CN118271475A - Preparation of novel glycan in dendrobium candidum flower and novel application of novel glycan in dendrobium candidum flower in regulating intestinal flora - Google Patents

Abstract

The application discloses a new glycan in dendrobium candidum flower in the technical field of medicine, which consists of rhamnose, galacturonic acid, galactose and arabinose, wherein the molar ratio is 1.00-1.50: 1.01 to 1.60:4.00 to 6.00:2.55 to 4.00. The polysaccharide is obtained by adopting a water extraction and alcohol precipitation method and separating and purifying an anion exchange column and a gel column, can promote the growth of human intestinal bacteroides, and can be used for preparing medicines or medicine compositions and functional foods for regulating intestinal flora.

Description

Preparation of novel glycan in dendrobium candidum flower and novel application of novel glycan in dendrobium candidum flower in regulating intestinal flora

Technical Field

The invention relates to the technical field of medicines, in particular to a preparation method of new glycan in dendrobium candidum flowers and a new application of the new glycan in regulating intestinal flora.

Background

The human intestinal tract is a large container for the microflora to inhabit. It is reported that there are 10% human cells and 90% bacterial cells in the intestinal tract. These bacteria play a vital role in regulating the immune, nervous system and metabolism. Changes in the structure of the intestinal microbiota occur in several diseases, such as diabetes, crohn's disease, alzheimer's Disease (AD), and the like. Thus, there is an urgent need to find microbiota-directed substance interventions.

In fact, there is increasing evidence that the main regulator of intestinal microbial composition is the host diet, especially complex polysaccharides. Dietary polysaccharides provide an opportunity to maximize health. Given the importance of polysaccharides as the primary nutrients for HGM, understanding the complex utilization of polysaccharides by these bacteria is a key prerequisite for successful dietary intervention. More and more studies explore the mechanism by which specific members of the HGM utilize selected polysaccharides. Bacteroides offer many benefits to the host, in particular energy is supplied from non-digestible polysaccharides. It has been reported that bacteroides uses 20% of their genome for digestion of complex polysaccharides in the diet. Taiao Tao Weimi Bacteroides VPI-5482 (BT), oval Bacteroides ATCC8483 (BO) and cellulose silicate WH2 (BC) are three species commonly used to study the mechanism by which a particular member utilizes a selected polysaccharide. These species evolve under tremendous stress, utilizing complex polysaccharides, mainly plant cell wall glycans such as RGI, RGII and xyloglucan, in our diet. Researchers found that bacteroides produced surface-internal action glycoside hydrolases or polysaccharide lyases, initiating degradation of specific polysaccharides. One skilled in the art recognizes that Bacteroides are windows into microbiome. Thus, the study of these species as models is critical for the exploration of the intestinal microbiota.

Arabinogalactan (AG) is a common feature of plant polysaccharides highly favored by HGMs. The sugar composition of AG includes a backbone of beta-1, 3-galactose units that are modified by beta-1, 6-galactose oligosaccharides, typically by a range of sugar modifications such as rhamnose, glucuronic acid and other arabinose units as well as arabinose units. However, at present, only arabinogalactan protein (AG) isolated from seaweed and red wine is used to study the function against bacteroides. The intramural galactanase enzyme in BT is reported to provide a key place for AGs degradation. In addition, bacteroides is another bacterium that metabolizes AGs sulfation in seaweed and red wine. Despite the large amount of AG isolated from herbal medicine, there is no report on the human intestinal microbiota. Therefore, it is necessary to investigate the effect of AG-like polysaccharide isolated from Chinese herbal medicines on HGM.

Dendrobium officinale (Dendrobium officinale Kimura et Migo) is used in China as a herbal medicine and a new food resource. Polysaccharide is one of the main bioactive substances of dendrobium candidum. To date, more than ten polysaccharides have been isolated and identified from Dendrobium officinale, however, AG-like polysaccharides have not been isolated from Dendrobium officinale.

Disclosure of Invention

Aiming at the defects of the prior art, the invention designs the method which can separate AG-like polysaccharide from dendrobium candidum and is used for adjusting intestinal flora.

