Gelsemium elegans homogenous polysaccharide GEP, preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and discloses a uniform GEP polysaccharide of Gelsemium elegans Benth, wherein the uniform GEP polysaccharide of Gelsemium elegans Benth is mainly composed of rhamnose, arabinose, galactose, glucose and galacturonic acid in a molar ratio of 18.0:19.8:23.0:13.5:25.7, and the weight average molecular weight of the uniform GEP polysaccharide of Gelsemium elegans Benth is 2.109 KDa. The obtained uniform GEP polysaccharide of Gelsemium elegans Benth has fixed components and high yield, has an anticancer effect, is safe, and provides a basis for developing a potential new drug for colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a homogeneous polysaccharide GEP from Gelsemium elegans, its preparation method, and its application. Background Technology

[0002] Gelsemium elegans (Gardn. & Champ.) Benth., commonly known as Gelsemium elegans, is a plant belonging to the Loganiaceae family and the Gelsemium genus. It was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and listed as a lower-grade herb. It has a pungent and bitter taste, is warm in nature, and is highly toxic (though less toxic to cattle, sheep, and pigs). It enters the heart, lung, and large intestine meridians. It possesses stomach-strengthening, insecticidal, anti-inflammatory, and analgesic effects. The main components of Gelsemium elegans are indole alkaloids, comprising approximately 100 compounds, which are its main active ingredients and can significantly inhibit central nervous system activity. Modern pharmacological studies have shown that Gelsemium alkaloids have various pharmacological activities, including anti-tumor effects through cell cycle regulation; enhancing macrophage phagocytic capacity, protecting leukocytes, and improving immune regulation; treating cancer pain and chronic pain; weakening myocardial contractility and vasodilation to lower blood pressure; and also playing a role in the treatment of anxiety and skin diseases.

[0003] Colorectal cancer (CRC) is one of the most common and deadliest malignant tumors worldwide. In my country, its incidence and mortality rates are second only to stomach cancer, esophageal cancer, and lung cancer. Treatment for CRC includes surgical resection, radiotherapy, and neoadjuvant and palliative chemotherapy. However, these treatment regimens have limited impact on cure rates and long-term survival. Increasing research has found that polysaccharides from traditional Chinese medicine have great potential in cancer treatment due to their immunomodulatory effects. Astragalus polysaccharides, Ganoderma lucidum polysaccharides, Lentinan, and ginseng polysaccharides, among others, have shown good clinical efficacy in cancer treatment, increasing cure rates and reducing adverse reactions.

[0004] Currently, there is limited research on the preparation and pharmacological activity of Gelsemium elegans polysaccharides, and the composition of polysaccharides obtained by different extraction methods varies significantly. Existing preparation processes for Gelsemium elegans polysaccharides have low extraction rates, which restricts their application in practical production. Therefore, developing a method for preparing Gelsemium elegans polysaccharides with a fixed composition and high yield would greatly promote the industrialization of Gelsemium elegans and expand its application scope. Summary of the Invention

[0005] This invention addresses the technical problem of limited applications of Gelsemium elegans polysaccharides by providing a homogeneous Gelsemium elegans polysaccharide (GEP), its preparation method, and its applications. The prepared homogeneous Gelsemium elegans polysaccharide has a fixed composition and a high yield, exhibits anticancer activity, and has good safety, providing a basis for the development of potential new drugs for colorectal cancer.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a homogeneous polysaccharide GEP from Gelsemium elegans, which is mainly composed of rhamnose, arabinose, galactose, glucose, and galacturonic acid in a molar ratio of 18.0:19.8:23.0:13.5:25.7. Its weight-average molecular weight is 2.109 kDa, and its structural unit has the following structural formula:

[0008]

[0009] This invention also provides a method for preparing the above-mentioned homogeneous polysaccharide GEP from Gelsemium elegans, comprising the following steps:

[0010] 1) Boil the chopped Gelsemium elegans in water, cool, filter, and obtain Gelsemium elegans polysaccharide extract;

[0011] 2) The extract of Gelsemium elegans polysaccharide was subjected to alcohol precipitation to obtain crude alcohol-precipitated polysaccharide;

[0012] 3) The crude polysaccharide was precipitated with alcohol and the protein was removed using the Sevag method to obtain total polysaccharide from Gelsemium elegans;

[0013] 4) The total polysaccharides of Gelsemium elegans were separated and purified by ion exchange column chromatography and gel column chromatography, then concentrated and freeze-dried to obtain homogeneous polysaccharide GEP.

