Dendrobium nobile protoplasm, and preparation method and application thereof

Abstract

The present application belongs to the technical field of plant plasma preparation, and particularly relates to a dendrobium candidum plasma, a preparation method and application thereof. In the preparation method, first, the dendrobium candidum is extracted by using 1% sodium bicarbonate solution. The 1% sodium bicarbonate solution can neutralize the acidic environment and inhibit the acid hydrolysis of polysaccharides in the extraction process. After the extraction by the 1% sodium bicarbonate solution, 50% ethanol solution is added to the dendrobium candidum residue for further extraction. Different polar solvent systems can be used to realize gradient separation of components, and the 50% ethanol solution can dissolve the acidic polysaccharides in the dendrobium candidum which are not dissolved by the alkaline solution, and can effectively extract flavonoids, glycosides and other moderately polar substances. Furthermore, water-saturated n-butanol solution is added to the extracted dendrobium candidum residue for extraction, so as to effectively extract the residual polysaccharides and saponins in the dendrobium candidum. Through the above steps, the plasma components in the dendrobium candidum can be effectively retained, and the dendrobium candidum has excellent anti-inflammatory effect.

Description

Technical Field

[0001] This invention belongs to the field of plant pulp preparation technology, specifically relating to a Dendrobium pulp, its preparation method, and its application. Background Technology

[0002] Dendrobium, a traditional Chinese medicine, has the effects of nourishing the stomach and promoting the production of body fluids, nourishing yin and clearing heat. It is often used to treat symptoms such as stomach yin deficiency, heat-related injury to body fluids, and kidney yin deficiency. Modern pharmacological studies have shown that Dendrobium contains active ingredients such as polysaccharides and alkaloids, which have immunomodulatory, antioxidant, and metabolism-promoting effects.

[0003] Dendrobium officinale extract is a concentrated liquid product extracted from Dendrobium officinale stems. It retains core components such as polysaccharides and amino acids, and boasts high absorption rates and convenience. However, in some studies known to the inventor, the preparation of Dendrobium officinale extract involved adding cellulase to the secondary extract. The added cellulase degraded and damaged the polysaccharides, the main component of the extract, failing to maintain the integrity of the original Dendrobium officinale components. In other studies known to the inventor, when preparing Dendrobium officinale extract using Dendrobium nobile as the raw material, the extract was added to an extraction tank and boiled at a controlled temperature of 80-100℃. This extraction method, exceeding 80℃, hydrolyzes and breaks glycosidic bonds, destroying the three-dimensional structure of polysaccharides and resulting in loss of activity, thus damaging the active ingredients in the Dendrobium officinale extract.

[0004] Therefore, there is an urgent need in this field to provide a method for preparing Dendrobium pulp that can completely preserve the active ingredients of Dendrobium. Summary of the Invention

[0005] The purpose of this invention is to provide a Dendrobium officinale pulp, its preparation method and application. The preparation method can retain more active ingredients in the Dendrobium officinale pulp, and it is stable in nature, while also having excellent anti-inflammatory effects.

[0006] This invention provides a method for preparing Dendrobium officinale pulp, comprising the following steps: 1) Mix Dendrobium powder with 1% sodium bicarbonate aqueous solution and heat to extract. Filter the extracted solution and concentrate the filtrate under reduced pressure to obtain the first Dendrobium pulp. 2) Mix the filter residue obtained in step 1) with a 50% ethanol solution and heat to extract. Filter the extracted solution and concentrate the filtrate under reduced pressure to obtain the second Dendrobium pulp. 3) The filter residue obtained in step 2) is mixed with water-saturated n-butanol for extraction, and the resulting extract is concentrated under reduced pressure to obtain the third Dendrobium pulp; 4) Mix the first Dendrobium pulp, the second Dendrobium pulp and the third Dendrobium pulp to obtain Dendrobium raw pulp.

[0007] Preferably, in step 1), the 1% sodium bicarbonate aqueous solution is 8 to 12 times the mass of Dendrobium powder.

[0008] Preferably, in step 1), the temperature of the heating extraction is ≤60℃, the heating extraction time is 1~3h, and the number of heating extractions is 1~3 times.

[0009] Preferably, in step 2), the ethanol solution is 8 to 12 times the volume of the filter residue; the temperature of the heating extraction is ≤60℃; the heating extraction time is 1 to 3 hours; and the heating extraction is performed 1 to 3 times.

