Method for purifying anti-inflammatory components of dendrobium candidum by chromatographic column

Abstract

The application discloses a method for purifying anti-inflammatory components of Dendrobium candidum by using a chromatographic column. Under the assistance of ultrasonic waves and microwaves, an extractant is used to extract anti-inflammatory effective components of Dendrobium candidum, a flocculating agent is used to simply remove particles and part of impurities, and a solubilizing agent is used to assist in back-extracting the effective components into an ethanol solution, and finally, the anti-inflammatory components of Dendrobium candidum are highly purified through two chromatographic columns.

Description

Technical Field

[0001] This invention relates to the field of health food preparation technology, and more specifically to a method for purifying the anti-inflammatory components of Dendrobium officinale using chromatographic columns. Background Technology

[0002] Dendrobium officinale has a long history of medicinal use and is considered the most effective among more than seventy varieties of Dendrobium in China. It is rich in polysaccharides and dendrobines, containing several times more than other varieties. It is a traditional and precious Chinese medicine with both medicinal and health-promoting effects. The stems of Dendrobium officinale contain various alkaloids, polysaccharides, amino acids, and starch. The pharmacological effects of Dendrobium officinale polysaccharides, alkaloids, and phenolic compounds are interconnected yet distinct. The pharmacological effects of alkaloids are mainly manifested in anti-tumor activity, inhibition of cardiovascular and gastrointestinal diseases, and analgesic and antipyretic effects.

[0003] Dendrobium officinale contains abundant chemical components. According to the analysis of relevant domestic and foreign literature, as of 2023, a total of 261 compounds have been extracted, isolated and identified from Dendrobium officinale. According to their structure, they can be divided into flavonoids, bibenzyl groups, alkaloids, phenylpropanoids and other components. Among them, there are 52 flavonoids, 58 bibenzyl groups, 7 alkaloids, 51 phenylpropanoids, 15 terpenoids, 5 quinones, 15 phenanthrenes, 8 nucleosides, 32 volatile oils and 18 other compounds, as well as 42 amino acids and trace elements. Since Dendrobium officinale is a resource that can be used for both medicinal and edible purposes, current research on its chemical components mainly focuses on the stems, leaves, and flowers. Flavonoids, bibenzyl, and phenylpropanoids are considered to be its main components, and these are precisely the effective anti-inflammatory components in Dendrobium officinale. This invention provides an excellent process for simultaneously extracting various anti-inflammatory components with different physical properties, such as flavonoids, bibenzyl, phenylpropanoids, polysaccharides, proteins, amino acids, and polypeptides. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the technical solution provided by the present invention includes the following steps: (1) Take Dendrobium officinale leaves, dry them at 60℃, pulverize them and sieve them through a 60-mesh sieve to obtain Dendrobium powder; (2) Take 10 parts of Dendrobium powder and 100-140 parts of extractant, mix them, and heat the mixture at 80℃ for 1 hour to allow it to penetrate. (3) After the permeation was completed, the sample was extracted for 1.5 h under microwave 350W, ultrasonic 50W and 60℃ conditions; (4) After extraction, mix 1-3 parts of flocculant and filter while hot to obtain filtrate A and solid particles; (5) Mix 120-180 parts of 50% ethanol solution with 5-13 parts of solubilizer and then mix with filtrate A for back extraction; (6) After back-extraction for 20 min, centrifugation was performed. The upper layer was a mixed ethanol solution and the lower layer was the extractant solution. (7) The mixed ethanol solution was purified by high performance liquid chromatography to obtain a crude extract; (8) The crude extract was further purified by semi-preparative liquid chromatography column to obtain Dendrobium officinale extract solution.

[0005] (9) The solvent was removed by fractional distillation, and the product was washed once with saturated saline and once with deionized water, and then dried to obtain Dendrobium officinale extract.

