina Baroni. 10 mg rutin standard was precisely weighed and added to 50% methanol to make a 0.2 mg / mL rutin standard solution. 1 mL, 2 mL, 3 mL, 4 mL, 5 mL and 6 mL of the rutin standard solution were precisely measured into 25 mL volumetric flasks, respectively, and 50% methanol was added to make up to 6 mL. 1 mL of 5% sodium nitrite solution was added, and the mixture was shaken and allowed to stand for 6 minutes. 1 mL of 10% aluminum nitrate solution was added, and the mixture was shaken and allowed to stand for 6 minutes. 10 mL of sodium hydroxide solution was added, and 50% methanol was added to the mark. The mixture was shaken and allowed to stand for 15 minutes. The corresponding solution was used as a blank, and the absorbance was measured at 510 nm. The standard curve was plotted with the absorbance as the ordinate and the mass concentration of flavonoids as the abscissa. Figure 1 The regression equation and the correlation coefficient were calculated as Y = 13.266X + 0.006 and R = 0.9984, respectively. 2

[0075] 1 mL of the extract of Hemerocallis citrina Baroni was taken, and its absorbance was determined according to the experimental method. The concentration of total flavonoids in Hemerocallis citrina Baroni was calculated from the regression equation, and the results are shown in Table 1. The content of total flavonoids in the extract of Hemerocallis citrina Baroni was determined to be 0.36 mg / mL, and the relative standard deviation RSD was 3.89%. The content of flavonoids in Hemerocallis citrina Baroni was calculated as the extract concentration multiplied by the total volume of the extract divided by the mass of Hemerocallis citrina Baroni, and the content of total flavonoids in Hemerocallis citrina Baroni was determined to be 14.4 μg / g.

[0076] Table 1 Determination results of the content of flavonoids in Hemerocallis citrina Baroni

[0077]

[0078] Rapid detection of flavonoid active substances

[0079] After the total flavonoids in Hemerocallis citrina Baroni were detected by UV-visible spectrophotometry, fluorescence detection was used to identify the results for the sake of rapidity and economy in batch production. Since C3-hydroxyl flavonoids have significant fluorescence, the extract of Hemerocallis citrina Baroni was dropped onto a filter paper, and a bright yellow-green fluorescence was observed under ultraviolet light, indicating the presence of C3-hydroxyl flavonoids, i.e., flavonol flavonoids.

[0080] 1.2 Extraction and determination of polysaccharides

[0081] The filtrate after extraction of flavonoids and the remaining residue were refluxed twice with 80% ethanol for 2 h each time. Monosaccharides and oligosaccharides were removed by suction filtration. The material-liquid ratio was 1:40, and the residue was soaked in hot water at 50°C for 2 h, followed by suction filtration. The residue was extracted again, and the combined filtrate was filtered through a microfiltration membrane with a pore size of 0.2 μm to remove macromolecular substances and impurities. The filtrate was successively ultrafiltered through membranes with molecular weight cutoffs of 6000, 20,000 and 100,000, respectively, for 3 times. The concentrated solution (the permeate was used for extracting saponins) was concentrated to 20% of the original volume by a rotary evaporator under vacuum, and then vacuum dried to obtain crude polysaccharides. ​

[0082] The standard curve equation of the sulfuric acid-phenol method was used to determine the polysaccharide content. According to the national standard SN / T 4260-2015, the absorbance value was measured at 490 nm, and the standard curve was drawn with the absorbance value as the vertical coordinate and the concentration of the glucose control solution as the horizontal coordinate. Figure 2 The regression equation and correlation coefficient Y = 0.0055x + 0.0007, R 2 = 0.9999 were calculated.

[0083] The polysaccharide content was expressed as a mass fraction ω, and the unit was grams per hundred grams (g / 100g). m1 was the sugar content in the sample determination solution obtained from the standard curve, and the unit was micrograms (μg). The sample was diluted to a volume V1, and the unit was milliliters (mL). The sample determination liquid was removed by colorimetric determination, and the volume V2 was milliliters (mL). The sample mass m2 was grams (g). The conversion factor of 0.9 was used to convert glucose to glucan.

