Dendrobium officinale polysaccharide quality evaluation method based on acetylation degree and application thereof

Abstract

The invention provides a dendrobium officinale polysaccharide quality evaluation method based on acetylation degree and application of the dendrobium officinale polysaccharide quality evaluation method, and belongs to the technical field of medicines. In the prior art, quality control of dendrobium officinale is mainly based on polysaccharide content and monosaccharide composition, and biological activity of dendrobium officinale is difficult to objectively reflect. According to the invention, an in-vitro fermentation model reveals that the acetylation degree level of dendrobium officinale polysaccharide is a key factor influencing the activity of prebiotics, and an acetylation index quality control method based on parameters such as acetyl content, infrared spectrum and nuclear magnetic spectrum characteristics is established. By means of the quality control method, rapid and accurate evaluation of the activity of the dendrobium officinale polysaccharide prebiotics is achieved, and powerful technical support is provided for standardized production and industrial application of the dendrobium officinale polysaccharide.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a quality evaluation method for Dendrobium officinale polysaccharides based on acetylation degree and application thereof. BACKGROUND

[0002] Dendrobium officinale is a precious Chinese medicinal material with functions of benefiting stomach, generating fluid, nourishing yin and clearing heat. It contains glycosides, shihuchai, phenols, polysaccharides and amino acids. Modern pharmacological studies have shown that Dendrobium officinale has immunomodulatory, antioxidant, anti-inflammatory and intestinal flora regulation activities. Among them, polysaccharides are the main components of Dendrobium officinale for exerting biological activities. According to the Chinese Pharmacopoeia, the polysaccharide content of Dendrobium officinale should not be less than 25%, and the mannose content should be 13.0%-38.0%, which are important indicators for measuring the quality of Dendrobium officinale.

[0003] Dendrobium officinale polysaccharide is a linear glucomannan composed of D-mannose and D-glucose connected by β-1,4 glycosidic bonds. The O-2 or O-3 position of 1,4-D-mannose residues in the main chain is often connected with acetyl groups, which is a natural acetylated polysaccharide. Studies have shown that the acetylation degree of polysaccharides can significantly affect their solubility, anti-inflammatory activity, immunomodulatory capacity and intestinal flora regulation function. It is worth noting that intestinal flora, as the “second genome” of the human body, is closely related to many diseases: dysbiosis has been proven to be directly related to the occurrence and development of obesity, diabetes, inflammatory bowel disease, autism, Alzheimer's disease and even cancer. Therefore, studying how polysaccharides affect intestinal flora and then regulate health status has become a hot spot in the research of functional foods and modernization of traditional Chinese medicines. Therefore, studying the regulation of Dendrobium officinale polysaccharides on intestinal flora through in vitro fermentation model can not only reveal its prebiotic mechanism, but also provide a theoretical basis for the prevention or adjuvant therapy of related diseases.

[0004] However, the existing quality control system of Dendrobium officinale is mainly based on polysaccharide content and monosaccharide composition, but does not consider the influence of acetylation degree on its function, which has certain defects. If polysaccharides with different acetylation degrees have significant differences in activity, then using polysaccharide content as the quality standard may not accurately reflect its biological function. Therefore, establishing a quality control method based on acetylation degree, combined with in vitro intestinal flora fermentation experiment, to clarify the influence of acetylation on the prebiotic activity of polysaccharides, is of great importance to the standardized production and precise application of Dendrobium officinale polysaccharides. SUMMARY

[0005] The present application aims to provide a quality evaluation method for Dendrobium officinale polysaccharide based on acetylation degree and application thereof. The present application proposes to prepare Dendrobium officinale polysaccharides with different acetylation degrees, verify their prebiotic activity by combining in-vitro fermentation model, and establish a quality control system containing acetylation index to fill the blank of the prior art.

[0006] The 'acetylation degree' of the present application refers to the acetyl content in Dendrobium officinale polysaccharide.

[0007] The 'content' of the present application refers to mass fraction.

[0008] The present application provides a quality evaluation method for Dendrobium officinale polysaccharide based on acetylation degree, comprising the following steps: determining the acetyl content in Dendrobium officinale polysaccharide sample, and dividing it into deacetylated Dendrobium officinale polysaccharide, Dendrobium officinale polysaccharide and acetylated modified Dendrobium officinale polysaccharide.

[0009] The method for determining the acetyl content in Dendrobium officinale polysaccharide sample is selected from one of the following methods:

[0010] 1) UV-visible spectrophotometry for determining acetyl content;

[0011] 2) infrared spectroscopy for determining acetyl content;

[0012] 3) nuclear magnetic spectroscopy for determining acetyl content;

[0013] Among them, the acetyl content of deacetylated Dendrobium officinale polysaccharide is 9% to 12%; the acetyl content of Dendrobium officinale polysaccharide is 26% to 32%; and the acetyl content of acetylated modified Dendrobium officinale polysaccharide is 42% to 45%.

