Polysaccharide component for inhibiting activity of alpha-glucosidase as well as preparation method and application of polysaccharide component

Abstract

The invention discloses a polysaccharide component for inhibiting activity of alpha-glucosidase in the technical field of traditional Chinese medicine, the polysaccharide component is HCPN or HCPA1, the HCPN is mainly heteropolysaccharide composed of four monosaccharides, and the monosaccharides comprise Man, Glc, Gal and Ara; the HCPA1 is heteropolysaccharide mainly composed of seven monosaccharides, and the monosaccharides of the HCPA1 comprise Man, GlcA, Rha, GalA, Glc, Gal and Ara. The alpha-glucosidase inhibition ratio of HCPN and HCPA1 is measured, and the result shows that the alpha-glucosidase inhibition ratio of HCPN and HCPA1 is higher than that of a positive drug acarbose.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a polysaccharide component for inhibiting the activity of α-glucosidase, a preparation method thereof, and an application thereof. Background Art

[0002] According to a report of the World Health Organization, more than 422 million people suffered from diabetes in 2014 [1]. As a common metabolic disease, diabetes is characterized by abnormal elevation of blood glucose levels due to lack of or ineffective use of insulin in the body. The World Health Organization mainly classifies diabetes into type I diabetes, type II diabetes, gestational diabetes and special type diabetes [2]. As a common metabolic disease, diabetes is characterized by abnormal elevation of blood glucose levels due to lack of or ineffective use of insulin in the body. Failure to manage the diabetes condition may lead to complications such as kidney diseases, nervous system diseases, leg amputations, heart diseases and even blindness.

[0003] Currently, the treatment of diabetes mainly uses insulin, α-glucosidase inhibitors, insulin sensitizers, meglitinide insulin secretagogues, metformin, GLP-1 and DPP-4 inhibitors, etc. Among them, α-glucosidase inhibitors can not only treat type I diabetes and type II diabetes, but also are one of the few oral hypoglycemic drugs that can be used to intervene in impaired glucose tolerance.

[0004] α-Glucosidase is a class of hydrolases present in the brush border of the small intestinal mucosa, which plays an important role in the cleavage of glycosidic bonds. α-Glucosidase inhibitors can delay the digestion of carbohydrates and prevent postprandial hyperglycemia by inhibiting such enzymes. Currently, the commercially available α-glucosidase inhibitors mainly include acarbose, voglibose, etc. Once these drugs are on the market, they are widely used in the treatment of diabetes due to their good therapeutic effects and low prices. However, they also have certain side effects such as gastrointestinal dysfunction, jaundice and elevated transaminases, which have an adverse impact on the life safety and quality of life of patients. Therefore, diabetic patients need a new type of α-glucosidase inhibitor that is safe, effective and inexpensive.

[0005] Houttuynia cordata Thunb, a herbaceous plant belonging to the Saururaceae family and the Houttuynia genus, is considered a medicinal and edible plant with great development potential by both the National Health Commission of the People's Republic of China and the Japanese Pharmacopoeia Guidelines. It is recorded in "Supplementary Records of Famous Physicians" that Houttuynia cordata Thunb is pungent in taste and slightly cold in nature, with the effects of clearing heat and detoxifying, eliminating carbuncles and discharging pus, and inducing diuresis for treating stranguria. Modern research shows that the extracts of Houttuynia cordata Thunb are effective against various diseases, including cancer, diabetes, obesity, pulmonary fibrosis, skin diseases, and severe acute respiratory syndrome, and also have antibacterial and antiviral effects against various pathogens. In recent years, polysaccharides extracted from natural resources have attracted much attention due to their low toxicity, low antigenicity, and various biological activities. Studies have found that polysaccharides have biological activities such as antiviral, anticancer, antioxidant, immunomodulatory, and hypoglycemic effects. Polysaccharides are also one of the important active components in Houttuynia cordata Thunb. In 2014, it was reported that the polysaccharides of Houttuynia cordata Thunb can increase the secretion of IL-1β, TNF-α, and MIP-1α, thereby regulating immune function. In 2018, it was reported that the polysaccharides of Houttuynia cordata Thunb may inhibit the growth of A549 lung cancer cells by inducing cell cycle arrest and promoting apoptosis through the caspase-3 and cyclin B1 signaling pathways. However, the current research on the polysaccharides of Houttuynia cordata Thunb mainly focuses on aspects such as anti-tumor, anti-inflammatory, and antiviral, and there are relatively few studies on the treatment of diabetes with the polysaccharides of Houttuynia cordata Thunb. Summary of the Invention

[0006] The present invention aims to provide a polysaccharide component for inhibiting α-glucosidase activity and a preparation method thereof.