[0010] Preferably, in step 3), the water-saturated n-butanol is 15 to 20 times the mass of the filter residue; and the extraction is performed 2 to 3 times.

[0011] Preferably, 95% of the Dendrobium powder contains powder with a particle size of ≥40 mesh.

[0012] Preferably, the dendrobium includes Dendrobium officinale and / or Dendrobium purpureus.

[0013] The present invention also provides a Dendrobium officinale pulp, which is prepared by the preparation method described in the above technical solution.

[0014] The present invention also provides the application of the Dendrobium officinale pulp described in the above technical solution in food and / or pharmaceuticals.

[0015] The present invention also provides the application of the Dendrobium officinale pulp described in the above technical solution in the preparation of anti-inflammatory products.

[0016] Beneficial effects: This invention provides a method for preparing Dendrobium officinale pulp. In this method, Dendrobium officinale is first thermally extracted using a 1% sodium bicarbonate aqueous solution. The 1% sodium bicarbonate solution neutralizes the acidic environment and inhibits acidic hydrolysis of polysaccharides during extraction. Glycosidic bonds in Dendrobium officinale polysaccharides are easily broken under acidic conditions, while neutral or weakly alkaline environments maintain their structural integrity. Furthermore, a 50% ethanol solution is added to the Dendrobium officinale residue after extraction with the 1% sodium bicarbonate aqueous solution for further extraction. This allows for gradient separation of components using solvent systems of different polarities. The 50% ethanol solution dissolves acidic polysaccharides in Dendrobium officinale that were not dissolved in alkaline water, and effectively extracts moderately polar substances such as flavonoids and glycosides, which have low solubility in weakly alkaline aqueous solutions. Furthermore, after extraction with a 50% ethanol solution, water-saturated n-butanol was added to the extracted Dendrobium residue for further extraction. n-Butanol is a food additive permitted for use in China and is safer than organic solvents such as chloroform and methanol. It also has a high extraction capacity for residual polysaccharides and saponins in Dendrobium. Through these steps, the active ingredients in Dendrobium can be effectively preserved, and it exhibits excellent anti-inflammatory effects. Attached Figure Description To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 This is a comparison of the effects of Dendrobium officinale extract on LPS-induced TNF-α secretion in RAW264.7 cells in Example 2. Figure 2 This is a comparison of the effects of Dendrobium officinale extract on LPS-induced IL-1β secretion in RAW264.7 cells in Test Example 2. Detailed Implementation

[0018] This invention provides a method for preparing Dendrobium officinale pulp, comprising the following steps: 1) Mix Dendrobium powder with 1% sodium bicarbonate aqueous solution and heat to extract. Filter the extracted solution and concentrate the filtrate under reduced pressure to obtain the first Dendrobium pulp. 2) Mix the filter residue obtained in step 1) with a 50% ethanol solution and heat to extract. Filter the extracted solution and concentrate the filtrate under reduced pressure to obtain the second Dendrobium pulp. 3) The filter residue obtained in step 2) is mixed with water-saturated n-butanol for extraction, and the resulting extract is concentrated under reduced pressure to obtain the third Dendrobium pulp; 4) Mix the first Dendrobium pulp, the second Dendrobium pulp and the third Dendrobium pulp to obtain Dendrobium raw pulp.

[0019] In one embodiment, the present invention sequentially washes, cuts, crushes, and sieves Dendrobium officinale strips to obtain Dendrobium officinale powder. In one embodiment, the Dendrobium officinale is *Dendrobium nobile* and / or *Dendrobium purpureus*. In one embodiment, the sieve aperture is 40 mesh.

[0020] After obtaining the Dendrobium powder, this invention mixes the Dendrobium powder with a 1% sodium bicarbonate aqueous solution and heats to extract it. The extracted solution is filtered, and the filtrate is concentrated under reduced pressure to obtain the first Dendrobium pulp. In this invention, the 1% sodium bicarbonate aqueous solution is non-corrosive, and residues can be removed by concentration under reduced pressure, meeting the requirements of green extraction. As one embodiment, the 1% sodium bicarbonate aqueous solution is 8-12 times, 9-11 times, or 10-11 times the mass of the Dendrobium powder. As one embodiment, this invention uses a filter cloth with a pore size of 200 mesh for filtration. As one embodiment, the temperature of the reduced pressure concentration is ≤60℃; as one embodiment, the temperature of the heat extraction is ≤60℃; the heat extraction temperature ensures the thermal stability of the Dendrobium polysaccharides. As one embodiment, the heat extraction time is 1-3 hours. As one embodiment, the number of heat extractions is 1-3 times; this invention preferably combines the extracts from multiple heat extractions before subsequent filtration.