[0014] Further, the quality evaluation standard for determining infrared spectrum characteristics is as follows:

[0015] The ratio of acetyl peak to internal standard band peak in infrared spectrum of deacetylated Dendrobium officinale polysaccharide is 0.048 to 0.072; the ratio of acetyl peak to internal standard band peak in infrared spectrum of Dendrobium officinale polysaccharide is 0.368 to 0.552; and the ratio of acetyl peak to internal standard band peak in infrared spectrum of acetylated modified Dendrobium officinale polysaccharide is 1.059 to 1.588;

[0016] Further, the ratio of acetyl peak to internal standard band peak in infrared spectrum of deacetylated Dendrobium officinale polysaccharide is 0.07; the ratio of acetyl peak to internal standard band peak in infrared spectrum of Dendrobium officinale polysaccharide is 0.47; and the ratio of acetyl peak to internal standard band peak in infrared spectrum of acetylated modified Dendrobium officinale polysaccharide is 1.37;

[0017] The acetyl peak is a band at 1736 cm-1; and the internal standard band peak is a band at 3420 cm-1. -1 -1 The acetyl peak is a band at 1736 cm-1; and the internal standard band peak is a band at 3420 cm-1.​

[0018] Further, the quality evaluation standard of the determination of the nuclear magnetic spectrum characteristics is as follows:

[0019] The integral ratio of the nuclear magnetic spectrum of the deacetylated Dendrobium officinale Kimura et Migo polysaccharide (peak area of chemical shift of 2.1-2.2ppm / 3) / (peak area of chemical shift of 3.15-4.30ppm / 6) is 0.15-0.20; the integral ratio of the nuclear magnetic spectrum of the Dendrobium officinale Kimura et Migo polysaccharide (peak area of chemical shift of 2.1-2.2ppm / 3) / (peak area of chemical shift of 3.15-4.30ppm / 6) is 0.34-0.38; and the integral ratio of the nuclear magnetic spectrum of the acetylated Dendrobium officinale Kimura et Migo polysaccharide (peak area of chemical shift of 2.1-2.2ppm / 3) / (peak area of chemical shift of 3.15-4.30ppm / 6) is 1.0-1.20.

[0020] Further, the integral ratio of the nuclear magnetic spectrum of the deacetylated Dendrobium officinale Kimura et Migo polysaccharide (peak area of chemical shift of 2.1-2.2ppm / 3) / (peak area of chemical shift of 3.15-4.30ppm / 6) is 0.16; the integral ratio of the nuclear magnetic spectrum of the Dendrobium officinale Kimura et Migo polysaccharide (peak area of chemical shift of 2.1-2.2ppm / 3) / (peak area of chemical shift of 3.15-4.30ppm / 6) is 0.35; and the integral ratio of the nuclear magnetic spectrum of the acetylated Dendrobium officinale Kimura et Migo polysaccharide (peak area of chemical shift of 2.1-2.2ppm / 3) / (peak area of chemical shift of 3.15-4.30ppm / 6) is 1.15.

[0021] The application also provides the use of the quality evaluation method of the Dendrobium officinale Kimura et Migo polysaccharide for evaluating the prebiotic activity of the Dendrobium officinale Kimura et Migo polysaccharide.

[0022] The application also provides a Dendrobium officinale Kimura et Migo polysaccharide which is composed of mannose and glucose, the molar ratio of the mannose and the glucose is 5-5.5:1, the total sugar content is 88%-97%, the O-acetyl content is 26%-32%, the ratio of the acetyl peak of the infrared spectrum to the internal standard band peak is 0.368-0.552, and the integral ratio of the nuclear magnetic spectrum (peak area of chemical shift of 2.1-2.2ppm / 3) / (peak area of chemical shift of 3.15-4.30ppm / 6) is 0.34-0.38.

[0023] Further, in the Dendrobium polysaccharide, the molar ratio of mannose and glucose is 4.78:1, the total sugar content is 92.6±4.1%, the O-acetyl content is 28.6±1.83%, the infrared spectrum acetyl peak / internal standard band peak ratio is 0.47, and the integral ratio of the peak area of the chemical shift of 2.1-2.2ppm / 3 to the peak area of the chemical shift of 3.15-4.30ppm / 6 in the nuclear magnetic spectrum is 0.35.

[0024] The application further provides a method for preparing the Dendrobium polysaccharide, comprising the following steps:

[0025] (1) taking Dendrobium powder, adding an alcohol solvent for ultrasonic extraction, centrifuging, and collecting the precipitate;

[0026] (2) taking the precipitate, adding water for heating extraction, and concentrating under reduced pressure to obtain a concentrate;

[0027] (3) taking the concentrate, adding amylase for heating reaction, centrifuging, and collecting the supernatant;

[0028] (4) taking the supernatant, adding an alcohol solvent for precipitation, centrifuging, redissolving the precipitate in water, dialyzing, and freeze-drying to obtain the Dendrobium polysaccharide.

[0029] Further, in the method one, step (1) needs to be repeated twice, and the heating extraction of step (2) needs to be repeated twice.

[0030] Further, in step (1), the ratio of the Dendrobium powder to the alcohol solvent is 1g:5-15mL; the alcohol solvent is 80% ethanol; the ultrasonic extraction time is 20-40min, and the ultrasonic power is 460-500w;

[0031] In step (2), the ratio of the precipitate to water is 1g:30-40mL; the heating extraction temperature is 80-100℃, and the time is 0.5-2h;

[0032] In step (3), the ratio of the concentrate to amylase is 40-80mL:1g; the amylase is α-high peak amylase; the heating reaction temperature is 60-100℃, and the time is 4-8h;

[0033] In step (4), the volume ratio of the supernatant to the alcohol solvent is 1:2-4; the alcohol solvent is 95% ethanol.

[0034] The present application also provides a Dendrobium officinale polysaccharide, which is composed of mannose and glucose, the molar ratio of the mannose and the glucose is 4.5-5:1, the total sugar content is 86%-93%, the O-acetyl content is 9%-12%, the ratio of the acetyl peak to the internal standard band peak in infrared spectrum is 0.048-0.072, and the integral ratio of the peak area of the chemical shift of 2.1-2.2ppm to 3 to the peak area of the chemical shift of 3.15-4.30ppm to 6 in nuclear magnetic spectrum is 0.15-0.20.

