Dendrobium officinale glycolipid as well as preparation method and anti-oxidation application thereof

Abstract

The invention discloses dendrobium candidum glycolipid as well as a preparation method and antioxidant application thereof, the dendrobium candidum glycolipid is diglyceride rich in glucose, mannose, linoleic acid and 9, 12, 15-octadecatrienoic acid, and the content of the mannose, the linoleic acid and the 9, 12, 15-octadecatrienoic acid is more than 1%. The invention also discloses an alcohol extraction process of the dendrobium officinale glycolipid, and application of the dendrobium officinale glycolipid in the aspects of improving abundance of acid-producing bacteroides in intestinal tracts, up-regulating piceatannol expression, down-regulating 1-naphthol expression and enhancing the activity of superoxide dismutase and glutathione peroxidase of organisms, so that the in-vivo oxidation resistance is finally improved.

Description

Technical Field

[0001] The invention belongs to the technical field of research on the structure and activity of Dendrobium officinale glycolipid, and particularly relates to Dendrobium officinale glycolipid and a preparation method and antioxidant application thereof. Background Art

[0002] Dendrobium candidum Dendrobium officinale ) is a perennial herbaceous plant of the genus Dendrobium in the family Orchidaceae, known for its medicinal and edible properties. Modern studies have shown that Dendrobium candidum is rich in a variety of bioactive ingredients, such as polysaccharides and alkaloids, which have potential effects such as enhancing immunity, regulating blood sugar levels, and anti-oxidation and anti-aging. Usually, these active ingredients are extracted by hot water and then separated and prepared by ethanol precipitation or column chromatography. However, the residue left after water extraction is often discarded and not effectively utilized, resulting in a waste of resources. Glycolipids are complex compounds composed of water-insoluble lipids and sugars. Structurally, glycolipids mainly consist of sphingosine fatty acids as the lipid part, and monosaccharides, oligosaccharides or polysaccharides as their sugar part connected by glycosidic bonds. In addition to maintaining normal cellular physiological functions, glycolipids are also involved in immune defense mechanisms and the development of diseases.

[0003] In normal physiological processes, human metabolic activities produce reactive oxygen species (ROS), which are essential for certain cellular functions (such as signaling pathways and immune responses), but if produced in excess, they may cause health problems. Under normal circumstances, the body's antioxidant defense system (including superoxide dismutase, catalase, and glutathione peroxidase, etc.) can effectively neutralize excess ROS. However, when the balance between the generation of reactive oxygen species and their removal by antioxidant enzymes is lost, these accumulated harmful substances will damage various cellular components such as proteins, lipids, and DNA, thereby destroying normal cell structure and function, and causing a series of health problems, including inflammatory bowel disease and diabetic ulcers. Therefore, effective management of oxidative damage is essential for maintaining optimal health and preventing / treating various diseases.

[0004] The intestinal microbiota refers to a group of microorganisms that inhabit the human gastrointestinal tract and coexist with the host. It not only participates in food digestion and absorption, but also plays an important role in key functions such as immune regulation, metabolic control, and psychological and behavioral regulation. The intestinal flora can convert indigestible cellulose and polysaccharides into easily absorbed beneficial metabolites such as short-chain fatty acids, providing an energy source for cell metabolism. It also plays an important role in regulating immune responses, promoting colon mucosal barrier function, and inhibiting the proliferation of pathogenic microecology. In addition, some specific types of intestinal flora can produce antioxidants such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) to reduce damage caused by oxidative stress. When the intestinal flora is unbalanced, it may lead to increased levels of oxidative stress.

[0005] At present, the preparation technology and biological activity of Dendrobium officinale glycolipids are obviously insufficient, which will limit its further application in the fields of food and medicine. Summary of the invention

[0006] The present invention provides a kind of Dendrobium officinale sugar lipid ( D. officinale The present invention discloses a dendrobium officinale glycolipid (DOG) and its preparation method and antioxidant application. The dendrobium officinale glycolipid is prepared by hot water extraction residue as raw material, and its structure is preliminarily analyzed. In addition, an oxidative damage animal model is established to study its antioxidant activity based on intestinal flora metabolism. These research results will provide valuable data support for the further development of DOG in the field of functional foods and promote the efficient utilization of dendrobium officinale raw materials.

[0007] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: a kind of Dendrobium officinale glycolipid, wherein the Dendrobium officinale glycolipid is a diglyceride formed by glycerol (20.43%), stearic acid (1.71%), D-mannose (7.42%), D-glucose (62.36%), linoleic acid (4.38%), 9,12,15-octadecatrienoic acid (2.78%) and octadecenoic acid (0.92%).