[0007] The polysaccharide component for inhibiting α-glucosidase activity in this solution is HCPN or HCPA1;

[0008] The HCPN is mainly a heteropolysaccharide composed of four monosaccharides, and its monosaccharide composition includes Man, Glc, Gal, and Ara;

[0009] The HCPA1 is mainly a heteropolysaccharide composed of seven monosaccharides, and its monosaccharide composition includes Man, GlcA, Rha, GalA, Glc, Gal, and Ara;

[0010] The polysaccharide component for inhibiting α-glucosidase activity described in the present invention is mainly obtained by subjecting Houttuynia cordata Thunb to water extraction and alcohol precipitation to obtain crude polysaccharides of Houttuynia cordata Thunb (HCP), and separating the HCPN and HCPA1 homogeneous charge fractions from the crude polysaccharides of Houttuynia cordata Thunb (HCP) using anion exchange column chromatography.

[0011] The main peak molecular weights of the homogeneous charge fractions HCPN, HCPA1, and HCPA2 were determined. Through a mobile phase of 0.2 M NaCl, a flow rate of 0.5 mL / min, an analytical column: TSK3000 PWXL, and a differential detector, it was finally obtained that there were two main peaks in HCPN, with molecular weights of 34.4 KDa and 15.1 KDa respectively, and the main peak molecular weight of HCPA1 was 66.07 KDa.

[0012] The present invention determined the inhibitory rates of HCPN and HCPA1 on α-glucosidase. Among them, the neutral sugar fraction HCPN had the best inhibitory rate on α-glucosidase, and under the experimental conditions, when the concentration > 5 mg / mL, its inhibitory rate on α-glucosidase was greater than that of the positive drug acarbose.

[0013] The present invention used the MTT method to determine the activities of the crude polysaccharide from Houttuynia cordata root (HCP) on cell lines such as HepG2, A-549, Tca-8113, and HCT-116. The results showed that within the experimental concentration range, the crude polysaccharide from Houttuynia cordata root (HCP) had no obvious activity on the four cell lines, indicating that the crude polysaccharide from Houttuynia cordata root (HCP) has no cytotoxicity and high safety, and can be used to prepare drugs for treating diabetes.

[0014] Furthermore, the molar ratio of the monosaccharide composition of HCPN is Man:Glc:Gal:Ara = 3.16:29.96:22.7:1.

[0015] Furthermore, the molar ratio of the monosaccharide composition of HCPA1 is Man:GlcA:Rha:GalA:Glc:Gal:Ara = 16.83:1:14.31:1.37:65.27:192.7:46.23.

[0016] Furthermore, a preparation method for the polysaccharide component that inhibits α-glucosidase activity includes the following steps:

[0017] Step 1: Crush the dried product of Houttuynia cordata root, add distilled water and soak overnight, decoct and separate multiple times, then combine and concentrate the separated filtrate, add ethanol to make the final ethanol concentration reach 70% - 80%, precipitate, centrifuge, redissolve the precipitate with distilled water, volatilize the residual ethanol, and then freeze-dry to obtain the crude polysaccharide from Houttuynia cordata root;

[0018] Step 2: Subject the obtained crude polysaccharide from Houttuynia cordata root to DEAE-Agarose anion exchange column chromatography, elute with distilled water and 0.1 M NaCl solution as the mobile phase, collect the mobile phase, and combine the same fractions by the phenol-sulfuric acid method and determination with an enzyme-linked immunosorbent assay. The obtained homogeneous charge fractions of Houttuynia cordata root are HCPN and HCPA1 respectively. Description of the Drawings

[0019] Figures 1 to 2This is the liquid chromatography diagram of the monosaccharide composition of the first fraction of the crude polysaccharide from Houttuynia cordata roots in the present invention;

[0020] Figure 3 This is the chromatogram of the molecular weight distribution of the homogeneous charge fraction of the crude polysaccharide from Houttuynia cordata roots in the present invention;

[0021] Figure 4 In the present invention, the MTT method was used to determine the activities of the crude polysaccharide (HCP) from Houttuynia cordata roots against HepG2, A–549, Tca–8113, and HCT–116 cell lines;

[0022] Figure 5 This is the experiment for determining the inhibitory activity of the homogeneous charge fraction of the crude polysaccharide from Houttuynia cordata roots against α–glucosidase in the present invention;