[0035] Further, in the Dendrobium officinale polysaccharide, the molar ratio of the mannose and the glucose is 5.22:1, the total sugar content is 89.5±3.2%, the O-acetyl content is 10.5±1.15%, the ratio of the acetyl peak to the internal standard band peak in infrared spectrum is 0.07, and the integral ratio of the peak area of the chemical shift of 2.1-2.2ppm to 3 to the peak area of the chemical shift of 3.15-4.30ppm to 6 in nuclear magnetic spectrum is 0.16.

[0036] The present application also provides a method for preparing the Dendrobium officinale polysaccharide, which comprises the following steps: dissolving the Dendrobium officinale polysaccharide prepared by the method in water, reacting with alkali, adjusting the pH to neutral, dialysis, and freeze-drying.

[0037] Further, the mass ratio of the Dendrobium officinale polysaccharide to the alkali is 1:3-5, the alkali is sodium carbonate, the reaction temperature is 10-40 DEG C, and the reaction time is 20-40 min.

[0038] The present application also provides the use of the Dendrobium officinale polysaccharide in the preparation of a medicine with prebiotic activity or a health-care food for helping to regulate the intestinal flora function.

[0039] Further, the medicine or the health-care food is a medicine or a health-care food for promoting the proliferation of probiotics, inhibiting the growth of pathogenic bacteria and promoting the generation of short-chain fatty acids.

[0040] Further, the probiotics are Bifidobacterium, the short-chain fatty acids are acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid, and the pathogenic bacteria are Escherichia.

[0041] The present application has the following beneficial effects:

[0042] (1) The present application extracts and prepares three kinds of Dendrobium officinale polysaccharides (DDOP, DOP and ADOP) with different acetylation degrees, which can all regulate the intestinal flora and promote the generation of short-chain fatty acids (SCFAs).

[0043] (2) The present application found that different acetylation degree of polysaccharide has significant difference in regulating intestinal flora and SCFAs metabolism. Among them, deacetylated dendrobium polysaccharide (DDOP) and dendrobium polysaccharide (DOP) show stronger prebiotic activity, which can significantly increase the abundance of beneficial bacteria bifidobacterium, inhibit the growth of pathogenic bacteria escherichia coli, and promote the generation of SCFAs. In contrast, the prebiotic effect of acetylated dendrobium polysaccharide (ADOP) is obviously reduced, the production of SCFAs is reduced, and the enrichment effect of probiotics is weakened.

[0044] (3) The present application found that the acetylation degree of dendrobium polysaccharide is the key factor affecting its prebiotic activity. Moderate acetylation is conducive to the proliferation of probiotics and the generation of SCFAs, while excessive acetylation may reduce its availability, thereby weakening its probiotic efficacy.

[0045] (4) The present application found that when developing functional food or prebiotic preparation based on dendrobium polysaccharide, the acetylation degree should be reasonably controlled to optimize its intestinal health promoting effect.

[0046] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modification, replacement or change can be made without departing from the above technical idea of the present application.

[0047] The above content of the present application will be further explained in detail by the following specific embodiments. However, it should not be understood that the scope of the above subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 Molecular weight chromatogram of dendrobium polysaccharide with different acetylation degree (DDOP, DOP and ADOP).

[0049] Figure 2 Monosaccharide composition chromatogram of dendrobium polysaccharide with different acetylation degree (DDOP, DOP and ADOP).

[0050] Figure 3 Infrared spectrum of dendrobium polysaccharide with different acetylation degree (DDOP, DOP and ADOP).

[0051] Figure 4 Nuclear magnetic resonance hydrogen spectrum of dendrobium polysaccharide with different acetylation degree (DDOP, DOP and ADOP).

[0052] Figure 5Figure of the bacterial community diversity and species composition of different acetylation degree of Dendrobium polysaccharides (DDOP, DOP and ADOP): (A) PCA plot of principal component analysis; (B) species composition circle plot at the door level; (C) species composition bar plot at the genus level; (D) relative abundance of Bifidobacterium; (E) relative abundance of Escherichia. DETAILED DESCRIPTION

[0053] The raw materials and equipment used in the present application are known products, which can be obtained by purchasing commercially available products.

[0054] The "room temperature" referred to in the present application means 25±5℃.

[0055] Example 1, Preparation of different acetylation degree of Dendrobium polysaccharides (DDOP, DOP and ADOP)

[0056] 1. Preparation of Dendrobium polysaccharide DOP

[0057] (1) Dry and powder Dendrobium officinale Kimura et Migo, and pass through a 60-mesh sieve. Add 80% ethanol according to a solid-liquid ratio of 1:10 (w / v, i.e., g / mL), and ultrasonically extract for 30 min (ultrasonic power: 480 w). Centrifuge at a speed of 4000 rpm for 10 min; collect the precipitate, and repeat this step twice.

[0058] (2) Add ultrapure water according to a solid-liquid ratio of 1:35 (w / v), and extract at 90℃ for 1 h, twice. Combine the supernatants, and concentrate under reduced pressure to 40% of the volume.

[0059] (3) Remove starch: add a certain amount of α-high peak amylase to the concentrated solution after concentration according to a ratio of 60:1 (v / w), and react in a water bath at 80℃ for 6 h. Centrifuge at a speed of 4000 rpm for 10 min; collect the supernatant.