[0008] Preferably, the carbon atoms at positions 1 and 2 of the glycerol in the Dendrobium officinale glycolipid are linked to sugar and fatty acid molecules.

[0009] The present invention also provides a method for preparing the Dendrobium officinale glycolipid, and the specific steps of the preparation method are as follows: S1, adding the residue components after hot water extraction of Dendrobium officinale to ethanol solution for extraction, with a liquid-to-solid ratio of 20 mL / g, an ethanol concentration of 70%, an extraction temperature of 70°C, and an extraction time of 2.5 h to obtain a concentrated solution; S2, the concentrated solution obtained in step S1 is treated by a vacuum rotary evaporator to remove ethanol, and then subjected to vacuum freeze-drying to obtain Dendrobium officinale glycolipid.

[0010] The present invention also provides application of the Dendrobium officinale glycolipid in the field of anti-oxidation.

[0011] The application in the field of anti-oxidation includes being used as a dietary supplement for patients with oxidative damage, immunodeficiency and tumor patients.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The Dendrobium officinale glycolipid of the present invention is a diglyceride, rich in carbohydrate components and unsaturated fatty acids, has good emulsification properties, can not only directly participate in the scavenging of free radicals, but also shows in animal experiments that it can increase the abundance of acidogenic Bacteroides in the gastrointestinal tract of mice, upregulate the expression of piceatannol, and downregulate the expression of 1-naphthol, thereby enhancing the activity of SOD and GSH-Px in mouse serum and reducing the content of MDA, which is malondialdehyde. Its excessive accumulation can cause cross-linking polymerization of life macromolecules such as proteins and nucleic acids, resulting in changes in the structure and function of cell membranes, and belongs to Class III carcinogens. Finally, a strong antioxidant effect is exerted. These results will be conducive to the further development and utilization of Dendrobium officinale glycolipid in the field of food and medicine.

[0013] Other features and advantages of the present disclosure will become apparent from the following detailed description of exemplary embodiments of the present disclosure with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure or related technologies, the drawings required for use in the embodiments or related technical descriptions will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0015] Figure 1 It is the ultraviolet full wavelength scanning spectrum of the Dendrobium officinale glycolipid of the present invention; Figure 2 This is the infrared spectrum of the Dendrobium officinale glycolipid of the present invention; Figure 3 This is a graph showing the direct scavenging rate of the Dendrobium officinale glycolipid of the present invention on ABTS free radicals; Figure 4 This is a graph showing the direct scavenging rate of the Dendrobium officinale glycolipid of the present invention on DPPH free radicals; Figure 5 This is a comparison chart of the effects of the Dendrobium officinale glycolipid of the present invention on the activity of serum SOD in mice with oxidative damage; Figure 6 This is a comparison diagram of the effects of the Dendrobium officinale glycolipid of the present invention on the activity of serum GSH-Px in mice with oxidative damage; Figure 7 This is a comparison chart of the effects of the Dendrobium officinale glycolipid of the present invention on the serum MDA content of mice with oxidative damage; Figure 8 This is a comparison diagram of the effects of the Dendrobium officinale glycolipid of the present invention on the total antioxidant capacity of serum of mice with oxidative damage; Fig. 9 The Venn diagram of the effect of the Dendrobium officinale glycolipid of the present invention on the intestinal flora of mice with oxidative damage; Fig.10This is a graph showing the effect of the Dendrobium officinale glycolipid of the present invention on the top 10 bacterial species in the intestinal flora of mice with oxidative damage; Fig.11 This is a ternary phase diagram of the effect of ascorbic acid on the top 10 bacterial species in the intestinal flora of mice with oxidative damage; Fig.12 The ternary phase diagram of the effect of the Dendrobium officinale glycolipid of the present invention on the top 10 bacterial species of the intestinal flora of mice with oxidative damage; Fig.13 This is a heat map of the effects of ascorbic acid on the metabolites of intestinal flora in mice with oxidative damage; Fig.14 This is a heat map showing the effects of the Dendrobium officinale glycolipid of the present invention on the metabolites of intestinal flora in mice with oxidative damage. DETAILED DESCRIPTION

[0016] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0017] See also Figure 1-14 , the present invention provides a technical solution: The dried Dendrobium officinale was crushed into uniform powder (300 mesh), then soaked in deionized water at 80℃ for 4 h, and water extracted. The extraction was repeated 3 times, and all the extracts were mixed to prepare polysaccharides. The residue after water extraction was dried and used as raw material for the preparation of Dendrobium officinale glycolipids. It was soaked in ethanol and heated to improve the dissolution efficiency. The liquid-to-solid ratio was 20 mL / g, the ethanol concentration was 70%, the extraction temperature was 70℃, and the extraction time was 2.5 h to obtain a concentrate. The concentrate was treated with a vacuum rotary evaporator to remove ethanol, and then vacuum freeze-dried to obtain Dendrobium officinale glycolipids (DOG). Under this process, the yield of DOG was 2.64±0.18%.

