Dendrobium polysaccharide, and preparation method and application thereof

Abstract

This invention provides a method for preparing Dendrobium officinale polysaccharides. First, Dendrobium huoshanense is pretreated by steam explosion. Then, water-soluble polysaccharides are extracted using hot water extraction. Next, the polysaccharides are precipitated using low-temperature alcohol precipitation. Proteins and pigments are then removed using the Sevag method and macroporous resin adsorption. Afterward, the polysaccharide solution is dialyzed through a regenerated cellulose dialysis bag to remove substances with a molecular weight less than 2000 Da. Finally, the solution is freeze-dried to obtain crude Dendrobium officinale polysaccharides, which are further purified to obtain refined Dendrobium officinale polysaccharides. In vitro experiments show that the refined Dendrobium officinale polysaccharides possess significant antioxidant and antitumor activities, showing promising application prospects in skincare and pharmaceutical fields.

Description

Technical Field

[0001] This invention relates to the field of natural product extraction and preparation technology, and in particular to a Dendrobium polysaccharide, its preparation method, and its application. Background Technology

[0002] Dendrobium nobile Lindl., also known as golden hairpin dendrobium, golden hairpin flower, and thousand-year-old dendrobium, is a plant belonging to the genus Dendrobium in the family Orchidaceae. Dendrobium is distributed in China, India, Nepal, and other regions, with its main distribution in subtropical areas such as Fujian and Hunan provinces in China. Dendrobium prefers warm, humid, and relatively shady environments, often growing alongside mosses, and is suitable for growing on tree trunks in mountain forests or on rocks in valleys. Dendrobium has the effects of nourishing the stomach and promoting the production of body fluids, nourishing yin and clearing heat. It is commonly used for conditions such as heat-related illnesses with fluid depletion, dry mouth and thirst, stomach yin deficiency, poor appetite and dry retching, persistent low-grade fever after illness, yin deficiency with exuberant fire, steaming bone fever, blurred vision, and weakness of muscles and bones.

[0003] Dendrobium contains a variety of chemical components, among which the main effective components include Dendrobium polysaccharides and alkaloids. Natural polysaccharides are extracted from herbaceous plants, have significant biological activity, and are used in the treatment of immune-related diseases. Researchers have been committed to the development of polysaccharide extraction methods. Common extraction methods include: (1) solvent extraction, which has a wide range of applications and can extract a variety of polysaccharides, but the solvent consumption is large and may pose environmental and safety problems; (2) ultrasonic-assisted extraction, which has high extraction efficiency and shortens the extraction time, but the equipment cost is high and industrial application is difficult; (3) microwave-assisted extraction, which has a fast extraction speed and can effectively extract heat-sensitive components, but high temperature may cause the loss of some components and the equipment cost is high; (4) enzymatic hydrolysis, which has strong selectivity and can efficiently extract target polysaccharides, but the enzyme cost is high, the reaction time is long, and the conditions need to be precisely controlled; hot water extraction is simple to operate, low in cost, has good repeatability, and has low requirements for equipment maintenance and operation, which makes hot water extraction more feasible in large-scale production, but the extraction efficiency is relatively low and may take a long time. Therefore, it is necessary to optimize the extraction process and purification method of Dendrobium polysaccharides to improve the extraction rate and purity of Dendrobium polysaccharides and further study their biological activity. Summary of the Invention

[0004] In view of this, the present invention proposes a Dendrobium polysaccharide, its preparation method and application, to solve the above problems.

[0005] The technical solution of this invention is implemented as follows:

[0006] A method for preparing Dendrobium polysaccharide includes the following steps:

[0007] S1. Pretreatment: Take the medicinal parts of Dendrobium and perform steam explosion treatment. The steam explosion treatment conditions are: temperature 160-180℃, pressure 1.0-1.2MPa, and steam explosion treatment time 1-2 minutes.

[0008] S2. Hot water extraction: The mixture after steam explosion is extracted with hot water, filtered, and the filtrate is concentrated; the conditions for hot water extraction are: liquid-to-solid ratio of 20-35:1mL / g, extraction temperature controlled at 80-100℃, and extraction time of 2-3 hours.

[0009] S3, alcohol precipitation: Add ethanol to the filtrate, carry out alcohol precipitation at low temperature, and collect the precipitate after centrifugation;

[0010] S4. Removal of protein and pigment: After redissolving the precipitate, remove protein with Sevage reagent and then remove pigment through macroporous adsorption resin.

[0011] S5. Dialysis: The solution from which proteins and pigments have been removed is dialyzed, and the dialysate is freeze-dried to obtain crude Dendrobium polysaccharide.