[0023] Figure 6 This is the experiment for determining the inhibitory activity of starch, sucrose, and lactose against α–glucosidase in the present invention. Specific embodiments

[0024] The following is a further detailed description through specific embodiments:

[0025] Preparation of Houttuynia cordata polysaccharides HCP, HCPN, HCPA1, and HCPA2

[0026] Take 1 Kg of Houttuynia cordata roots, crush them, add 25 L of distilled water, and soak overnight. After soaking, bring to a boil over high heat and then turn to low heat and continue boiling for 2.5 h. After boiling, separate the filtrate and the residue. The residue is boiled with 25 L of distilled water for 2.5 h again. Repeat this operation three times. Combine the filtrates and concentrate the combined filtrates of the three times to 5 L. Add 26.66 L of 95% ethanol, precipitate overnight, and centrifuge. The precipitate obtained by centrifugation is redissolved in distilled water, the residual ethanol is removed by evaporation, and then freeze-dried to obtain the crude polysaccharide (HCP) of Houttuynia cordata.

[0027] Weigh 2 g of crude Houttuynia cordata polysaccharide, dissolve it in 20 ml of distilled water, take the supernatant after centrifugation, and fractionate it using DEAE-Agross chromatography (518 mL). Elute it with distilled water, 0.1 mol / L, and 0.23 mol / L NaCl solutions at a flow rate of 1 mL / min. Collect each fraction with an automatic collector, take 100 μL from each tube, add 200 μL of 5% phenol and 1 mL of concentrated sulfuric acid, mix well, react at room temperature for 15 min, finally take 100 μL from each tube, add it to a 96-well plate, measure its absorbance value at a wavelength of OD490 nm with an enzyme-linked immunosorbent assay (ELISA) reader, draw an elution curve, and combine the same fractions. Finally, obtain homogeneous charge fractions HCPN (eluted with distilled water), HCPA1 (eluted with 0.1 M NaCl), and HCPA2 (eluted with 0.23 M NaCl). Concentrate the combined solution of the obtained homogeneous charge fractions with a rotary evaporator, dialyze it with a dialysis bag with a molecular weight cut-off of 500, and then lyophilize it to obtain HCPA1 and HCPA2. The yields of HCP and the yields of HCPN, HCPA1, and HCPA2 are as follows:

[0028] Name HCP HCPN HCPA1 HCPA2 Yield 11.7％ 47.95％ 6.34％ 11.11％

[0029] Determine the monosaccharide composition of HCPN, HCPA1, and HCPA2

[0030] Complete acid hydrolysis: Weigh 10 mg of polysaccharide samples (HCP, HCPA1N, HCPA1, HCPA2) into an ampoule, add 5 ml of 2 M trifluoroacetic acid, seal it with an alcohol burner, and react in an incubator at 110 °C for 3 h. After the reaction is completed, neutralize it to neutral with NaOH, and finally concentrate the volume to 1 ml with a rotary evaporator. PMP derivatization: Take 100 μL of the above completely acid-hydrolyzed solution, add 100 μL of 0.6 M NaOH solution, mix well, add 200 μL of 0.5 M PMP methanol solution (prepared freshly), seal it, and vortex to mix well. React in a water bath at 70 °C for 100 min, cool it to room temperature, add 200 μL of 0.3 M HCl to neutralize it, and make up to 1 mL with distilled water. Then add 1 mL of chloroform for extraction, draw the upper aqueous phase, and repeat the extraction 3 times.

[0031] Take 20 μL of the supernatant after centrifugation and inject it into an HPLC for detection and analysis. Take 100 μL of a 10 mg / mL mixture of monosaccharide standards (Man, Rha, Gal, GalA, Glc, GlcA, Xyl, Ara, Fuc), and analyze it according to the method of PMP derivatization and analysis of the sample. Ultraviolet absorption wavelength: 254 nm; mobile phase: phosphate buffer: acetonitrile = 82:18; flow rate: 1 mL / min. The results show that:

[0032] (1) HCPN is a heteropolysaccharide mainly composed of four monosaccharides, and the molar ratio of its monosaccharide composition is Man:Glc:Gal:Ara = 3.16:29.96:22.7:1;

[0033] (2) HCPA1 is a heteropolysaccharide mainly composed of seven monosaccharides, and its molar ratio is Man:GlcA:Rha:GalA:Glc:Gal:Ara = 16.83:1:14.31:1.37:65.27:192.7:46.23;

[0034] (3) HCPA2 is a heteropolysaccharide mainly composed of six monosaccharides, and its molar ratio is Man:GlcA:Rha:Glc:Gal:Ara = 1:1.19:2.92:1.49:23.2:6.14. The liquid chromatogram of the monosaccharide composition of the crude polysaccharide from Houttuynia cordata roots at the first fraction is as Figure 1 and Figure 2 shown.