[0018] The UV full wavelength scanning spectrum of DOG was measured, and the results are as follows Figure 1 As shown. The absorption of saturated fatty acids is relatively weak, and their absorption peaks are usually located in the lower wavelength range (200 ~ 210 nm). In contrast, unsaturated fatty acids have a stronger ability to absorb ultraviolet light, and their absorption peaks are located in the higher wavelength range (220-230 nm). In addition, the UV spectrum shows absorption near 280 nm and 410 nm, indicating that double bonds and conjugation may exist in DOG.

[0019] The characteristic functional groups of DOG were determined by infrared spectroscopy. Figure 2 shown. Figure 2 The infrared spectrum of DOG showed that DOG contained both saturated fatty acids and unsaturated fatty acids. -1 The huge absorption peak at 2923.37 cm indicates that it has a certain hydrophilicity and can be attributed to OH stretching. -1 and 2852.05 cm -1 The signals at 1740.40 cm -1 and 1632.50 cm -1 The absorption at 1465.68 cm belongs to the stretching vibration of the carbonyl group (C=O), suggesting the existence of an ester structure. -1 and 1384.11 cm -1 The bands appearing at 1262.33 cm can be attributed to the antisymmetric and symmetric bending vibrations of the CH3 group. -1 The characteristic absorption peak at 1165.31 cm -1 and 1076.23 cm -1 The bands found at 4400 Å may be related to the vibrations of COC and COH. Therefore, DOG has the characteristic functional groups of glycolipids.

[0020] Table 1 shows the results of GC-MS analysis of DOG after hydrolysis and acetylation. As shown in the table, the content of 1,2-diacetylglycerol in DOG is 20.43%, which indicates that the main component of DOG is diglycerol. The presence of D-glucitol hexaacetate (62.36%) and D-mannitol hexaacetate (7.42%) in DOG indicates that the glycosyl part of DOG is mainly composed of glucose and mannose. The detection of specific fatty acids such as palmitic acid (1.71%), linoleic acid (4.38%), 9,12,15-octadecatrienoic acid (2.78%), and oleic acid (0.92%) is consistent with the previous test results. The structural framework of glyceroglycolipids consists of a glycerol skeleton covalently linked to monosaccharide residues and fatty acids through O-glycosidic bonds. It is widely present in plants, animals, and bacteria, and is involved in processes such as intercellular communication, immune response regulation, and photosynthetic electron transfer, and has antioxidant potential.

[0021] Table 1 Monomer composition of DOG Serial number Compound Description Molecular formula Retention time (min) Molecular weight (Da) Proportion(%) 1 Glycerol 1,2-diacetate <![CDATA[C7H 12 O5]]> 4.79 176.07 20.43% 2 Ethyl stearate <![CDATA[C 18 H 36 O2]]> 10.55 284.27 1.71% 3 D-Mannitol, hexaacetate <![CDATA[C 18 H 26 O 12 ]]> 12.45 434.14 7.42% 4 D-Glucol, hexaacetate <![CDATA[C 18 H 26 O 12 ]]> 12.66 434.14 62.36% 5 Ethyl linoleate <![CDATA[C 20 H 36 O2]]> 13.02 308.27 4.38% 6 9,12,15-Octadecatrienoic acid, ethyl ester <![CDATA[C 20 H 34 O2]]> 13.13 306.26 2.78% 7 Octadecenoic acid, ethyl ester <![CDATA[C 20 H 40 O2]]> 13.42 312.30 0.92% Figure 3 and Figure 4The scavenging rate of DOG on ABTS and DPPH free radicals is shown, with ascorbic acid as a positive control. ABTS free radical means that if the absorbance at 734nm decreases after a substance is added to the ABTS free radical solution, it means that the substance has free radical scavenging activity and is an antioxidant. This method is called the ABTS free radical scavenging method, which can be used to evaluate the antioxidant capacity of plants (or Chinese herbal extracts) and pure compounds. DPPH free radical is a stable free radical and is widely used to quantitatively determine the in vitro antioxidant capacity of biological samples, pure compounds, and extracts. As shown in the figure, as the concentration of DOG and ascorbic acid increases from 0.25 to 4.00 mg / mL, their scavenging effects on ABTS and DPPH free radicals show similar trends. Ascorbic acid has strong antioxidant activity in vitro. Even at the lowest concentration (0.25 mg / mL), it can achieve about 90% of the free radical scavenging capacity. Further increase in concentration cannot continue to significantly improve its antioxidant activity. However, with the increase of DOG concentration, the average scavenging rate of ABTS free radicals increased from 30.97% to 88.78%, and the average scavenging rate of DPPH free radicals increased from 39.56% to 83.09%, which indicated that DOG also had a certain ability to directly scavenge free radicals.