[0012] S6. Purification of Dendrobium polysaccharides: After dissolving the crude polysaccharide, centrifuge and elute the supernatant with NaCl solution. Collect the polysaccharide components and freeze-dry them to obtain refined Dendrobium polysaccharides.

[0013] Preferably, the Dendrobium species used in step S1 is Dendrobium huoshanense, and the medicinal parts are the stems and leaves of Dendrobium.

[0014] Preferably, in step S2, 3 to 5 times the volume of 75 to 95 vol% ethanol is added to the filtrate; the alcohol precipitation under low temperature conditions specifically involves standing at 4 degrees Celsius for 12 to 24 hours.

[0015] Preferably, step S5 dialysis specifically involves placing the solution after removing proteins and pigments into a dialysis bag with a molecular weight cutoff of 2000 Da, and dialyzing with distilled water at 4°C for 48–72 hours, changing the water every 4 hours.

[0016] Preferably, step S6, the separation and purification of the crude polysaccharide, specifically involves dissolving the crude polysaccharide in deionized water, centrifuging, loading the supernatant onto a DEAE-Cellulose 52 column, eluting with 0.05M NaCl solution, collecting the eluent using an automatic collector, monitoring the sugar-containing components using the sulfuric acid-phenol method, preparing an elution chromatogram, collecting the main components in the eluent based on the elution chromatogram, dialyzing, and lyophilizing to obtain the polysaccharide components.

[0017] Preferably, the further purification of the polysaccharide component in step S6 specifically involves: loading the polysaccharide component onto a Sephadex G-75 gel column, eluting it with distilled water at a flow rate of 0.4 ml / min, collecting the eluent, and lyophilizing it to obtain refined Dendrobium polysaccharide.

[0018] The present invention also provides a Dendrobium polysaccharide obtained by the above-described method for preparing Dendrobium polysaccharide.

[0019] The present invention also provides an application of Dendrobium polysaccharide, wherein the Dendrobium polysaccharide is used as an antioxidant in pharmaceuticals or skin care products.

[0020] This invention also provides an application of Dendrobium polysaccharide in the preparation of inhibitory drugs for cancer cell lines HePG2, BGC-823, LoVo, A-549 and CAL-62.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] This invention provides a method for preparing Dendrobium officinale polysaccharides. First, Dendrobium officinale is pretreated with steam explosion, which effectively breaks down plant cell walls, increasing the release of polysaccharides and making cell tissues more soluble through physical action, thereby improving the extraction efficiency of polysaccharides. Then, water-soluble polysaccharides are extracted using hot water extraction. Next, the polysaccharides are precipitated using low-temperature alcohol precipitation. Proteins and pigments are then removed using the Sevag method and macroporous resin adsorption. Afterward, the polysaccharide solution is dialyzed through a dialysis bag to remove substances with a molecular weight less than 2000 Da. Finally, the solution is freeze-dried to obtain crude Dendrobium officinale polysaccharides, which are further purified to obtain refined Dendrobium officinale polysaccharides. Furthermore, in vitro experiments have demonstrated that the obtained Dendrobium officinale polysaccharides possess significant antioxidant and antitumor activities, especially against human liver cancer cell lines (HePG2), human colon cancer cells (LoVo), and human thyroid cancer cell lines (CAL-62), exhibiting strong inhibitory activity against cancer cell proliferation. This method shows promising application prospects in skincare and pharmaceutical fields. Detailed Implementation

[0023] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0024] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0026] Example 1

[0027] A method for preparing Dendrobium polysaccharide includes the following steps:

[0028] S1. Pretreatment: Take the stems and leaves of Dendrobium huoshanense and steam explosion treatment, set the temperature to 170℃, the pressure to 1.2MPa, and the steam explosion treatment time to 90s;

[0029] S2. Hot water extraction: Add deionized water to the mixture after steam explosion at a material-to-liquid ratio of 30:1 mL / g, extract at 95 degrees Celsius for 180 min, filter and concentrate to 1 / 3 of the filtrate volume.

[0030] S3, alcohol precipitation: Add 3 times the volume of 95% ethanol to the concentrated filtrate, let it stand at 4 degrees Celsius for 12 hours, and collect the precipitate after centrifugation.

[0031] S4. Removal of protein and pigment: The precipitate is reconstituted with ultrapure water, centrifuged, and the supernatant is filtered under reduced pressure. Then, the protein is removed by using one-fifth of the volume of Sevage reagent (chloroform: n-butanol = 4:1 volume ratio) (repeated several times until no protein layer is visible). The solution after protein removal is depigmented by passing it through D101 macroporous adsorption resin, concentrated under reduced pressure to remove Sevage reagent, and then filtered under reduced pressure through a 0.2 μm filter membrane to remove bacteria.