[0035] Determination of the molecular weight distribution of HCPN, HCPA1 and HCPA2

[0036] In this experiment, high performance gel permeation chromatography (HPGPC) was used to determine the molecular weights of HCPN, HCPA1 and HCPA2. HPGPC detection conditions: use a TSK g3000 SWXL gel column and a differential detector; the mobile phase is 0.2 M NaCl; the flow rate: 0.5 mL / min; system calibration: accurately weigh dextran standard products with known molecular weights and dissolve them in 0.2 M NaCl to prepare a 2 mg / mL solution, centrifuge at 10000 rpm for 10 min, take the supernatant for auto-sampling analysis. Using the logarithm of the molecular weights of each standard product as the ordinate and the corresponding elution volume as the abscissa, draw a standard curve. Calculate their molecular weights according to the retention times of the main peaks of HCPN, HCPA1 and HCPA2 brought into the standard curve. As Figure 3 shown, the results show that there are two main peaks for HCPN, and their molecular weights are 34.4 KDa and 15.1 KDa respectively. The molecular weight of the main peak of HCPA1 is 66.07 KDa, and the molecular weight of the main peak of HCPA2 is 134.59 KDa.

[0037] MTT method for determining the activities of crude polysaccharides from Houttuynia cordata roots (HCP) against HepG2, A–549, Tca–8113, HCT–116 cell lines

[0038] 1. Collect the logarithmic-phase cells of HepG2, A–549, Tca–8113, HCT–116 cell lines, adjust the cell suspension concentration, so that the cell density is adjusted to 3000–5000 cells / well, add 100 μL to each well and plate in a 96-well plate (fill the edge with sterile PBS), incubate at 2.5% CO2 and 37 °C for about 12 h.

[0039] 2. Pre-configure the drug concentration at the highest concentration (2 mg / ml) in advance, vortex each drug for 3 min, and shake overnight for standby

[0040] 3. On the next day, centrifuge the drug at 10,000 rpm for 10 min, filter it through a 0.22-μm filter membrane, dilute the sample drug according to the set drug concentration gradient, and process each drug at 0, 125, 250, 500, 1000, and 2000 μg / mL. Add 100 μL to each well, set 5 replicates, and continue to incubate for 48 h.

[0041] 4. After 48 h, under light-proof conditions, add 20 μL of MTT solution (5 mg / ml, i.e., 10% MTT) to each well and continue to culture for 4 h.

[0042] 5. After culturing for 4 h, terminate the culture and aspirate the culture medium in the wells.

[0043] 6. Under light-proof conditions, add 150 μL of DMSO to each well and place it on a shaker to shake at low speed for 20 min to fully dissolve the crystals.

[0044] 7. Measure the absorbance value of each well at OD490 nm with an enzyme-linked immunosorbent detector. The results are processed as Figure 4 shown. The results show that HCP has no obvious inhibitory effect on the four cell lines of HepG2, A–549, Tca–8113, and HCT–116, indicating that the use of HCP is safe.

[0045] Determination of the activities of HCP, HCPN, HCPA1, and HCPA2 against α-glucosidase

[0046] Solution preparation: 0.1 M PBS with pH = 6.8, 0.01 M PNPG substrate solution, and 0.8 U / mL α-glucosidase.

[0047] Sample solution preparation: Dissolve HCP, HCPN, HCPA1, HCPA2, and the positive drug Acarbose at a maximum concentration of 10 mg / mL with PBS buffer. After dissolution, centrifuge at 10,000 rpm / min for 10 min. The supernatant after centrifugation is filtered through a 0.22-μm filter membrane and diluted to PBS solutions with concentrations of 0, 0.5, 1, 2, 5, and 10 mg / mL.

[0048] Experimental procedure: Mix 100 μL of the sample solution, 60 μL of PBS, and 20 μL of α-glucosidase, react at 37 °C for 10 min, add 20 μL of PNPG solution, and continue to react at 37 °C for 30 min. After the reaction is completed, detect its absorbance value at a wavelength of 405 nm. The experimental results are as Figure 5As shown, both HCP, the neutral sugar fraction HCPN, and the acidic sugar fraction HCPA1 have inhibitory effects on α-glucosidase in a concentration-dependent manner. When the concentration of the neutral sugar fraction HCPN is greater than 5 mg / mL, its inhibitory activity against α-glucosidase is greater than that of the positive drug acarbose. The acidic sugar fraction HCPA1 also exhibits good activity at high concentrations.