[0021] After obtaining the first Dendrobium pulp, the present invention mixes the filter residue obtained from the above steps with a 50% ethanol solution and heats it for extraction. The extracted solution is then filtered, and the filtrate is concentrated under reduced pressure to obtain the second Dendrobium pulp. In one embodiment, the ethanol solution is 8-12 times the mass of the filter residue. In another embodiment, the heating extraction temperature is ≤60℃; this heating extraction temperature ensures the thermal stability of Dendrobium polysaccharides. In another embodiment, the present invention uses a filter cloth with a pore size of 200 mesh for filtration. In another embodiment, the temperature of the reduced pressure concentration is ≤60℃; this reduced pressure concentration removes ethanol from the Dendrobium pulp. In another embodiment, the heating extraction time is 1-3 hours. In another embodiment, the heating extraction is performed 1-3 times; preferably, the extracts from multiple heating extractions are combined before subsequent filtration.

[0022] After obtaining the second Dendrobium pulp, the present invention mixes the filter residue obtained from the above steps with water-saturated n-butanol for extraction, and concentrates the resulting extract under reduced pressure to obtain the third Dendrobium pulp. In one embodiment, the water-saturated n-butanol is 15-20 times the mass of the filter residue. In one embodiment, the extraction is performed 2-3 times. In one embodiment, the extraction time is 1.0-2.0 hours. In one embodiment, the temperature of the reduced pressure concentration is ≤60℃; the reduced pressure concentration can remove organic solvents from the Dendrobium pulp.

[0023] After obtaining the third Dendrobium pulp, the present invention mixes the first Dendrobium pulp, the second Dendrobium pulp and the third Dendrobium pulp to obtain Dendrobium raw pulp.

[0024] This invention also provides a Dendrobium officinale extract, prepared using the method described above. The Dendrobium officinale extract of this invention contains abundant polysaccharides, retains relatively complete active ingredients, and possesses excellent anti-inflammatory effects.

[0025] This invention also provides the application of the Dendrobium officinale pulp described in the above-described technical solution in food and / or pharmaceuticals. As one embodiment, the food can be one or more of health foods, functional foods, and ordinary foods.

[0026] This invention also provides the application of the Dendrobium officinale pulp described in the above-mentioned technical solution in the preparation of anti-inflammatory products. As one embodiment, the product can be a pharmaceutical product.

[0027] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1 A method for preparing Dendrobium officinale pulp, comprising the following steps: 1) Pretreatment: Take 1000g of fresh Dendrobium officinale strips, wash the surface with clean water to remove impurities and dust. Cut the Dendrobium officinale strips into small sections of about 1-2cm, then grind them with a grinder and pass them through a 40-mesh sieve to obtain Dendrobium officinale powder.

[0029] 2) Extraction of water-soluble components: Add 8 times the mass of 1% sodium bicarbonate aqueous solution to Dendrobium powder, and extract twice at 50℃, 1.5h each time. Combine the two extracts, filter through a 200-mesh filter cloth to remove residue. Concentrate the filtered extract under reduced pressure at 50℃ to 100g to obtain Dendrobium pulp 1.

[0030] 3) Extraction of alcohol-soluble components: Add 8 times the mass of 50% ethanol solution to the Dendrobium residue after extraction in step 2), and extract twice at 50℃, 1.5h each time. Combine the two extracts, filter through a 200-mesh filter cloth to remove the residue. Remove the ethanol from the filtered extract under reduced pressure at 50℃, and concentrate to 75g to obtain Dendrobium pulp 2.

[0031] 4) Extraction of organic solvent-soluble components: Add 15 times the mass of water-saturated n-butanol to the Dendrobium residue after extraction in step 3), and extract twice at room temperature, 1 hour each time. Combine the two extracts, remove the organic solvent under reduced pressure at 40°C, and concentrate to 25g to obtain Dendrobium pulp 3.

