Physicochemical property changes of Dendrobium officinale leaf polysaccharide LDOP‐A and it promotes GLP‐1 secretion in NCI‐H716 cells by simulated saliva‐gastrointestinal digestion

Abstract A polysaccharide LDOP‐A with a molecular weight of 9.9 kDa was isolated and purified from Dendrobium officinale leaves by membrane separation, cellulose column, and dextran gel column. The Smith degradable products, methylation products, and nuclear magnetic resonance analysis showed that LDOP‐A may be composed of →4)‐Glc‐(1→, →3,6)‐Man‐(1→, and →6)‐Glc‐(1→sugar residues. In vitro, simulated digestion assays showed that LDOP‐A could be partially digested in the stomach and small intestine, and produced a large amount of acetic acid and butyric acid during colonic fermentation. Further cell experiment results illustrated that LDOP‐A‐I (LDOP‐A digested by gastrointestinal tract) could induce glucagon‐like peptide‐1 (GLP‐1) secretion in NCI‐H716 cells without showing any cytotoxicity.

studies have proved that the activity of polysaccharides is related to the composition, molecular weight, residue type, and repeat unit of monosaccharides (Xie et al., 2020). As a natural active ingredient that is difficult to be digested and utilized, the structural changes and active structure of polysaccharides in gastrointestinal tract need further study (Koh et al., 2016). Short-chain fatty acids (SCFA), a microbial fermentation product derived from dietary fiber, play an important medium in intestinal flora to affect host health. The available evidence suggests that polysaccharides can slow down or reverse the decrease of butyric-acid-producing bacteria and intestinal butyric acid levels in T2DM patients (Kumar et al., 2021;Qin et al., 2021). Some studies have shown that butyric acid can increase the secretion of Glucagon-like peptide-1 (GLP-1) and peptide YY in the intestine, thus promoting the secretion of insulin in T2DM patients (Fan & Pedersen, 2020). GLP-1 is a kind of incretin secreted under the stimulation of eating, which is released by L cells of the distal ileum and colon (Pan et al., 2021).
Previous studies have indicated that plant polysaccharides have an excellent hypoglycemic effect by increasing the levels of insulin and GLP-1 in streptozotocin-induced diabetic rats (Lee et al., 2018).
GLP-1 can control the fluctuation of blood sugar by increasing insulin secretion, reducing glucagon level and appetite, and also can promote pancreatic islets β-cell proliferation, differentiation, and reduction of β apoptosis (Kuang et al., 2020;Xie et al., 2020;Yang et al., 2021).
In order to detect the in vitro anti-diabetes effect of the polysaccharide extracted from Dendrobium officinale leaf named LDOP-A, the present study investigated the structure and simulated digestion of LDOP-A in vitro and then explored secretion of GLP-1 from endocrine L cells by direct stimulation of digested LDOP-A .

| Extraction of Dendrobium officinale leaf polysaccharides
Dendrobium officinale leaves were purchased from Yueqing, Zhejiang Province. Eighty-five percent ethanol was added into the dry leaf powder at the ratio of 1:10 and stirred by magnetic force for 8 h.
The filter residue was collected. The decolorized powder was dissolved in distilled water at a dilution rate of 2:3 (v/v), extracted at 70°C for 2 h, repeated twice, combined with the filtrate, and centrifuged at 7140 g for 15 min. The supernatant was then concentrated and precipitated with 80% ethanol, standing at 4°C overnight, and centrifuged to obtain precipitation. Freeze dry the precipitate and dissolve it with distilled water under the condition of pressure of 0.2 MPa and flow rate of 30 L/h, and continuous ultrafiltration with 100 kDa membrane is carried out 5-10 times until the maximum permeation yield is reached. The filtrate was collected and freeze-dried as described above.

| Purification of polysaccharides
Five-hundred milligram of crude polysaccharides were dissolved in 20 mL distilled water and fractionated through a DEAE cellulose column (2.6 cm × 60 cm). Use distilled water, and 0.2 mol/L, 0.4 mol/L, and 0.8 mol/L NaCl solution to conduct gradient elution in turn. The elution rate is 1 mL/min. Collect 6 mL in each tube and 200 tubes in total. Dilute the eluent in the tube and measure the absorbance value at 490 nm according to the phenol-sulfuric acid method. Draw the elution curve with OD value as the ordinate, and combine the eluents of main peaks according to the curve. The collected DEAE samples were further purified by Sephadex G-100 gel permeation column chromatography. Elute with distilled water at a flow rate of 0.5 mL/min, collect the eluent (5 mL/tube), determine the sugar content with phenol sulfuric acid method, and draw the elution curve. Collect the fraction containing main carbohydrate and concentrate and dry it to obtain LDOP-A.