[0022] Fifty six-week-old Kunming mice (weight 25±2 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. The mice were raised in an environment with a relative humidity of 45%~55% and a temperature controlled at 20~25°C. After the adaptation period, the mice were randomly divided into five groups (10 mice in each group): blank group, model group, ascorbic acid group, low-dose DOG treatment group (DOG-L, 50 mg / kg) and high-dose DOG treatment group (DOG-H, 100 mg / kg). The specific experimental process is as follows: At the beginning of the experiment, the blank group and the model group were gavaged with 0.2 mL of normal saline (0.9%) daily, while the ascorbic acid group was gavaged with 50 mg / kg ascorbic acid. The DOG-L and DOG-H groups were given corresponding doses of DOG for 21 days. On the 8th day, except for the blank group, mice in each experimental group received 60 mg / kg cyclophosphamide (CTX) injection for 3 days. On day 22, serum and feces samples were collected from mice for further analysis.

[0023] Figure 5 and Figure 6 The activity of SOD and GSH-Px in the serum of mice in each group is shown. As shown in the figure, compared with the blank group, the activity of SOD and GSH-Px in the serum of mice in the model group was significantly reduced ( p<0.01), indicating that intraperitoneal injection of cyclophosphamide caused serious damage to the body's antioxidant system. No significant improvement was observed after ascorbic acid intervention, indicating that ascorbic acid cannot effectively enhance the activity of antioxidant enzymes in vivo. On the contrary, after DOG gavage intervention, the activities of these two antioxidant enzymes in mouse serum increased to varying degrees, indicating that DOG can improve the overall antioxidant level by increasing the activity of antioxidant enzymes.

[0024] Figure 7 The MDA content in the serum of mice in each group is shown. As shown in the figure, compared with the blank group, the MDA content in the serum of mice in the model group increased significantly ( p <0.01), suggesting that cyclophosphamide inhibited the body's antioxidant system. After treatment with ascorbic acid and DOG, the MDA content of mice in the model group was significantly reduced, indicating that they have certain antioxidant effects in vivo, but their mechanisms of action may be different. DOG exerts its antioxidant function in vivo by enhancing the activity of related enzymes, while ascorbic acid achieves its antioxidant effect by directly scavenging free radicals.

[0025] Figure 8 The results showed that there was no significant difference in the direct free radical scavenging ability of the serum of each group of mice, which indicates that the peripheral blood of the body has good self-coordination ability.

[0026] Fig. 9 The changes in the abundance of intestinal flora in each group of mice are shown. As shown in the figure, there are 461 common operational taxonomic units (OTUs) in these groups, while the unique OTUs in the blank group, model group, ascorbic acid group and DOG group are 121, 137, 69 and 124, respectively, indicating that various intervention treatments have a significant effect on the diversity of intestinal microorganisms in mice with oxidative damage.

[0027] Fig.10The changes in the content of the top 10 intestinal bacteria in each group of mice are shown in the figure. As shown in the figure, compared with the blank group, the content of Clostridium papyrosolvens, Bacteroides vulgatus and Helicobacter hepaticus in the model group increased significantly, while the content of Escherichia coli and Bacteroides acidifaciens decreased significantly, indicating that the mice treated with cyclophosphamide had colitis and increased oxidative stress. Compared with the model group, the content of Escherichia coli and Helicobacter MIT01-6451 in the ascorbic acid group mice increased significantly, while the content of Clostridium papyrosolvens and Bacteroides acidifaciens decreased. At the same time, the DOG group contained higher content of Helicobacter MIT 01-6451 and Bacteroides acidifaciens, and the proportion of Bacteroides acidifaciens and Clostridium papyrosolvens was lower. These results show that DOG and ascorbic acid have different antioxidant effects.