[0014] In one technical solution, the boiling time in step 1) is 1 to 2 hours.

[0015] In one technical solution, the alcohol precipitation in step 2) uses ethanol with a concentration of 80-95%, the precipitation temperature is 4-25℃, and the time is 2-12h.

[0016] In one technical solution, when removing protein using the Sevag method in step 3), a mixture of chloroform and n-butanol is used for extraction; the volume ratio of chloroform to n-butanol is 3-5:1.

[0017] In one technical solution, the ion exchange chromatography column in step 4) is a DEAE-agarose gel FF, the flow rate of the ion exchange column chromatography is 50 mL / h, and the gradient elution program is: 0–20 min water, 20–40 min 0.2 mol / L NaCl, 40–60 min 0.5 mol / L NaCl, 60–80 min 2 mol / L NaCl; the packing material of the gel column is Superdex-200.

[0018] The present invention also provides the application of the above-mentioned Gelsemium homogeneous polysaccharide GEP in the preparation of cancer treatment drugs.

[0019] In one technical solution, the present invention relates to the application of Gelsemium elegans homogeneous polysaccharide GEP in the preparation of a drug for treating colorectal cancer.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The Gelsemium elegans homogeneous polysaccharide GEP of this invention is a novel homogeneous polysaccharide compound discovered for the first time. Its composition is fixed and its yield is high, providing a basis for the quality control and standardized production of Gelsemium elegans homogeneous polysaccharide.

[0022] The homogeneous polysaccharide GEP prepared by this invention has anticancer activity and no obvious toxicity at a concentration of 400 μg / ml, providing a basis for the development of potential new drugs for colorectal cancer. Attached Figure Description

[0023] Figure 1 The hydrogen spectrum of GEP, a homogeneous polysaccharide from Gelsemium elegans;

[0024] Figure 2 The carbon spectrum of GEP, a homogeneous polysaccharide from Gelsemium elegans;

[0025] Figure 3 HH-COSY map of GEP (a homogeneous polysaccharide from Gelsemium elegans);

[0026] Figure 4 HSQC map of GEP, a homogeneous polysaccharide from Gelsemium elegans;

[0027] Figure 5 HMBC map of GEP (a homogeneous polysaccharide from Gelsemium elegans);

[0028] Figure 6 The NOESY map of GEP, a homogeneous polysaccharide from Gelsemium elegans.

[0029] Figure 7 This describes the toxic effects of Gelsemium elegans polysaccharide in normal colon cells.

[0030] Figure 8 This study investigated the inhibitory effect of Gelsemium elegans polysaccharide on different colorectal cancer cell lines.

[0031] Figure 9 Gelsemium polysaccharide inhibits the formation of colorectal cancer cell clones.

[0032] Figure 10 This study aimed to enhance the ability of Gelsemium elegans polysaccharide to inhibit the metastasis and invasion of colorectal cancer cells. Detailed Implementation

[0033] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the test methods in the following embodiments are conventional methods.

[0034] Example 1: Extraction of homogeneous polysaccharide GEP from Gelsemium elegans

[0035] (1) The specific method for extracting total polysaccharides from Gelsemium elegans is as follows:

[0036] The dried whole plant of *Gelsemium elegans* from Zhejiang Province was collected. 5 kg of *Gelsemium elegans* was chopped, added to 10 times its volume of distilled water, and decocted at 100℃ for 1 hour. After cooling, the mixture was filtered, and the filtrate was collected to obtain a *Gelsemium elegans* polysaccharide extract. The *Gelsemium elegans* polysaccharide extract was concentrated using a rotary thin-film evaporator to obtain a fluid extract. The fluid extract was dispersed and dissolved in 1 L of distilled water, and 90% ethanol was added. After standing for 24 hours, the mixture was centrifuged to obtain a precipitate. The ethanol was evaporated to obtain crude polysaccharide precipitated by alcohol precipitation.

[0037] The crude polysaccharide was precipitated with alcohol, and then extracted with 1 / 5 volume of chloroform-n-butanol (4:1 volume ratio) mixture according to the Sevag method. The mixture was shaken on a shaker for 20 min, transferred to a separatory funnel, allowed to stand, and the supernatant was collected and centrifuged for 1 min to remove residual protein precipitate. This process was repeated 3–5 times to obtain a crude polysaccharide solution of *Gelsemium elegans* after protein removal. The solution was concentrated under reduced pressure and dried to obtain 0.82 kg of total polysaccharide from *Gelsemium elegans*.

