Dendrobium polysaccharide as well as preparation method and application thereof

Abstract

The invention provides a preparation method of dendrobium huoshanense polysaccharide, which comprises the following steps: firstly, pretreating dendrobium huoshanense through steam explosion, then extracting water-soluble polysaccharide by using a hot water extraction method, then separating out the polysaccharide by using a low-temperature alcohol precipitation method, removing protein and pigment by using a Sevag method and a macroporous resin adsorption method, and then separating out the dendrobium huoshanense polysaccharide. Dialyzing the polysaccharide solution through a regenerated cellulose dialysis bag to remove substances with the molecular weight less than 2000Da, finally freeze-drying to obtain a dendrobium polysaccharide crude product, and further separating and purifying to obtain the refined dendrobium polysaccharide. In-vitro experiments show that the refined dendrobium polysaccharide has remarkable antioxidant activity and antitumor activity, and has a better application prospect in the fields of skin care and medicines.

Description

Technical Field

[0001] The invention relates to the technical field of natural product extraction and preparation, and in particular to a dendrobium polysaccharide and a preparation method and application thereof. Background Art

[0002] Dendrobium nobile Lindl., also known as Golden Hairpin Dendrobium, Golden Hairpin Flower, Millennium Run, etc., is a plant of the genus Dendrobium in the family Orchidaceae. Dendrobium is distributed in China, India, Nepal and other places. In China, it is mainly distributed in subtropical areas such as Fujian and Hunan. Dendrobium likes warm, humid and relatively cool environments. It often grows with mosses and is suitable for growing on tree trunks in mountain forests or on rocks in valleys. Dendrobium has the effects of benefiting the stomach and producing fluid, nourishing yin and clearing heat. It is often used for damage to body fluid caused by febrile diseases, dry mouth and thirst, insufficient stomach yin, poor appetite and dry retching, persistent deficiency heat after illness, yin deficiency and excessive fire, bone steaming and fatigue fever, blurred vision, and weak tendons and bones.

[0003] Dendrobium contains a variety of chemical components, among which the main active ingredients include dendrobium polysaccharides and alkaloids. Natural polysaccharides are extracted from herbal 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 amount of solvent used is large and there may be environmental and safety issues; (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, which is simple to operate, low cost, good reproducibility, and low equipment maintenance and operation requirements, makes hot water extraction more practical in large-scale production, but the extraction efficiency is relatively low and it 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 and a preparation method and application thereof to solve the above problems.

[0005] The technical solution of the present invention is achieved in this way:

[0006] A method for preparing dendrobium polysaccharide comprises the following steps:

[0007] S1. Pretreatment: steam explosion treatment is performed on the medicinal part of Dendrobium officinale. The steam explosion treatment conditions are: temperature of 160-180° C., pressure of 1.0-1.2 MPa, and steam explosion treatment time of 1-2 minutes.

[0008] S2, hot water extraction: extract the mixture after steam explosion with hot water, filter, and concentrate the filtrate; the conditions of the hot water extraction are: liquid-to-solid ratio is 20-35:1mL / g, the extraction temperature is controlled at 80-100°C, and the extraction time is 2-3 hours;

[0009] S3, alcohol precipitation: adding ethanol to the filtrate, performing alcohol precipitation under low temperature conditions, and collecting the precipitate after centrifugation;

[0010] S4, remove protein and pigment: after re-dissolving the precipitate, use sevage reagent to remove protein, and then use macroporous adsorption resin to remove pigment;

[0011] S5, dialysis: dialyzing the solution from which the protein and pigment are removed, and freeze-drying the dialysate to obtain a crude product of dendrobium polysaccharide;

[0012] S6. Purification of Dendrobium polysaccharide: The crude polysaccharide is dissolved and then centrifuged, the supernatant is eluted with a NaCl solution, and the polysaccharide components are collected. The polysaccharide components are further purified and freeze-dried to obtain refined Dendrobium polysaccharide.

[0013] Preferably, in step S1, the species of Dendrobium 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; and the alcohol precipitation under low temperature conditions is specifically: standing at 4 degrees Celsius for 12 to 24 hours.