| Analysis of Smith degradation products of LDOP-A
Fifteen milligram LDOP-A was dissolved in 8 mL 30 M sodium periodate solution, then allowed reaction under 4°C without light.
Samples were taken every 12 h and measured the absorbance at UV 233 nm. After the OD value is stable, 0.4 mL ethylene glycol is added to the system to stop the reaction. The reaction solution was dialysised with distilled water for 24 h, and then concentrate it to 2-3 mL. 100 mg NaBD 4 was added into the concentrated solution, and placed at room temperature for 12 h, then neutralized with glacial acetic acid. Twice the volume of methanol was added to the solution and dried at 45°C under reduced pressure.
TFA (3 mL, 2M) was added into the reduced product and then hydrolyzed at 110°C for 5 h, evaporate it with methanol. Four milliliter acetic anhydride-pyridine (1:1) was mixed into the hydrolyzate, filled with nitrogen, and then placed at 110°C for 1 h for acetylation reaction. After the reaction, the mixture was extract 3 times with n-hexane, the extraction liquid was combined and evaporated to dryness under reduced pressure, the residue was dissolving with water and passed 0.22 μm membrane filtration to conduct GC-MS analysis. The chromatographic column is HP-5MS (30 m × 0.25 mm × 0.25 μm), with high-purity helium as carrier gas, and the flow rate is controlled to 1 mL/min. The sample injection volume is 0.2 μ 50. The injection port temperature is set to 210°C. The heating procedure is as follows: the initial temperature is 140°C and maintained for 6 min; first, raise the temperature to 190°C at the rate of 2°C/min; then, the temperature was raised to 210°C at the rate of 1°C/min and maintained for 5 min.
2.2.2 | Analysis of methylation products of LDOP-A Fifteen milligram LDOP-A was completely dissolved in 3 mL of DMSO. NaOH powder (100 mg) was added into the reaction system; after reaction at 18-20°C for 30 min, the reaction is ended by freezing in ice bath. Then, 2 mL methyl iodide was slowly added to the mixture under nitrogen atmosphere, and stand for 16 h to ensure complete methylation, repeat 2-3 times. The reaction solution was washed repeatedly with 2 mL chloroform for four times and the organic phase were combined and dried with anhydrous sodium sulfate. TFA (2 mL, 2M) was added to the residue. After reacting at 120°C for 2 h, two times the volume of methanol was added to the mixture and steam three times. 2-3 mL distilled water was added into the evaporated system, the pH was adjusted between 10 and 12 with 0.1 mol/L NaOH solution, then 100 mg NaBD 4 , was added and reacted for 2 h, the glacial acetic acid was used to neutralize excess NaBD 4 until there are no bubbles. The hydrolyzate was acetylated and analyzed by GC-MS. Column and analysis procedure are the same as above.

| 1 H-NMR and 13 C-NMR analysis of LDOP-A
Thirty milligram LDOP-A was dissolved in 0.5 mL of D 2 O to conduct the structural identifications by 1 H-NMR and 13 C-NMR analysis and obtain one-dimensional NMR spectrum.

| Simulated digestion
The in vitro simulated saliva-gastrointestinal digestion of LDOP-A was performed based on previous methods (Wu, Nie, et al., 2021;. Briefly, Saliva samples (age range 20-25 years) were provided by two volunteers who had not received antibiotic treatment within 3 months. Volunteers were asked not eat or drink at morning until saliva was collected. LDOP-A solution (8 mg/mL) was mixed with 100.0 mL simulated saliva at equal volume and oscillated at a rate of 100 rpm in water bath oscillator at 37°C.
After 0.25, 0.5, and 1.0 h of incubation, 5.0 mL digested solution was collected and boiled to inactivate the enzyme and investigate the content of reducing sugar using 3,5-dinitrosalicylic acid method. In addition, a part of mixed liquid after 1.0 h was centrifuged, precipitated with ethanol, redissolved, and dialyzed to obtain LDOP-A-S.
After salivary digestion, 50 mL of simulated gastric solution (112.5 mg gastric lipase and 106.2 mg pepsin added to 450 mL gastric electrolyte solution) was added to 50.0 mL mixture after saliva digestion at the ratio of 1:1 (v/v) (Jagadeesan et al., 2022), and the pH was immediately adjusted to pH 3.0 using 0.1 M HCl. After 0.5, 1.0, 2.0, 4.0, and 6.0 h of incubation at 37°C, 5 mL digested solution was again determined, and LDOP-A-G was obtained.
Add 200.0 g bile salt solution (4%, w/w), 100.0 g trypsin solution (7%, w/w), and 13.0 mg trypsin into 100.0 g intestinal electrolyte solution, and fully mix them to obtain a simulated small intestine solution (Jagadeesan et al., 2022). The pH of the mixture after salivagastric digestion was adjusted to pH 7.0 by 0.1 M NaOH, and the simulated small intestine solution was added at a ratio of 3: 10 (v/v).
2.3.2 | Determination of reducing sugar content and SCFAs content LDOP-A-I was further used for fermentation. The mixed fecal samples of healthy C57 mice were collected and diluted to 10% (w/v) with normal saline solution, and the diluent is filtered with sterile cotton cloth. 1.0 mL of filtrate was add to a 9.0 mL of fermentation medium containing 1.0 mg LDOP-A-I, and the mixture was cultured in a constant temperature shaking flask at 37°C. Blank control group was designed; 1.0 mL of filtrate was added into 9.0 mL fermentation medium. The fermentation samples were collected at 0 h and 48 h, respectively, and the content of SCFAs was analyzed by gas chromatography. The temperature rise procedure of GC is as follows: the temperature rises from 60°C to 100°C at the rate of 5°C/min, and then rises to 145°C at the rate of 10°C/min (Dou et al., 2019).

