Preparation method and application of dendrobium officinale polysaccharide

Abstract

The invention provides a preparation method and application of dendrobium officinale polysaccharide. Belongs to the technical field of plant polysaccharide preparation and application. The preparation method comprises the following steps: 1) performing water extraction on dendrobium officinale to obtain a dendrobium officinale extracting solution; 2) inoculating lactobacillus plantarum into the dendrobium officinale extracting solution for fermentation, performing solid-liquid separation after fermentation is finished, and collecting a liquid-phase component as fermentation liquor; (3) sequentially carrying out alcohol precipitation and deproteinization on the fermentation liquor to obtain dendrobium officinale crude polysaccharide; and 4) dialyzing and purifying the crude dendrobium officinale polysaccharide to obtain the purified dendrobium officinale polysaccharide. Through fermentation, the in-vitro free radical scavenging capacity of the dendrobium officinale polysaccharide is remarkably enhanced, the activity of cell antioxidant enzymes is promoted through the dendrobium officinale polysaccharide, and the anti-oxidation defense capacity of nematodes is remarkably improved. The optimal fermentation condition is provided for maximizing the biological activity of the dendrobium officinale polysaccharide, the potential of the dendrobium officinale polysaccharide serving as an anti-aging agent is emphasized, and a solid foundation is laid for future transformation research.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant polysaccharide preparation and application, and particularly relates to a preparation method and application of polysaccharides from Dendrobium officinale Background Art

[0002] Dendrobium officinale is a traditional Chinese herbal medicine, renowned for its medicinal properties and nutrition. In the Taoist classic "Daozang" during the Kaiyuan period of the Tang Dynasty, Dendrobium officinale was listed as "the top of the nine greatest fairy herbs in China". Dendrobium officinale belongs to the genus Dendrobium, and its main components include polysaccharides, alkaloids, etc. Polysaccharides are sugar chains linked by glycosidic bonds. As one of the basic molecules constituting living organisms, polysaccharides are widely distributed in plants, animals, fungi, and microorganisms, and participate in physiological activities such as cell differentiation, proliferation, and signal transduction. A large number of studies have shown that polysaccharides from various natural sources have biological functions such as reducing blood sugar, anticoagulation, anti-tumor, immunomodulation, antioxidant, and regulating the intestinal flora (Wu Haoshu, Xu Jianhua, Chen Lizuan, etc. Research on the hypoglycemic effect and mechanism of Dendrobium officinale [J]. China Journal of Chinese Materia Medica, 2004, (02): 69 - 72.; Bian C, Wang Z and Shi J. Extraction Optimization, Structural Characterization, and Anticoagulant Activity of Acidic Polysaccharides from Auricularia auricula-judae [J]. Molecules, 2020, 25(3): 710.; Liu L, Li M, Yu M et al. Natural polysaccharides exhibit anti-tumor activity by targeting gut microbiota [J]. International Journal of Biological Macromolecules, 2018, 121: 743 - 751.; Jingwen, Wang, Xuexi et al. Polysaccharides from Hedysarum polybotrys Promotes Splenic Lymphocytes Proliferation and Reveals Immunoregulatory effect [J]. Journal of Biomaterials and Tissue engineering, 2018, 8(6): 900 - 905.; Okolie C L, C.K. Rajendran S R, Udenigwe C C et al. Prospects of brown seaweed polysaccharides (BSP) as prebiotics and potential immunomodulators [J].Journal of Food Biochemistry, 2017, 41(5): e12392; Zhang X, Qi C, Guo Y et al. Toll-like receptor 4-related immunostimulatory polysaccharides: Primary structure, activity relationships, and possible interaction models[J]. Carbohydrate Polymers, 2016, 149: 186-206. And natural polysaccharides play roles as drug carriers, in biomaterials, and participate in biological imaging functions due to their unique characteristics such as non-toxicity, good biocompatibility, and biodegradability (Debele T A, Mekuria S L and Tsai H-C. Polysaccharide based nanogels in the drug delivery system: Application as the carrier of pharmaceutical agents[J]. Materials Science and engineering C-Materials for Biological Applications, 2016, 68: 964-981; Su C, Chen Y, Tian S et al. Research Progress on emerging Polysaccharide Materials Applied in Tissue engineering[J]. Polymers, 2022, 14(16): 3268-3268; Saravanakumar G, Jo D G and Park J H. Polysaccharide-Based Nanoparticles: A Versatile Platform for Drug Delivery and Biomedical Imaging[J]. Current Medicinal Chemistry, 2012, 19(19): 3212-3229.)。Natural source polysaccharides have a hemiacetal hydroxyl group structure and strong free radical scavenging ability, which endows polysaccharides with strong antioxidant capacity. Some studies have found that gallnut polysaccharides have strong ability to scavenge DPPH free radicals, and seabuckthorn polysaccharides show certain ability to scavenge DPPH free radicals, superoxide anion free radicals and hydroxyl free radicals, exhibiting good antioxidant activity. Dendrobium officinale polysaccharides have strong scavenging effects on various free radicals such as superoxide anion free radicals and hydroxyl free radicals, and also have significant inhibitory effects on the linoleic acid oxidation system induced by alkyl free radicals, showing significant antioxidant activity.

[0003] Some studies have found that natural plants can degrade toxic substances and release a large number of active substances after being fermented by strains (Ai Su, Tang Wei, Guo Ruolin, etc. Research progress on microbial fermentation of Chinese herbal medicines and their active substances [J]. China Journal of Chinese Materia Medica, 2019(6): 48-56). During the fermentation process, the interaction between microorganisms and substrates can significantly change the structure of polysaccharides, thereby affecting their biological activities.

