Extraction, Purification, and Gastroprotective Activity of Glucomannan from Bletilla Striata

Research Background

The gastric secretion of protein hydrolytic enzymes and hydrochloric acid plays an important role in aiding digestion. In daily life, many risk factors such as stress, alcohol, and non-steroidal anti-inflammatory drugs (NSAIDs) can lead to gastric diseases. Currently, many synthetic drugs are widely used as first-line treatments for gastric diseases, including proton pump inhibitors, anticholinergics, histamine receptor antagonists, and antacids, but they can cause many adverse reactions. Therefore, there is a need to develop an efficient and safe gastroprotective drug to address this issue. Extensive research has shown that natural ingredients such as flavonoids, polysaccharides, and polyphenols can exhibit good gastroprotective activity. Among them, Bletilla striata polysaccharide (BSP) has low toxicity and high biological activity, making it a subject of extensive research in the field of phytochemistry. Studies have indicated that BSP can alleviate oxidative stress induced by H₂O₂ in porcine chondrocytes, which may be related to the antioxidant and anti-inflammatory activities of BSP. Additionally, BSP’s adhesive oral films can promote the healing of acetic acid-induced oral ulcers. However, information regarding the gastroprotective effects of BSP and its protective mechanisms is limited and requires further exploration. This study employed DEAE-52 cellulose and Sephadex G-150 column chromatography to separate and purify BSP, and established in vitro and in vivo ethanol injury models to evaluate its gastroprotective effects.

Research Content

Result 1: Extraction and Purification of Bletilla Striata Polysaccharide

The authors extracted 100g of pre-treated powder with 3000 mL of water at 80 °C for 4 hours, then precipitated with 4 times the volume of 95% ethanol at 4 °C for 24 hours. The precipitate was then re-dissolved in distilled water and underwent 10 cycles of freezing and thawing to remove proteins from the solution, followed by dialysis in distilled water for 72 hours to further remove small molecules. Finally, the non-dialyzable liquid was collected and freeze-dried to obtain crude polysaccharide.

Subsequently, the authors re-dissolved the crude polysaccharide in distilled water (10 mg/mL) and loaded it onto a DEAE-52 cellulose column (5.0×60 cm) for chromatography, eluting with a gradient of 0~0.4 M NaCl at a flow rate of 1 mL/min. The total sugar content was determined using the phenol-sulfuric acid method. Finally, the eluent was collected, dialyzed, freeze-dried, and further purified using Sephadex G-150 column (5.0×60 cm). Figure 1 shows a main elution peak (BSP) obtained from DEAE-52 cellulose anion exchange column (Figure 1A) and Sephadex G-150 column chromatography (Figure 1B).

Figure 1: Elution profile of crude polysaccharides from dried Bletilla striata pseudobulbs (A) DEAE-52 cellulose anion exchange chromatography and (B) Sephadex G-150 gel filtration chromatography.

Result 2: Preliminary Characterization of Bletilla Striata Polysaccharide

The authors then conducted some preliminary characterizations of Bletilla striata polysaccharide, including monosaccharide composition, molar mass, and chemical structure. Figure 2 (A-B) shows that BSP consists solely of mannose and glucose in a molar ratio of 7.45:2.55. Many studies have indicated that BSP is a glucomannan. However, their molar percentages vary significantly, which may be caused by various factors such as extraction processes, purification levels, and the source of Bletilla.

Based on Figure 2 (C), the average molecular weight was calculated to be approximately 1.7×10⁵ Da using log Mw= -1.1287t+13.579 (R2=0.9952), with a number-average molecular weight of approximately 1.11×10⁵ Da. Additionally, the polydispersity index (Mw/Mn) of BSP was 1.53. The spectral bands at 323 cm⁻¹ and 2890 cm⁻¹ are considered to be O-H stretching vibrations and C-H asymmetric stretching vibrations, respectively, while the C=O stretching vibration at 1644 cm^-1 and the peak at 1250 cm^-1 indicate that BSP may be acetylated. The spectral band at 890 cm^-1 indicates that Bletilla striata polysaccharide has β-glycosidic bonds, while the bands at 818 cm^-1 and 813 cm^-1 are due to mannose residues, which are consistent with the results shown in monosaccharide composition.

Figure 2 (A) Gas chromatography of BSP and (B) monosaccharide standards (C) Fourier-transform infrared spectroscopy and (D) high-performance liquid chromatography (HPLC) peaks: (1) Rha (2) Fur (3) Ara (4) Xyl (5) Man (6) GLC (7) Gal.

Result 3: In Vitro Gastroprotective Effects of Bletilla Striata Polysaccharide

3.1 Ethanol-Induced Cell Injury Model

The authors cultured GES-1 cells for 24 hours and then incubated the cells in ethanol (50-1600µM) for 3-24 hours, as shown in Figure 3 (A). The results in Figure 3 (B) indicate that cell viability significantly decreased with increasing concentration (100-1600 µM). Furthermore, as the treatment time with ethanol (3−6 h) was extended, cell viability was significantly inhibited. However, after prolonged treatment (12-24 h), cell viability recovered, likely due to the volatility of ethanol and the self-repairing ability of the cells. Notably, after 6 hours of treatment with 100 µM ethanol, cell survival rate was close to 50%. Therefore, this experiment adopted the GES-1 cell injury model induced by ethanol (100 µM, 6 h).