[0084]

[0085] An appropriate amount of Hemerocallis polysaccharide was taken to prepare a solution, and the absorbance value was measured. The concentration of Hemerocallis polysaccharide glucose was calculated from the regression equation, and the polysaccharide content was calculated according to the formula provided in the national standard SN / T 4260-2015 based on the sample glucose concentration. The results are shown in Table 2. From Table 2, it can be seen that the glucose concentration in the Hemerocallis extract measured by this method was 78.53 μg / mL, and the relative standard deviation RSD was 3.55%. Then, the polysaccharide content in Hemerocallis was calculated according to the formula, which was 11.31 g / 100g.

[0086] Table 2 Polysaccharide content measured by the sulfuric acid-phenol method

[0087]

[0088] Optimization of flavonoid and polysaccharide extraction process:

[0089] To further optimize the step-by-step extraction process of flavonoids and polysaccharides, ultrasonic-assisted extraction process optimization can be considered in future work. The factors affecting the test results were selected and the corresponding levels were orthogonalized to screen the optimal extraction process of flavonoids.

[0090] During the process of 1.2, after the extraction of crude polysaccharide, the polysaccharide purification process can be increased before the single sugar and oligosaccharide are removed by suction filtration. The alcohol-sedimented Hemerocallis polysaccharide is separated from ethanol by vacuum suction filtration. After the precipitate is broken up by centrifugation as much as possible, it is washed with anhydrous ethanol, acetone, diethyl ether and ethyl acetate respectively for three times, each time is centrifuged at 20°C, 4000 rpm for 20 min, and then the solvent is evaporated and placed in a vacuum drying oven at 60°C until the constant weight.

[0091] 1.3 Extraction and content determination of saponin

[0092] After the polysaccharide extraction is ultrafiltered, the permeate is vacuum concentrated to 20% of the original volume by a rotary evaporator, and dried under reduced pressure to obtain crude saponin. The saponin is dissolved with deionized water, passed through an AB-8 macroporous adsorption resin column, eluted with ethanol solution, and collected and concentrated to dryness to obtain Hemerocallis saponin.

[0093] The total saponin content is determined by vanillin glacial acetic acid colorimetric method. 20, 50, 100, 150 and 200 μL of 2.0 mg / mL ginsenoside Re are taken respectively, and the absorbance is determined at 560 nm according to the "Health Food Inspection and Evaluation Technical Specification (2003 Edition)" XXIII, Determination of Total Saponin in Health Food. The standard curve is drawn with the absorbance as the vertical coordinate and the total saponin content (μg) as the horizontal coordinate. Figure 3 The regression equation and correlation coefficient Y = 0.0011X + 0.0164, R 2 = 0.9969 are calculated.

[0094] 1 mL of Hemerocallis extract solution is taken in five portions, and the absorbance is determined according to the experimental method. The total saponin content (μg) in Hemerocallis is calculated from the regression equation, and the results are shown in Table 3. From Table 3, the total saponin content in the Hemerocallis extract solution determined by this method is 437.64 μg, and the relative standard deviation RSD = 2.79%, and the polysaccharide content in Hemerocallis is 1.75 g / 100 g.

[0095] Table 3 Determination of total saponin content by vanillin glacial acetic acid colorimetric method

[0096]

[0097] Example Two:

[0098] Spectral analysis of Hemerocallis polysaccharide

[0099] 2.1 Ultraviolet spectrum analysis

[0100] The appropriate amount of Hemerocallis polysaccharide was dissolved in water to prepare a 1.0 g / L aqueous solution, and the UV-visible spectrophotometry was used to scan in the range of 190-400 nm. The absorption spectrum only had a fine absorption peak at 200 nm, and the spectrum showed that Hemerocallis polysaccharide had the characteristics of polysaccharide, and there was no obvious UV absorption peak at 220 nm and 260 nm, which was a flat background. The results showed that the extracted and purified Hemerocallis polysaccharide did not contain nucleic acid, protein and polypeptide components, and the results are shown in Figure 4 .