[0006] Furthermore, the high-performance liquid chromatography (HPLC) conditions are as follows: Nova-Pak C18 column (150 mm x 3.9 mm, 4 μm); mobile phase I is a mixture of 50 mmol / L sodium acetate buffer, methanol, and acetonitrile in a volume ratio of 40:40:20; mobile phase I1 is a mixture of 50 mmol / L sodium acetate buffer and methanol in a volume ratio of 80:20; gradient elution program is as follows: 5% I changes to 45% I within 30 min, then 45% I changes to 60% I within 15 min, and finally 60% I changes to 100% I within the last 5 min; detection wavelength is 260 nm.

[0007] Furthermore, the semi-preparative liquid chromatography column conditions are as follows: column: Silica C18M 10E (250mm x 10mm, 5μm); mobile phase: acetonitrile (A): water-glacial acetic acid-ammonia (B) = 12:88; detection wavelength: 254 nm; column temperature: 25℃.

[0008] Furthermore, the extractant is 1-ethyl-3-methylpyridine tetrafluoroborate.

[0009] Furthermore, the flocculant consists of 3% aluminum zinc polysilicic acid and 97% distilled water.

[0010] Furthermore, the solubilizer is sodium dialkyl dicarboxylate.

[0011] The beneficial effects of this invention are as follows: (1) In order to fully extract various anti-inflammatory components from Dendrobium officinale, ionic liquids are used as extractants, which can be well compatible with both polar and non-polar components.

[0012] (2) In this invention, 1-ethyl-3-methylpyridine tetrafluoroborate ionic liquid is used as the extractant in this process. While possessing the excellent properties of ionic liquid, its continuous and regular molecular arrangement similar to crystals and the formation of hydrogen bond network can form a stable force after binding with the components in Dendrobium officinale, thus more effectively extracting various effective components of Dendrobium officinale.

[0013] (3) In this process, after the extraction of the extractant is completed, a flocculant is needed to assist in the separation of particulate impurities in the extractant, which has unique characteristics. In this invention, polyaluminum chloride zinc is selected. It relies on the van der Waals forces and hydrogen bonds between the anionic active groups on the molecular chain and the surface of colloidal particles to induce adsorption bridging. At the same time, its chain network structure retains high-efficiency adsorption in special solvent systems, which can effectively avoid the influence of free anions and cations on the flocculation effect in the system, and just right to fully separate the impurities in the system.

[0014] (4) After extraction, the active ingredients are dissolved in the extractant. Then, back-extraction is required to recover the extractant and prepare for subsequent chromatographic column purification. For mixed back-extraction of multiple substances, ethanol aqueous solution is used to accommodate the effects of water solubility and organic solvent. However, under this system, the back-extraction effect of some components is still not ideal. Therefore, a surfactant is needed to reduce surface tension, increase solubility and improve extraction efficiency.

[0015] (5) In this invention, sodium dialkyl dicarboxylate, a Gemini surfactant, is selected as a solubilizer to increase the solubility and solubility efficiency of various anti-inflammatory components of Dendrobium officinale in the solvent. Its structure is a long straight chain containing ether bonds, with hydrophilic groups located on the side, which can effectively coat organic groups and facilitate their separation from the extractant ion system, protect them from denaturation and other losses during back-extraction, and improve the yield of anti-inflammatory components. Attached Figure Description

[0016] Figure 1 The test chromatogram for Example 1; Figure 2 This is the test chromatogram for Example 2; Figure 3 The test chromatogram for Example 3; Figure 4 The test chromatogram is for Comparative Example 1; Figure 5 The test chromatogram for Comparative Example 2 is shown below. Figure 6 The test chromatogram is for Comparative Example 3; Figure 7 The test chromatogram for Comparative Example 4 is shown below. Figure 8 The test chromatogram is for Comparative Example 5; Figure 9 The test chromatogram for Comparative Example 6 is shown below. Figure 10 This is the test chromatogram for Comparative Example 7. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to the embodiments.