Figure 3 (A) Ethanol-induced GES-1 cell injury model (B) Survival rate of GES-1 cells under different ethanol concentrations and treatment times.

3.2 Study on Cytotoxicity and Gastroprotective Effects of BSP

The authors first measured the cytotoxicity of BSP at different concentrations (0.25-8 mg/mL) on GES-1 cells. Figure 4 (A) shows that BSP only negatively affected cell survival at the highest concentration (8 mg/mL). Therefore, a series of sample concentrations (0.25−4 mg/mL) were selected for further experiments. Based on this, the protective effect of BSP against ethanol-induced GES-1 cell injury was observed. Figure 4 (B) shows that the survival rate of cells in the model group was significantly lower than that of the control group, at 53.8%. However, pre-treatment with BSP significantly increased cell survival rate in a concentration-dependent manner. At a concentration of 4 mg/mL, the cell survival rate significantly increased to 83.8%.

Subsequently, the authors used calmodulin-AM (green fluorescence) staining for live cells and PI (red fluorescence) staining for dead cells to visually observe and analyze the above results. The results in Figure 4 (C) show that cells in the model group exhibited stronger red fluorescence compared to those in the control group, confirming that GES-1 cells underwent ethanol-induced injury. Compared to the model group, the red fluorescence weakened with increasing BSP concentration (0.25−4 mg/mL), indicating significantly enhanced cell protective ability. The authors demonstrated the gastroprotective effect of BSP through in vitro models, while the in vivo gastroprotective effect of BSP in mice requires further investigation.

Figure 4 (A) Cytotoxicity (B) Protective effect of BSP on GES-1 cells (C) Live/dead staining of cells after different treatments.

Result 4: Experimental Study on In Vivo Gastroprotective Effects

4.1 Macroscopic Inhibition of Gastric Ulcers

The authors first measured the cytotoxicity of BSP at different concentrations (0.25-8 mg/mL) on GES-1 cells. Excessive alcohol gavage can induce acute gastric injury, characterized by mucosal edema, bleeding, and epithelial cell death. The macroscopic observations of the stomachs of rats in different groups are shown in Figure 5. The stomach mucosa of NC group rats was smooth and without bleeding, while the MC group rats exhibited severe bleeding and edema in the gastric mucosa. The BSP treatment group showed a significant reduction in gastric ulcers, especially in the high dose group (100 mg/kg).

Figure 5: Representative images of gastric tissues from different groups.

4.2 In Vivo Antioxidant Activity

Many literatures indicate that excessive ethanol increases the levels of reactive oxygen species, which attack all biomacromolecules (DNA, proteins, and lipids), ultimately leading to cellular and tissue damage. Compared to the NC group, the antioxidant enzyme activities (SOD, CAT, and GSH-Px) were reduced in the treatment group, but significantly increased compared to the model group, demonstrating a reversal of activity.

Figure 6: Antioxidant enzyme activities in gastric tissues from different groups: (A) SOD (B) CAT (C) GSH-Px.

Figure 7 shows that an increase in MDA and LPO was observed in the MC group, indicating oxidative stress induced by ethanol. However, after pre-treatment with BSP, the aforementioned phenomena were reversed in a dose-dependent manner. The levels of MDA and LPO were significantly lower than those of the MC group by 38.0% and 19.8%, respectively. Notably, in the high-dose BSP group, the antioxidant enzyme activities were enhanced, and the lipid products were not lower than those produced by the positive drug (omeprazole). These findings suggest that BSP can enhance antioxidant enzyme activities, eliminate free radicals, and inhibit lipid peroxidation.

Figure 7: Oxidative products in gastric tissues from different groups (A) MDA and (B) LPO.

4.3 Histopathological Results

The H&E staining results in Figure 8 (A) show that the gastric mucosa structure of the NC group was intact, with no abnormal epithelial necrosis. The gastric mucosa of the MC group rats was severely damaged, with destruction of surface epithelial cells and disordered gastric mucosa structure, along with infiltration of inflammatory cells. However, pre-treatment with BSP or omeprazole alleviated gastric damage, reduced inflammatory cell infiltration, and protected the gastric mucosa structure. These results indicate that BSP possesses activity similar to that of omeprazole. Figure 8 (B) shows a large number of apoptotic cells in the MC group. In contrast, the green fluorescence in BSP-L, BSP-M, BSP-H, and PC groups dimmed, indicating reduced apoptosis of gastric tissue cells. Correspondingly, the apoptosis index was also calculated based on the number of apoptotic cells in the microscopic sections (Figure 8C). The apoptosis index was 2.7±3.0%, 36.4±2.1%, 32.3±2.4%, 28.9±2.6%, 20.4±3.2%, and 16.7±3.9% for NC group, MC group, BSP-L group, BSP-M group, BSP-H group, and PC group, respectively. Interestingly, no significant difference was observed between the high-dose BSP group and the PC group. This suggests that Bletilla striata polysaccharide may alleviate alcoholic gastric ulcers by reducing cell apoptosis.

Figure 8: H&E (A), TUNEL (B) staining images and apoptosis index (C) of gastric tissues from different groups.

Summary and Reflection