[0049] The calculation formula is: Inhibition rate % = [1 - (A 样品 - A 背景 ) / A 空白 × 100%

[0050] A 样品 = Phosphate buffer + α-glucosidase + polysaccharide solution + PNPG substrate;

[0051] A 背景 = Phosphate buffer + polysaccharide solution + PNPG substrate;

[0052] A 空白 = Phosphate buffer + α-glucosidase + PNPG substrate.

[0053] Determination of the activities of potato starch, sucrose, lactose, etc. against α-glucosidase

[0054] To exclude the interference of starch or disaccharides that may exist in HCP and its homogeneous charge fractions as hydrolysis substrates of α-glucosidase on the inhibitory activity of Houttuynia cordata polysaccharide against α-glucosidase, starch, sucrose, and lactose were selected as research objects to test whether they affect the hydrolysis of the PNPG substrate by α-glucosidase.

[0055] Solution preparation: 0.1 M PBS (pH = 6.8), 0.01 M PNPG substrate solution, and 0.8 U / mL α-glucosidase.

[0056] Sample solution preparation: Starch, Sucorse, Lactose, and the positive drug Acarbose were dissolved in PBS buffer at a maximum concentration of 10 mg / mL. After dissolution, the solution was centrifuged at 10000 rpm for 10 min. The supernatant after centrifugation was filtered through a 0.22 μm filter membrane and diluted to PBS solutions with concentrations of 0, 0.5, 1, 2, 5, and 10 mg / mL. The test results are as Figure 6 shown. Starch, sucrose, and galactose did not show inhibitory effects on α-glucosidase, and the positive drug acarbose showed high inhibitory activity against α-glucosidase, indicating that the experimental results are reliable and that starch, sucrose, and galactose did not affect the hydrolysis of the PNPG substrate by α-glucosidase.

Claims

1. A polysaccharide component that inhibits α-glucosidase activity, characterized in that: The polysaccharide component is HCPN or HCPA1; The HCPN is a heteropolysaccharide mainly composed of four monosaccharides, and its monosaccharide composition includes Man, Glc, Gal, and Ara; The HCPA1 is a heteropolysaccharide mainly composed of seven monosaccharides, and its monosaccharide composition includes Man, GlcA, Rha, GalA, Glc, Gal, and Ara.

2. The polysaccharide component for inhibiting α-glucosidase activity according to claim 1, characterized in that: The molar ratio of the monosaccharide composition of the HCPN is Man:Glc:Gal:Ara = 3.16:29.96:22.7:

1.

3. The polysaccharide component for inhibiting α-glucosidase activity according to claim 1, characterized in that: The molar ratio of the monosaccharide composition of the HCPA1 is Man:GlcA:Rha:GalA:Glc:Gal:Ara = 16.83:1:14.31:1.37:65.27:192.7:46.

23.

4. The preparation method of the polysaccharide component for inhibiting α-glucosidase activity according to any one of claims 1 to 3, characterized in that: The polysaccharide is the crude polysaccharide of Houttuynia cordata obtained by the water extraction and alcohol precipitation method. The crude polysaccharide of Houttuynia cordata is separated by anion exchange column chromatography to obtain the homogeneous charge fractions of HCPN and HCPA1.

5. The preparation method of the polysaccharide component for inhibiting α-glucosidase activity according to claim 4, characterized in that It includes the following steps: Step 1: Crush the dry product of Houttuynia cordata roots, add distilled water and soak overnight. After multiple decoctions and separations, the separated filtrates are combined and concentrated. Add ethanol to make the final concentration of ethanol reach 70% - 80%. After precipitation and centrifugation, the precipitate is redissolved with distilled water and the residual ethanol is volatilized, and then freeze-dried to obtain the crude polysaccharide of Houttuynia cordata; Step 2: The obtained crude polysaccharide of Houttuynia cordata is eluted through a DEAE-Agarose anion exchange column chromatography with distilled water and 0.1M Nacl solution as the mobile phase. The mobile phase is collected and the same fractions are combined by the phenol-sulfuric acid method and the determination results of the microplate reader. The homogeneous charge fractions of each Houttuynia cordata are the above-mentioned HCPN and HCPA1 respectively.

6. Use of the polysaccharide component for inhibiting α-glucosidase activity according to any one of claims 1 to 3 in the preparation of a medicament for treating diabetes.

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