[0018] Unless otherwise specified, the raw materials used in the embodiments of this invention are obtained through conventional commercial channels. Example 1

[0019] (1) Take 3 parts of polysilicic acid zinc chloride and mix it with 97 parts of distilled water to obtain a flocculant; (2) Take Dendrobium officinale leaves, dry them at 60℃, pulverize them and sieve them through a 60-mesh sieve to obtain Dendrobium powder; (3) Take 10 parts of Dendrobium powder and 120 parts of 1-ethyl-3-methylpyridine tetrafluoroborate and mix them. Heat the mixture at 80°C for 1 hour to allow it to penetrate. (4) After the permeation is complete, extract for 1.5 h under microwave 350W, ultrasonic 50W and 60℃ conditions; (5) After extraction, mix 2 parts of flocculant and filter while hot to obtain filtrate A and solid particles; (6) Mix 150 parts of 50% ethanol solution with 9 parts of sodium dialkyl dicarboxylate and then mix with filtrate A for back-extraction; (7) After back-extraction for 20 min, centrifugation was performed. The upper layer was a mixed ethanol solution and the lower layer was an ionic solution. (8) The mixed ethanol solution was purified by high performance liquid chromatography (HPLC). The HPLC conditions were as follows: Nova-Pak C18 column (150 mm x 3.9 mm, 4 μm); mobile phase I was a mixture of 50 mmol / L sodium acetate buffer, methanol, and acetonitrile in a volume ratio of 40:40:20; mobile phase I1 was a mixture of 50 mmol / L sodium acetate buffer and methanol in a volume ratio of 80:20; the gradient elution program was as follows: 5% I was changed to 45% I within 30 min, then 45% I was changed to 60% I within 15 min, and finally 60% I was changed to 100% I within 5 min; the detection wavelength was 260 nm, and the crude extract was obtained. (9) The crude extract was further purified by semi-preparative liquid chromatography (SLC). The SLC conditions were: column: Silica C18M 10E (250 mm x 10 mm, 5 μm); mobile phase: acetonitrile (A): water-glacial acetic acid-ammonia (B) = 12:88; detection wavelength: 254 nm; column temperature: 25℃. The resulting Dendrobium officinale extract solution was obtained.

[0020] (10) The solvent was removed by fractional distillation, and the product was washed once with saturated saline and once with deionized water, and then dried to obtain Dendrobium officinale extract. Example 2

[0021] (1) Take 3 parts of polysilicic acid zinc chloride and mix it with 97 parts of distilled water to obtain a flocculant; (2) Take Dendrobium officinale leaves, dry them at 60℃, pulverize them and sieve them through a 60-mesh sieve to obtain Dendrobium powder; (3) Take 10 parts of Dendrobium powder and 100 parts of 1-ethyl-3-methylpyridine tetrafluoroborate and mix them. Heat the mixture at 80°C for 1 hour to allow it to penetrate. (4) After the permeation is complete, extract for 1.5 h under microwave 350W, ultrasonic 50W and 60℃ conditions; (5) After extraction, mix 3 parts of flocculant and filter while hot to obtain filtrate A and solid particles; (6) Mix 150 parts of 50% ethanol solution with 13 parts of sodium dialkyl dicarboxylate ester and then mix with filtrate A for back-extraction; (7) After back-extraction for 20 min, centrifugation was performed. The upper layer was a mixed ethanol solution and the lower layer was an ionic solution. (8) The mixed ethanol solution was purified by high performance liquid chromatography (HPLC). The HPLC conditions were as follows: Nova-Pak C18 column (150 mm x 3.9 mm, 4 μm); mobile phase I was a mixture of 50 mmol / L sodium acetate buffer, methanol, and acetonitrile in a volume ratio of 40:40:20; mobile phase I1 was a mixture of 50 mmol / L sodium acetate buffer and methanol in a volume ratio of 80:20; the gradient elution program was as follows: 5% I was changed to 45% I within 30 min, then 45% I was changed to 60% I within 15 min, and finally 60% I was changed to 100% I within 5 min; the detection wavelength was 260 nm, and the crude extract was obtained. (9) The crude extract was further purified by semi-preparative liquid chromatography (SLC). The SLC conditions were: column: Silica C18M 10E (250 mm x 10 mm, 5 μm); mobile phase: acetonitrile (A): water-glacial acetic acid-ammonia (B) = 12:88; detection wavelength: 254 nm; column temperature: 25℃. The resulting Dendrobium officinale extract solution was obtained.