[0101] 2.2 Infrared spectrum analysis

[0102] 1 mg of dried Hemerocallis polysaccharide sample was taken, and 100 mg of dried potassium bromide powder was uniformly ground under an infrared lamp in a corundum mortar, and was pressed into a thin slice for infrared spectrum determination, and the results are shown in Figure 5 .

[0103] From the infrared spectrum, the structures and groups contained in the Hemerocallis polysaccharide molecules and their characteristic absorption peaks are as follows: 3427 cm -1 hydroxyl stretching vibration peak, 2925 cm -1 methylene stretching vibration peak, 1636 cm -1 hydroxyl rotation vibration peak, 1379 cm -1 methylene shear vibration peak, 1119 cm -1 shear vibration peak of secondary alcohol group, 1030 cm -1 stretching vibration peak of ether bond on sugar ring, 936 cm -1 symmetrical stretching vibration peak of fructofuranose ring, 867 cm -1 fructofuranose methylene transverse vibration peak, 817 cm -1 fructofuranose methylene shear vibration peak, wherein 1119, 1020 cm -1 are characteristic absorption peaks of ketal characteristic groups, 936, 867, 817 cm -1 are fructan related absorption peaks. Based on the comprehensive infrared spectrum characteristic absorption peaks and fructose composition and proportion data, it can be considered that the obtained product is a mixed fructan.

[0104] Example Three:

[0105] In vitro detection method for the use of Hemerocallis polysaccharide in anti-neuritis

[0106] Establishment of cell model and grouping

[0107] BV2 microglial cells were cultured in DMEM high glucose medium containing 50 mL / L fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, in a 37°C, 5% CO2 incubator. BV2 microglial cells were treated with 100 ng / mL LPS for 12 h. The active substance of BCHCB was used to protect the cells 24 h before LPS stimulation. The BV2 microglial cells were divided into 4 groups: normal control group, model group (LPS stimulation), BCHCB group, and BCHCB combined with LPS treatment group.

[0108] 3.1 MTT method for detecting the survival rate of BV2 microglial cells

[0109] Logarithmic growth phase cells were taken, and 24 h later, the cells were divided into control group, LPS group, BCHCB group, and LPS + BCHCB group at 1 × 10 4 The cells were inoculated in 96-well plates and cultured at 37°C, 5% CO2, with 6 replicates per group. The LPS group was stimulated with LPS 1 mg / L for 24 h; the BCHCB group was added with 1, 10, 30, and 60 mg / L BCHCB for 24 h; the LPS + BCHCB group was incubated with LPS 1 mg / L and BCHCB 1, 10, 30, and 60 mg / L for 24 h; and the normal control group was not treated. After drug treatment, the supernatant was discarded, 100 μL of MTT 0.5 g / L solution was added to each well, and the cells were cultured at 37°C, 5% CO2 for 4 h. The supernatant was discarded, 100 μL of DMSO was added to each well, and the cells were shaken until the crystals dissolved. The absorbance (A) value was measured at 490 nm using a microplate reader. Cell survival rate (%) = (experimental group A490nm / cell control group A490nm) × 100%.

[0110] MTT method for detecting the survival rate of BV2 microglial cells

[0111] The MTT results (as shown in Figure 6 ) showed that after BCHCB was incubated with 1.5 g / L LPS for 24 h, there was no statistically significant difference in cell survival rate between each group and the control group, and BCHCB alone or in combination with LPS had no obvious toxicity to BV2 microglial cells.

[0112] 3.2 Detection of NO levels in BV2 microglial cell culture medium

[0113] 96-well plates were inoculated with 4 × 10 5BV2 microglial cells, 100 μL. The experimental groups were control group, 1 mg / L LPS group, 10 mg / L BCHCB combined with 1 mg / L LPS group, 30 mg / L BCHCB combined with 1 mg / L LPS group, 60 mg / L BCHCB combined with 1 mg / L LPS group, 6 replicates in each group. After the cells were adhered, the control group was not treated, and 10, 30, and 60 mg / L BCHCB were added to the LBP treatment groups, respectively, and cultured in an incubator for 12 h; except for the control group, the cells were collected after 1 mg / L LPS was added to each well and cultured for 24 h. According to the kit method, the absorbance (A) of each group was detected at 540 nm.