| Determination of structural changes of polysaccharides before and after simulated digestion
The monosaccharide composition of LDOP-A was determined by high-performance liquid chromatography (HPLC) (Xie et al., 2022).
Cold field-emission scanning electron microscope was used at 2 kV accelerating voltage and the sample surface was observed at ×2000 image magnification.

| Cell experiment
2.4.1 | Cell culture NCI-H716 cell was obtained from ATCC. The cell growth medium was RPMI 1640 medium, in which 10% FBS and 1% penicillin/streptomycin were added.

| Cell viability assay
Cytotoxicity was assessed by Cell Counting Kit-8 (CCK-8) assay. The operation is the same as reported (Pan et al., 2021). The number of multiple holes in each group is set to 5. Incubate at 37°C for 24 h, wash with preheated PBS three times, and add 100 μL of culture medium containing 0.5 mg/mL CCK-8 into each well. After incubated at 37°C for 24 h and washed with preheated PBS three times, the cells were incubated with 100 μL of culture medium containing 0.5 mg/mL CCK-8 at 37°C in the dark for 2 h. Read the absorbance at 490 nm on the microplate reader.

| GLP-1 secretion assay
The secretion level of GLP-1 was detected after the NCI-

| Simulated digestion of LDOP-A in vitro
In the process of polysaccharide digestion in vivo, the breaking of glycosidic bonds will lead to more reduction ends exposed as a whole, which will increase the content of reducing sugar (Kasipandi et al., 2019). The main function of saliva is to partially digest the starch in food. As shown in Figure 3a Generally speaking, the glycosidic bond on the branch chain of the segment is easier to break, while polysaccharide with larger molecular weight is more difficult to be absorbed and utilized in the digestive tract, which may be related to the relatively regular and orderly structure and its low solubility and easy formation of gel (Payling et al., 2020). During in vitro digestion, the monosaccharide composition of LDOP-A after oral digestion is almost no different from that of untreated polysaccharides, which is consistent with the change in reducing sugar content. However, after simulated gastric digestion, the monosaccharide composition of LDOP-A changed significantly (Figure 3c). The monosaccharide composition of LDOP-A-G and LDOP-A-I was determined as

F I G U R E 1 Elution curves of LDOP-A by DEAE column (a) and Sephadex G column (b); GC-MS chromatograms of Smith degradable products of LDOP-A (c).
glucose, and there were also trace amounts of mannose and glucuronic acid. Compared with undigested LDOP-A, the mannose peak basically disappeared after gastric digestion, which may be because mannose on the polysaccharide branch chain can be hydrolyzed at low pH (Wu, Nie, et al., 2021;.
Combined with the results of the glycosidic bond composition of LDOP-A, we can predict that after digestion in the stomach and small intestine, LDOP-A-I will become a glycan composed of →4)-Glc-(1→ and →6)-Glc-(1→sugar residues. It can be seen from the scanning electron microscope in Figure 3d that a large number of pore structures can still be seen in the LDOP-A-S after saliva digestion, while the pore structure of polysaccharide in LDOP-A-G basically disappears and becomes fine flakes ( Figure 3d). After simulating small intestine digestion, the polysaccharide is further degraded, and small pores appear on the surface of LDOP-A-I. This may be related to the branched-chain structure of LDOP-A is decomposed by gastric juice, which is consistent with the disappearance of mannose in the monosaccharide composition of LDOP-A-G.

| In vitro fermentation of polysaccharides
On basis of simulated digestion in vitro, the effect of indigestible polysaccharide LDOP-A-I on SCFAs production of intestinal microorganisms was further explored. In general, non-digestible polysaccharides enter the intestine and are decomposed and metabolized as a carbon source for the microbiota in the intestine. The derived end products from anaerobic fermentation of microorganisms include gases and SCFAs, mainly acetate, propionate, and butyrate, and their proportions have many beneficial effects in several different tissues (Koh et al., 2016). Therefore, SCFAs can reflect the relationship between the decomposition of polysaccharides and the structure of intestinal microbiota.