[0032] 5) Combine Dendrobium pulp 1, Dendrobium pulp 2 and Dendrobium pulp 3, stir well, and you will get 200g of Dendrobium raw pulp.

[0033] Example 2 A method for preparing Dendrobium officinale pulp, comprising the following steps: 1) Pretreatment: Take 1000g of fresh Dendrobium officinale strips, wash the surface with clean water to remove impurities and dust. Cut the Dendrobium officinale strips into small sections of about 1-2cm, then grind them with a grinder and pass them through a 40-mesh sieve to obtain Dendrobium officinale powder.

[0034] 2) Extraction of water-soluble components: Add 12 times the mass of 1% sodium bicarbonate aqueous solution to Dendrobium powder, and extract twice at 55℃, 2 hours each time. Combine the two extracts, filter through a 200-mesh filter cloth to remove residue. Concentrate the filtered extract under reduced pressure at 50℃ to 100g to obtain Dendrobium pulp 1.

[0035] 3) Extraction of alcohol-soluble components: Add 10 times the mass of 50% ethanol solution to the Dendrobium residue after extraction in step 2), and extract twice at 55℃, 2 hours each time. Combine the two extracts, filter through a 200-mesh filter cloth to remove the residue. Remove the ethanol from the filtered extract under reduced pressure at 50℃, and concentrate to 75g to obtain Dendrobium pulp 2.

[0036] 4) Extraction of organic solvent-soluble components: Add 20 times the mass of water-saturated n-butanol to the Dendrobium residue after extraction in step 3), and extract twice at room temperature, 1.5 h each time. Remove the organic solvent from the extract under reduced pressure at 40°C, and concentrate to 25 g to obtain Dendrobium pulp 3.

[0037] 5) Combine Dendrobium pulp 1, Dendrobium pulp 2 and Dendrobium pulp 3, stir well, and you will get 200g of Dendrobium pulp.

[0038] Comparative Example 1 Referring to the "Preparation Method of Dendrobium officinale Pulp Beverage" disclosed in Chinese Patent CN103300450A, the Dendrobium officinale pulp is prepared in the following steps: 1) Take 1000g of Dendrobium officinale stems, bake them at 90℃ for 15 minutes, and then cut them into stem segments with a length of 0.1cm; 2) Take Dendrobium officinale stem segments with a material-to-water ratio of 1:5 and clean water, and pulp them to obtain the initial pulp; 3) Keep the initial slurry at 55℃ for 120 minutes; after keeping it at 55℃, add 8 times the amount of water as Dendrobium officinale raw material, boil for 3 minutes, filter, and store the filtrate; 4) Repeat the process of "adding water and pulping - boiling - filtering" three times on the filter residue. Take the filter residue, add 4 times the amount of water, and pulp to obtain a secondary slurry. 5) Add cellulase to the secondary slurry at a rate of 15 U / g of Dendrobium officinale raw material, and enzymatically hydrolyze for 10 min at pH 5.0 and 52℃ to obtain the enzymatic hydrolysate; 6) Boil the enzymatic hydrolysate for 3 minutes, then filter and store the filtrate; 7) Repeat the process of "adding water and pulping - boiling - filtering" three times on the filter residue. Add 3 times the amount of water to the filter residue, pulp, filter, and collect the filtrate. 8) Combine all the filtrates and concentrate them under reduced pressure to 200g to obtain Dendrobium officinale pulp.

[0039] Comparative Example 2 A method for preparing Dendrobium officinale pulp, comprising the following steps: Compared to Example 1, pure water was added in step 2 instead of 1% sodium bicarbonate aqueous solution, while other conditions remained unchanged.

[0040] 1) Pretreatment: Take 1000g of fresh Dendrobium officinale strips, wash the surface with clean water to remove impurities and dust. Cut the Dendrobium officinale strips into small sections of about 1-2cm, then grind them with a grinder and pass them through a 40-mesh sieve to obtain Dendrobium officinale powder.

[0041] 2) Extraction of water-soluble components: Add 8 times the mass of pure water to the Dendrobium powder and extract twice at 50℃, each extraction lasting 1.5 hours. Combine the two extracts and filter through a 200-mesh filter cloth to remove residue. Concentrate the filtered extract under reduced pressure at 50℃ to 100g to obtain Dendrobium pulp 1.