[0114] BCHCB inhibits LPS-induced NO release from BV2 microglial cells

[0115] The results of the Griess method showed that, compared with the control group, a large amount of NO was released from BV2 microglial cells after LPS stimulation, and the difference was statistically significant (P<0.01, Figure 7 ), compared with the LPS group, the release of NO from BV2 microglial cells treated with different doses of BCHCB and LPS was significantly reduced in a dose-dependent manner (P<0.01, Figure 7 ).

[0116] 3.3 Determination of the contents of inflammatory factors IL-1β, IL-6, TNF-α and anti-inflammatory factor IL-10 in cell culture medium by ELISA method

[0117] The BV2 microglial cell culture medium was collected, and the control and treatment groups were the same as above. According to the requirements of the ELISA kit, the absorbance (A) of each group was detected at 405 nm by a microplate reader.

[0118] BCHCB reduces the secretion of inflammatory factors and increases the secretion of anti-inflammatory factors in LPS-induced BV2 microglial cells The secretion of inflammatory factors and anti-inflammatory factors in each group of cell culture medium was detected by ELISA method. The results showed that, compared with the control group, the secretion of TNF-α, IL-1β, and IL-6 in the LPS group was significantly increased ( Figure 8 ), and the secretion of IL-10 was significantly reduced ( Figure 8 ). After 24 h of co-treatment of LPS with different doses of BCHCB, the secretion of TNF-α, IL-1β, and IL-6 was reduced, and the secretion of IL-10 was increased, and the correlation with the dose was shown, and the difference was statistically significant (P<0.01, Figure 8 ). This indicates that BCHCB has the effect of inhibiting the secretion of TNF-α, IL-1β, and IL-6 and promoting the secretion of IL-10.

[0119] LPS treatment group 1 mg / L; BCHCB combined with LPS 1 mg / L treatment group BCHCB dose was 10, 30, 60 mg / L. b P<0.01 vs control group; c P<0.01 vs LPS treatment group.

[0120] 3.4 RT-qPCR detection of BV2 microglial cells in inflammatory factors TNF-α, IL-1β, IL-6 and anti-inflammatory factor IL-10 mRNA expression levels

[0121] Logarithmic growth phase cells were inoculated in 60 mm culture dishes, and the cells were treated as before. The cells were collected and mRNA was extracted using conventional reagents or commercially available kits. According to the instructions, mRNA was reverse transcribed to obtain the corresponding cDNA, and real-time quantitative PCR (RT-qPCR) was used to detect the mRNA expression of TNF-α, IL-1β, IL-6, IL-10 and β-Actin. Reaction conditions: 95℃ for 5 min pre-denaturation; 95℃ for 10 s, 60℃ for 30 s, 40 cycles of reaction; 95℃ for 15 s, 60℃ for 60 s, 95℃ for 15 s. β-Actin as an internal reference, 2 -△△Ct The relative expression level of the target gene mRNA was calculated. The primer sequences are shown in Table 4.

[0122] Table 4 RT-qPCR primer sequences

[0123]

[0124] Statistical analysis

[0125] The experimental results are expressed as ± s, and the data are analyzed using one-way ANOVA. P<0.05 is statistically significant.

[0126] RT-qPCR results Figure 9 showed that compared with the cell control group, the mRNA expression levels of TNF-α, IL-1β and IL-6 in the LPS group were significantly increased (P<0.01). Compared with the LPS group, the expression levels of TNF-α, IL-1β and IL-6 in the LPS+BCHCB treatment groups were significantly reduced (P<0.01), and the mRNA expression level of IL-10 was significantly increased (P<0.01).

[0127] LPS treatment group 1 mg / L; BCHCB combined with LPS 1 mg / L treatment group BCHCB dose was 10, 30, 60 mg / L. b P<0.01 vs control group;c P<0.01 vs LPS treated group.