[0038] (2) Isolation and purification of homogeneous polysaccharides: A DEAE-Sepharose FF column was used, connected to a fraction collector and a peristaltic pump. The total polysaccharides of *Gelsemium elegans* were eluted sequentially with distilled water, 0.2, 0.4, and 2 mol / L NaCl at a flow rate of 50 mL / h. The eluent was collected in 10 mL test tubes and labeled. The absorbance was measured at 490 nm using the phenol-sulfuric acid method, and a scatter plot was plotted. The eluent (0–20 Tubes of 0–0.2 mol / L NaCl) was collected, concentrated, dialyzed through a 3500 Da dialysis bag, and freeze-dried to obtain the eluted fraction Fr.A, weighing 182 g.

[0039] Take 100 mg of Fr.A polysaccharide fraction, dissolve it in 3 mL of distilled water, centrifuge (12000 rpm) for 10 min, and further separate and purify the supernatant by passing it through a dextran gel Superdex-200 column. Combine the solutions and concentrate them by rotary evaporation and freeze-dry to obtain the fraction named GEP, with a mass of 35 mg.

[0040] Example 2: Structural Identification of Gelsemium Homogeneous Polysaccharide GEP

[0041] (1) The molecular weight of polysaccharides was determined by HPGPC. The optimized conditions were: Shimadzu LC-10A high-performance liquid chromatograph, RI-20A differential detector, BRT105-103-101 tandem gel chromatography column (8×300mm); mobile phase: 0.2mol / L NaCl solution, flow rate: 0.8mL / min, column temperature: 40℃; injection volume: 25μL. Regression curves were established using dextran standards of different molecular weights, and the molecular weight was calculated. Dextran standards (molecular weights: 1152, 5000, 11600, 23800, 48600, 80900, 148000, 273000) were prepared into 5 mg / mL solutions with the mobile phase immediately before use. A standard curve was plotted with lg(molecular weight) on the x-axis and retention time (RT) on the y-axis. The equation for the lgMp-RT calibration curve was: Y = -0.207X + 11.06 (R² = 0.999); the equation for the lgMw-RT calibration curve was: Y = -0.204X + 10.99 (R² = 0.999). 2 =0.999); The equation of the lgMn-RT correction curve is: Y = -0.205X + 11.01(R) 2 =0.999); GEP was prepared using the same method, and its molecular weight was calculated using the regression equation of the standard curve. The weight-average molecular weight of GEP was found to be approximately 2.109 kDa.

[0042] (2) Analysis of GEP monosaccharide composition by ion chromatography:

[0043] a. A mixture of monosaccharide standards including fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid (GalA), glucuronic acid (GlcA), galactosyl hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid (GulA), and mannuronic acid (ManA) were used as controls.

[0044] b. Accurately weigh 5 mg of sample into an ampoule, add 2 mL of 3M trifluoroacetic acid (TFA), and hydrolyze at 120℃ for 3 h. Accurately pipette the acid hydrolysis solution into a tube and blow it dry under nitrogen. Add 5 mL of water and vortex to mix. Pipe 200 μL of the solution into 800 μL of deionized water and centrifuge at 12000 rpm for 5 min. Take the supernatant and analyze it using an ion chromatograph (ICS5000). Chromatographic conditions: Dionex Carbopac™ PA20 column (3×150 mm), column temperature 30℃, injection volume 25 μL, flow rate: 0.3 mL / min, mobile phase: A: H2O; B: 15 mM NaOH; C: 15 mM NaOH & 100 mM NaOAC, detector: electrochemical detector.

[0045] The results showed that the homogeneous polysaccharide GEP of Gelsemium elegans was mainly composed of rhamnose, arabinose, galactose, glucose, and galacturonic acid in a molar ratio of 18.0:19.8:23.0:13.5:25.7.

[0046] (3) Gas chromatography-mass spectrometry (GC-MS) analysis of samples after methylation, hydrolysis, and acetylation, as well as infrared spectroscopy and 1H NMR spectroscopy. Figure 1 ), 13C NMR spectrum ( Figure 2 Using DEPT135 one-dimensional and two-dimensional spectra, the glycosidic bond signals of the polysaccharide were assigned, and the main glycosidic bond structure of the polysaccharide was determined to be as follows: the main chain connection mode is α-L-Rha-(1→4)-α-D-GalAp-(1→2)- ...A raf-(1→5)-α-L-Araf-(1→), β-D-Galp-(1→3)-β-D-Galp-(1→3)-β-D-Galp-(1→), β-D-Glcp-(1→3)-β-D-Glcp-(1→4-β-D-Glcp-(1→) and α-D-GalAp-(1→) are connected to the main chain via O-4 and O-3 bonds of →2,4)-α-L-Rha-(1→ and →3,4)-α-D-GalAp-(1→), respectively.