[0032] S5. Dialysis: The solution with proteins and pigments removed is placed in a dialysis bag with a molecular weight cutoff of 2000 Da for dialysis. The solution is dialyzed with distilled water at 4℃ for 48 hours, and the water is changed every 4 hours. The dialysate is freeze-dried to obtain crude Dendrobium polysaccharide.

[0033] S6. Refined Dendrobium polysaccharide: The crude polysaccharide was dissolved in deionized water, centrifuged, and the supernatant was loaded onto a DEAE-Cellulose 52 column. Elution was performed with 0.05M NaCl solution, and the eluent was collected using an automatic collector (flow rate 1.2 mL / min). The sugar content was monitored using the sulfuric acid-phenol method, and an elution chromatogram was prepared. Based on the elution chromatogram, the main components in the eluent were collected, dialyzed, and lyophilized to obtain the polysaccharide component. The polysaccharide component was loaded onto a Sephadex G-75 gel column, eluted with distilled water (flow rate 0.4 mL / min), and the eluent was collected and lyophilized to obtain refined Dendrobium polysaccharide.

[0034] The total sugar content of the refined Dendrobium polysaccharide was 92.12%, and the yield of Dendrobium polysaccharide was 25.11%.

[0035] Example 2

[0036] A method for preparing Dendrobium polysaccharide includes the following steps:

[0037] S1. Pretreatment: The stems and leaves of Dendrobium huoshanense were subjected to steam explosion treatment. The temperature was set at 160℃, the pressure at 1.2MPa, and the steam explosion treatment time was 60s.

[0038] S2. Hot water extraction: Add deionized water to the mixture after steam explosion at a material-to-liquid ratio of 20:1 mL / g, extract at 100 degrees Celsius for 120 min, filter and concentrate to 1 / 3 of the filtrate volume.

[0039] S3, alcohol precipitation: Add 5 times the volume of 75% ethanol to the concentrated filtrate, let it stand at 4 degrees Celsius for 24 hours, and collect the precipitate after centrifugation.

[0040] S4. Removal of protein and pigment: The precipitate is reconstituted with ultrapure water, centrifuged, and the supernatant is filtered under reduced pressure. Then, the protein is removed by using one-fifth of the volume of Sevage reagent (chloroform: n-butanol = 4:1 volume ratio) (repeated several times until no protein layer is visible). The solution after protein removal is depigmented by passing it through D101 macroporous adsorption resin, concentrated under reduced pressure to remove Sevage reagent, and then filtered under reduced pressure through a 0.2 μm filter membrane to remove bacteria.

[0041] S5. Dialysis: The solution with proteins and pigments removed is placed in a dialysis bag with a molecular weight cutoff of 2000 Da for dialysis. The solution is dialyzed with distilled water at 4℃ for 72 hours, and the water is changed every 4 hours. The dialysate is freeze-dried to obtain crude Dendrobium polysaccharide.

[0042] S6. Refined Dendrobium polysaccharide: The crude polysaccharide was dissolved in deionized water, centrifuged, and the supernatant was loaded onto a DEAE-Cellulose 52 column. Elution was performed with 0.05M NaCl solution, and the eluent was collected using an automatic collector (flow rate 1.2 mL / min). The sugar content was monitored using the sulfuric acid-phenol method, and an elution chromatogram was prepared. Based on the elution chromatogram, the main components in the eluent were collected, dialyzed, and lyophilized to obtain the polysaccharide component. The polysaccharide component was loaded onto a Sephadex G-75 gel column, eluted with distilled water (flow rate 0.4 mL / min), and the eluent was collected and lyophilized to obtain refined Dendrobium polysaccharide.

[0043] The total sugar content of the refined Dendrobium polysaccharide was 91.06%, and the yield of Dendrobium polysaccharide was 24.78%.

[0044] Example 3

[0045] A method for preparing Dendrobium polysaccharide includes the following steps:

[0046] S1. Pretreatment: Take the stems and leaves of Dendrobium huoshanense and steam explosion treatment, set the temperature to 180℃, the pressure to 1.0MPa, and the steam explosion treatment time to 180s;

[0047] S2. Hot water extraction: Add deionized water to the mixture after steam explosion at a material-to-liquid ratio of 35:1 mL / g, extract at 80 degrees Celsius for 180 min, filter and concentrate to 1 / 3 of the filtrate volume.