[0022] (10) The solvent was removed by fractional distillation, and the product was washed once with saturated saline and once with deionized water, and then dried to obtain Dendrobium officinale extract. Example 3

[0023] (1) Take 3 parts of polysilicic acid zinc chloride and mix it with 97 parts of distilled water to obtain a flocculant; (2) Take Dendrobium officinale leaves, dry them at 60℃, pulverize them and sieve them through a 60-mesh sieve to obtain Dendrobium powder; (3) Take 10 parts of Dendrobium powder and 140 parts of 1-ethyl-3-methylpyridine tetrafluoroborate and mix them. Heat the mixture at 80°C for 1 hour to allow it to penetrate. (4) After the permeation is complete, extract for 1.5 h under microwave 350W, ultrasonic 50W and 60℃ conditions; (5) After extraction, mix 1 part of flocculant and filter while hot to obtain filtrate A and solid particles; (6) Mix 150 parts of 50% ethanol solution with 5 parts of sodium dialkyl dicarboxylate and then mix with filtrate A for back-extraction; (7) After back-extraction for 20 min, centrifugation was performed. The upper layer was a mixed ethanol solution and the lower layer was an ionic solution. (8) The mixed ethanol solution was purified by high performance liquid chromatography (HPLC). The HPLC conditions were as follows: Nova-Pak C18 column (150 mm x 3.9 mm, 4 μm); mobile phase I was a mixture of 50 mmol / L sodium acetate buffer, methanol, and acetonitrile in a volume ratio of 40:40:20; mobile phase I1 was a mixture of 50 mmol / L sodium acetate buffer and methanol in a volume ratio of 80:20; the gradient elution program was as follows: 5% I was changed to 45% I within 30 min, then 45% I was changed to 60% I within 15 min, and finally 60% I was changed to 100% I within 5 min; the detection wavelength was 260 nm, and the crude extract was obtained. (9) The crude extract was further purified by semi-preparative liquid chromatography (SLC). The SLC conditions were: column: Silica C18M 10E (250 mm x 10 mm, 5 μm); mobile phase: acetonitrile (A): water-glacial acetic acid-ammonia (B) = 12:88; detection wavelength: 254 nm; column temperature: 25℃. The resulting Dendrobium officinale extract solution was obtained.

[0024] (10) The solvent was removed by fractional distillation, and the product was washed once with saturated saline and once with deionized water, and then dried to obtain Dendrobium officinale extract.

[0025] Comparative Example 1 The difference between this comparative example and Example 1 is that the 1-ethyl-3-methylpyridine tetrafluoroborate in step 3 is N-ethylpyridine tetrafluoroborate, and the rest of the implementation is the same as in Example 1.