[0060] (4) Add 3 times the volume of ethanol (95%) to the supernatant to precipitate, stand for 12 h, centrifuge at 4000 g for 10 min, and reserve the precipitate.

[0061] (5) After resuspension of the precipitate, use a 3500 Da dialysis bag to dialyze for 3 d, and replace the deionized water every 4 h. Dialyze until the conductivity is stable (below 3). Finally, freeze-dry the product to obtain purified Dendrobium officinale Kimura et Migo polysaccharide, which is named Dendrobium officinale Kimura et Migo polysaccharide (hereinafter referred to as DOP).

[0062] 2. Preparation of deacetylated Dendrobium polysaccharide DDOP

[0063] DOP was dissolved in deionized water to a concentration of 5 mg / mL, and an equal volume of 0.2 M Na2CO3 was added. The reaction was stirred at room temperature for 30 min, and then neutralized with 1 M HCl. Subsequently, dialysis was performed using a 3500 Da dialysis membrane, and freeze-drying was performed. Deacetylated dendrobium officinale polysaccharide (hereinafter referred to as DDOP) was obtained.

[0064] 3. Preparation of acetylated sample dendrobium officinale polysaccharide ADOP

[0065] DOP was dissolved in formamide at 60°C to a final concentration of 16 mg / mL, and 1 / 2 volume of acetic anhydride (containing 4.5 mg / mL 4-dimethylaminopyridine (DMAP)) was added. The reaction was stirred at 60°C for 20 h, cooled, neutralized with 0.1 M NaOH, dialyzed using a 3500 Da dialysis membrane, and freeze-dried. An acetylated sample was obtained, which was named acetylated dendrobium officinale polysaccharide (hereinafter referred to as ADOP).

[0066] Example 2, Analysis of Dendrobium officinale polysaccharides with different acetylation degrees (DDOP, DOP and ADOP)

[0067] 1. Experimental method

[0068] (1) Determination of total sugar and acetyl content

[0069] The total polysaccharide content of DDOP, DOP and ADOP samples was determined by the phenol-sulfuric acid method, and glucose was selected as the standard to draw a standard curve. The sample concentration was 0.1 mg / mL. In addition, the acetyl content in the polysaccharide sample was determined according to the Pharmacopoeia (2020) O-acetyl determination method.

[0070] (2) Molecular weight analysis

[0071] The molecular weight distribution of DDOP, DOP and ADOP was determined by high performance liquid chromatography coupled with a charged aerosol detector (HPLC-CAD). Six dextran reference substances with molecular weights of 50000, 80000, 150000, 270000, 410000 and 670000 Da were precisely weighed and prepared into a standard solution with a concentration of 2.0 mg / mL. The 0.02 M ammonium acetate was used as the mobile phase, and the Acclaim SEC-1000, 7 μm, Analytical, 7.8 x 300 mm gel column was used for separation of the standard and sample at 40°C with a flow rate of 0.6 mL / min. Finally, the logarithm of the relative molecular mass of the standard dextran logMw was used as the Y coordinate, and the retention time Tr was used as the X coordinate, and the results were regressed.

[0072] (3) Composition sugar analysis

[0073] The monosaccharide composition of DDOP, DOP and ADOP was determined by high performance liquid chromatography combined with 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization. 6.0 mg of polysaccharide sample was weighed and hydrolyzed with trifluoroacetic acid (2.0 mol / L) at 95°C for 10 h. Subsequently, the trifluoroacetic acid was volatilized, and the hydrolysate was dissolved in 1.0 mL of deionized water. 50.0 μL of the hydrolysate was mixed with 50.0 μL of NaOH (0.6 mol / L) and 100 μL of PMP (0.5 mol / L) at 70°C for 100 min. Then, the mixture was neutralized with hydrochloric acid solution and diluted with ultrapure water to 1.0 mL. Subsequently, the mixture was repeatedly extracted with chloroform 4 times, and the organic layer was removed after the layers were separated. Finally, the mixture was filtered through a 0.22 μm organic filter membrane, and the monosaccharide composition was determined. The mixed standard was prepared by proportioning mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose. At a working temperature of 30°C, 20 μL of the derivative was injected into a high performance liquid chromatography system equipped with a ZORBAX Eclipse XDB-C18 chromatographic column (4.6×250 mm, i.d. 5 μm, Agilent Technologies Inc., CA, USA), and a mixture of phosphate buffer and acetonitrile (82:18, v / v) was used as the eluent at a flow rate of 1.0 mL / min. The wavelength of the DAD was set to 245 nm.

[0074] (4) Infrared spectrum analysis

[0075] The infrared spectrum of DDOP, DOP and ADOP was determined by Fourier infrared spectrometer. 1.0 mg of sample was accurately weighed and mixed with 100.0 mg of dry potassium bromide, and then carefully ground and pressed into a tablet. The absorption value of the sample in the wave number range of 4000-500 cm -1 was detected by the infrared spectrometer.

[0076] (5) Nuclear magnetic resonance spectrum analysis

[0077] 20 mg of DDOP, DOP and ADOP sample was respectively weighed and dissolved in 1.0 mL of D2O, and then left at room temperature overnight. After freeze-drying, 1.0 mL of D2O was added and repeated 3 times. Finally, the sample was dissolved in 1.0 mL of D2O and placed in a nuclear magnetic tube. The 1 H nuclear magnetic resonance spectrum of the polysaccharide sample was determined by nuclear magnetic resonance spectrometer.