One of the purposes of the invention is to provide a novel glycan TF112 in dendrobium officinale flowers, which mainly comprises rhamnose, galacturonic acid, galactose and arabinose, and the molar ratio is 1.00-1.50: 1.01 to 1.60:4.00 to 6.00:2.55 to 4.00.

Further, the weight average molecular weight of the novel polysaccharide TF112 is 10-70 kDa, the number average molecular weight is 10-70 kDa, and the dispersion coefficient D is 1-1.6.

The second purpose of the invention is to provide a preparation method of the novel polysaccharide in dendrobium candidum flowers, which comprises the following steps:

1) Soaking: immersing dendrobium candidum flowers in water, and soaking for 12-24 hours at room temperature;

2) Decocting: heating and keeping micro-boiling for 2-4 hours/time, decocting for 2 times, and combining the filtrates;

3) And (3) dialysis: concentrating the filtrate to 1/10-1/20 of the original volume, naturally cooling to room temperature, dialyzing the concentrated solution against flowing water by using cellophane for 2-3 days;

4) Alcohol precipitation: concentrating the dialysate to 1/5-1/10 of the original volume, naturally cooling to room temperature, centrifuging at 4000-8000 rpm for 10-30 min, taking the supernatant, adding 95% ethanol with the volume 3-6 times that of the supernatant, stirring, and standing overnight;

5) And (3) freeze-drying: centrifuging the ethanol precipitation solution at 4000-8000 rpm for 10-30 min, adding water into the precipitate for re-dissolving, heating to volatilize residual ethanol, freezing, and freeze-drying by a freeze dryer to obtain dendrobium candidum flower water extraction crude polysaccharide TF;

6) Separating: separating crude polysaccharide TF by DEAE anion exchange, eluting with 0.1M NaCl to obtain component TF1, and purifying TF1 by gel permeation chromatography column to obtain new polysaccharide TF112.

Further, the ratio of water to dendrobium candidum flowers is 1:10-1:50.

Further, the dendrobium candidum flower is selected from dried or fresh dendrobium candidum flower buds.

Further, the crude polysaccharide obtained in the step 6) is dissolved in 60-100 mL of deionized water through an anion exchange column DEAE FAST FLOW separation loading amount ranging from 6g to 10 g.

Further, TF1 in the step 6) is purified by a gel column, and the sample loading amount range is 100-200 mg, and the solution is dissolved in 2.5-5 mL of deionized water.

The invention also aims to provide the application of the novel polysaccharide TF112 in preparing medicines, pharmaceutical compositions or functional foods for regulating intestinal flora.

In particular, the novel glycan TF112 can promote the growth of human intestinal bacteroides.

More specifically, the concentration of the novel polysaccharide TF112 is 2.5 mg/mL-10 mg/mL.

The fourth object of the present invention is to provide a pharmaceutical composition comprising neoglycan TF112 and pharmaceutically acceptable excipients.

The working principle and the beneficial effects of the invention are as follows:

The invention separates and purifies a new glycan TF112 from the flower of dendrobium candidum, and monosaccharide composition analysis by PMP pre-column derivatization shows that TF112 contains rhamnose, galacturonic acid, galactose and arabinose, and the molar ratio is 1.00-1.50: 1.01 to 1.60:4.00 to 6.00:2.55 to 4.00.

According to researches, TF112 of 2.5-10 mg/mL can promote the growth of human intestinal bacteroides, and at the moment, the bacteroides can metabolize to produce short-chain fatty acid acetate and propionate beneficial to human bodies, and meanwhile, the neoglycans can be degraded into mono-saccharides and oligosaccharides by the bacteroides. Can be used for preparing medicines or pharmaceutical compositions for regulating intestinal bacteria and functional foods.

Drawings

FIG. 1 is a diagram showing HPGPC detection results of TF 112;

FIG. 2 is a schematic diagram of a TF112 monosaccharide composition analysis;

FIG. 3 is a schematic diagram showing the growth curve of Bacteroides and the measurement of TF112 degradation products;

FIG. 4 is a schematic representation of the collection of supernatant SCFA from Bacteroides grown on TF112 for detection by GC-MS.