[0128] From the above experimental results, it is found that the Hemerocallis citrina Baroni active substance can significantly inhibit the production and release of TNF-α, IL-1β, IL-6, etc. inflammatory factors and anti-inflammatory factor IL-10 in the BV2 microglial cell inflammation model induced by LPS. This shows that Hemerocallis citrina Baroni active substance has good anti-inflammatory effect.

[0129] Example Four:

[0130] Use of Hemerocallis citrina Baroni polysaccharide against neuroinflammation

[0131] Animals: SPF healthy male C57BL / 6 mice, body weight 22-25g;

[0132] Drugs: BCHCB; LPS;

[0133] 4.1 Grouping, dosing, modeling and sampling:

[0134] The experiment used 9-11 week old C57BL / 6 male mice, which were adaptively fed for 1 week and then randomly divided into 5 groups, with 10 mice in each group. The blank group, model group and high, medium and low three different concentrations of BCHCB drug treatment groups were set up. According to the group, the corresponding high, medium and low three different concentrations of drugs BCHCB (240, 120, 60 mg / kg) were given for three consecutive weeks, and the blank group and the model group were given normal saline, which were all given by gavage, with a dose of 0.2 mL, once a day. From the 15th day, except for the blank group, the other groups of mice were injected with lipopolysaccharide (LPS) 400 μg / kg, 10 mL / kg to establish a neuroinflammatory model, and LPS was injected for 7 consecutive days. The gavage was still continued, and after three weeks of administration, the brain tissue was taken 4 hours after the last injection of the day under anesthesia, and 7 mice in each group were stored at -20°C, and the rest were fixed with 4% paraformaldehyde.

[0135] 4.2 HE staining of mouse brain tissue:

[0136] The mouse brain tissue fixed in paraformaldehyde was dehydrated, transparentized, immersed in wax, embedded, sectioned, and deparaffinated, and then HE staining was performed, neutral gum was sealed, and the morphology of nerve cells was observed under a light microscope.

[0137] If the hippocampal structure in the brain tissue of the normal control group mice is relatively complete, the morphology of the neuronal cells is regular, the arrangement is relatively compact, the cytoplasm is red-stained, the nucleus is large and round, and there is no other obvious change. The hippocampal cells in the brain tissue of the model group mice are sparse and disordered, a large number of neuronal cells are pyknotic, the nucleus is small, and the intercellular space is increased. The BCHCB combined with LPS treatment group can significantly improve the changes caused by LPS, indicating that BCHCB has a protective effect on LPS-induced neuronal damage.

[0138] 4.3 Detection of NO content in mouse brain tissue:

[0139] The frozen mouse brain tissue was washed with pre-cooled PBS (0.01 M, pH = 7.4) to remove residual blood, weighed, and cut into small pieces. The cut tissue was thoroughly ground with 9 times the volume of PBS solution (V / W) in an ice bath. Finally, the homogenate was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected. The NO content of each group was determined according to the instructions of the NO assay kit.

[0140] If the NO content in the brain of the model group mice is significantly increased compared with the normal control group, and the BCHCB combined with LPS treatment group can significantly reduce the NO level in the brain of the LPS-induced neuroinflammatory model mice compared with the model group, it indicates that BCHCB has an inhibitory effect on the release of NO in the brain of the LPS-induced neuroinflammatory model mice.

[0141] 4.4 ELISA detection of IL-10, TNF-α, IL-1β, IL-6 content in mouse brain tissue:

[0142] The frozen mouse brain tissue was added with PBS solution and ground in an ice bath to prepare 10% tissue homogenate. Finally, the homogenate was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected. The IL-10, TNF-α, IL-1β, and IL-6 contents of each group were detected according to the instructions of the corresponding ELISA kit.