[0078] 2. Experimental results

[0079] (1) Determination of total sugar and acetyl content

[0080] The results are shown in Table 1: the total sugar contents of DDOP, DOP and ADOP measured by colorimetry were 89.5%, 92.6% and 86.1%, respectively; the acetyl contents were 10.5%, 28.6% and 43.6%, respectively. It is shown that the Dendrobium polysaccharides with different acetylation degrees are successfully prepared by alkali treatment and acetic anhydride modification.

[0081] Table 1 Total sugar content and acetyl content of Dendrobium polysaccharides with different acetylation degrees

[0082]

[0083] (2) Molecular weight analysis

[0084] The molecular weight chromatograms of the standard and sample are shown in Figure 1 Fig. 1. DDOP, DOP and ADOP are all one component. Based on the regression curve of the dextran standard, the molecular weights of DDOP, DOP and ADOP are calculated to be 3.48 x 10 5 , 3.5 x 10 5 Da and 3.38 x 10 5 Da, respectively. The results show that the molecular weights of Dendrobium polysaccharides with different acetylation degrees have no significant difference.

[0085] (3) Analysis of constituent sugars

[0086] The results are shown in Figure 2 Fig. 2: DDOP, DOP and ADOP are both composed of mannose and glucose, and the mannose / glucose molar ratios are 5.22:1, 4.78:1 and 4.92:1, respectively.

[0087] (4) Infrared spectrum analysis

[0088] The results are shown in Figure 3 Fig. 3: the infrared spectra of DDOP, DOP and ADOP are relatively similar. There is a very strong absorption band at 3420 cm -1 , which represents the stretching vibration of hydroxyl. The absorption peak at 2889 cm -1 is caused by the asymmetric stretching vibration of C-H. The asymmetric stretching vibration of C-H of CH3 is at 1378 cm -1 . The vibration at 876 cm -1 indicates that Dendrobium polysaccharides contain mannopyranose rings. The absorption peak at 1736 cm -1 is caused by the C=O stretching vibration of O-acetyl, and 1250 cm -1 is related to the C-O stretching vibration of O-acetyl. Among them, the relative peak intensity is 1736 cm -1 and 1250 cm -1The signal of DDOP, DOP and ADOP was enhanced with the increase of acetyl group, which confirmed the successful preparation of Dendrobium polysaccharide samples with different acetyl degrees.

[0089] 1 H nuclear magnetic resonance spectrum as shown in Figure 2: the signal peaks of DDOP, DOP and ADOP at δ 5.51, δ 2.1-2.2 and δ 4.5-4.6 were enhanced with the increase of acetyl group. Figure 4 1 The H-NMR spectrum showed that the resonance peak signals appeared at δ 5.51 and δ 2.1-2.2, both of which represented the presence of acetyl group. The signal intensity of δ 2.1-2.2 was enhanced with the increase of acetyl group. In addition, the Dendrobium polysaccharide of the present application mainly includes the following residues:

[0090] The specific analysis is as follows: the signal peak at δ 5.51 belongs to H-1 of β-1, 4-Linked-2-O-acetyle-Manp. The signal peak at δ 4.5 belongs to H-1 of β-1, 4-Linked-Glcp. The signal peak at δ 4.11 belongs to H-2 of β-1, 4-Linked-Manp. It is shown that the main chains of DDOP, DOP and ADOP are composed of (1→4)-β-D-Glcp, (1→4)

[0091] -β-D-Manp, (1→4)-2-O-acetyl-β-D-Manp.

[0092] Example 3, method for determining the acetyl degree of Dendrobium polysaccharide

[0093] According to Example 1, the preparation method of Dendrobium polysaccharide with different acetyl degrees (DDOP, DOP and ADOP) was repeated three times to prepare three batches of Dendrobium polysaccharide with different acetyl degrees. The acetyl content range, infrared spectrum characteristics and nuclear magnetic spectrum characteristics of the three batches of Dendrobium polysaccharide with different acetyl degrees were obtained according to the method of Example 2, and the acetyl degree determination method of different Dendrobium polysaccharide was established according to the above test results of the three batches of Dendrobium polysaccharide with different acetyl degrees.

[0094] (1) The acetyl degree determination method of Dendrobium polysaccharide based on the acetyl content determined by colorimetry is shown in Table 2:

[0095] Table 2 Acetyl degree determination method of Dendrobium polysaccharide based on acetyl content

[0096]

[0097] (2) The acetyl degree determination method of Dendrobium polysaccharide based on infrared spectrum analysis

[0098] The acetyl content of Dendrobium polysaccharide was semi-quantitatively determined by infrared spectrum method. When the acetyl content of Dendrobium polysaccharide was less than 0.5%, the infrared spectrum showed that the signal intensity of 3420 cm -1 ​The stretching vibration band of 1736 cm -1 was selected as the internal standard band, and the band of 1740 cm 1736 was selected as the acetyl peak. The peak height ratio of 1740 cm 3420 / 1736 cm 1736 was calculated. 3420 The greater the ratio, the more acetyl groups the sample contained. Based on the DDOP, DOP and ADOP of the three batches of D. candidum polysaccharides, the peak height ratios of 1740 cm 1 / 1736 cm AC were calculated, and the ratios were 0.07, 0.47 and 1.37, respectively. Based on the results of the acetylation degree of the three batches of D. candidum polysaccharides determined by infrared spectrum analysis, ±20% of the average value calculated was taken as the range, and the determination method is shown in Table 3. 2-6 AC Table 3 Determination method of the acetylation degree of D. candidum polysaccharides based on infrared spectrum analysis 2-6 1 AC 2-6 e (3) Determination method of the acetylation degree of D. candidum polysaccharides based on nuclear magnetic resonance spectrum analysis d a The integral ratio of the peak area of H c (2.1-2.2 ppm) and H e (3.15-4.30 ppm) in the H c nuclear magnetic resonance spectrum can further compare the acetyl content in the sample, and the formula is (H b / 3) / (H b / 6). The greater the ratio, the higher the acetylation degree. According to the DDOP, DOP and ADOP of the three batches of D. candidum polysaccharides, the (H d / 3) / (H c / 6) ratios calculated by H b NMR were 0.16, 0.35 and 1.15, respectively. The determination method of the acetylation degree of the three batches of D. candidum polysaccharides based on nuclear magnetic resonance spectrum analysis is shown in Table 4. a c Table 4 Determination method of the acetylation degree of D. candidum polysaccharides based on nuclear magnetic resonance spectrum analysis a b b a a The beneficial effects of the present application are demonstrated by the following experimental examples. b a Experimental Example 1, Effects of D. candidum polysaccharides with different acetylation degrees (DDOP, DOP and ADOP) on the composition of healthy adult flora and SCFAs bc a 1. Experimental method d a (1) In vitro fermentation