[0042] 3) Extraction of alcohol-soluble components: Add 8 times the mass of 50% ethanol solution to the Dendrobium residue after extraction in step 2), and extract twice at 50℃, 1.5h each time. Combine the two extracts, filter through a 200-mesh filter cloth to remove the residue. Remove the ethanol from the filtered extract under reduced pressure at 50℃, and concentrate to 75g to obtain Dendrobium pulp 2.

[0043] 4) Extraction of organic solvent-soluble components: Add 15 times the mass of water-saturated n-butanol to the Dendrobium residue after extraction in step 3), and extract twice at room temperature, 1 hour each time. Combine the two extracts, remove the organic solvent under reduced pressure at 40°C, and concentrate to 25g to obtain Dendrobium pulp 3.

[0044] 5) Combine Dendrobium pulp 1, Dendrobium pulp 2 and Dendrobium pulp 3, stir well, and you will get 200g of Dendrobium raw pulp.

[0045] Comparative Example 3 A method for preparing Dendrobium officinale pulp, comprising the following steps: Compared to Example 1, the 50% ethanol in step 3 was replaced with pure water, while other conditions remained unchanged.

[0046] 1) Pretreatment: Take 1000g of fresh Dendrobium officinale strips, wash the surface with clean water to remove impurities and dust. Cut the Dendrobium officinale strips into small sections of about 1-2cm, then grind them with a grinder and pass them through a 40-mesh sieve to obtain Dendrobium officinale powder.

[0047] 2) Extraction of water-soluble components: Add 8 times the mass of pure water to the Dendrobium powder and extract twice at 50℃, each extraction lasting 1.5 hours. Combine the two extracts and filter through a 200-mesh filter cloth to remove residue. Concentrate the filtered extract under reduced pressure at 50℃ to 100g to obtain Dendrobium pulp 1.

[0048] 3) Pure water replacement for alcohol-soluble extraction: Add 8 times the mass of pure water to the Dendrobium residue after extraction in step 2), and extract twice at 50℃, 1.5h each time. Combine the two extracts, filter through a 200-mesh filter cloth to remove residue. Concentrate the filtered extract under reduced pressure at 50℃ to 75g to obtain Dendrobium pulp 2.

[0049] 4) Extraction of organic solvent-soluble components: Add 15 times the mass of water-saturated n-butanol to the Dendrobium residue after extraction in step 3), and extract twice at room temperature, 1 hour each time. Combine the two extracts, remove the organic solvent under reduced pressure at 40°C, and concentrate to 25g to obtain Dendrobium pulp 3.

[0050] 5) Combine Dendrobium pulp 1, Dendrobium pulp 2 and Dendrobium pulp 3, stir well, and you will get 200g of Dendrobium pulp.

[0051] Comparative Example 4 A method for preparing Dendrobium officinale pulp, comprising the following steps: Compared with Example 1, step 4 is removed, while other conditions remain unchanged.

[0052] 1) Pretreatment: Take 1000g of fresh Dendrobium officinale strips, wash the surface with clean water to remove impurities and dust. Cut the Dendrobium officinale strips into small sections of about 1-2cm, then grind them with a grinder and pass them through a 40-mesh sieve to obtain Dendrobium officinale powder.

[0053] 2) Extraction of water-soluble components: Add 8 times the volume of pure water to the Dendrobium micropowder and extract twice at 50℃, each extraction lasting 1.5 hours. Combine the two extracts and filter through a 200-mesh filter cloth to remove residue. Concentrate the filtered extract under reduced pressure at 50℃ to 125g to obtain Dendrobium pulp 1.

[0054] 3) Extraction of alcohol-soluble components: Add 8 times the volume of pure water to the Dendrobium residue after extraction in step 2), and extract twice at 50°C, each time for 1.5 hours. Combine the two extracts, filter through a 200-mesh filter cloth to remove the residue. Concentrate the filtered extract under reduced pressure at 50°C to 75g to obtain Dendrobium pulp 2.

[0055] 4) Combine Dendrobium pulp 1 and Dendrobium pulp 2, stir well, and you will get 200g of Dendrobium raw pulp.