Detailed Description

The following is a further detailed description of the embodiments:

a first part: polysaccharide extraction

Soaking: soaking dried/fresh dendrobium candidum flower buds and water in a ratio of 1:10-1:30 (g/mL) at room temperature for 12-24 h;

Decocting: micro-boiling for 2-4 hours/time, 2 times;

and (3) dialysis: concentrating the decoction to 1/10-1/20 of the original volume, naturally cooling to room temperature, and dialyzing with cellophane for 2-3 days;

Alcohol precipitation: concentrating the dialysate to 1/5-1/10 of the original volume, naturally cooling to room temperature, centrifuging at 4000-8000 rpm for 10-30 min, taking the supernatant, adding 95% ethanol with the volume 3-6 times that of the supernatant, stirring, and standing overnight;

and (3) freeze-drying: centrifuging the ethanol precipitation solution at 4000-8000 rpm for 10-30 min, adding 0.5-3L of water into the precipitate for re-dissolving, uniformly mixing, heating to volatilize residual ethanol, freezing, and then freeze-drying by a freeze dryer to obtain the dendrobium candidum flower water extraction crude polysaccharide TF.

A second part: analysis of polysaccharide purity and monosaccharide composition

1. Polysaccharide purity determination:

precisely weighing 2-6 mg of polysaccharide sample, dissolving in 300-600 mu L of 0.1M sodium nitrate, oscillating to fully dissolve the sample, centrifuging at 4000-8000 rpm for 5-10 min, filtering the supernatant with 0.22 mu M aqueous phase filter membrane, and measuring the purity.

The special gel chromatographic column for polysaccharide is connected in series with Shodex KS 804 ((8.0 mm×300mm, exclusion limit 4×105 Da) and Shodex KS 802 ((8.0 mm×300mm, exclusion limit 1×104 Da)) the chromatographic condition is that the mobile phase is 0.1M sodium nitrate, the flow rate is 0.5mL/min, the sample injection amount is 10-20 mu L, the column temperature is 35 ℃, the ultraviolet absorption wavelength is 280nm, and the differential detector temperature is 35 ℃ for 40-60 min/sample.

2. Polysaccharide monosaccharide composition analysis:

Complete acid hydrolysis: weighing 2-4 mg of the sample, dissolving in 2mL of distilled water, carrying out vortex oscillation to assist dissolution, dissolving the sample (which can be heated or ultrasonically) as much as possible, adding the sample solution into a heart bottle (specification 50/19), then adding 2mL of 4M trifluoroacetic acid (TFA), uniformly mixing, adding a hollow plug matched with the heart bottle, sealing a contact opening between the plug and the heart bottle by using medical rubber paste, and heating and hydrolyzing for 2-5 h at 110 ℃. After hydrolysis, the mixture was cooled, methanol was added and the mixture was evaporated to dryness under reduced pressure several times to remove TFA. 200. Mu.L of distilled water was added to dissolve the hydrolysate.

PMP derivatization process: mu.L of the hydrolysate was added to 10mL of the EP tube from 200. Mu.L of the above step, and 100. Mu.L of a 0.6M NaOH solution was added thereto and mixed well. 200. Mu.L of 0.5M PMP was added, the mixture was sealed and mixed well, and heated in a water bath at 70℃for 1h+40min, after the completion of the reaction, the mixture was cooled to room temperature, then 200. Mu.L of 0.3M HCl was added, and 400. Mu.L of deionized water was added to make the total volume of the system 1mL.

Standard solution preparation: the method comprises the steps of preparing 9mg/mL of deionized water for each monosaccharide, respectively taking 100 mu L of deionized water for mixing, obtaining 9 monosaccharide standard substance solutions, wherein the concentration of each monosaccharide is 1mg/mL, and taking 100 mu L of standard substance mixed solution for derivatization.

Extraction: the shaker was shaken for 3min, centrifuged at 8000rpm for 5min, and left standing at room temperature for 30min. The upper water phase is reserved, the chloroform is used for repeated extraction for 3 times in the same operation, and the upper water phase is filtered by a microporous filter membrane with the thickness of 0.22 mu m.

HPLC analysis: the analytical column is a C18 reverse phase column, the mobile phase is phosphate buffer (the volume ratio of the phosphate buffer with pH 7.0 to acetonitrile is 33:7), the column temperature is 35 ℃, the flow rate is 1mL/min, the ultraviolet absorption wavelength is 245 or 254nm, the sample injection amount is 10 mu L, and the detection time is 1 h/sample.