[0109] Six healthy adult volunteers aged 20 to 30 years old were selected, with no record of antibiotic use for three months, and their written informed consent was obtained before the experiment. The volunteers picked up at least 5g of middle stool with little oxygen contact when defecating using a sterile stool sampling box, and the test was performed within 6 hours. According to the ratio of 1g / 10mL, add sterile 0.2M PBS buffer (containing cysteine-HCl 0.5g / L) and homogenize for 5min, centrifuge at 500xg, 4°C for 1min to remove impurities. The supernatant was centrifuged at 8000xg, 4°C for 10min to collect the bacterial precipitate, which was suspended in sterile anaerobic PBS (cysteine-HCl 0.5g / L) to prepare a 15% bacterial suspension inoculum. 1mL of bacterial suspension was inoculated into the following 9mL culture groups: blank medium (CK), positive control (containing 6mg / mL inulin, IN), low acetyl degree group (6mg / mL DDOP), medium acetyl degree group (6mg / mL DOP) and high acetyl degree group (6mg / mL ADOP). Anaerobic culture at 37°C for 48h.

[0110] Culture medium preparation: 0.5g / L bile salts, 1.5g / L peptone water, 1.0g / L yeast extract, 0.1g / L NaCl, 0.04g / L K2HPO4, 0.04g / L KH2PO4, 0.01g / L MgSO4·7H2O, 0.01g / L CaCl2·6H2O, 2g / L NaHCO3, 0.002g / L hematin, 10μL vitamin K1, 2mL Tween-80 and 0.5g / L l-cysteine-HCl. At 0, 12, 24, 36 and 48h of culture, samples were taken and centrifuged at 8000g, and the supernatant was used to determine the fermentation rate, reducing sugar and short-chain fatty acids (SCFAs). The precipitate was subjected to microbiota sequencing analysis.

[0111] (2) Fermentation rate and reducing sugar content determination

[0112] The total sugar of samples at different fermentation time periods was determined by the phenol-sulfuric acid method, and the fermentation rate was calculated. The dinitrosalicylic acid method was used to determine the change of reducing sugar.

[0113] (3) Third-generation full-length 16S amplicon

[0114] The bacterial precipitate after 48h of fermentation was sent to Shanghai Meiji Company for third-generation full-length 16S amplicon sequencing, and the DNA sample was amplified by specific 16S primers (27F and 1492R).

[0115] (4) SCFAs determination

[0116] This invention uses gas chromatography-mass spectrometry (GC-MS) to determine the content of SCFAs after 48 hours of fermentation. Acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid, and isovaleric acid standards were prepared using 50% methanol solution. The fermentation supernatant or standard solution was added to an equal volume of 2-ethylbutyric acid solution, mixed, and filtered through a 0.22 μm organic filter membrane. 1 μL of the mixture was then loaded into the GC-MS instrument. Helium was used as the carrier gas at a total flow rate of 34.0 mL / min, a pressure of 9.38 psi, and a split ratio of 30:1. The detector and syringe temperatures were both 280 °C. An HPINNOWax column (30 m × 250 μm × 0.25 μm, Agilent Technologies Inc., Santa Clara, CA, USA) was selected for detection. The initial column temperature was 80 °C, held for 1 min, increased to 120 °C at a rate of 10 °C / min, and then increased to 220 °C at a rate of 20 °C / min, held for 1 min.

[0117] 2. Experimental Results

[0118] 2.1 Fermentation rate and reducing sugar content

[0119] The total sugar content of samples at different fermentation time periods was determined by the phenol-sulfuric acid method, and the fermentation rate was calculated accordingly. The change in reducing sugar content was determined by the dinitrosalicylic acid method. The results are shown in Table 5.

[0120] Table 5. Changes in fermentation rate and reducing sugar during fermentation.

[0121]

[0122] 2.2 Effects on gut microbiota abundance

[0123] (1) β-diversity analysis

[0124] This study investigated the effects of DDOP, DOP, and ADOP on gut microbiota structure using 16S diversity sequencing technology. Principal coordinate analysis (PCA) represented the differences in gut microbiota among groups. Figure 5 As shown in Figure A, the differences among the five groups were significant, indicating that the gut microbiota in the fermentation broth underwent significant changes after 48 hours of fermentation. Furthermore, the ADOP and CK groups were close in position along the PC1 axis, suggesting a similarity in their regulatory effects on the gut microbiota. Meanwhile, DOP and DDOP were closer to the positive control group (IN) and farther from CK, indicating that the low- and medium-acetylation polysaccharides had a similar regulatory effect on the gut microbiota as inulin.