[0004] Aging is a natural and irreversible biological state. With the aging of the global population, the burden of age-related diseases is increasing day by day. Understanding and intervening in the mechanism of cellular aging can help delay the development of geriatric diseases and improve the quality of life of the elderly. At present, the free radical theory of aging, as one of the most widely accepted theories, can explain the damage caused by free radicals to organisms during the aging process. However, the body's own antioxidant defense system is not sufficient to completely prevent oxidative damage caused by excessive free radicals. Supplementing antioxidants can reduce the degree of aging and related oxidative damage and extend the lifespan of the body. Therefore, finding a natural, safe and efficient exogenous free radical scavenger is of great significance for delaying the aging of the body and developing health care products and skin care products related to anti-aging. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a preparation method and application of Dendrobium officinale polysaccharides; the fermented Dendrobium officinale polysaccharides obtained by the preparation method have excellent antioxidant and anti-aging activities and can be applied to the preparation of antioxidant drugs, anti-aging health care products, anti-aging skin care products, etc.

[0006] The present invention provides a preparation method of Dendrobium officinale polysaccharides, comprising the following steps:

[0007] 1) Extract Dendrobium officinale with water to obtain Dendrobium officinale extract;

[0008] 2) Inoculate Lactobacillus plantarum into the Dendrobium officinale extract for fermentation. After the fermentation is completed, perform solid-liquid separation, and collect the liquid phase component as the fermentation broth;

[0009] 3) The fermentation broth is successively subjected to alcohol precipitation and protein removal to obtain crude Dendrobium officinale polysaccharides;

[0010] 4) Dialyze and purify the crude polysaccharide of Dendrobium officinale to obtain purified Dendrobium officinale polysaccharide.

[0011] Preferably, the Lactobacillus plantarum is BNCC 339790.

[0012] Preferably, the temperature of the fermentation in step 2) is 36 - 38 °C, the time of the fermentation is 40 - 60 h, oscillation is accompanied during the fermentation, and the rotation speed of the oscillation is 100 - 200 rpm.

[0013] Preferably, the temperature of the water extraction in step 1) is 85 - 95 °C, the time of the water extraction is 1.5 - 2.5 h, and the mass of the water used for the water extraction is 25 - 35 times the mass of Dendrobium officinale.

[0014] Preferably, the alcohol precipitation in step 3) is carried out with ethanol, the volume ratio of the ethanol to the fermentation broth is (2 - 4):1, the temperature of the alcohol precipitation is 3 - 5 °C, and the time of the alcohol precipitation is 40 - 60 h.

[0015] Preferably, the dialysis is carried out with a dialysis bag, the cut-off molecular weight of the dialysis bag is 3500 Da; the time of the dialysis is 60 - 84 h, and the water is changed every 7 - 9 h.

[0016] Preferably, the purification in step 4) is carried out with a DEAE-52 ion cellulose column and a SephadexG-100 dextran gel column.

[0017] The present invention provides the Dendrobium officinale polysaccharide obtained by the described preparation method.

[0018] The present invention provides the application of the described Dendrobium officinale polysaccharide in the preparation of antioxidant drugs, anti-aging health products or anti-aging cosmetics.

[0019] The present invention provides the application of the described Dendrobium officinale polysaccharide in the preparation of drugs for improving the locomotor ability of nematodes, enhancing the heat stress resistance of nematodes or extending the lifespan of nematodes.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a preparation method of Dendrobium officinale polysaccharide, ferments the extraction solution of Dendrobium officinale with Lactobacillus plantarum, significantly enhances the free radical scavenging ability of Dendrobium officinale polysaccharide in vitro, the obtained Dendrobium officinale polysaccharide promotes the activity of cellular antioxidant enzymes, and significantly improves the antioxidant defense ability of nematodes. In addition, the Dendrobium officinale polysaccharide obtained by the present invention also significantly enhances the locomotor ability and heat tolerance of nematodes and extends their lifespan. The present invention provides the optimal fermentation conditions for maximizing the biological activity of Dendrobium officinale polysaccharide, emphasizes its potential as an anti-aging agent, and lays a solid foundation for future translational research. Description of the Drawings

[0021] Figure 1 For the comparison of the total sugar content before and after the fermentation of polysaccharides from Dendrobium officinale; the total sugar content of polysaccharides from Dendrobium officinale decreased after fermentation by three fermentation methods, and there was no statistical difference.

[0022] Figure 2 In vitro antioxidant capacity of fermented Dendrobium officinale polysaccharides; (A)-(C) show the changes in the free radical scavenging ability of fermented Dendrobium officinale polysaccharides in each group during fermentation, where (A) is the OH free radical scavenging ability; (B) is the DPHH free radical scavenging ability; (C) is the ABTS free radical scavenging ability; (D)-(F) show the production amounts of MDA (D), LDH (E), and SOD (F) after different concentrations of LP-fermented Dendrobium officinale polysaccharides intervened in the Caco-2 oxidation model. Among them, unfermented Dendrobium officinale polysaccharides are represented as Unif, Dendrobium officinale polysaccharides fermented by Lactobacillus plantarum are represented as LP, Dendrobium officinale polysaccharides fermented by baker's yeast are represented as SC, and Dendrobium officinale polysaccharides fermented by compound lactobacilli are represented as CL.