[0047] The analysis results are shown in Table 1-2.

[0048] Table 1. Analysis of GEP methylated sugar alcohol acetyl ester (PMAA) results

[0049]

[0050]

[0051] Table 2. Hydrogen and Carbon Signal Assignments for GEP

[0052]

[0053]

[0054]

[0055] The present invention also performed a series of structural characterizations, including: methylation assays to analyze its sugar residues and NMR spectra analysis to determine the glycosidic bond linkage mode. Figure 3 HH-COSY map of GEP, a homogeneous polysaccharide from Gelsemium elegans. Figure 4The HSQC map of GEP, a homogeneous polysaccharide from Gelsemium elegans. Figure 5 The HMBC map of GEP, a homogeneous polysaccharide from Gelsemium elegans. Figure 6 The NOESY spectrum of Gelsemium elegans homogeneous polysaccharide GEP is shown. The structural formulas of the GEP homogeneous polysaccharide structural units were obtained through various analytical methods, and are as follows:

[0056]

[0057] Example 3: Inhibitory effect of Gelsemium elegans homogeneous polysaccharide on colorectal cancer cells.

[0058] 3 materials

[0059] 3.1 Colon cancer cell line

[0060] The colon cancer cell lines used in this invention are all derived from ATCC (including normal human colon fibroblasts CCD-18Co, human colon cancer cells HCT15, and HCT116).

[0061] 3.2 Reagents

[0062] PRIM-1640 medium (Procell), McCoy's 5A medium (BI), FBS fetal bovine serum (BI), penicillin-dipgs antibody (Solepro), CCK-8 (Solepro), PBS, and Gelsemium elegans homogenate were dissolved and diluted in complete cell culture medium and prepared fresh for use.

[0063] 3.3 Consumables and Instruments

[0064] 3.3.1 Haier Medical Low-Temperature Storage Box (Qingdao Haier Special Electric Appliances Co., Ltd.). Analytical Balance (Restaurant Scientific Instruments Co., Ltd.), Thermo Clean Bench, Water-jacketed CO2 Cell Culture Incubator (Thermo), Microplate Reader.

[0065] 3.3.2 Pipettes, pipettes (5ml and 10ml respectively), 6-well plates, 96-well plates, and Transwell 24-well plates.

[0066] 3.4 Experimental Methods

[0067] 3.4.1 Cell proliferation experiment study:

[0068] Colon cancer cells HCT15 and HCT116 were seeded into 96-well plates with 1000 cells per well and cultured in a 37°C, 5% CO2 incubator. After 24 hours of cell adhesion, the cells were treated with Gelsemium elegans homogenate. Then, at 0h, 24h, 48h and 72h, 10 μL of CCK-8 reagent was added and the plates were incubated in a 37°C, 5% CO2 incubator for 1h. Cell proliferation was then detected using a microplate reader at a wavelength of 450 nm.

[0069] 3.4.2 Non-anchoring experimental study:

[0070] Non-anchored growth is one of the important characteristics of tumor cell growth. In this invention, the inhibitory effect of Gelsemium elegans homogeneous polysaccharide on the growth of colorectal cancer cells was observed at different concentrations (0 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL) (the lower gel was prepared by adding the corresponding concentration of Gelsemium elegans homogeneous polysaccharide to 0.6% Agar, and the upper gel was prepared by adding the corresponding concentration of Gelsemium elegans homogeneous polysaccharide to 0.3% Agar). The images were taken after two weeks, and then the inhibition efficiency was calculated.

[0071] 3.4.3 Experimental studies on cell transfer and invasion:

[0072] Cancer cell invasion and metastasis are major causes of cancer treatment failure. This invention investigates the inhibitory effect of Gelsemium elegans homogeneous polysaccharide on the growth, metastasis, and invasion of colorectal cancer cells at different concentrations (0 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL). The lower compartment is filled with 600 μl of culture medium containing 10% fetal bovine serum. 8 × 10⁸ g of the medium is added to the upper compartment. 4 Cells were cultured in 100 μl of medium containing 1% FBS. After 12 h, 100 μl of homogenized Gelsemium elegans polysaccharide of different concentrations was added to the upper chamber. After 24 h of culture, cells were fixed with methanol for 15 min, and the unmigrated cells in the upper layer were wiped off with a cotton swab. Invading cells were stained with 0.1% crystal violet. In the invasion assay, Matrigel (BD Company) was pre-coated in the chamber, and 8 × 10⁸ g / L gel was added to the upper part of the chamber. 4 Cells. Other steps were performed in the same manner as the migration assay. Five fields of view were randomly selected from each group under a microscope to count the stained cells.