[0056] Test Example 1 The effective components in the Dendrobium officinale pulp prepared in Examples 1-2 and Comparative Examples 1-4 were determined, including: total polysaccharides were determined according to GB / T 18672, mannose and dendrobine were determined by high performance liquid chromatography, and total solids content was determined by drying method. The results are shown in Table 1.

[0057] Table 1. Content of active ingredients in Dendrobium officinale pulp prepared in Examples 1-2 and Comparative Examples 1-4

[0058] Table 1 shows that the total polysaccharide content of the Dendrobium pulp extracted using the method described in this invention is higher, and the contents of mannose, dendrobine, and total solids are also increased to varying degrees compared with the control group.

[0059] Test Example 2 The anti-inflammatory properties of the Dendrobium officinale pulp in Example 1 were determined by Yunnan Zhicui Testing and Inspection Co., Ltd., and the steps are as follows: 1. Principles and methods: LPS-induced RAW264.7 is a classic cell model for studying inflammatory factors. LPS binds to antigen recognition receptors on the surface of macrophages, inducing macrophages to secrete various inflammatory factors such as TNF-α and IL-1β. TNF-α and IL-1β can activate three signaling pathways: caspase, JNK, and the transcription factor NF-κB, thereby achieving their biological functions such as cytotoxicity, antiviral activity, immune regulation, and apoptosis.

[0060] This method evaluates the inhibitory effect of the sample on TNF-α and IL-1β secretion by comparing the differences in TNF-α and IL-1β secretion levels between the model group and the sample group after drug administration. The levels of inflammatory factors were measured using enzyme-linked immunosorbent assay (ELISA). The principle is as follows: inflammatory factors specifically bind to antibodies coated on an ELISA plate, which then bind to substrate-labeled antibodies. The substrate is catalyzed to generate a colored product with absorbance at 450 nm. The levels of inflammatory factors are positively correlated with the OD value. The levels of TNF-α and IL-1β were calculated.

[0061] 2. Reagents, consumables and instruments (1) Reagents and consumables: PBS buffer; DMEM high glucose basal medium; cell proliferation and toxicity test kit (CCK-8); mouse TNF-α ELISA kit and mouse IL-1β ELISA kit; programmed cooling box; DMSO cell culture grade; cell cryopreservation solution; cell cryopreservation tubes; fetal bovine serum; trypsin / EDTA solution; phosphate buffer; penicillin-streptomycin 100X; mycoplasma scavenger; primary water; carbon dioxide tank; T25 and T75 cell culture flasks (ventilated caps); centrifuge tube rack; cell counting chamber; timer; counter; and other routine laboratory instruments and equipment: alcohol lamp, anhydrous ethanol, waste liquid container, aluminum foil, iron or aluminum lunch box, gloves, mask, alcohol-resistant marker, 75% ethanol, alcohol spray bottle, etc.

[0062] (2) Instruments: electric constant temperature water bath; low speed centrifuge; inverted microscope; ultra-clean workbench; medical refrigerator; ultra-low temperature refrigerator; carbon dioxide incubator; high pressure steam sterilizer; electronic balance; enzyme reader; vortex mixer.

[0063] 3. Experimental Procedure: 3.1 Cell Culture: RAW264.7 cells were cultured to the second generation before experiments were conducted.

[0064] 3.2 CCK-8 Cytotoxicity Assay: The in vitro toxicity of the test samples to RAW264.7 cells was investigated using the CCK-8 (Cell Counting Kit 8) assay. Cells were seeded at a specific concentration in 96-well plates. After 24 hours of incubation, the culture medium was discarded, and serially diluted test samples were added. The plates were then incubated for another 24 hours, the supernatant was discarded, and fresh culture medium and CCK-8 reagent were added. Incubation was then performed for 2 hours, followed by absorbance measurement at 450 nm. Cell viability was calculated using the following formula. Note: A cell viability ≥ 80% was considered to have no effect on cell proliferation and was regarded as a safe cell concentration. ; 3.3 Sample preparation and induction drug administration: (1) Preparation of irritant solution: Preparation of LPS stock solution (2 mg / mL): Dissolve 10 mg LPS in 5000 μL PBS, vortex mix for 5 min, let stand for 30 min, dispense into 100 μL / EP tubes, and store at -20℃.