[0023] Figure 3 For the effect of treatment with fermented Dendrobium officinale polysaccharides on the antioxidant capacity in N2 wild-type Caenorhabditis elegans. (A) is a representative photo of the ROS content in nematodes labeled with H2DCFDA after treatment with fermented Dendrobium officinale polysaccharides in each group; (B) is the release amount of ROS in nematodes detected by an enzyme-labeling instrument over time; (C) is the statistical result of the ROS release amount in nematodes labeled with H2DCFDA after treatment with fermented Dendrobium officinale polysaccharides in each group. (D)-(F) show the effects of fermented Dendrobium officinale polysaccharides in each group on antioxidant enzymes in nematodes, where (D) is the change in CAT content, (E) is the change in SOD content, and (F) is the change in GR content. Among them, the group without treatment with Dendrobium officinale polysaccharides is represented as Ctrl, the group treated with unfermented Dendrobium officinale polysaccharides is represented as Unif, the group treated with Dendrobium officinale polysaccharides fermented by Lactobacillus plantarum is represented as LP, the treatment with Dendrobium officinale polysaccharides fermented by baker's yeast is represented as SC, and the group treated with Dendrobium officinale polysaccharides fermented by compound lactobacilli is represented as CL.

[0024] Figure 4 For the effects of treatment with fermented Dendrobium officinale polysaccharides on the vitality and lifespan of N2 wild-type Caenorhabditis elegans. (A) is the number of arc-shaped swings of nematodes within 30 s; (B) is the change in the heat stress resistance of nematodes after being treated at 35 °C for 2 h; (C) is the change in the lifespan of nematodes. Detailed Implementation Modes

[0025] The present invention provides a method for preparing polysaccharides from Dendrobium officinale, comprising the following steps: 1) extracting Dendrobium officinale with water to obtain an extract of Dendrobium officinale; 2) inoculating Lactobacillus plantarum into the extract of Dendrobium officinale for fermentation, and after the fermentation is completed, separating the solid and liquid phases, and collecting the liquid phase component as the fermentation broth; 3) obtaining crude polysaccharides from Dendrobium officinale by successively subjecting the fermentation broth to alcohol precipitation and protein removal; 4) dialyzing and purifying the crude polysaccharides from Dendrobium officinale to obtain purified polysaccharides from Dendrobium officinale.

[0026] In the present invention, Dendrobium officinale is extracted with water to obtain an extract of Dendrobium officinale. In the present invention, the Dendrobium officinale is preferably Dendrobium officinale powder; it is obtained by drying fresh Dendrobium officinale stems and then grinding. In the present invention, the temperature of the water extraction is preferably 85 - 95°C, more preferably 88 - 92°C, and most preferably 90°C; the time of the water extraction is preferably 1.5 - 2.5 h, more preferably 1.7 - 2.3 h, and most preferably 2 h; the mass of the water used for the water extraction is 25 - 35 times the mass of Dendrobium officinale, preferably 28 - 32 times, and most preferably 30 times. After the water extraction in the present invention, solid-liquid separation is preferably carried out to obtain the supernatant as the extract of Dendrobium officinale; the solid-liquid separation is preferably carried out by centrifugation.

[0027] In the present invention, Lactobacillus plantarum is inoculated into the extract of Dendrobium officinale for fermentation, and after the fermentation is completed, the solid and liquid phases are separated, and the liquid phase component is collected as the fermentation broth. In the present invention, the extract of Dendrobium officinale is preferably sterilized before inoculating Lactobacillus plantarum, the sterilization treatment is preferably high-temperature and humid heat sterilization, the temperature of the sterilization treatment is preferably 121°C, the pressure of the sterilization treatment is preferably 1.2 MPa; the time of the sterilization treatment is preferably 20 - 30 min; the pH is adjusted to 6.0 after the sterilization treatment in the present invention; then Lactobacillus plantarum is inoculated. In the present invention, the Lactobacillus plantarum is preferably BNCC 339790; the inoculation amount of the Lactobacillus plantarum is preferably 4% - 6% (v / v), more preferably 4.5% - 5.5%, and most preferably 5%; the temperature of the fermentation is preferably 36 - 38°C, preferably 37°C, the time of the fermentation is preferably 40 - 60 h, further preferably 45 - 50 h, and more preferably 48 h; the fermentation process is accompanied by oscillation, and the rotation speed of the oscillation is preferably 100 - 200 rpm, more preferably 120 - 180 rpm, and most preferably 150 rpm. In the present invention, after the fermentation is completed, solid-liquid separation is carried out, preferably by centrifugation, and there are no special limitations on the rotation speed and time of the centrifugation in the present invention, and the conventional parameters for centrifuging bacteria in the art can be used.

[0028] In the present invention, the fermented liquid is successively subjected to alcohol precipitation and protein removal to obtain crude Dendrobium officinale polysaccharide. In the present invention, the alcohol precipitation is preferably carried out with ethanol, the volume ratio of the ethanol to the fermented liquid is preferably (2-4):1, more preferably 3:1, the temperature of the alcohol precipitation is preferably 3-5°C, more preferably 4°C, and the time of the alcohol precipitation is preferably 40-60 h, more preferably 45-50 h, and most preferably 48 h. In the present invention, the protein removal is preferably carried out with Sevage reagent.

[0029] After obtaining the crude Dendrobium officinale polysaccharide in the present invention, the crude Dendrobium officinale polysaccharide is dissolved, dialyzed and purified to obtain purified Dendrobium officinale polysaccharide. In the present invention, the dialysis is preferably carried out with a dialysis bag, and the cut-off molecular weight of the dialysis bag is preferably 3500 Da; the time of the dialysis is preferably 60-84 h, more preferably 70-75 h, and most preferably 72 h. During the dialysis process, it is preferably to change the water every 7-9 h. In the present invention, the purification is preferably carried out with a DEAE-52 ion cellulose column and a Sephadex G-100 dextran gel column. After the purification is completed, the present invention collects the eluate and performs vacuum freeze-drying to obtain Dendrobium officinale polysaccharide.

[0030] The present invention provides the Dendrobium officinale polysaccharide prepared by the preparation method described above.

[0031] The present invention provides the application of the Dendrobium officinale polysaccharide in the preparation of an antioxidant drug, an anti-aging health product or an anti-aging cosmetic.