[0028] Fig.11 and Fig.12 The ternary phase diagram of the top 10 intestinal bacterial species in the blank group-model group-ascorbic acid group and the blank group-model group-DOG group is shown. As shown in the figure, it can be more intuitively seen that Clostridium paper-soluble and Bacteroides vulgaris in the model group mainly play a role by inhibiting the body's antioxidant system, while Helicobacter MIT 01-6451 in the ascorbic acid group and DOG group may reflect the level of antioxidant and immune regulation in the body. Escherichia coli mainly plays the role of an important indicator in mice with oxidative damage after ascorbic acid intervention, while Bacteroides acidophilus can enhance DOG-induced antioxidant activity by protecting liver metabolism. This is consistent with the analysis results in the previous article.

[0029] Table 2 shows the information of differential metabolites in the intestine of mice in the ascorbic acid group and the model group. The results showed that 25 metabolites were significantly differentially expressed, of which 15 were down-regulated and 10 were up-regulated.

[0030] Table 2 Differential expression of metabolites in the ascorbic acid group compared with the model group Metabolite ID Compound Description Molecular formula Retention time (min) P-value Express C879 PG(18:3(9Z,12Z,15Z) / 18:3(6Z,9Z,12Z)) <![CDATA[C 42 H 71 O 10 P]]> 4.67 0.0192 Downward adjustment C271 8-isoprostaglandin F2a <![CDATA[C 20 H 34 O5]]> 5.55 0.0341 Downward adjustment C287 ACar 15:1 <![CDATA[C 22 H 42 NO4]]> 8.19 0.0194 Downward adjustment C897 Progesterone <![CDATA[C 21 H 30 O2]]> 6.76 0.0235 Downward adjustment C959 Urobilin <![CDATA[C 33 H 46 N4O6]]> 10.18 0.0338 Downward adjustment C298 ACar 20:2 <![CDATA[C 27 H 50 NO4]]> 9.36 0.0262 Downward adjustment C820 PC (22:6e / 18:5) <![CDATA[C 48 H 76 NO7P]]> 7.68 0.0343 Downward adjustment C711 N-Propyl-phenylalanine <![CDATA[C 12 H 15 NO4]]> 1.17 0.0179 Downward adjustment C106 2-(1H-1,2,3-Benzotriazole-1-yl)-N-(2,3-dihydro-1H-inden-2-yl)acetamide <![CDATA[C 17 H 16 N 4 O]]> 13.14 0.0305 Downward adjustment C437 DL-o-Tyrosine <h2 style=";text-align:left;direction:ltr"><![CDATA[C9H <h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> NO3]]><h2 style=";text-align:left;direction:ltr"> 2.87 0.0239 Downward adjustment C936 SM(d18:0 / 16:0) <![CDATA[C 39 H 81 N2O6P]]> 4.67 0.0459 Downward adjustment C672 N,N-Dimethylsphing-4-malvidin-3-glucoside <![CDATA[C 20 H 41 NO2]]> 9.15 0.0041 Downward adjustment C666 N-(3-Oxohexanoyl)homoserine lactone <![CDATA[C 10 H 15 NO4]]> 9.18 0.0372 Downward adjustment C362 Ceramide (d18:1 / 16:0) <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 34 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 67 <h2 style=";text-align:left;direction:ltr"> NO3]]><h2 style=";text-align:left;direction:ltr"> 10.06 0.0159 Downward adjustment C64 Ketocholesterol <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 19 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 28 <h2 style=";text-align:left;direction:ltr"> O3]]><h2 style=";text-align:left;direction:ltr"> 13.87 0.0444 Downward adjustment C673 Dihydrocapsaicin <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 29 <h2 style=";text-align:left;direction:ltr"> NO3]]><h2 style=";text-align:left;direction:ltr"> 13.72 0.0057 Increase C548 L-2-Amino-3-oxobutyric acid <![CDATA[C4H7NO3]]> 0.56 0.0174 Increase C236 5,7-Dimethyl-2-phenylpyrazolo[1,5-a]pyrimidine <![CDATA[C 14 H 13 N3]]> 13.24 0.0223 Increase C320 All-trans 13,14-dihydroretinol <![CDATA[C 20 H 32 O]]> 5.11 0.0362 Increase C384 Creatinine <![CDATA[C4H7N3O]]> 0.57 0.0274 Increase C246 5-Methoxyindoleacetic acid <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> NO3]]><h2 style=";text-align:left;direction:ltr"> 4.06 0.0185 Increase C615 Lysolecithin (14:0) <![CDATA[C 22 H 46 NO7P]]> 6.51 0.0340 Increase C385 Cryptotanshinone <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 19 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 20 <h2 style=";text-align:left;direction:ltr"> O3]]><h2 style=";text-align:left;direction:ltr"> 4.3 0.0102 Increase C118 2,4-Dihydroxybenzoic acid <![CDATA[C7H6O4]]> 0.83 0.0004 Increase C192 3-Methoxycinnamic acid <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> O3]]><h2 style=";text-align:left;direction:ltr"> 4.27 0.0126 Increase