[0048] S3, alcohol precipitation: Add 5 times the volume of 95% ethanol to the concentrated filtrate, let it stand at 4 degrees Celsius for 12 hours, and collect the precipitate after centrifugation.

[0049] S4. Removal of protein and pigment: The precipitate is reconstituted with ultrapure water, centrifuged, and the supernatant is filtered under reduced pressure. Then, the protein is removed by using one-fifth of the volume of Sevage reagent (chloroform: n-butanol = 4:1 volume ratio) (repeated several times until no protein layer is visible). The solution after protein removal is depigmented by passing it through D101 macroporous adsorption resin, concentrated under reduced pressure to remove Sevage reagent, and then filtered under reduced pressure through a 0.2 μm filter membrane to remove bacteria.

[0050] S5. Dialysis: The solution with proteins and pigments removed is placed in a dialysis bag with a molecular weight cutoff of 2000 Da for dialysis. The solution is dialyzed with distilled water at 4℃ for 48 hours, and the water is changed every 4 hours. The dialysate is freeze-dried to obtain crude Dendrobium polysaccharide.

[0051] S6. Refined Dendrobium polysaccharide: The crude polysaccharide was dissolved in deionized water, centrifuged, and the supernatant was loaded onto a DEAE-Cellulose 52 column. Elution was performed with 0.05M NaCl solution, and the eluent was collected using an automatic collector (flow rate 1.2 mL / min). The sugar content was monitored using the sulfuric acid-phenol method, and an elution chromatogram was prepared. Based on the elution chromatogram, the main components in the eluent were collected, dialyzed, and lyophilized to obtain the polysaccharide component. The polysaccharide component was loaded onto a Sephadex G-75 gel column, eluted with distilled water (flow rate 0.4 mL / min), and the eluent was collected and lyophilized to obtain refined Dendrobium polysaccharide.

[0052] The total sugar content of the refined Dendrobium polysaccharide was 91.34%, and the yield of Dendrobium polysaccharide was 24.89%.

[0053] Comparative Example 1

[0054] The difference between this comparative example and Example 1 is that the preparation method of Dendrobium polysaccharide did not involve steam explosion pretreatment. Instead, the stems and leaves of Dendrobium huoshanense were pulverized and directly extracted with hot water. All other steps and parameters were the same as in Example 1.

[0055] The total sugar content of the refined Dendrobium polysaccharide was 82.04%, and the yield of Dendrobium polysaccharide was 12.82%.

[0056] Comparative Example 2

[0057] The difference between this comparative example and Example 1 is that the steam explosion treatment conditions in step S1 are different (the set temperature is 170°C, the pressure is 0.8MPa, and the steam explosion treatment time is 240s), while the other steps are the same.

[0058] The total sugar content of the refined Dendrobium polysaccharide was 88.34%, and the yield of Dendrobium polysaccharide was 18.26%.

[0059] Comparative Example 3

[0060] The difference between this comparative example and Example 1 is that the hot water extraction conditions in step S1 are different (the mixture after steam explosion is added to deionized water at a material-to-liquid ratio of 30:1 mL / g, and extracted at 70 degrees Celsius for 240 min), while the rest of the steps are the same.

[0061] The total sugar content of the refined Dendrobium polysaccharide was 89.45%, and the yield of Dendrobium polysaccharide was 19.97%.

[0062] Experimental Example 1: Determination of the in vitro antioxidant activity of Dendrobium polysaccharides

[0063] DPPH free radical scavenging ability experiment: The Dendrobium polysaccharide obtained in Example 1 was prepared into aqueous solutions of 100, 200, 400, 600, 800 and 1000 mg / L and stored for later use.

[0064] Take 0.5 mL of each Dendrobium polysaccharide solution of different concentrations and mix them thoroughly with 0.5 mL of 0.1 mmol / L DPPH solution. Let them stand in the dark for 30 min. Use deionized water as a blank control and vitamin C (ascorbic acid) as a positive control. Measure the absorbance at 517 nm. Then, calculate the DPPH free radical scavenging rate (%) according to the following formula: DPPH free radical scavenging rate (%) = [1 - (A - A0) / A1] × 100; where A is the absorbance of the mixture of Dendrobium polysaccharide solution and DPPH solution, A0 is the absorbance of Dendrobium polysaccharide solution and anhydrous ethanol (same volume as DPPH solution), and A1 is the absorbance of the mixture of deionized water and DPPH solution.

[0065] The results are shown in Table 1. The ability of Dendrobium polysaccharides to scavenge DPPH free radicals gradually increased in a concentration-dependent manner, and they showed significant antioxidant capacity in vitro. They can be used as antioxidants in drugs and skin care products.