[0032] The present invention provides the application of the Dendrobium officinale polysaccharide in the preparation of a drug for improving the motility of nematodes, enhancing the heat stress resistance of nematodes or prolonging the lifespan of nematodes.

[0033] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] 1) The colon adenocarcinoma cell line Caco-2 was purchased from ATCC;

[0035] 2) The high-glucose DMEM medium (containing double antibodies) was purchased from KeyGen Biotech, and the product number was KGL1206-500;

[0036] 3) The 1X phosphate buffer solution (1X PBS buffer solution) was purchased from Solarbio Co., Ltd., China, and the product number was P1020.

[0037] 4) The fetal bovine serum was purchased from Gibco, USA, and the product number was 10099-141.

[0038] 5) Pancreatic enzyme cell digestion solution (containing EDTA, 0.25%: 0.02%) was purchased from Keygen Biotechnology with the catalog number VC2005.

[0039] 6) NGM nematode growth medium was purchased from Top Biotechnology with the catalog number M2328B.

[0040] 7) M9 buffer is prepared in the laboratory. The preparation method is as follows: 3 g KH2PO4, 15 g K2HPO4·12H2O, 5 g NaCl, dissolved in 1 L water, and then sterilized by high pressure. After sterilization, add 1 mL 1M MgSO4. After mixing, filter with a 0.45 micron filter.

[0041] 8) BCA Protein Assay Kit was purchased from China Kangrun Company with the catalog number E162-01.

[0042] 9) SOD kit was purchased from Nanjing Jiancheng Bioengineering Institute, catalog number A001-3

[0043] 10) GR kit was purchased from Nanjing Jiancheng Bioengineering Institute, catalog number A062-1-1

[0044] 11) CAT kit was purchased from Nanjing Jiancheng Bioengineering Institute, catalog number A007-1-1

[0045] 12) H2DCFDA fluorescent probe was purchased from MCE, item number: HY-D0940

[0046] 13) Fresh Dendrobium officinale strips (Huoshan wild Dendrobium) were purchased from Hainan Xinwang Biotechnology Co., Ltd.

[0047] 14) Lactobacillus plantarum (BNCC 339790) and Lactobacillus delbrueckiisubsp (BNCC 187941) were purchased from BNCC.

[0048] 15) Baker’s yeast (Saccharomyces cerevisiae) was purchased from Angel Yeast Co., Ltd.

[0049] 16) Caenorhabditis elegans was provided by the International Research Center for Aging and Cancer, Hainan Medical University.

[0050] Example 1

[0051] The fresh Dendrobium officinale stems are dried and ground into powder, which is mixed with distilled water at a mass ratio of 1:30, extracted at 90 °C for 2 h, centrifuged at 1000 rpm for 5 min after cooling to obtain the supernatant, which is the Dendrobium officinale extract. After the Dendrobium officinale extract is autoclaved for 20 min, the pH is adjusted to 6, and 5% (V / V) solutions of Lactobacillus plantarum, Lactobacillus farciminis, and Lactobacillus casei (Lactobacillus plantarum: Lactobacillus bulgaricus = 1:1) activated according to the instructions are added respectively, and then placed in a shaker and cultured at 150 rpm for 48 h. The supernatant is collected by centrifugation to obtain 3 kinds of Dendrobium officinale fermentation broths. Anhydrous ethanol with a volume 3 times that of the fermentation broth is added to the fermentation broth, and alcohol precipitation is carried out in a 4 °C ice box for 48 h. The precipitate is separated by centrifugation and vacuum freeze-dried to obtain the fermented Dendrobium powder.

[0052] Accurately weigh 3.5 g of crude polysaccharide from Dendrobium officinale, dissolve it in an appropriate amount of deionized water to obtain a polysaccharide solution. If the dissolution is difficult, a vortex shaker can be used. Then add Sevage reagent (chloroform: n-butanol = 4:1), and mix the polysaccharide solution and Sevage reagent at a volume ratio of 3:1 and shake vigorously for 30 min. Centrifuge at 5000 r / min for 10 min, remove the upper organic layer and the middle white precipitate layer, collect the lower aqueous solution, and repeat the above operation steps until the precipitate completely precipitates and the resulting solution is divided into two layers without a middle white precipitate. The lower aqueous solution is collected, which is the protein-depleted polysaccharide aqueous solution. The lower aqueous solution is vacuum freeze-dried to obtain the crude polysaccharide from fermented Dendrobium officinale. The crude polysaccharide from fermented Dendrobium officinale is dissolved in a small amount of distilled water to form an aqueous solution (ratio: 1 g: 2 ml), dialyzed with a 3500 Da dialysis bag against deionized water for 72 h, with water changed every 8 h, and then freeze-dried to obtain the further purified crude polysaccharide from Dendrobium officinale. Further separation and purification are carried out through a DEAE-52 ion cellulose column and a Sephadex G-100 dextran gel column. After collecting the eluate, it is vacuum freeze-dried to obtain the polysaccharide from Dendrobium officinale, which is also called the fermented Dendrobium polysaccharide in the following examples.

[0053] Purification steps of the DEAE-52 ion cellulose column: Accurately weigh 5 g of the protein-depleted crude polysaccharide from fermented Dendrobium officinale, dissolve it in 10 mL of ultrapure water, centrifuge at 12000 rpm for 5 min, and remove the precipitate to retain the supernatant. Before loading, filter the supernatant with a hydrophilic filter membrane (0.45 μm), then slowly drip the sample along the inner wall of the column with a dropper, and at the same time open the lower outlet. After the sample completely penetrates into the packing, fill the empty space at the upper end of the column with ultrapure water. The eluent of the separation column is ultrapure water. Collect the eluted liquid and then freeze-dry it.