As shown in fig. 1, column Shodex KS 804 is connected in series with KS 802, a: TF112 differential detection map; b: GPC analysis showed that TF112 molecular weight was obtained. The weight average molecular weight (Mw) of TF112 is 10-70 kDa, the number average molecular weight (Mn) is 10-70 kDa, and the dispersion coefficient D is 1-1.6 as determined by HPGPC purity analysis.

As shown in fig. 2, wherein a: the peak sequence of monosaccharide composition is measured by the pre-column derivatization of 9 monosaccharide standard substances PMP; b: TF112 monosaccharide composition analysis (Man: mannose; rha: rhamnose; glcA: glucuronic acid; galA: galacturonic acid; glc: glucose; gal: galactose; xyl: xylose; ara: arabinose).

Monosaccharide composition analysis by PMP pre-column derivatization shows that TF112 contains rhamnose, galacturonic acid, galactose and arabinose in a molar ratio of 1.00-1.50: 1.01 to 1.60:4.00 to 6.00:2.55 to 4.00.

Third section: determination of polysaccharide intestinal flora-related Activity

1. Growth curve measurement of intestinal bacterial proliferation

Three bacteroides BT, BO and BC which are frozen at the temperature of minus 80 ℃ are revived by using a TSB culture medium containing 5% of fetal bovine serum, the bacteroides is cultured for 24 hours in an anaerobic incubator filled with gas (10% CO2, 10% H2 and 80% N2) with a special proportion, when the growth of bacteria reaches the logarithmic phase, the rotation speed is 3000rpm, the bacteria are centrifugated for 5 minutes, the lower layer of bacteria are collected, the bacteria are washed by the MM culture medium, and then the concentration of the bacteria is diluted to the concentration by taking the MM culture medium as a solvent according to the concentration when the absorbance OD600 of the bacteria is approximately equal to 0.2. Finally, 100. Mu.L of the diluted bacterial liquid and an equal volume of a polysaccharide solution with a specific concentration (3 replicates per well) are added to each well of the 96-well plate, respectively, so that the absorbance value of the final concentration of the bacterial cells in each well OD600 is approximately equal to 0.1. When the influence of polysaccharide on the proliferation and growth curve of intestinal bacteria is screened, the mass concentration of the selected polysaccharide culture is usually 2.5mg/mL and 5mg/mL. The 96-well plate with the added thalli and polysaccharide is placed in a constant temperature anaerobic incubator at 37 ℃ for co-cultivation for 72 hours, and every 12 hours, the OD600 value at the moment needs to be recorded by using a Micro PLATES READER. And taking time as an abscissa and absorbance value as an ordinate scatter diagram to obtain the intestinal bacteria.

2. Quantitative analysis of short chain fatty acids in bacterial degradation products

Precision measuring 50. Mu.L of acetic acid and propionic acid, and 20. Mu.L of isobutyric acid, butyric acid, isovaleric acid and valeric acid, and using deionized water to reach a volume of 10mL (ready-to-use), obtaining a mother solution with the concentration of 5mL/L of acetic acid and propionic acid. And the mother liquor concentration of isobutyric acid, butyric acid, isovaleric acid and valeric acid is 2mL/L, different standard substance solutions are sucked according to the following table, deionized water is used for fixing the volume and 5mL, and 6 standard substance solutions with different concentrations of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid standard substances are obtained (the standard substances have certain volatility and need to be prepared at present). Respectively precisely measuring 500 mu L of standard substances and bacterial liquid supernatants with different concentrations in the table, adding 100 mu L of 50% (v/v) sulfuric acid in an acidification reaction, precisely measuring 500 mu L of diethyl ether in an extraction reaction, adding the solution into the solution, carrying out vortex oscillation for 30 seconds, rotating at 10,000rpm, and centrifuging for 5min (4 ℃). The upper diethyl ether layer is sucked through a 0.22 μm organic phase filter membrane for gas chromatography, and the short chain fatty acid content extracted by diethyl ether is preferably measured at the present time because diethyl ether is easy to volatilize.