[0065] Preparation of LPS working solution (5 μg / mL): Take 100 μL of LPS with a concentration of 2 mg / mL and add it to 900 μL of culture medium to obtain a working solution of 200 μg / mL.

[0066] (2) Preparation of positive control solution: Preparation of bisabolol (1%) stock solution: Take 10 μL of bisabolol, add DMSO to prepare a 1% bisabolol solution, and then filter it through a 0.22 μm filter membrane for sterilization.

[0067] Preparation of working solution of bisabolol (0.1%): Take 1 μL of stock solution and dilute it to 1 mL with culture medium to obtain 0.1% bisabolol working solution 1.

[0068] (3) Sample preparation: Take Dendrobium officinale pulp and filter it through a 0.22μm microporous membrane for sterilization, and set it aside.

[0069] (4) Cell seeding: Dilute the cells with cell culture medium to the seeding density, seed 1000 μL in each well of a 12-well plate, and incubate in a CO2 incubator for 24 h.

[0070] (5) Induction and drug administration: After the cells reached 45-60% confluence, the culture medium was discarded, and the samples, culture medium, and bisabolol were used to treat the corresponding groups, respectively, and incubated at 37°C in a 5% CO2 incubator for 2 hours. Subsequently, except for the blank / solvent control wells, LPS working solution was added to the other groups, mixed well, and incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0071] (6) Collection of cell supernatant: Collect the cell culture supernatant into a 1.5 mL sterile centrifuge tube, centrifuge at 3000 r / min for 10 min, and then freeze the supernatant at -80℃.

[0072] 3.4 ELISA detection: Perform the procedure according to the kit instructions.

[0073] 3.5 Result Interpretation: The OD value of each standard and sample should be subtracted from the OD value of the blank group. A standard fitting curve should be plotted with the standard concentration on the x-axis and the OD value on the y-axis. Then, the concentration of the sample can be calculated based on its OD value.

[0074] 4. Data Processing: (1) Content Standard solutions of TNF-α and IL-1β were prepared separately and then serially diluted to obtain standard solutions of varying concentrations. The OD values ​​were then measured using the ELISA method described above. 450 ), fit a standard curve, and then based on the OD of the sample 450 The TNF-α and IL-1β levels in the samples were calculated. Each group was repeated three times in parallel, and the mean and standard deviation (SD) were calculated. One-way ANOVA was performed on the results using Prism 10.1.2 statistical software. A p-value < 0.05 was considered statistically significant.

[0075] (2) Inhibition rates of TNF-α and IL-1β Inhibition rate (%) = (1 - T / C) × 100%; T represents the average levels of TNF-α and IL-1β in each sample group; C represents the average levels of TNF-α and IL-1β in the model group.

[0076] 5. Test Results 5.1 CCK-8 cytotoxicity test results: The effect of the samples on the viability of RAW264.7 cells was tested using the CCK-8 kit. The results are shown in Table 2.

[0077] Table 2. Effects of different concentrations of test samples on the viability of RAW264.7 cells.

[0078] According to Table 2, the selected Dendrobium officinale pulp concentration should be able to ensure a cell survival rate of ≥90%. Therefore, samples with a concentration of 1% or less were selected for subsequent experiments.

[0079] 5.2 Anti-inflammatory efficacy test 5.2.1 Results of the detection of the content and inhibition rate of the inflammatory factor TNF-α are as follows: Figure 1 As shown in Table 3, in Figure 1 In the middle, compared with the blank group, #P <0.05, ##P <0.01, ###P <0.001; compared with the model group, P <0.05, P <0.01, P <0.001.

[0080] Table 3. Inhibition of the effect of Dendrobium officinale extract on LPS-induced secretion of the inflammatory cytokine TNF-α in RAW264.7 cells in Example 1.

[0081] Note: Inhibition rate (%) = (1-T / C)×100%; T - average TNF-α content in the sample group; C - average TNF-α content in the model group.

[0082] Figure 1The effects of each test group on the LPS-induced TNF-α secretion level and inhibition rate of RAW264.7 cells were compared with those in Table 3. After 16 h of LPS stimulation of RAW264.7 cells, the TNF-α level in the model group was significantly increased by 1.30 times compared with the blank control group. P <0.001 indicates that the inflammation model was established under these experimental conditions. In the RAW264.7 cell inflammation model treated with the positive control (0.1% bisabolol), the TNF-α content decreased to 1093.14 pg / mL, significantly lower than that in the model group ( P <0.001), the TNF-α inhibition rate was 56.35%. After treating LPS-induced cells with Dendrobium officinale pulp, the TNF-α content secreted by the cells was significantly reduced to 1453.80 pg / mL and 1212.80 pg / mL, respectively, and the calculated TNF-α inhibition rates were 41.99% and 51.59%, respectively.