[0054] Purification steps of Sephadex G-100 dextran gel column: The pre-treatment and loading method of the sample are basically the same as those of the separation column operation. The eluent is ultrapure water after ultrasonic degassing of bubbles. The flow rate is controlled at 0.5 mg / mL, and the collection time for each tube is 10 min. After concentration under reduced pressure and then freeze-drying, Dendrobium officinale polysaccharide is obtained.

[0055] The total sugar content was measured by the phenol-sulfuric acid method

[0056] Accurately weigh 10 mg of the Dendrobium officinale polysaccharide prepared above, dissolve it and make up the volume to 100 mL with a volumetric flask. Take 1 mL and dilute it 10 times to obtain a crude Dendrobium officinale polysaccharide solution with a concentration of 0.01 mg / mL. Weigh 200 mg of dry glucose, dissolve it in deionized water and make up the volume. Take 1 mL of the polysaccharide solution after volume determination and add deionized water to dilute it to a standard solution with a concentration of 0.04 mg / mL.

[0057] Add 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8 mL of the glucose standard solution to the dried test tubes in sequence and make up to 2.0 mL with deionized water. Then add 1.0 mL of 6% phenol solution and concentrated sulfuric acid. Shake to mix the liquid evenly, then place it at room temperature for 20 min and measure its absorbance at a wavelength of 490 nm. Use 2.0 mL of deionized water instead of the glucose solution as the blank. With the glucose concentration as the abscissa and the absorbance as the ordinate, draw a standard curve.

[0058] The total sugar content is calculated according to the following formula:

[0059] The experimental results are as Figure 1 described. By comparing the total sugar content of Dendrobium officinale polysaccharide before and after fermentation, it can be seen that the total sugar content of Dendrobium officinale polysaccharide decreases after fermentation by the three fermentation methods, and there is no statistical difference.

[0060] Example 2

[0061] Determination of the free radical scavenging ability of fermented crude Dendrobium officinale polysaccharide

[0062] Experimental setup: The Dendrobium officinale polysaccharide fermented by Lactobacillus plantarum in Example 1 is denoted as LP, the Dendrobium officinale polysaccharide fermented by Saccharomyces cerevisiae in Example 1 is denoted as SC, the Dendrobium officinale polysaccharide fermented by compound lactobacillus in Example 1 is denoted as LP + LB, and the unfermented one is denoted as Unfermented. The following Dendrobium officinale polysaccharide solutions are LP, SC, LP + LB and Unfermented.

[0063] Hydroxyl radical determination

[0064] 1. Experimental procedure

[0065] In the experimental group, 0.5 mL of 0.15 mol / L FeSO4 solution, 2 mL of 0.002 mol / L salicylic acid solution and 2.5 mL of distilled water were added. In the administration group Am and the blank group A0, 1 mL of the fermented Dendrobium officinale polysaccharide solution to be tested was added, in the control group An, 1 mL of distilled water was added, and then 1 mL of 0.006 mol / L H2O2 was added to the administration group and the control group, and 1 mL of distilled water was added to the blank group. After reacting in a water bath at 37 °C for 1 h, it was centrifuged at 3700 rpm for 15 min in a centrifuge, and the supernatant was taken to measure the absorbance value at 510 nm.

[0066] The scavenging rate was calculated according to the following formula:

[0067] 2. Experimental results

[0068] The experimental results are as Figure 2 shown in A below. Compared with the unfermented Dendrobium officinale polysaccharide and the fermented Dendrobium officinale polysaccharides of other groups, the OH free radical scavenging ability of the Lactobacillus plantarum-fermented Dendrobium officinale polysaccharide generally increased with the increase of fermentation time.

[0069] DPPH free radical determination

[0070] 1. Experimental procedure

[0071] 4 mg of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) was dissolved in a small amount of 95% ethanol solution to obtain a DPPH solution. In the administration group Am, 1 mL of the fermented Dendrobium officinale polysaccharide solution to be tested and 3 mL of DPPH solution were added, in the blank group A0, 1 mL of 95% ethanol solution and 3 mL of DPPH solution were added, and in the control group An, 1 mL of the Dendrobium officinale polysaccharide solution to be tested and 3 mL of 95% ethanol solution were added. After mixing, it was allowed to stand at room temperature for 20 min, centrifuged at 3700 rpm for 15 min, and the supernatant was taken to measure its absorbance value at 517 nm.

[0072] The scavenging rate was calculated according to the following formula:

[0073] 2. Experimental results

[0074] The experimental results are as Figure 2 shown in B below. Compared with the unfermented Dendrobium officinale polysaccharide and the fermented Dendrobium officinale polysaccharides of other groups, the DPPH free radical scavenging ability of the Lactobacillus plantarum-fermented Dendrobium officinale polysaccharide generally increased with the increase of fermentation time.

[0075] ABTS free radical determination

[0076] 1. Experimental procedure

[0077] 0.0384g of 2,2-azino-di(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) was prepared into 7mmol / L ABTS solution with distilled water, and 0.0065g of potassium persulfate was dissolved in distilled water to prepare 2.4mmol / L potassium persulfate solution. The prepared ABTS solution and potassium persulfate solution were mixed and shaken, and allowed to stand at 4℃ for 12h to form ABTS+ solution. The ABTS+ solution was diluted with 95% ethanol solution to make its absorbance at 734nm 0.700±0.001 as the standard solution. The drug administration group Am took 1mL of the fermented Dendrobium polysaccharide solution to be tested and reacted with 9mL ABTS+ standard solution, and the control group An took 1mL of polysaccharide solution of different concentrations and mixed with 9mL distilled water. After 30min, the absorbance was measured at 735nm.