F I G U R E 2 GC-MS chromatograms of methylation product of LDOP-A (a), 1H NMR (b), and 13C NMR (c) spectrum of LDOP-A.
More and more evidence has shown that the occurrence of diabetes is related to the insufficient production of butyrate in the intestine because butyrate may play a key role in maintaining the integrity of intestinal epithelium and the metabolic balance of human body and intestinal microorganisms (Kumar et al., 2021). As shown in Table 1, acetic acid is the most abundant organic acid produced F I G U R E 3 Reducing sugar content (a), infrared spectrum (b), monosaccharide composition (c), and surface structure (d) during digestion of LDOP-A.

| Effects of LDOP-A-I on GLP-1 secretion in NCI-H716 cells
The results of GLP-1 secretion showed that a certain amount of sodium acetate (4-8 mmol/L) and sodium butyrate ( Recent studies have shown that some natural polysaccharides can promote GLP-1 secretion through direct interaction with intestinal epithelial cells (Pan et al., 2021). In the present study, NCI-H716 cells It can be inferred that LDOP-A can have an effect on the increase of insulin after meals, reducing glucagon level, or reducing appetite.

| CON CLUS ION
The activity of natural polysaccharides is related to its structure.
Polysaccharides from plants are usually administered orally, and the digestive tract (especially the intestine) has been proven to be an important part of polysaccharides regulating body functions.
Polysaccharide was the main component in Dendrobium Officinale leaf, the contents were measured as 8.99-16.55%, respectively (Zhou et al., 2014), while the total flavonoid contents in D. officinale leaves collected only ranged from 7.66 to 9.50 mg/g dried weight (Zhong et al., 2022). Therefore, understanding the digestive characteristics of polysaccharides is conducive to the in-depth study of their functions. As a natural macromolecular substance, plant polysaccharides are mainly fermented and utilized by intestinal flora.
SCFAs, as metabolites of flora, are important mediators for intestinal flora to affect host health (Ulven, 2012). The remission of T2D by polysaccharides may be through a variety of ways, one of which is to stimulate the secretion of GLP-1 by intestinal L cells. Therefore, the present study explored the structural changes in LDOP-A after oral administration and its effect on promoting SCFAs production and GLP-1 secretion, thus providing new evidence for LDOP-A as anti-T2DM medicated diet for patients.
In this preliminary work, we presented a comprehensive understanding of the structural change process of LDOP-A after oral administration and explored the GLP-1 secretion-promoting effect of digested polysaccharides, which will provide a theoretical basis for the clinical application of LDOP-A. It has been proved that after oral administration, LDOP-A is partially digested in the stomach and small intestine, the mannose fragments are absorbed and recycled into the blood, and the indigestible part can produce a large amount of acetic acid and butyric acid through intestinal fermentation.
Moreover, polysaccharides in the gut can promote the secretion of GLP-1 by NCI-H716 cells. However, the interaction between polysaccharides and NCI-H716 cells still needs further systematic study.
In the future, the digestion of polysaccharides will be explored with animal experiments in vivo. In addition, whether the degradation

F I G U R E 5 LDOP-A (a) promotes the secretion of GLP-1 by NCI-H716 cells; binding analysis of LDOP-A and NCI-H716 cells (b).
products (smaller fragments) of LDOP-A or the flora secondary metabolites change induced by LDOP-A plays a more important role in ameliorating T2DM is also our future research direction.

ACK N O WLE D G E M ENTS
The authors are highly thankful to Zhejiang Hospital of Integrated Traditional Chinese and Western Medicine for the financial support.
Authors are also thankful to the Department of Food Science and Technology, Zhejiang, China, for providing the laboratory facility in conducting a part of the experimental work of this study.

This work was supported by the Hangzhou Science and Technology
Bureau (20201203B175) and the Chinese Medicine Science and Technology Project of Zhejiang Province (2023ZL558).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data are contained within the article.

I N FO R M E D CO N S E NT
Informed consent was obtained from all subjects involved in the study.