[0143] If the TNF-α, IL-1β, and IL-6 contents in the brain of the model group mice are significantly increased compared with the normal control group, and the IL-10 content is significantly reduced, and the BCHCB combined with LPS treatment group, BCHCB 60 mg / kg, 120 mg / kg, and 240 mg / kg dose groups can significantly reduce the TNF-α, IL-1β, and IL-6 levels in the brain of the LPS-induced neuroinflammatory model mice, and the IL-10 level is increased, it indicates that BCHCB has an inhibitory effect on the release of TNF-α, IL-1β, and IL-6 in the brain of the LPS-induced neuroinflammatory model mice, and has a promoting effect on the release of IL-10.

[0144] 4.5 Western blot detection of iNOS, COX-2, p65, p-IkB, p-Ikka, p-p38, PKC, Myd88, and AQP4 protein expression:

[0145] The frozen mouse brain tissue was cut into small pieces, and RIPA lysis buffer was added to the tissue pieces and ground on ice for 30 min. The lysis buffer was centrifuged at 12000 rpm for 5 min at 4°C, and the protein concentration of each group was determined by BCA method. The protein solution was boiled with protein loading buffer for 10 min, and the proteins were separated by SDS-PAGE gel electrophoresis (5% concentrated gel, 10% separation gel, constant voltage 80V120V). The PVDF membrane was soaked in TBST containing 5% skimmed milk (blocking solution), and blocked on a shaking bed at room temperature for 2 h. Phosphorylated proteins were blocked with 1% BSA. One iNOS (1:1000), one COX-2 (1:500), one p-IkB (1:2000), one p-Ikka (1:1000), one p-p38 (1:1000), one p65 (1:2000), one Myd88 (1:5000), one AQP4 (1:1000), one PKC (1:5000), and one β-actin (1:5000) were added, and incubated at 4°C overnight. The primary antibody was washed with TBST, and the corresponding HRP-labeled secondary antibody (1:10000) was added and incubated at room temperature for 2 h. The secondary antibody was washed with TBST, and the PVDF membrane was soaked in ECL chemiluminescence solution for several minutes. The film was scanned and imaged under a multifunctional imaging system, and the gray value of the film was analyzed using IPP software.

[0146] If the expression of inflammatory proteins iNOS, COX-2, p65, p-IkB, p-Ikka, p-p38, PKC, Myd88, AQP4 in the brain of the model group mice is significantly higher than that of the normal control group, it indicates that LPS induces inflammation in the brain of mice. Compared with the model group, BCHCB combined with LPS treatment group BCHCB 60mg / kg, 120mg / kg, 240mg / kg dose group can significantly reduce the expression of inflammatory proteins iNOS, COX-2, p65, p-IkB, p-Ikka, p-p38, PKC, Myd88, AQP4 in the brain of LPS-induced neuroinflammatory model mice, indicating that BCHCB can inhibit neuroinflammatory response.

[0147] LPS is a product of gram-negative bacteria, which can activate intracellular NF-κB, MAPK and other inflammatory signaling pathways, causing increased expression of inflammatory factors such as TNF-α and inflammatory mediators such as NO in microglial cells. It is widely used to induce BV2 microglial cell inflammation model to study the anti-neuroinflammatory effect and mechanism of BCHCB.

[0148] Experiments have shown that after LPS stimulation, the PI3K / AKT and NF-κB pathways in BV2 microglial cells are activated, and the expression of NO and inflammatory factors is significantly increased, which provides a reference for further exploring the accurate chemical composition of BCHCB to regulate the inflammatory response signaling pathway in cells.

[0149] TNF-α plays a regulatory role in the physiological processes of the central nervous system. When it binds to the receptor, it can increase the intracellular calcium ion level, activate the p38-MAPK signaling pathway, up-regulate the NF-κB signal, and induce cell survival or apoptosis. IL-1β produced by microglial cells is a key pro-inflammatory factor. TNF-α and IL-1β can stimulate the abnormal release of glutamate in neurons through mitochondrial glutamate, leading to nerve cell death, and can also activate transcription factors to increase the production of pro-inflammatory cytokine IL-6, further exacerbating inflammation. The results showed that after LPS stimulation of BV2 microglial cells, the mRNA levels of TNF-α, IL-1β and IL-6 increased, and the secretion of TNF-α, IL-1β and IL-6 also increased significantly. BCHCB can significantly inhibit the expression of TNF-α, IL-1β and IL-6 mRNA in BV2 microglial cells induced by LPS, and inhibit the secretion of TNF-α, IL-1β and IL-6. Compared with the control group, different doses of BCHCB can inhibit the secretion of the above inflammatory factors in BV2 microglial cells after LPS stimulation, while promoting the secretion of anti-inflammatory factor IL-10, and the results have a dose-dependent effect. At the same time, other studies have shown that different amounts of polysaccharide active substances combined with LPS together act on BV2 microglial cells, and the cells gradually recover from the activated inflammatory form to the resting form.