[0078] The clearance rate was calculated according to the formula:

[0079] 2. Experimental results

[0080] The experimental results are as follows Figure 2 As shown in Figure C, compared with unfermented Dendrobium polysaccharides and other groups of fermented Dendrobium polysaccharides, the overall ABTS free radical scavenging ability of Dendrobium polysaccharides fermented by Lactobacillus plantarum increased with the increase of fermentation time. Figure 2 The results of A and B in the figure show that the antioxidant capacity of Dendrobium polysaccharides was significantly enhanced after fermentation by Lactobacillus plantarum.

[0081] Example 3

[0082] An oxidative stress model was constructed by inducing Caco-2 cells with H2O2, and the relationship between the antioxidant capacity and dosage of Dendrobium polysaccharides fermented by Lactobacillus plantarum was explored.

[0083] 1. Experimental Procedure

[0084] Caco-2 cells were cultured at 5 × 10 4 The cells were inoculated at a density of 100 μM / mL in a 24-well culture plate, and the cells were divided into a sample group, a blank group, and a positive control group, with 3 replicates in each group. The cells were cultured at 37°C and 5% CO2 for 24 hours, and the culture medium was discarded. The sample group was added with 1 ml of the basic culture medium containing different concentrations (50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL) of plant lactobacillus fermented dendrobium polysaccharide, and the positive control group and the blank group were added with the same amount of culture medium containing 100 μM H2O2 and the basic culture medium, respectively, and cultured at 37°C and 5% CO2 for 24 hours. The cell culture medium was discarded, and the cells were treated with TritonX-100 cell lysis buffer, centrifuged at 1000 rpm for 5 minutes, and the supernatant was collected to determine the MDA, SOD and LDH activities of each group.

[0085] 2. Experimental results

[0086] The effect of Lactobacillus plantarum-fermented Dendrobium officinale polysaccharide (LP) on the intracellular MDA content in the H2O2-induced oxidative model of Caco-2 cells is shown as Figure 2 shown in D of the figure. Compared with the blank group, the MDA content in Caco-2 cells increased significantly after H2O2 stimulation (p < 0.05). Compared with the model group, in the concentration range of 50 - 250 μg / mL, LP could significantly reduce the intracellular MDA content (p < 0.05) and showed a dose-dependent relationship. When the LP concentration was 50 - 250 μg / mL, the intracellular MDA contents in the Caco-2 cell oxidative model were 157.13 ± 18.12 nmol / mgprot, 132.69 ± 11.26 nmol / mg prot, 114.72 ± 12.23 nmol / mgprot, 93.86 ± 10.04 nmol / mgprot, 84.21 ± 15.27 nmol / mg prot, which were 87.61%, 73.98%, 63.96%, 52.33%, 46.95% of the model group. When the LP concentration was 250 μg / mL, the intracellular MDA content decreased by 53.05%. The results showed that LP had the effect of reducing the content of the oxidation product MDA in Caco-2 cells.

[0087] The effect of Lactobacillus plantarum-fermented Dendrobium officinale polysaccharide (LP) on the intracellular LDH content in the H2O2-induced oxidative model of Caco-2 cells is shown as Figure 2 shown in E of the figure. Compared with the blank group, the LDH content in Caco-2 cells increased significantly after H2O2 stimulation (p < 0.05). Compared with the model group, in the concentration range of 50 - 250 μg / mL, the fermented Dendrobium officinale polysaccharide could significantly reduce the intracellular LDH content (p < 0.05) and showed a dose-dependent relationship. When the LP concentration was 50 - 250 μg / mL, the intracellular LDH contents in the Caco-2 cell oxidative model were 806.64 ± 15.25 U / L, 764.15 ± 4.26 U / L, 725.43 ± 2.1 U / L, 670.18 ± 1.66 U / L, 617.47 ± 3.89 U / L, which were 92.92%, 88.47%, 83.99%, 77.59%, 71.49% of the model group. When the LP concentration was 250 μg / mL, the intracellular LDH content decreased by 28.51%. The results showed that LP had the effect of reducing the LDH content in Caco-2 cells.

[0088] The effect of LP on the intracellular SOD content in the H2O2-induced oxidative model of Caco-2 cells is shown as Figure 2As shown by F in []. Compared with the blank group, the content of SOD in Caco-2 cells decreased significantly after H2O2 stimulation (p<0.05). Compared with the model group, in the concentration range of 50-250 μg / mL, LP could significantly increase the content of SOD in cells (p<0.05) and showed a dose relationship. When the concentration of LP was 50-250 μg / mL, the SOD contents in the oxidative model of Caco-2 cells were 298.57±7.40 U / mgprot, 313.26±6.98 U / mgprot, 368.75±12.18 U / mgprot, 406.17±16.22 U / mgprto, and 487.64±18.15 U / mgprot respectively. They were 127.13%, 133.39%, 157.02%, 172.95%, and 207.64% of the model group. Compared with the SOD content of 608.14±8.23 U / mgpro in the blank group cells, the SOD in the model group cells decreased by 373.3 U / mgprot. When the LP concentration was 250 μg / mL, compared with the blank group, the SOD content in cells increased by 120.5 U / mgprot, and the decreased content was 32.27% of the model group. The results showed that LP had the effect of increasing the SOD content in Caco-2 cells.