[0083] 5.2.2 Results of the detection of the content and inhibition rate of the inflammatory factor IL-1β are as follows: Figure 2 As shown in Table 4, compared with the blank group, #P <0.05, ##P <0.01, ###P <0.001; compared with the model group, P <0.05, P <0.01, P <0.001.

[0084] Table 4. Inhibition of LPS-induced secretion of the inflammatory cytokine IL-1β by Dendrobium officinale extract in Example 1.

[0085] Note: Inhibition rate (%) = (1-T / C)×100%; T - average IL-1β content in the sample group; C - average IL-1β content in the model group.

[0086] Figure 2 The effects of each test group on the LPS-induced IL-1β secretion level and inhibition rate of RAW264.7 cells were compared with those in Table 4. After 16 h of LPS stimulation of RAW264.7 cells, the IL-1β level in the model group was significantly increased by 1.46 times compared with the blank control group. P<0.001 indicates that the inflammation model was established under these experimental conditions. In the RAW264.7 cell inflammation model treated with the positive control (0.1% bisabolol), the IL-1β level decreased to 356.0 pg / mL, significantly lower than that in the model group ( P <0.001), the IL-1β inhibition rate was 38.44%. After treating LPS-induced cells with Dendrobium officinale extract, the IL-1β levels secreted by the cells were 593.83 pg / mL and 467.33 pg / mL, respectively, with calculated IL-1β inhibition rates of -2.68% and 19.19%. Therefore, 1% Dendrobium officinale extract has a significant inhibitory effect on the IL-1β levels secreted by LPS-induced RAW264.7 cells. P <0.05).

[0087] The above results show that the Dendrobium officinale extract prepared by the method of this invention has a significant inhibitory effect on the levels of TNF-α and IL-1β secreted by LPS-induced RAW264.7 cells. P The value was <0.05, thus proving that Dendrobium officinale pulp has anti-inflammatory effects.

[0088] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Dendrobium officinale extract, characterized in that, It is prepared by the following steps: 1) Mix Dendrobium powder with a 1% sodium bicarbonate aqueous solution and heat to extract. Filter the extracted solution and concentrate the filtrate under reduced pressure to obtain the first Dendrobium pulp. The 1% sodium bicarbonate aqueous solution is 8-12 times the mass of the Dendrobium powder. The extraction temperature is ≤60℃, the extraction time is 1-3 hours, and the extraction is performed 1-3 times. 2) Mix the filter residue obtained in step 1) with a 50% ethanol solution and heat to extract. Filter the extracted solution and concentrate the filtrate under reduced pressure to obtain the second Dendrobium pulp. The ethanol solution is 8 to 12 times the mass of the filter residue; the temperature of the heating extraction is ≤60℃, the heating extraction time is 1 to 3 hours, and the heating extraction is performed 1 to 3 times. 3) The filter residue obtained in step 2) is mixed with water-saturated n-butanol for extraction, and the resulting extract is concentrated under reduced pressure to obtain the third Dendrobium pulp; the water-saturated n-butanol is 15 to 20 times the mass of the filter residue; the extraction is performed 2 to 3 times. 4) Mix the first Dendrobium pulp, the second Dendrobium pulp and the third Dendrobium pulp to obtain Dendrobium raw pulp.

2. The Dendrobium officinale pulp according to claim 1, characterized in that, 95% of the Dendrobium powder has a particle size ≥40 mesh.

3. The Dendrobium officinale pulp according to claim 1, characterized in that, The Dendrobium species include Dendrobium officinale and / or Dendrobium purpureus.

4. The application of Dendrobium officinale pulp according to any one of claims 1 to 3 in food.

5. The use of Dendrobium officinale pulp according to any one of claims 1 to 3 in the preparation of pharmaceuticals.

6. The use of Dendrobium officinale pulp according to any one of claims 1 to 3 in the preparation of anti-inflammatory products.

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