[0031] Fig.13 A heat map analysis of these metabolites was performed in a more intuitive way. Fig.13In the figure, the color gradient from blue to red indicates the increase in metabolite concentration, making the expression differences between different metabolites clearer. In addition, through the signal pathway enrichment analysis of these metabolites, two significantly changed pathways were found: progesterone (C897)-mediated oocyte meiosis and maturation. Studies have shown that progesterone can promote the expression of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPX) in cells, especially oocytes, and improve their antioxidant activity through related signal pathways. However, the results of this study showed that ascorbic acid intervention led to a significant downregulation of progesterone levels in oxidative stress mice, indicating that the signal pathway mediated by progesterone was also inhibited, which is consistent with previous studies that ascorbic acid cannot enhance the activity of antioxidant enzymes.

[0032] Table 3 shows the information of differential metabolites in the intestine of mice in the DOG group and the model group. The results showed that a total of 63 metabolites showed significant differences, of which 35 were downregulated and 28 were upregulated.

[0033] Table 3 Differential expression of metabolites in the DOG group compared with the model group Metabolite ID Compound Description Molecular formula Retention time (min) P-value Express C1014 (2,3,9,17,22R)-2,3,14,20,22-Pentahydroxyergost-7-en-6-one <![CDATA[C 28 H 46 O6]]> 6.28 0.0025 Downward adjustment C576 LPC 14:0 <![CDATA[C 22 H 46 NO7P]]> 8.60 0.0019 Downward adjustment C97 1-Naphthol <![CDATA[C 10 H8O]]> 13.00 0.0080 Downward adjustment C537 Inosine <![CDATA[C 10 H 12 N4O5]]> 4.05 0.0006 Downward adjustment C108 2-(1H-Indol-3-yl)acetic acid <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 10 <h2 style=";text-align:left;direction:ltr"> H9NO2<h2 style=";text-align:left;direction:ltr"> 15.19 0.0085 Downward adjustment C92 1-Methyladenosine <![CDATA[C 11 H 15 N5O4]]> 0.55 0.0034 Downward adjustment C959 Urobilin <![CDATA[C 33 H 46 N4O6]]> 10.18 0.0182 Downward adjustment C480 FRH <![CDATA[C 21 H 30 N8O4]]> 10.88 0.0435 Downward adjustment C179 3-Hydroxy-5, 8-carnitine <![CDATA[C 19 H 25 BN4O4]]> 6.17 0.0366 Downward adjustment C809 PC (19:0 / 19:1) <![CDATA[C 46 H 90 NO8P]]> 10.45 0.0359 Downward adjustment C840 PC(18:1(11Z) / 20:0) <![CDATA[C 46 H 90 NO8P]]> 13.30 0.0341 Downward adjustment C287 ACar 15:1 <![CDATA[C 22 H 42 NO4]]> 8.19 0.0350 Downward adjustment C1105 (13E,16E,19E)-Docosatri-13,16,19-enoursolic acid <![CDATA[C 22 H 38 O2]]> 9.62 0.0286 Downward adjustment C188 3-Indoleacrylic acid <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> H9NO2<h2 style=";text-align:left;direction:ltr"> 3.22 0.0251 Downward adjustment C437 DL-o-Tyrosine <h2 style=";text-align:left;direction:ltr"><![CDATA[C9H <h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> NO3]]><h2 style=";text-align:left;direction:ltr"> 2.87 0.0157 Downward adjustment C282 ACar 12:1 <![CDATA[C 19 H 36 NO4]]> 5.93 0.0184 Downward adjustment C883 Choline Phosphate <![CDATA[C5H 14 NO4P]]> 13.19 0.0348 Downward adjustment C1147 PC (18:2e / 20:4) <![CDATA[C 46 H 82 NO7P]]> 13.17 0.0152 Downward adjustment C823 PC (9:0 / 9:0) <![CDATA[C 26 H 52 NO8P]]> 8.43 0.0390 Downward adjustment C906 Protectin D1 <![CDATA[C 22 H 32 O4]]> 6.73 0.0224 Downward adjustment C699 N-Arachidonic acid-L-serine <![CDATA[C 23 H 37 NO4]]> 13.02 0.0420 Downward adjustment C112 2-(3,5-Dimethyl-1H-pyrazol-4-yl)-5-methoxybenzoic acid <![CDATA[C 13 H 14 N 2 O3 ]]> 4.05 0.0314 Downward adjustment C578 LPC 15:0 <![CDATA[C 23 H 48 NO7P]]> 6.87 0.0168 Downward adjustment C711 N-Propyl-phenylalanine <![CDATA[C 12 H 15 NO4]]> 1.17 0.0013 Downward adjustment C1103 (+ / -)11(12)-EET <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 20 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 32 <h2 