[0089] Example 4

[0090] Detect the effects of fermented Dendrobium officinale polysaccharide treatment on the activities of CAT, GR, and SOD in Caenorhabditis elegans

[0091] 1. Experimental procedure

[0092] Caenorhabditis elegans were synchronously cultured for 5 days in normal NGM medium, NGM medium supplemented with 200 μg / mL unfermented Dendrobium officinale polysaccharide, and NGM medium supplemented with 200 μg / mL fermented Dendrobium officinale polysaccharide by 3 different strains. All Caenorhabditis elegans were transferred to a centrifuge tube using sterile M9 buffer, centrifuged at 2000 rpm for 1 min at room temperature, the supernatant was discarded, and the worms were resuspended in 1 ml of M9 buffer. The worms were washed repeatedly with M9 buffer, centrifuged at 2000 rpm for 1 min at room temperature, and the supernatant was discarded. The worms were resuspended in 1 ml of M9 buffer, and PMSF and protease inhibitors were added. An appropriate amount of grinding beads were added and ground using a high-throughput tissue grinder (SCIENTZ-48, Xinzhi Biotechnology, China) at 1000 rpm for 1 min. The samples were taken out and cooled on ice, and the operation was repeated 2 times. The supernatant was taken after centrifugation at 2000 rpm for 5 min at room temperature. The protein concentration was quantified using a BCA kit, and the reductase contents in the worms were measured using a CAT kit, a GR kit, and an SOD kit.

[0093] 2. Experimental results

[0094] The experimental results are asFigure 3 As shown in A - C of Figure 3 , the CAT contents of the Control, Unfermented, LP, SC, and LB+LP treatment groups were 6.61±1.77 U / mgprot, 11.69±0.61 U / mgprot, 14.52±1.28 U / mgprot, 12.75±1.45 U / mgprot, and 13.40±1.04 U / mgprot, respectively, which were 1.77 - fold, 2.20 - fold, 1.93 - fold, and 2.03 - fold higher than that of the control group ( Figure 3 in A); the SOD contents of the Control, Unfermented, LP, SC, and LB+LP treatment groups were 48.14±22.91 U / mgprot, 63.37±16.55 U / mgprot, 85.29±17.43 U / mgprot, 40.55±6.68 U / mgprot, and 74.43±12.63 U / mgprot, respectively, which were 1.32 - fold, 1.77 - fold, 0.84 - fold, and 1.55 - fold higher than that of the control group ( Figure 3 in B); the GR contents of the Control, Unfermented, LP, SC, and LB+LP treatment groups were 19.97±7.93 U / mgprot, 31.53±8.34 U / mgprot, 166.03±1.82 U / mgprot, 15.76±0 U / mgprot, and 11.56±3.64 U / mgprot, respectively, which were 1.58 - fold, 8.32 - fold, 0.79 - fold, and 0.58 - fold higher than that of the control group (

[0095] Example 5

[0096] Detect the effect of fermented Dendrobium officinale polysaccharide treatment on the reactive oxygen species release of Caenorhabditis elegans

[0097] 1. Experimental procedure

[0098] Synchronize Caenorhabditis elegans, pick a sufficient amount of eggs onto a 9-cm NGM plate seeded with OP50, and observe the hatching of the nematodes. After the nematodes hatch and grow to the L4 stage, pick 30 nematodes each onto NGM plates of the normal NGM plate group (Control), the group added with unfermented Dendrobium officinale polysaccharide (Unfermented), the group added with Dendrobium officinale polysaccharide fermented by Lactobacillus plantarum (LP), the group added with Dendrobium officinale polysaccharide fermented by Lactobacillus brevis (SC), and the group added with Dendrobium officinale polysaccharide fermented by compound lactobacillus (LP+LB), and add an appropriate amount of 5-fluorouracil. Incubate Caenorhabditis elegans at 20 °C for 5 d. Wash the nematodes with 1 mL of M9 buffer, centrifuge at 1000 rpm at room temperature for 1 min. Subsequently, transfer the nematodes to a black 96-well plate with 50 μL of M9 buffer, with 20 worms per well, and add 100 μM of H2DCFDA fluorescent probe in a 1:1 ratio. Prepare a blank well control by adding 50 μL of M9 buffer and 50 μL of H2DCFDA, and set 3 replicates.

[0099] Place the 96-well plate in an incubator at a constant temperature of 30 °C, set the shaker speed to 30 rpm, and induce oxidative stress at high temperature for 2 h. Immediately insert the 96-well plate into a multifunctional microplate reader and gently shake for 30 s. Set the fluorescence intensity with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Measure the fluorescence signal every 0.5 h and continuously detect for 4 h. Keep the temperature of the microplate reader at 30 °C all the time. Summarize the data and use SPSS to analyze the differences between groups.

[0100] 2. Experimental results

[0101] The experimental results are as Figure 3 shown in D-E of the figure. The fermented Dendrobium officinale polysaccharide reduced the accumulation of ROS in Caenorhabditis elegans. Quantify the release amount of ROS, and it was found that the ROS in the Unfermented, LP, SC, and LB+LP treatment groups decreased to 72.78%, 47.40%, 58.34%, and 66.24% of the Control group, respectively.

[0102] Example 6

[0103] Detect the effects of four different Dendrobium officinale polysaccharides on the locomotor ability of nematodes

[0104] 1. Experimental procedure

[0105] Pick nematodes with comparable states from the control group and the experimental groups and place them on a 2% agarose gel. First, let the nematodes move for 2 min to adapt to the environment, observe the number of head swings of the nematodes under a stereomicroscope for 30 s and record it, which is used as an index to evaluate the locomotor ability of the nematodes. One head swing refers to the bow-shaped movement of the nematode's head, and 20 worms are observed in each group.

[0106] 2. Experimental results

[0107] The experimental results are as follows Figure 4 shown in A of []. After culturing nematodes with polysaccharides from Dendrobium officinale fermented by Lactobacillus plantarum for 5 days, the motility of nematodes was increased by 1.36 times compared with that of nematodes cultured on normal NGM medium for 5 days. Although there was no significant change in the motility of other treatment groups, there were also different degrees of improvement. The Unfermented group, SC group, and LB+LB group were increased by 1.15 times, 1.26 times, and 1.07 times respectively.