Claims

1. A method for extracting a hemerocallis active material, characterized by, The different physical and chemical properties of the active substances of Hemerocallis citrina are utilized to extract flavonoids, polysaccharides and saponins in sequence, and the specific steps are as follows: (1) Freshly washed Hemerocallis citrina is steamed, cooled to room temperature, dried and made into Hemerocallis citrina dry product coarse powder; (2) The Hemerocallis citrina dry product coarse powder is precisely weighed, soaked in a container with ethanol, and then transferred into a distillation flask after ultrasonic treatment; (3) The solution in the distillation flask in step (2) is refluxed several times with a water bath, filtered and heated to recover ethanol, and the concentrated filtrate is stopped heating when it reaches a small volume and has no alcohol smell, and then flavonoids are obtained; the filtrate and the Hemerocallis citrina residue are reserved for later use; (4) The filtrate and the Hemerocallis citrina residue in step (3) are refluxed with ethanol; (5) The residue after refluxing in step (4) is soaked with hot water, and monosaccharides and oligosaccharides are removed by suction filtration; the filtered material after suction filtration is extracted again, and the two filtrates are combined and filtered through a microfiltration membrane to remove macromolecular substances and impurities; (6) The combined filtrate in step (5) is sequentially subjected to ultrafiltration through ultrafiltration membranes with different molecular weight cut-offs, and the concentrated liquid retained by the ultrafiltration membranes and the permeate liquid passing through the ultrafiltration membranes are obtained through repeated ultrafiltration; (7) The concentrated liquid obtained in step (6) is vacuum concentrated by a concentration device to obtain crude polysaccharides; (8) The permeate liquid obtained in step (6) is concentrated to a small volume by rotary evaporation and vacuum concentration, and dried under reduced pressure to obtain crude saponins; (9) The crude saponins are dissolved in deionized water, passed through an adsorption resin column, and eluted with an ethanol solution to obtain an eluate; (10) The eluate in step (9) is collected and concentrated to dryness to obtain saponins.

2. A method for detecting a hemerocallis active material, characterized by, The rapid detection of flavonoid compounds, polysaccharides and saponins is realized by fluorescence and ultraviolet-visible spectrophotometry in sequence; and the specific steps are as follows: Step one, rapid detection of flavonoid compounds 1.1 A standard curve is established by ultraviolet-visible spectrophotometry to quantitatively detect Hemerocallis citrina total flavonoids; 1.2 Qualitative detection of flavonoids by fluorescence: Hemerocallis citrina extract is dropped on filter paper, and a bright yellow-green color is observed under ultraviolet light, which indicates that C3-hydroxyl flavonoids, i.e. flavonol flavonoids, are contained; Step two, rapid detection of polysaccharides A standard curve equation is established by ultraviolet-visible spectrophotometry to quantitatively detect Hemerocallis citrina polysaccharides; Step three, rapid detection of saponins A standard curve equation is established by ultraviolet-visible spectrophotometry to quantitatively detect Hemerocallis citrina saponins.

3. A method for detecting a hemerocallis active material, characterized by, The rapid detection of flavonoids in step one is to detect rutin standard solution by a spectrophotometer to establish a regression equation standard curve; The rapid detection of polysaccharides in step two is to determine the polysaccharide content by the sulfuric acid-phenol method standard curve equation; The rapid detection of saponins in step three is to determine the total saponin content by the vanillin glacial acetic acid solution colorimetric method.