[0015] Preferably, the dialysis in step S5 is specifically as follows: placing the solution from which the protein and pigment have been removed in a dialysis bag with a molecular weight cutoff of 2000 Da, and dialyzing it with 4° C. distilled water for 48 to 72 hours, with the water being changed every 4 hours.

[0016] Preferably, the step S6 of separating and purifying the crude polysaccharide comprises dissolving the crude polysaccharide in deionized water, centrifuging and loading the supernatant onto a DEAE-Cellulose 52 column, eluting with a 0.05 M NaCl solution, collecting the eluate with an automatic collector, monitoring the sugar-containing components with a sulfuric acid-phenol method, preparing an elution profile, collecting the main components in the eluate according to the elution profile, dialyzing, and freeze-drying to obtain a polysaccharide component.

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

[0018] The present invention also provides a dendrobium polysaccharide obtained by the preparation method of the dendrobium polysaccharide.

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

[0020] The present invention also provides an application of dendrobium polysaccharide, which is an application of the dendrobium polysaccharide in preparing inhibitory drugs for cancer cell lines HePG2, BGC-823, LoVo, A-549 and CAL-62.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a method for preparing a dendrobium polysaccharide. First, the Huoshan dendrobium is pretreated by steam explosion, which can effectively break the plant cell wall, increase the release amount of polysaccharides, and make the cell tissue more soluble through physical action, thereby improving the extraction efficiency of polysaccharides; then, water-soluble polysaccharides are extracted by hot water extraction, and then, polysaccharides are precipitated by low-temperature alcohol precipitation, and then proteins and pigments are removed by Sevag method and macroporous resin adsorption method, and then the polysaccharide solution is dialyzed by dialysis bag to remove substances with a molecular weight of less than 2000Da, and finally, a crude dendrobium polysaccharide is obtained by freeze drying, and refined dendrobium polysaccharides are obtained by further separation and purification. In addition, in vitro experiments have shown that the obtained Huoshan dendrobium polysaccharide has obvious antioxidant and anti-tumor activities at the same time, especially has strong inhibitory activity on cancer cell proliferation against human liver cancer cell line (HePG2), human colon cancer cell line (LoVo) and human thyroid cancer cell line (CAL-62), and has good application prospects in the fields of skin care and medicine. DETAILED DESCRIPTION

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

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

[0025] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0026] Example 1

[0027] A method for preparing dendrobium polysaccharide comprises the following steps:

[0028] S1. Pretreatment: The stems and leaves of Dendrobium huoshanense were subjected to steam explosion treatment, with the temperature set to 170°C, the pressure to 1.2 MPa, and the steam explosion treatment time to 90 s;

[0029] S2, hot water extraction: the mixture after steam explosion was added with deionized water at a solid-liquid ratio of 30:1 mL / g, extracted at 95 degrees Celsius for 180 minutes, filtered and concentrated to 1 / 3 of the filtrate volume;

[0030] S3, alcohol precipitation: add 3 volumes of 95% ethanol to the concentrated filtrate, let stand at 4 degrees Celsius for 12 hours, and collect the precipitate after centrifugation;

[0031] S4. Removal of protein and pigment: The precipitate was reconstituted with ultrapure water, and the supernatant was filtered under reduced pressure after centrifugation. Then, one-fifth of the volume of sevage reagent (chloroform: n-butanol = 4:1 volume ratio) was used for protein removal (repeated several times until there was no protein layer). The solution after protein removal was depigmented by D101 macroporous adsorption resin, and the sevage reagent was removed by vacuum concentration, and then the bacteria were removed by vacuum filtration using a 0.2um filter membrane;

[0032] S5, dialysis: the solution after removing protein and pigment is placed in a dialysis bag with a molecular weight cutoff of 2000Da for dialysis, and dialyzed with 4°C distilled water for 48 hours, and the water is changed every 4 hours. The dialysate is freeze-dried to obtain a crude product of dendrobium polysaccharide;

[0033] S6. Refined Dendrobium polysaccharide: dissolve the crude polysaccharide in deionized water, centrifuge and load the supernatant onto a DEAE-Cellulose 52 column, elute with 0.05 M NaCl solution, collect the eluate with an automatic collector (flow rate 1.2 mL / min), monitor the sugar-containing components using the sulfuric acid phenol method, prepare an elution spectrum, collect the main components in the eluate according to the elution spectrum, dialyze, and freeze-dry to obtain a polysaccharide component; load the polysaccharide component onto a Sephadex G-75 gel column, elute with distilled water (flow rate 0.4 ml / min), collect the eluate, and freeze-dry to obtain refined Dendrobium polysaccharide.