[0026] Comparative Example 2 (1) Take 3 parts of polysilicic acid zinc chloride and mix it with 97 parts of distilled water to obtain a flocculant; (2) Take Dendrobium officinale leaves, dry them at 60℃, pulverize them and sieve them through a 60-mesh sieve to obtain Dendrobium powder; (3) Take 10 parts of Dendrobium powder and 80 parts of 1-ethyl-3-methylpyridine tetrafluoroborate, mix them, and heat the mixture at 80°C for 1 hour to allow it to penetrate. (4) After the permeation is complete, extract for 1.5 h under microwave 350W, ultrasonic 50W and 60℃ conditions; (5) After extraction, mix 2 parts of flocculant and filter while hot to obtain filtrate A and solid particles; (6) Mix 150 parts of 50% ethanol solution with 9 parts of sodium dialkyl dicarboxylate and then mix with filtrate A for back-extraction; (7) After back-extraction for 20 min, centrifugation was performed. The upper layer was a mixed ethanol solution and the lower layer was an ionic solution. (8) The mixed ethanol solution was purified by high performance liquid chromatography (HPLC). The HPLC conditions were as follows: Nova-Pak C18 column (150 mm x 3.9 mm, 4 μm); mobile phase I was a mixture of 50 mmol / L sodium acetate buffer, methanol, and acetonitrile in a volume ratio of 40:40:20; mobile phase I1 was a mixture of 50 mmol / L sodium acetate buffer and methanol in a volume ratio of 80:20; the gradient elution program was as follows: 5% I was changed to 45% I within 30 min, then 45% I was changed to 60% I within 15 min, and finally 60% I was changed to 100% I within 5 min; the detection wavelength was 260 nm, and the crude extract was obtained. (9) The crude extract was further purified by semi-preparative liquid chromatography (SLC). The SLC conditions were: column: Silica C18M 10E (250 mm x 10 mm, 5 μm); mobile phase: acetonitrile (A): water-glacial acetic acid-ammonia (B) = 12:88; detection wavelength: 254 nm; column temperature: 25℃. The resulting Dendrobium officinale extract solution was obtained.

[0027] (10) The solvent was removed by fractional distillation, and the product was washed once with saturated saline and once with deionized water, and then dried to obtain Dendrobium officinale extract.

[0028] Comparative Example 3 (1) Take 3 parts of polysilicic acid zinc chloride and mix it with 97 parts of distilled water to obtain a flocculant; (2) Take Dendrobium officinale leaves, dry them at 60℃, pulverize them and sieve them through a 60-mesh sieve to obtain Dendrobium powder; (3) Take 10 parts of Dendrobium powder and 160 parts of 1-ethyl-3-methylpyridine tetrafluoroborate and mix them. Heat the mixture at 80°C for 1 hour to allow it to penetrate. (4) After the permeation is complete, extract for 1.5 h under microwave 350W, ultrasonic 50W and 60℃ conditions; (5) After extraction, mix 2 parts of flocculant and filter while hot to obtain filtrate A and solid particles; (6) Mix 150 parts of 50% ethanol solution with 9 parts of sodium dialkyl dicarboxylate and then mix with filtrate A for back-extraction; (7) After back-extraction for 20 min, centrifugation was performed. The upper layer was a mixed ethanol solution and the lower layer was an ionic solution. (8) The mixed ethanol solution was purified by high performance liquid chromatography (HPLC). The HPLC conditions were as follows: Nova-Pak C18 column (150 mm x 3.9 mm, 4 μm); mobile phase I was a mixture of 50 mmol / L sodium acetate buffer, methanol, and acetonitrile in a volume ratio of 40:40:20; mobile phase I1 was a mixture of 50 mmol / L sodium acetate buffer and methanol in a volume ratio of 80:20; the gradient elution program was as follows: 5% I was changed to 45% I within 30 min, then 45% I was changed to 60% I within 15 min, and finally 60% I was changed to 100% I within 5 min; the detection wavelength was 260 nm, and the crude extract was obtained. (9) The crude extract was further purified by semi-preparative liquid chromatography (SLC). The SLC conditions were: column: Silica C18M 10E (250 mm x 10 mm, 5 μm); mobile phase: acetonitrile (A): water-glacial acetic acid-ammonia (B) = 12:88; detection wavelength: 254 nm; column temperature: 25℃. The resulting Dendrobium officinale extract solution was obtained.

[0029] (10) The solvent was removed by fractional distillation, and the product was washed once with saturated saline and once with deionized water, and then dried to obtain Dendrobium officinale extract.

[0030] Comparative Example 4 The difference between this comparative example and Example 1 is that the polyaluminum chloride zinc in step 1 is polyaluminum chloride, and the rest of the implementation is the same as in Example 1.

[0031] Comparative Example 5 The difference between this comparative example and Example 1 is that the sodium dialkyl dicarboxylate in step 6 is an amide-based gemini quaternary ammonium salt, and the rest of the implementation is the same as in Example 1.