[0108] Example 7

[0109] Detect the effects of four different polysaccharides from Dendrobium officinale on the heat tolerance of nematodes

[0110] 1. Experimental procedure

[0111] The normal culture and grouping of nematodes were as above, with 60 worms in each group. After normal culture for 5 days, the nematodes were transferred to a constant temperature incubator at 35°C. The status of nematodes was observed under a dissecting microscope. The death criterion was that when the nematode was gently touched with a picker, the head or body of the nematode did not swing or bend. The number of dead nematodes was recorded every two hours, and the survival curve of nematodes under heat stress at 35°C was plotted to explore the average lifespan of Caenorhabditis elegans under heat stress at 35°C, so as to study the effect of fermented polysaccharides from Dendrobium officinale on heat tolerance.

[0112] 2. Experimental results

[0113] The experimental results are as follows Figure 4 shown in B of []. Under heat stress at 35°C, the average lifespan of control nematodes was 6.90 h, and the average lifespans of nematodes in the Unfermented group, LP group, SC group, and LP+LB group were 8.74 h, 10.18 h, 8.44 h, and 9.65 h respectively. Compared with the control group, the average survival time after treatment in the Unfermented group, LP group, SC group, and LP+LB group increased by 1.27 times, 1.48 times, 1.22 times, and 1.40 times respectively.

[0114] Example 8

[0115] Detect the effects of four different polysaccharides from Dendrobium officinale on the lifespan of nematodes

[0116] 1. Experimental procedure

[0117] Synchronized nematodes were randomly selected for the experiment and evenly inoculated into the Control group, Unfermented group, LP group, SC group, and LP+LB group. The concentration of polysaccharides from Dendrobium officinale in the experimental groups was 200 μg / mL. There were 20 nematodes in each group, with 3 replicates. They were cultured in a constant temperature and humidity incubator at 20 °C, which was recorded as the 0th day of the nematode lifespan. The medium covered with OP50 was changed every day. The survival status of the nematodes was observed under a microscope. The death criterion was that when the nematode was gently touched with a picker, its head or body did not swing or bend. Starting from the 7th day, the number of dead nematodes on each plate was recorded in detail every day, and the survival rate was calculated. Nematodes that burrowed into the medium and those that died due to other factors were excluded from the statistical data. A survival curve was plotted with the percentage of survival rate as the vertical axis and the survival time as the horizontal axis. The survival time of the last dead nematode was considered the maximum lifespan of the nematodes.

[0118] 2. Experimental results

[0119] The experimental results are as Figure 4 shown in C of the figure. After the treatment with polysaccharides from Dendrobium officinale, the survival curve of the larvae shifted to the right. The average lifespans of the nematodes in the Control group, Unfermented group, LP group, SC group, and LP+LB group were 12.21 d, 13.54 d, 15.13 d, 13.22 d, and 14.40 d, respectively. Compared with the control group, the average survival times after treatment in the Unfermented group, LP group, SC group, and LP+LB group increased by 1.11 times, 1.24 times, 1.08 times, and 1.18 times, respectively.

[0120] As can be seen from the above examples, the polysaccharides from Dendrobium officinale prepared by the preparation method of the present invention significantly enhanced the motility and heat stress resistance of nematodes and extended their lifespan. The present invention provides the optimal fermentation conditions for maximizing the biological activity of polysaccharides from Dendrobium officinale, emphasizes its potential as an anti-aging agent, and lays a solid foundation for future translational research.

[0121] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of dendrobium officinale polysaccharide, characterized in that, It includes the following steps: 1) Extract Dendrobium officinale by water extraction to obtain Dendrobium officinale extract; 2) Inoculate Lactobacillus plantarum into the Dendrobium officinale extract for fermentation. After fermentation, separate the solid and liquid phases, and collect the liquid phase component as the fermentation broth; 3) The fermentation broth is successively subjected to alcohol precipitation and protein removal to obtain crude Dendrobium officinale polysaccharide; 4) Dialyze and purify the crude Dendrobium officinale polysaccharide to obtain purified Dendrobium officinale polysaccharide.

2. The preparation method according to claim 1, characterized in that, The Lactobacillus plantarum is BNCC 339790.

3. The preparation method according to claim 2, wherein In step 2), the fermentation temperature is 36 - 38 °C, the fermentation time is 40 - 60 h, the fermentation process is accompanied by oscillation, and the oscillation speed is 100 - 200 rpm.

4. The preparation method according to claim 1, characterized in that, In step 1), the water extraction temperature is 85 - 95 °C, the water extraction time is 1.5 - 2.5 h, and the mass of water used for water extraction is 25 - 35 times the mass of Dendrobium officinale.

5. The preparation method according to claim 1, wherein In step 3), the alcohol precipitation is carried out with ethanol, the volume ratio of ethanol to the fermentation broth is (2 - 4):1, the alcohol precipitation temperature is 3 - 5 °C, and the alcohol precipitation time is 40 - 60 h.

6. The preparation method according to claim 5, wherein The dialysis is carried out using a dialysis bag, and the cut-off molecular weight of the dialysis bag is 3500 Da; the dialysis time is 60 - 84 h, and the water is changed every 7 - 9 h.

7. The preparation method according to claim 5, characterized in that In step 4), the purification is carried out using a DEAE-52 ion cellulose column and a SephadexG-100 dextran gel column.

8. Dendrobium officinale polysaccharide prepared by the preparation method according to any one of claims 1 - 7.

9. Use of the Dendrobium officinale polysaccharide according to claim 8 in the preparation of an antioxidant drug, an anti-aging health product or an anti-aging cosmetic.

10. Use of the Dendrobium officinale polysaccharide according to claim 8 in the preparation of a drug for improving the locomotor ability of nematodes, enhancing the heat stress resistance of nematodes or prolonging the lifespan of nematodes.

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