[0034] The total sugar content in the refined dendrobium polysaccharide is 92.12%, and the yield of dendrobium polysaccharide is 25.11%.

[0035] Example 2

[0036] A method for preparing dendrobium polysaccharide comprises the following steps:

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

[0038] S2, hot water extraction: the mixture after steam explosion was added with deionized water at a solid-liquid ratio of 20:1 mL / g, extracted at 100 degrees Celsius for 120 minutes, filtered and concentrated to 1 / 3 of the filtrate volume;

[0039] S3, alcohol precipitation: add 5 volumes of 75% ethanol to the concentrated filtrate, let stand at 4 degrees Celsius for 24 hours, and collect the precipitate after centrifugation;

[0040] S4. Removal of protein and pigment: The precipitate was reconstituted with ultrapure water, and the supernatant was filtered under reduced pressure after centrifugation. Then, one-fifth of the volume of sevage reagent (chloroform: n-butanol = 4:1 volume ratio) was used for protein removal (repeated several times until there was no protein layer). The solution after protein removal was depigmented by D101 macroporous adsorption resin, and the sevage reagent was removed by vacuum concentration, and then the bacteria were removed by vacuum filtration using a 0.2um filter membrane;

[0041] S5, dialysis: the solution with the protein and pigment removed is placed in a dialysis bag with a molecular weight cutoff of 2000Da for dialysis, and dialyzed with 4°C distilled water for 72 hours, with the water changed every 4 hours. The dialysate is freeze-dried to obtain a crude product of dendrobium polysaccharide;

[0042] S6. Refined Dendrobium polysaccharide: dissolve the crude polysaccharide in deionized water, centrifuge and load the supernatant onto a DEAE-Cellulose 52 column, elute with 0.05 M NaCl solution, collect the eluate with an automatic collector (flow rate 1.2 mL / min), monitor the sugar-containing components using the sulfuric acid phenol method, prepare an elution spectrum, collect the main components in the eluate according to the elution spectrum, dialyze, and freeze-dry to obtain a polysaccharide component; load the polysaccharide component onto a Sephadex G-75 gel column, elute with distilled water (flow rate 0.4 ml / min), collect the eluate, and freeze-dry to obtain refined Dendrobium polysaccharide.

[0043] The total sugar content in the refined dendrobium polysaccharide is 91.06%, and the yield of dendrobium polysaccharide is 24.78%.

[0044] Example 3

[0045] A method for preparing dendrobium polysaccharide comprises the following steps:

[0046] S1. Pretreatment: The stems and leaves of Dendrobium huoshanense were subjected to steam explosion treatment, with the temperature set to 180°C, the pressure to 1.0 MPa, and the steam explosion treatment time to 180 s;

[0047] S2, hot water extraction: the mixture after steam explosion was added with deionized water at a solid-liquid ratio of 35:1 mL / g, extracted at 80 degrees Celsius for 180 minutes, filtered and concentrated to 1 / 3 of the filtrate volume;

[0048] S3, alcohol precipitation: add 5 volumes of 95% ethanol to the concentrated filtrate, let stand at 4 degrees Celsius for 12 hours, and collect the precipitate after centrifugation;

[0049] S4. Removal of protein and pigment: The precipitate was reconstituted with ultrapure water, and the supernatant was filtered under reduced pressure after centrifugation. Then, one-fifth of the volume of sevage reagent (chloroform: n-butanol = 4:1 volume ratio) was used for protein removal (repeated several times until there was no protein layer). The solution after protein removal was depigmented by D101 macroporous adsorption resin, and the sevage reagent was removed by vacuum concentration, and then the bacteria were removed by vacuum filtration using a 0.2um filter membrane;