[0125] (2) Intestinal flora composition

[0126] like Figure 5As shown in B, based on the in vitro fermentation model, each group could change the composition of intestinal flora at the door level. Compared with CK and ADOP groups, the relative abundance of Actinomycetota in DOP, DDOP and IN groups increased significantly, and the relative abundance of Pseudomonadota decreased significantly. Actinomycetota contains probiotics such as Bifidobacterium. Pseudomonadota includes many pathogenic bacteria, such as Escherichia coli, Shigella, etc., which are easy to cause intestinal microbial imbalance. Further study on the regulation of sample groups on flora at the genus level. As shown in Figure 5 As shown in C, the relative abundance of harmful bacteria Escherichia in CK group was as high as 50%, and DOP and DDOP significantly reduced this kind of potential harmful bacteria. In addition, DOP and DDOP increased the relative abundance of Bifidobacterium, indicating that they may have better prebiotic effect Figure 5 D, 5E). This kind of effect is close to the recognized prebiotic inulin. Compared with CK group, although high acetylation ADOP can adjust the abundance of Escherichia and Bifidobacterium to a certain extent, but the adjustment effect is far lower than that of DOP and DDOP groups. It shows that high acetylation may weaken the utilization rate of polysaccharides by intestinal flora, affecting its prebiotic activity. Therefore, the degree of acetylation of Dendrobium polysaccharide may be an important factor affecting intestinal flora, which should be paid attention to in the development and quality control process.

[0127] 2.3, SCFAs content

[0128] Table 6 shows the changes in SCFAs content produced by low (DDOP), medium (DOP), high (ADOP) acetylated Dendrobium polysaccharides after 48 h in vitro fermentation. SCFAs are key products of gut microbiota metabolizing polysaccharides, which play an important role in regulating intestinal microecology, maintaining intestinal barrier function and improving host health. The CK group (control group) produced the lowest total amount of SCFAs (71.54 pg / mL), with acetic acid (48.24 pg / mL) as the main component. The IN group (positive control, inulin) significantly promoted the production of SCFAs, with a total amount of 375.05 pg / mL, mainly acetic acid (334.79 pg / mL), and a small amount of butyric acid (12.71 pg / mL). The DDOP group (low acetylated polysaccharides) produced a higher total amount of SCFAs (455.91 pg / mL), with acetic acid (379.79 pg / mL) being the highest, and the contents of propionic acid (33.88 pg / mL) and butyric acid (19.49 pg / mL) being higher than those in the CK and IN groups. The DOP group (medium acetylated polysaccharides) produced the highest SCFAs (536.58 pg / mL), with acetic acid (446.26 pg / mL) being significantly higher than that in other groups, indicating that this polysaccharide has strong prebiotic activity. The total amount of SCFAs in the ADOP group (high acetylated polysaccharides) decreased significantly (178.69 pg / mL), with acetic acid (102.61 pg / mL) being significantly lower than that in the DOP and DDOP groups, but the content of butyric acid (42.89 pg / mL) was the highest. However, the decrease in the overall production of SCFAs may limit its overall prebiotic effect. In summary, the DOP and DDOP groups significantly promoted the production of total SCFAs, especially the contents of acetic acid and isovaleric acid, which helped to maintain intestinal flora homeostasis and improve intestinal health. The total amount of SCFAs in the ADOP group was lower, which may be due to the high acetylation affecting the availability of polysaccharides, leading to a decrease in the metabolic capacity of the flora.

[0129] Table 6 SCFAs content (pg / mL) produced after 48 h in vitro fermentation

[0130] Sample Acetic acid Propionic acid Isobutyric acid Butyric acid Isovaleric acid Valeric acid Total SCFAs CK 48.24 ± 5.21 e ]] ND ND 3.88 ± 0.21 d ]] 15.63 ± 2.25 a ]] 3.78 ± 0.24 c ]] 71.54 ± 5.68 e ]]> IN 334.79 ± 8.33 c ]] 25.69 ± 2.25 b ]] ND ND 12.71 ± 1.36 b ]] 1.85 ± 0.75 d ]] 375.05 ± 8.77 c ]] DDOP 379.79 ± 10.31 b ]] 33.88±.17 a ]]> ND 19.49 ± 2.35 c ]] 15.13 ± 2.24 a ]] 7.60 ± 1.01 b ]] 455.91 ± 10.92 b ]] DOP 446.26 ± 8.27 a ]] 35.31 ± 3.33 a ]] ND 31.89 ± 3.37 b ]] 17.48 ± 1.65 a ]] 5.63 ± 1.35 bc ]] 536.58 ± 9.77 a ]] ADOP 102.61 ± 4.23 d ]] ND 1.53±2.24 42.89 ± 3.23 a ]] 15.65 ± 1.92 a ]] 16.01 ± 3.29 a ]] 178.69 ± 6.92 d ]]

[0131] Note: Lowercase letters (a, b, c, d, e) represent significant differences (p < 0.05) between different groups at the same time point; ND: not detected.

[0132] In summary, the application provides a quality evaluation method for Dendrobium officinale polysaccharide based on acetylation degree and application thereof. In the prior art, quality control of Dendrobium officinale is mainly based on polysaccharide content and monosaccharide composition, which is difficult to objectively reflect the biological activity. The application reveals that the acetylation degree of Dendrobium officinale polysaccharide is a key factor affecting the prebiotic activity through an in vitro fermentation model, and establishes a quality control method based on acetyl content, infrared spectrum characteristics and nuclear magnetic spectrum characteristics and other parameters of the acetylation index. Through the quality control method of the application, rapid and accurate evaluation of the prebiotic activity of Dendrobium officinale polysaccharide is realized, which provides strong technical support for the standardized production and industrial application of Dendrobium officinale polysaccharide.