[0032] Comparative Example 6 (1) Take 3 parts of polysilicic acid zinc chloride and mix it with 97 parts of distilled water to obtain a flocculant; (2) Take Dendrobium officinale leaves, dry them at 60℃, pulverize them and sieve them through a 60-mesh sieve to obtain Dendrobium powder; (3) Take 10 parts of Dendrobium powder and 120 parts of 1-ethyl-3-methylpyridine tetrafluoroborate and mix them. Heat the mixture at 80°C for 1 hour to allow it to penetrate. (4) After the permeation is complete, extract for 1.5 h under microwave 350W, ultrasonic 50W and 60℃ conditions; (5) After extraction, mix 2 parts of flocculant and filter while hot to obtain filtrate A and solid particles; (6) Mix 150 parts of 50% ethanol solution with 2 parts of sodium dialkyl dicarboxylate and then mix with filtrate A for back-extraction; (7) After back-extraction for 20 min, centrifugation was performed. The upper layer was a mixed ethanol solution and the lower layer was an ionic solution. (8) The mixed ethanol solution was purified by high performance liquid chromatography (HPLC). The HPLC conditions were as follows: Nova-Pak C18 column (150 mm x 3.9 mm, 4 μm); mobile phase I was a mixture of 50 mmol / L sodium acetate buffer, methanol, and acetonitrile in a volume ratio of 40:40:20; mobile phase I1 was a mixture of 50 mmol / L sodium acetate buffer and methanol in a volume ratio of 80:20; the gradient elution program was as follows: 5% I was changed to 45% I within 30 min, then 45% I was changed to 60% I within 15 min, and finally 60% I was changed to 100% I within 5 min; the detection wavelength was 260 nm, and the crude extract was obtained. (9) The crude extract was further purified by semi-preparative liquid chromatography (SLC). The SLC conditions were: column: Silica C18M 10E (250 mm x 10 mm, 5 μm); mobile phase: acetonitrile (A): water-glacial acetic acid-ammonia (B) = 12:88; detection wavelength: 254 nm; column temperature: 25℃. The resulting Dendrobium officinale extract solution was obtained.

[0033] (10) The solvent was removed by fractional distillation, and the product was washed once with saturated saline and once with deionized water, and then dried to obtain Dendrobium officinale extract.

[0034] Comparative Example 7 (1) Take 3 parts of polysilicic acid zinc chloride and mix it with 97 parts of distilled water to obtain a flocculant; (2) Take Dendrobium officinale leaves, dry them at 60℃, pulverize them and sieve them through a 60-mesh sieve to obtain Dendrobium powder; (3) Take 10 parts of Dendrobium powder and 120 parts of 1-ethyl-3-methylpyridine tetrafluoroborate and mix them. Heat the mixture at 80°C for 1 hour to allow it to penetrate. (4) After the permeation is complete, extract for 1.5 h under microwave 350W, ultrasonic 50W and 60℃ conditions; (5) After extraction, mix 2 parts of flocculant and filter while hot to obtain filtrate A and solid particles; (6) Mix 150 parts of 50% ethanol solution with 16 parts of sodium dialkyl dicarboxylate ester and then mix with filtrate A for back-extraction; (7) After back-extraction for 20 min, centrifugation was performed. The upper layer was a mixed ethanol solution and the lower layer was an ionic solution. (8) The mixed ethanol solution was purified by high performance liquid chromatography (HPLC). The HPLC conditions were as follows: Nova-Pak C18 column (150 mm x 3.9 mm, 4 μm); mobile phase I was a mixture of 50 mmol / L sodium acetate buffer, methanol, and acetonitrile in a volume ratio of 40:40:20; mobile phase I1 was a mixture of 50 mmol / L sodium acetate buffer and methanol in a volume ratio of 80:20; the gradient elution program was as follows: 5% I was changed to 45% I within 30 min, then 45% I was changed to 60% I within 15 min, and finally 60% I was changed to 100% I within 5 min; the detection wavelength was 260 nm, and the crude extract was obtained. (9) The crude extract was further purified by semi-preparative liquid chromatography (SLC). The SLC conditions were: column: Silica C18M 10E (250 mm x 10 mm, 5 μm); mobile phase: acetonitrile (A): water-glacial acetic acid-ammonia (B) = 12:88; detection wavelength: 254 nm; column temperature: 25℃. The resulting Dendrobium officinale extract solution was obtained.