style=";text-align:left;direction:ltr"> O3]]><h2 style=";text-align:left;direction:ltr"> 7.44 0.0266 Downward adjustment C1000 Uridine <![CDATA[C9H 12 N2O6]]> 0.58 0.0339 Downward adjustment C541 Kahweol <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 20 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 26 <h2 style=";text-align:left;direction:ltr"> O3]]><h2 style=";text-align:left;direction:ltr"> 2.98 0.0397 Downward adjustment C1115 2-Arachidylglycerol <![CDATA[C 23 H 38 O4]]> 9.03 0.0009 Downward adjustment C776 PC (17:1 / 17:2) <![CDATA[C 42 H 78 NO8P]]> 9.77 0.0010 Downward adjustment C1109 12-HETE <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 20 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 32 <h2 style=";text-align:left;direction:ltr"> O3]]><h2 style=";text-align:left;direction:ltr"> 7.26 0.0328 Downward adjustment C449 D-Phenylalanine <![CDATA[C9H 11 NO2]]> 1.43 0.0202 Downward adjustment C452 Ecgonine <h2 style=";text-align:left;direction:ltr"><![CDATA[C9H <h2 style=";text-align:left;direction:ltr"> 15 <h2 style=";text-align:left;direction:ltr"> NO3]]><h2 style=";text-align:left;direction:ltr"> 3.25 0.0166 Downward adjustment C64 11-Ketocholanolone <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 19 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 28 <h2 style=";text-align:left;direction:ltr"> O3]]><h2 style=";text-align:left;direction:ltr"> 13.87 0.0366 Downward adjustment C534 Indoline-2-carboxylic acid <![CDATA[C9H9NO2]]> 1.35 0.0391 Downward adjustment C888 Pimelyl Carnitine <![CDATA[C 14 H 25 NO6]]> 5.25 0.0136 Downward adjustment C279 9-Oxo-ODE <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 18 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 30 <h2 style=";text-align:left;direction:ltr"> O3]]><h2 style=";text-align:left;direction:ltr"> 6.44 0.0136 Increase C28 (9Z)-(7S,8S)- Octadecadienoic acid dihydroxy ester <![CDATA[C 18 H 34 O4]]> 6.07 0.0136 Increase C1021 12(13)-DiHOME <![CDATA[C 18 H 34 O4]]> 9.23 0.0147 Increase C115 2,3-Dihydroxypropyl-12-methyltridecanoate <![CDATA[C 17 H 34 O4]]> 6.79 0.0192 Increase C446 Dodecanoylcarnitine <![CDATA[C 19 H 37 NO4]]> 7.78 0.0353 Increase C320 All-trans 13,14-dihydroretinol <![CDATA[C 20 H 32 O]]> 5.11 0.0136 Increase C23 (6R,7S)-6,7-Epoxyoctadecanoic acid <![CDATA[C 16 H 26 O5]]> 7.41 0.0136 Increase C595 LPE 18:2 <![CDATA[C 23 H 44 NO7P]]> 6.93 0.0462 Increase C1 (+ / -)12(13)-DiHOME <![CDATA[C 18 H 34 O4]]> 6.47 0.0092 Increase C316 adrenaline <![CDATA[C 22 H 36 O2]]> 0.59 0.0087 Increase C12 (2R)-(9Z,12Z,15Z)-2-Hydroperoxyoctadecatri-9,12,15-enoursolic acid <![CDATA[C 20 H 40 O2]]> 6.03 0.0489 Increase C1129 D-(+)-Maltose <![CDATA[C 12 H 22 O 11 ]]> 4.32 0.0249 Increase C236 5,7-Dimethyl-2-phenylpyrazolo[1,5-a]pyrimidine <![CDATA[C 14 H 13 N3]]> 13.24 0.0259 Increase C283 ACar 12:3 <![CDATA[C 19 H 32 NO4]]> 5.27 0.0004 Increase C277 9-HpODE <![CDATA[C 18 H 32 O4]]> 6.5 0.0354 Increase C926 SM (d17:0 / 23:1) <![CDATA[C 45 H 91 N2O6P]]> 3.38 0.0015 Increase C228 4-Cresol <![CDATA[C7H8O]]> 3.69 0.0094 Increase C1123 9-HpODE <![CDATA[C 18 H 32 O4]]> 6.53 0.0270 Increase C24 (7S,8S)-DiHODE <![CDATA[C 18 H 32 O4]]> 6.44 0.0261 Increase C599 LPE 20:5 <![CDATA[C 25 H 42 NO7P]]> 6.93 0.0409 Increase C652 Matricaria <![CDATA[C 17 H 22 O5]]> 5.43 0.0290 Increase C493 Ginkgolic acid <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 22 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 34 <h2 style=";text-align:left;direction:ltr"> O3]]><h2 style=";text-align:left;direction:ltr"> 8.89 0.0307 Increase C294 ACar 18:2 <![CDATA[C 25 H 46 NO4]]> 7.41 0.0269 Increase C4 (11E,15Z)-9,10,13-trihydroxyoctadeca-11,15-dienoic acid <![CDATA[C 18 H 32 O5]]> 4.78 0.0121 Increase C319 all-cis-4,7,10,13,16-Docosapentaenoic acid <![CDATA[C 22 H 34 O2]]> 6.47 0.0033 Increase C994 Quinoline dimethacrylate <![CDATA[C 14 H 18 O4]]> 5.78 0.0445 Increase C758 PC (16:0 / 17:0) <![CDATA[C 41 H 82 NO8P]]> 8.25 0.0358 Increase C1167 Piceatannol <![CDATA[C 14 H 12 O4]]> 0.58 0.0204 Increase