4. Use of a hemerocallis active substance, characterized in that, The use of flavonoids, polysaccharides and saponins in resisting neurogenic inflammation is obtained by detecting and analyzing the effects of active substances on resisting neurogenic inflammation; a cell inflammation model is established by stimulating BV2 microglial cells with lipopolysaccharide, Hemerocallis citrina active substances are added, and the cell activity, secretion of inflammatory factors and gene expression are compared with those of the control group to detect and evaluate the effects of Hemerocallis citrina active substances on resisting neurogenic inflammation in vitro and in vivo.

5. Use of the herbaceous daylily active substance according to claim 4, characterized in that, The specific steps for detecting and proving the anti-neuroinflammatory effect of the in vitro stage are as follows: The BV2 microglial cells are cultured for 12 hours in contact with LPS; the active substance of BCHCB is used to protect the cells before LPS stimulation; the BV2 microglial cells are divided into four groups: a normal control group, a model group (LPS stimulation), a BCHCB group, and a BCHCB combined with LPS treatment group; wherein, Step 1.1: MTT method is used to detect the cell survival rate; Step 1.2: Griess method is used to detect NO (nitric oxide) release; Step 1.3: ELISA method is used to detect the content of inflammatory factors and anti-inflammatory factors; Step 1.4: RT-qPCR method is used to detect the mRNA expression level of inflammatory factors and anti-inflammatory factors.

6. Use of the herbaceous plant of Hemerocallis according to claim 5, characterized in that, In step 1.1, the BV2 microglial cells are treated according to the grouping; the absorbance (A) value is measured at 490 nm by a microplate reader; the cell survival rate (%) = (experimental group A490nm / cell control group A490nm) x 100%; In step 1.2, the BV2 microglial cells are collected according to the aforementioned grouping; the absorbance (A) value of each group is detected at 540 nm; the wavelength set may vary depending on different markers or substrates; In step 1.3, the cell culture solution is collected according to the grouping; The absorbance (A) value of each group is detected by a microplate reader at 405 nm; Based on the experimental data, it is evaluated whether BCHCB has a protective effect on LPS-induced BV2 microglial cell damage. The specific steps for detecting and proving the anti-neuroinflammatory effect of the in vivo stage are as follows:

7. Use of the Hemerocallis active substance according to claim 4, characterized in that, Step 2.1: Brain tissue pathological structure examination is performed to evaluate the damage degree of hippocampal structure in the brain tissue; Step 2.2: The content of NO (carbon monoxide) in the brain tissue is detected; Step 2.3: ELISA method is used to detect the content of inflammatory factors in the brain tissue; Step 2.4: Western blot method is used to detect the expression of inflammation-related proteins. The anti-neuroinflammatory effect of BCHCB is detected by animal experiment method; the animals are grouped, dosed, modeled, and sampled according to appropriate conditions; the animal grouping method is similar to the in vitro detection of BV2 microglial cells; 8. Use of the Hemerocallis active substance as claimed in claim 7, characterized in that, BCHCB is divided into high, medium, and low concentration groups; the experimental operations in steps 2.1, 2.2, and 2.3 are performed at low temperature; the frozen brain tissue is washed with pre-cooled buffer, which can be PBS (0.01M, pH=7.4), and is thoroughly ground and homogenized in an ice bath; by comprehensively evaluating the in vivo detection results of this stage, it is determined whether an inflammatory reaction has occurred, and whether BCHCB can inhibit neuroinflammatory reaction; wherein, Step 2.1: The brain tissue can be stained with HE, and the morphology of nerve cells in the brain tissue is observed under a light microscope; Step 2.2: The absorbance (A) value of each group is detected by a microplate reader at 405 nm; the wavelength set may vary depending on different markers or substrates; Step 2.3: The content of inflammatory factors in the brain tissue homogenate of each group of experimental animals is directly detected; ​ Step 2.4: The brain tissue block was grinded on ice for 30 min in RIPA lysis buffer, and the lysis buffer was centrifuged at 12000 rpm for 5 min at 4°C. The protein concentration of each group was determined by BCA method.

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