As shown in fig. 3, wherein a, b. The growth curves of bacteroides grown on 2.5mg/mL (n=3) and 5mg/mL (n=2) TF112, respectively. c. Degradation products produced by BT at each time point were analyzed by HPGPC. d. Degradation products produced by BO at each time point were analyzed by HPGPC. e. The degradation products produced by BC at each time point were analyzed by HPGPC. HPAEC-PAD detects monosaccharides produced by Bacteroides grown on TF 112.

FIG. 4 is a collection of supernatant from Bacteroides grown on TF112 and analysis of the supernatant for SCFA.

The results show that: 6.2.5 mg/mL-10 mg/mL TF112 can promote the growth of human intestinal bacteroides, at this time, the bacteroides can metabolize to produce short-chain fatty acid acetate and propionate beneficial to human body, and at the same time, the polysaccharide can be degraded into mono-and oligosaccharides by the bacteroides.

The foregoing is merely exemplary embodiments of the present application, and specific structures and features that are well known in the art are not described in detail herein. It should be noted that modifications and improvements can be made by those skilled in the art without departing from the structure of the present application, and these should also be considered as the scope of the present application, which does not affect the effect of the implementation of the present application and the utility of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

Claims (10)

1. The novel glycan TF112 in the dendrobium candidum flower is characterized in that the novel glycan TF112 mainly comprises rhamnose, galacturonic acid, galactose and arabinose, and the molar ratio is 1.00-1.50: 1.01 to 1.60:4.00 to 6.00:2.55 to 4.00.

2. The novel polysaccharide TF112 in dendrobium officinale flowers according to claim 1, wherein the novel polysaccharide TF112 has a weight average molecular weight of 10-70 kDa, a number average molecular weight of 10-70 kDa and a dispersion coefficient D of 1-1.6.

3. A process for the preparation of novel glycans TF112 according to any of claims 1 or 2, characterized in that it comprises the following steps:

1) Soaking: immersing dendrobium candidum flowers in water, and soaking for 12-24 hours at room temperature;

2) Decocting: heating and keeping micro-boiling for 2-4 hours/time, decocting for 2 times, and combining the filtrates;

3) And (3) dialysis: concentrating the filtrate to 1/10-1/20 of the original volume, naturally cooling to room temperature, dialyzing the concentrated solution against flowing water by using cellophane for 2-3 days;

4) Alcohol precipitation: concentrating the dialysate to 1/5-1/10 of the original volume, naturally cooling to room temperature, centrifuging at 4000-8000 rpm for 10-30 min, taking the supernatant, adding 95% ethanol with the volume 3-6 times that of the supernatant, stirring, and standing overnight;

5) And (3) freeze-drying: centrifuging the ethanol precipitation solution at 4000-8000 rpm for 10-30 min, adding water into the precipitate for re-dissolving, heating to volatilize residual ethanol, freezing, and freeze-drying by a freeze dryer to obtain dendrobium candidum flower water extraction crude polysaccharide TF;

6) Separating: separating crude polysaccharide TF by DEAE anion exchange, eluting with 0.1M NaCl to obtain component TF1, and purifying TF1 by gel permeation chromatography column to obtain new polysaccharide TF112.

4. A method of preparation according to claim 3, characterized in that: the feed liquid ratio of water to dendrobium candidum flowers is 1:10-1:50.

5. The method of manufacturing according to claim 4, wherein: the dendrobium candidum flower is selected from dried or fresh dendrobium candidum flower buds.

6. The method of manufacturing according to claim 5, wherein: the crude polysaccharide of the step 6) is dissolved in 60-100 mL deionized water through an anion exchange column DEAE FAST FLOW separation loading amount range of 6-10 g.

7. The method of manufacturing according to claim 6, wherein: the TF1 of the step 6) is purified by a gel column, and the sample loading amount range is 100-200 mg and dissolved in 2.5-5 mL of deionized water.

8. Use of a novel glycan TF112 according to any one of claims 1 or 2 for the preparation of a medicament, pharmaceutical composition or functional food for modulating intestinal flora.

9. The use according to claim 8, characterized in that: the administration concentration of the novel polysaccharide TF112 is 2.5 mg/mL-10 mg/mL.

10. A pharmaceutical composition comprising the neoglycan TF112 according to any one of claims 1 or 2, and a pharmaceutically acceptable adjuvant.