Claims

1. A method for evaluating the quality of Dendrobium officinale polysaccharides based on acetylation degree, characterized in that, Includes the following steps: The acetyl content in Dendrobium officinale polysaccharide samples was determined, and they were classified into deacetylated Dendrobium officinale polysaccharide, Dendrobium officinale polysaccharide, and acetylated modified Dendrobium officinale polysaccharide. The method for determining the acetyl content in Dendrobium officinale polysaccharide samples is selected from one of the following methods: 1) Determination of acetyl content by ultraviolet-visible spectrophotometry; 2) Determination of acetyl content by infrared spectroscopy; 3) Determination of acetyl content by nuclear magnetic resonance spectroscopy; Among them, the acetyl content of deacetylated Dendrobium officinale polysaccharide is 9%–12%; the acetyl content of Dendrobium officinale polysaccharide is 26%–32%; and the acetylated Dendrobium officinale polysaccharide has an acetyl content of 42%–45%.

2. The method for evaluating the quality of Dendrobium officinale polysaccharides according to claim 1, characterized in that, The quality evaluation criteria for measuring infrared spectral characteristics are as follows: The peak ratio of acetyl group to internal standard in the infrared spectrum of deacetylated Dendrobium officinale polysaccharide was 0.048–0.072; the peak ratio of acetyl group to internal standard in the infrared spectrum of Dendrobium officinale polysaccharide was 0.368–0.552; and the peak ratio of acetyl group to internal standard in the infrared spectrum of acetylated Dendrobium officinale polysaccharide was 1.059–1.

588. The acetyl peak is 1736 cm⁻¹. -1 The spectral band; the internal standard band peak is 3420 cm⁻¹. -1 The spectral bands.

3. The method for evaluating the quality of Dendrobium officinale polysaccharides according to claim 1, characterized in that, The quality evaluation criteria for the determination of NMR spectral characteristics are as follows: The integral ratio of the NMR spectrum of deacetylated Dendrobium officinale polysaccharide (peak area of ​​2.1–2.2 ppm / 3) / (peak area of ​​3.15–4.30 ppm / 6) was 0.15–0.20; the integral ratio of the NMR spectrum of Dendrobium officinale polysaccharide (peak area of ​​2.1–2.2 ppm / 3) / (peak area of ​​3.15–4.30 ppm / 6) was 0.34–0.38; and the integral ratio of the NMR spectrum of acetylated Dendrobium officinale polysaccharide (peak area of ​​2.1–2.2 ppm / 3) / (peak area of ​​3.15–4.30 ppm / 6) was 1.0–1.

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4. The method for evaluating the quality of Dendrobium officinale polysaccharides according to any one of claims 1 to 3 is used to evaluate the prebiotic activity of Dendrobium officinale polysaccharides.

5. A polysaccharide from Dendrobium officinale, characterized in that, It is composed of mannose and glucose, with a molar ratio of mannose to glucose of 5 to 5.5:1, a total sugar content of 88% to 97%, an O-acetyl content of 26% to 32%, an infrared spectrum acetyl peak / internal standard band peak ratio of 0.368 to 0.552, and an NMR spectrum integral ratio of (peak area with chemical shift of 2.1 to 2.2 ppm / 3) / (peak area with chemical shift of 3.15 to 4.30 ppm / 6) of 0.34 to 0.

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6. A method for preparing the Dendrobium officinale polysaccharide according to claim 5, characterized in that, The method includes the following steps: (1) Take Dendrobium officinale powder, add alcohol solvent for ultrasonic extraction, centrifuge, and collect the precipitate; (2) Take the precipitate, add water and heat to extract, concentrate under reduced pressure to obtain the concentrate; (3) Take the concentrate, add amylase, heat and react, centrifuge, and collect the clear liquid; (4) Take the clear liquid, add an alcohol solvent to precipitate, centrifuge, redissolve the precipitate in water, dialyze, freeze dry, and the product is obtained.

7. A polysaccharide from Dendrobium officinale, characterized in that, It is composed of mannose and glucose, with a molar ratio of mannose to glucose of 4.5 to 5:1, a total sugar content of 86% to 93%, an O-acetyl content of 9% to 12%, an infrared spectrum acetyl peak / internal standard band peak ratio of 0.048 to 0.072, and an NMR spectrum integral ratio of (peak area at chemical shift of 2.1 to 2.2 ppm / 3) / (peak area at chemical shift of 3.15 to 4.30 ppm / 6) of 0.15 to 0.

20.

8. A method for preparing the Dendrobium officinale polysaccharide according to claim 7, characterized in that, The method includes the following steps: dissolving the Dendrobium officinale polysaccharide prepared by the method of claim 6 in water, reacting with alkali, adjusting the pH to neutral, dialyzing, and freeze-drying to obtain the final product.

9. The use of Dendrobium officinale polysaccharide according to claim 5 or 7 in the preparation of pharmaceuticals with prebiotic activity or in the preparation of health foods that help regulate intestinal flora function.

10. The use according to claim 9, characterized in that, The medicine or health food mentioned is a medicine or health food that promotes the proliferation of probiotics, inhibits the growth of pathogenic bacteria, and promotes the production of short-chain fatty acids.

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