[0034] Fig.14 A heat map analysis of these metabolites was performed in a more intuitive way. Fig.14 In the figure, the color gradient from blue to red indicates the increase in metabolite concentration, making the expression differences between different metabolites clearer. In addition, through the signal pathway enrichment analysis of these metabolites, four pathways were found: 4-methylphenol (C228)-mediated nitrotoluene degradation, piceatannol (C1167)-mediated flavonoids, diphenylheptane compounds and turmeric ketones, and 1-naphthol (C97)-mediated cytochrome P450 and naphthalene family metabolism of exogenous substances. In this application, DOG mainly exerts its antioxidant effect by enhancing the activity of related enzymes in the body, reducing the potential direct free radical scavenging effect of 4-methylphenol. Studies have shown that some microorganisms can synthesize piceatannol during fermentation and enhance the activity of antioxidant enzymes in the body. In contrast, 1-naphthol and its metabolites can induce the massive production of ROS in the body, destroying the inherent redox balance and triggering oxidative stress. Therefore, the antioxidant mechanism of DOG may be related to upregulating the expression of piceatannol and downregulating the expression of 1-naphthol, thereby increasing the activity of antioxidant enzymes and ultimately exerting a protective effect against cyclophosphamide-induced oxidative damage.

[0035] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A Dendrobium officinale glycolipid, characterized in that: The Dendrobium officinale sugar lipid is a diglyceride formed by 20.43% glycerol, 1.71% stearic acid, 7.42% D-mannose, 62.36% D-glucose, 4.38% linoleic acid, 2.78% 9,12,15-octadecatrienoic acid and 0.92% octadecenoic acid.

2. The Dendrobium officinale sugar lipid according to claim 1, characterized in that: The carbon atoms at positions 1 and 2 of the glycerol in the Dendrobium officinale glycolipid link the sugar and fatty acid molecules.

3. A method for preparing a Dendrobium officinale glycolipid as described in any one of claims 1-2, characterized in that: The specific steps of the preparation method are as follows: S1, adding the residue components after hot water extraction of Dendrobium officinale to ethanol solution for extraction, with a liquid-to-solid ratio of 20 mL / g, an ethanol concentration of 70%, an extraction temperature of 70°C, and an extraction time of 2.5 h to obtain a concentrated solution; S2, the concentrated solution obtained in step S1 is treated by a vacuum rotary evaporator to remove ethanol, and then subjected to vacuum freeze-drying to obtain Dendrobium officinale glycolipid.

4. Use of the Dendrobium officinale glycolipid as described in any one of claims 1-2 in the field of anti-oxidation.

5. The use of a Dendrobium officinale glycolipid in the field of anti-oxidation according to claim 4, characterized in that: The application in the field of anti-oxidation includes being used as a dietary supplement for patients with oxidative damage, immunodeficiency and tumor patients.

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