[0073] 3.5 Experimental Results

[0074] Toxicity screening of homogeneous polysaccharide from *Gelsemium elegans* against the normal colon cell line CCD-18Co showed that homogeneous polysaccharide at 500 μg / ml had no significant toxicity to normal colon cells. Figure 7 ).

[0075] The growth inhibitory effect of Gelsemium elegans homogeneous polysaccharide on colon cancer cell lines HCT15 and HCT116 (cell growth was measured after treatment for 0, 24, 48, and 72 hours, in three independent experiments) is shown in the figure. Figure 8 The results showed that as the concentration of homogeneous polysaccharide from Gelsemium elegans increased, the viability of both colon cancer cells gradually decreased, and the decrease in viability became more pronounced over time.

[0076] In the non-anchoring assay, the inhibitory effect of Gelsemium elegans homogeneous polysaccharide on the clonal formation and size of colon cancer cell lines HCT15 and HCT116 was investigated. The results are shown in [Figure number missing]. Figure 9 The results showed that as the concentration of Gelsemium elegans homogeneous polysaccharide increased, the number of clones of both colon cancer cells decreased significantly, indicating that high concentrations of Gelsemium elegans homogeneous polysaccharide were more effective in inhibiting the growth of colon cancer cells.

[0077] The inhibitory effect of Gelsemium elegans homogeneous polysaccharide on the growth, metastasis, and invasion of colorectal cancer cells was investigated. Results are shown in […]. Figure 10 The results showed that high concentrations of Gelsemium elegans homogeneous polysaccharide were more effective in inhibiting the growth, metastasis, and invasion of colon cancer cells.

[0078] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. A homogeneous polysaccharide GEP from Gelsemium elegans, characterized in that, The homogeneous polysaccharide of *Gelsemium elegans* is mainly composed of rhamnose, arabinose, galactose, glucose, and galacturonic acid in a molar ratio of 18.0:19.8:23.0:13.5:25.7, with a weight-average molecular weight of 2.109 kDa. Its structural unit has the following structural formula: 。 2. The method for preparing the homogeneous polysaccharide GEP from Gelsemium elegans according to claim 1, characterized in that, Includes the following steps: 1) Boil the chopped Gelsemium elegans in water, cool, filter, and obtain Gelsemium elegans polysaccharide extract; 2) The extract of Gelsemium elegans polysaccharide was subjected to alcohol precipitation to obtain crude alcohol-precipitated polysaccharide; 3) The crude polysaccharide was precipitated with alcohol and the protein was removed using the Sevag method to obtain total polysaccharide from Gelsemium elegans; 4) The total polysaccharides of *Gelsemium elegans* were sequentially separated and purified by ion exchange chromatography and gel column chromatography, then concentrated and freeze-dried to obtain homogeneous polysaccharide GEP. The ion exchange chromatography column was a DEAE-agarose gel FF, the flow rate of the ion exchange column chromatography was 50 mL / h, and the gradient elution program was: 0–20 min water, 20–40 min 0.2 mol / L NaCl, 40–60 min 0.4 mol / L NaCl, 60–80 min 2 mol / L NaCl. The gel column packing material was Superdex-200.

3. The method for preparing GEP (Gelsemium elegans homogeneous polysaccharide) according to claim 2, characterized in that, The boiling time mentioned in step 1) is 1-2 hours.

4. The method for preparing GEP (Gelsemium elegans homogeneous polysaccharide) according to claim 2, characterized in that, The alcohol precipitation in step 2) uses ethanol with a concentration of 80-95%, the precipitation temperature is 4-25℃, and the time is 2-12h.

5. The method for preparing GEP (Gelsemium elegans homogeneous polysaccharide) according to claim 2, characterized in that, In step 3), when removing protein using the Sevag method, a mixture of chloroform and n-butanol is used for extraction; the volume ratio of chloroform to n-butanol is 3~5:

1.

6. The use of the Gelsemium elegans homogeneous polysaccharide GEP according to claim 1 in the preparation of a drug for treating colorectal cancer.

Images