[0035] (10) The solvent was removed by fractional distillation, and the product was washed once with saturated saline and once with deionized water, and then dried to obtain Dendrobium officinale extract.

[0036] Product testing: Samples of anti-inflammatory components from Dendrobium officinale were prepared according to Examples 1-3 and Comparative Examples 1-7, respectively, and the extraction effect of anti-inflammatory components was judged with flavonoids and alkaloids as references. 1) The solutions obtained from the permeation extraction and the samples were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) to obtain the flavonoid content. The flavonoid extraction rate (extraction rate: content in the permeation extraction solution / mass of Dendrobium officinale raw material) and flavonoid yield (yield: content in the sample / content in the permeation extraction solution) were calculated. The results are shown in Table 1, and the chromatograms of each sample are shown in the figure. Figures 1-10 As shown; 2) The solutions obtained from the permeation extraction and the samples were analyzed by ultraviolet-visible spectrophotometry to obtain the alkaloid extraction rate and alkaloid yield. The results are shown in Table 1. 3) Anti-inflammatory effect determination: RAW 264.7 cells were seeded at a density of 5 x 10 cells / mL in 24 plates and cultured for 24 h in a 37°C incubator containing 5% CO2. The supernatant was discarded, and the resulting solution was used as the experimental group (5 g / mL LPS + 500 g / mL sample). 1 mL of the corresponding test solution (or blank culture medium) was added to each group. After 24 h of culture, the NO content in the samples was measured according to the kit instructions. In the LPS-induced macrophage inflammation model, NO secretion significantly increased. The NO content was used as the basis for the anti-inflammatory effect. The results are shown in Table 1.

[0037] Table 1. Data obtained from the measurement

Claims

1. A method for purifying anti-inflammatory components of Dendrobium officinale using chromatographic columns, characterized in that, The preparation steps are as follows: (1) Take Dendrobium officinale leaves, dry them at 60℃, pulverize them and sieve them through a 60-mesh sieve to obtain Dendrobium powder; (2) Take 10 parts of Dendrobium powder and 100-140 parts of extractant, mix them, and heat the mixture at 80℃ for 1 hour to allow it to penetrate. (3) After the permeation was completed, the sample was extracted for 1.5 h under microwave 350W, ultrasonic 50W and 60℃ conditions; (4) After extraction, mix 1-3 parts of flocculant and filter while hot to obtain filtrate A and solid particles; (5) Mix 120-180 parts of 50% ethanol solution with 5-13 parts of solubilizer and then mix with filtrate A for back extraction; (6) After back-extraction for 20 min, centrifugation was performed. The upper layer was a mixed ethanol solution and the lower layer was the extractant solution. (7) The mixed ethanol solution was purified by high performance liquid chromatography to obtain a crude extract; (8) The crude extract was further purified by semi-preparative liquid chromatography column to obtain Dendrobium officinale extract solution; (9) The solvent was removed by fractional distillation, and the product was washed once with saturated saline and once with deionized water, and then dried to obtain the anti-inflammatory component extract of Dendrobium officinale.

2. The method for purifying anti-inflammatory components of Dendrobium officinale using a chromatographic column as described in claim 1, characterized in that: The extractant is 1-ethyl-3-methylpyridine tetrafluoroborate.

3. The method for purifying anti-inflammatory components of Dendrobium officinale using a chromatographic column as described in claim 1, characterized in that: The flocculant consists of 3% aluminum zinc polysilicate and 97% distilled water.

4. The method for purifying anti-inflammatory components of Dendrobium officinale using a chromatographic column as described in claim 1, characterized in that: The solubilizer is sodium dialkyl dicarboxylate.

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