[0050] S5, dialysis: the solution after removing protein and pigment is placed in a dialysis bag with a molecular weight cutoff of 2000Da for dialysis, and dialyzed with 4°C distilled water for 48 hours, and the water is changed every 4 hours. The dialysate is freeze-dried to obtain a crude product of dendrobium polysaccharide;

[0051] S6. Refined Dendrobium polysaccharide: dissolve the crude polysaccharide in deionized water, centrifuge and load the supernatant onto a DEAE-Cellulose 52 column, elute with 0.05 M NaCl solution, collect the eluate with an automatic collector (flow rate 1.2 mL / min), monitor the sugar-containing components using the sulfuric acid phenol method, prepare an elution spectrum, collect the main components in the eluate according to the elution spectrum, dialyze, and freeze-dry to obtain a polysaccharide component; load the polysaccharide component onto a Sephadex G-75 gel column, elute with distilled water (flow rate 0.4 ml / min), collect the eluate, and freeze-dry to obtain refined Dendrobium polysaccharide.

[0052] The total sugar content in the refined dendrobium polysaccharide is 91.34%, and the yield of dendrobium polysaccharide is 24.89%.

[0053] Comparative Example 1

[0054] The difference between this comparative example and Example 1 is that the preparation method of Dendrobium polysaccharide does not perform steam explosion pretreatment, the stems and leaves of Dendrobium huoshanense are crushed and directly extracted with hot water, and the remaining steps and parameters are the same as those in Example 1.

[0055] The total sugar content in the refined dendrobium polysaccharide is 82.04%, and the yield of dendrobium polysaccharide is 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 0.8 MPa, and the steam explosion treatment time is 240 s), and the other steps are the same.

[0058] The total sugar content in the refined dendrobium polysaccharide is 88.34%, and the yield of dendrobium polysaccharide is 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 (deionized water is added to the mixture after steam explosion at a material-liquid ratio of 30:1 mL / g, and the extraction is carried out at 70 degrees Celsius for 240 minutes), and the other steps are the same.

[0061] The total sugar content in the refined dendrobium polysaccharide is 89.45%, and the yield of dendrobium polysaccharide is 19.97%.

[0062] Test Example 1 Determination of in vitro antioxidant activity of Dendrobium polysaccharides

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

[0064] Take 0.5 mL of each dendrobium polysaccharide solution of different concentrations, mix them evenly with 0.5 mL of 0.1 mmol / L DPPH solution, keep them in the dark for 30 min, use deionized water as a blank control and Vc (ascorbic acid) as a positive control, measure the absorbance at 517 nm, and then calculate according to the following formula: DPPH free radical scavenging rate (%) = [1-(A-A0) / A1]×100; wherein A is the absorbance of the mixture of dendrobium polysaccharide solution and DPPH solution, A0 is the absorbance of the mixture of dendrobium polysaccharide solution and anhydrous ethanol (with the same volume as the 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 polysaccharide to scavenge DPPH free radicals gradually increases in a concentration-dependent manner, and it exhibits significant antioxidant capacity in vitro. It can be used as an antioxidant in medicines and skin care products.

[0066] Table 1 Antioxidant activity test of Dendrobium polysaccharides

[0067]

[0068]

[0069] The polysaccharide obtained in Example 1 of the present invention and the dendrobium polysaccharide obtained in Comparative Examples 1-3 were respectively prepared into 1000 mg / L aqueous solutions, and the dendrobium officinale polysaccharide was extracted according to the preparation method of dendrobium polysaccharide disclosed in Example 1 Treatment 1 of patent CN113880960A and was also prepared into 1000 mg / L aqueous solution; the scavenging ability of DPPH free radicals was detected according to the above method, and the results are shown in Table 2. The results in Table 2 show that the dendrobium polysaccharide obtained by the preparation method of the present invention is significantly higher than other dendrobium polysaccharides in antioxidant effect.