[0066] Table 1. Test of antioxidant activity of Dendrobium polysaccharides

[0067]

[0068]

[0069] The polysaccharides obtained in Example 1 of this invention and the Dendrobium polysaccharides obtained in Comparative Examples 1-3 were each prepared into 1000 mg / L aqueous solutions. The Dendrobium officinale polysaccharide extracted according to the preparation method of Dendrobium polysaccharide in Example 1 of patent CN113880960A was also prepared into a 1000 mg / L aqueous solution. The DPPH free radical scavenging ability was tested according to the above method, and the results are shown in Table 2. Table 2 shows that the Dendrobium polysaccharides obtained by the preparation method of this invention have significantly higher antioxidant effects than other Dendrobium polysaccharides.

[0070] Table 2 Comparison of antioxidant activity of different Dendrobium polysaccharides

[0071]

[0072] Experimental Example 2: Antitumor Activity Test of Dendrobium Polysaccharides

[0073] The antitumor activity of Dendrobium polysaccharide prepared in Example 1 of this invention against human hepatocellular carcinoma cell line (HePG2), human gastric carcinoma cell line (BGC-823), human colon cancer cell line (LoVo), human lung cancer cell line (A-549), and human thyroid cancer cell line (CAL-62) was tested using the MTT assay with VX-680 as a positive control. The results are shown in Table 3 (all results are expressed as mean ± SD, and n = 3 for each group).

[0074] Table 3. Inhibitory activity of Dendrobium polysaccharides on tumor cell proliferation

[0075]

[0076] The results showed that Dendrobium polysaccharides exhibited certain cytotoxicity against the tested HePG2, BGC-823, LoVo, A-549 and CAL-62 cell lines, especially showing strong inhibitory activity against human liver cancer cell line (HePG2), human colon cancer cell line (LoVo) and human thyroid cancer cell line (CAL-62).

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of Dendrobium polysaccharide in the preparation of an inhibitory drug for the human thyroid cancer cell line CAL-62, characterized in that, The preparation method of the Dendrobium polysaccharide includes the following steps: S1. Pretreatment: The medicinal parts of Dendrobium are subjected to steam explosion treatment. The steam explosion treatment conditions are: temperature 170℃, pressure 1.2MPa, and steam explosion treatment time 90s. S2. Hot water extraction: The mixture after steam explosion is extracted with hot water, filtered, and the filtrate is concentrated; the conditions for hot water extraction are: liquid-to-solid ratio of 30:1mL / g, extraction temperature controlled at 95℃, and extraction time of 180min. S3, alcohol precipitation: Add ethanol to the filtrate, carry out alcohol precipitation at low temperature, and collect the precipitate after centrifugation; S4. Removal of protein and pigment: After redissolving the precipitate, remove protein with Sevage reagent and then remove pigment through macroporous adsorption resin. S5. Dialysis: The solution from which proteins and pigments have been removed is dialyzed, and the dialysate is freeze-dried to obtain crude Dendrobium polysaccharide. S6. Purification of Dendrobium polysaccharide: After dissolving the crude polysaccharide, centrifuge the solution, take the supernatant and elute with NaCl solution, collect the polysaccharide fraction, and freeze-dry the polysaccharide fraction to obtain refined Dendrobium polysaccharide. In step S1, the Dendrobium species is Dendrobium huoshanense, and the medicinal parts are the stems and leaves of Dendrobium. In step S3, three times the volume of 95 vol% ethanol is added to the filtrate; the alcohol precipitation under low temperature conditions specifically involves standing at 4 degrees Celsius for 24 hours. The specific steps of step S5 dialysis are as follows: the solution from which proteins and pigments have been removed is placed in a dialysis bag with a molecular weight cutoff of 2000 Da, and dialyzed with distilled water at 4°C for 48-72 hours, with the water changed every 4 hours. The purification of Dendrobium polysaccharides in step S6 specifically involves dissolving the crude polysaccharide in deionized water, centrifuging, and then loading the supernatant onto a DEAE plate. Cellulose 52 column, eluted with 0.05M NaCl solution, collected the eluent with an automatic collector, monitored the sugar components using the sulfuric acid-phenol method, prepared an elution chromatogram, collected the main components in the eluent based on the elution chromatogram, dialyzed, and lyophilized to obtain the polysaccharide components; The further purification of the polysaccharide component in step S6 specifically involves loading the polysaccharide component onto Sephadex G. The solution was eluted with distilled water at a flow rate of 0.4 mL / min on a 75 gel column, and the eluent was collected and freeze-dried to obtain refined Dendrobium polysaccharide.

Images