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

[0071]

[0072] Test Example 2 Antitumor Activity Test of Dendrobium Polysaccharide

[0073] The antitumor activity of the polysaccharide prepared in Example 1 of the present invention against human liver cancer cell line (HePG2), human gastric cancer 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 by MTT method with VX-680 as positive control. The results are shown in Table 3 (all results are expressed as mean ± SD, 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 showed certain cytotoxicity to the tested HePG2, BGC-823, LoVo, A-549 and CAL-62 cell lines, especially had strong inhibitory activity on cancer cell proliferation in 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing dendrobium polysaccharide, characterized in that: The following steps are involved: S1. Pretreatment: steam explosion treatment is performed on the medicinal part of Dendrobium officinale. The steam explosion treatment conditions are: temperature of 160-180° C., pressure of 1.0-1.2 MPa, and steam explosion treatment time of 1-2 minutes. S2, hot water extraction: extract the mixture after steam explosion with hot water, filter, and concentrate the filtrate; the conditions of the hot water extraction are: liquid-to-solid ratio is 20-35:1mL / g, the extraction temperature is controlled at 80-100°C, and the extraction time is 2-3 hours; S3, alcohol precipitation: adding ethanol to the filtrate, performing alcohol precipitation under low temperature conditions, and collecting the precipitate after centrifugation; S4, remove protein and pigment: after re-dissolving the precipitate, use sevage reagent to remove protein, and then use macroporous adsorption resin to remove pigment; S5, dialysis: dialyzing the solution from which the protein and pigment are removed, and freeze-drying the dialysate to obtain a crude product of dendrobium polysaccharide; S6. Purification of Dendrobium polysaccharide: The crude polysaccharide is dissolved and then centrifuged, the supernatant is eluted with a NaCl solution, and the polysaccharide components are collected. The polysaccharide components are further purified and freeze-dried to obtain refined Dendrobium polysaccharide.

2. The method for preparing a dendrobium polysaccharide according to claim 1, characterized in that: In step S1, the species of Dendrobium is Dendrobium huoshanense, and the medicinal parts are the stems and leaves of Dendrobium.

3. The method for preparing a dendrobium polysaccharide according to claim 1, characterized in that: In the 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 is specifically: standing at 4 degrees Celsius for 12 to 24 hours.

4. The method for preparing a dendrobium polysaccharide according to claim 1, characterized in that: The dialysis in step S5 is specifically as follows: placing the solution from which the protein and pigment have been removed in a dialysis bag with a molecular weight cutoff of 2000 Da, and dialysis with 4° C. distilled water for 48 to 72 hours, with the water being changed every 4 hours.

5. The method for preparing a dendrobium polysaccharide according to claim 1, characterized in that: The step S6 of separating and purifying the crude polysaccharide comprises dissolving the crude polysaccharide in deionized water, centrifuging and loading the supernatant onto a DEAE-Cellulose 52 column, eluting with a 0.05 M NaCl solution, collecting the eluate with an automatic collector, monitoring the sugar-containing components with a sulfuric acid-phenol method, preparing an elution spectrum, collecting the main components in the eluate according to the elution spectrum, dialyzing, and freeze-drying to obtain the polysaccharide component.

6. The method for preparing a dendrobium polysaccharide according to claim 1, characterized in that: The further purification of the polysaccharide component in step S6 is specifically as follows: the polysaccharide component is loaded onto a Sephadex G-75 gel column, eluted with distilled water at a flow rate of 0.4 ml / min, the eluate is collected, and the refined Dendrobium polysaccharide is obtained after freeze-drying.

7. A dendrobium polysaccharide, characterized in that: The polysaccharide is prepared by the preparation method of the dendrobium polysaccharide according to any one of claims 1 to 6.

8. Use of the dendrobium polysaccharide according to claim 7 as an antioxidant in medicine or skin care products.

9. The use of a dendrobium polysaccharide according to claim 7, characterized in that: The dendrobium polysaccharide is used in preparing inhibitory drugs for cancer cell lines HePG2, BGC-823, LoVo, A-549 and CAL-62.

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