Gynostemma pentaphyllum polysaccharide as well as extraction process and application thereof

Abstract

The invention belongs to the technical field of polysaccharide extraction, and particularly relates to gynostemma pentaphyllum polysaccharide as well as an extraction process and application thereof. A gynostemma pentaphyllum raw material produced in Sichuan is adopted, and the structure of gynostemma pentaphyllum polysaccharide is shown in the specification. The number-average molecular weight of the gynostemma pentaphyllum polysaccharide is 65.8 kDa, the weight-average molecular weight of the gynostemma pentaphyllum polysaccharide is 77.0 kDa, and the z-average molecular weight of the gynostemma pentaphyllum polysaccharide is 97.9 kDa. Through precise parameter control, high-efficiency and low-loss extraction of the gynostemma pentaphyllum polysaccharide is realized, the activity of the obtained gynostemma pentaphyllum polysaccharide is better than that of polysaccharide obtained in the prior art, and reliable technical support is provided for subsequent development of medicinal polysaccharide preparations.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide extraction technology, specifically relating to Gynostemma pentaphyllum polysaccharide, its extraction process, and its applications. Background Technology

[0002] Diabetes mellitus is a chronic disease characterized by hyperglycemia, caused by insufficient insulin secretion and impaired insulin utilization. With improved living standards, diabetes is showing a trend of increasing incidence and affecting younger people. Researching effective drugs for blood sugar control is of great significance for managing the progression of diabetes.

[0003] Gynostemma pentaphyllum Makino, a perennial climbing herbaceous plant belonging to the Cucurbitaceae family, is also known as Seven-Leaf Ginseng, Small Bitter Medicine, Heavenly Grass, Root-Growing Plant, Five-Leaf Ginseng, or Seven-Leaf Ginseng. Gynostemma pentaphyllum is commonly used medicinally as a whole plant or rhizome. It has good preventive and therapeutic effects on cardiovascular diseases such as hyperlipidemia, hypertension, and coronary heart disease, as well as diabetes and tumors. Long-term use has no toxic side effects.

[0004] Gynostemma pentaphyllum contains various chemical components, among which polysaccharides are one of its active ingredients. Studies have shown that Gynostemma pentaphyllum polysaccharides have a hypoglycemic effect. However, the effective components vary among Gynostemma pentaphyllum from different origins, and different extraction processes result in differences in the types and amounts of polysaccharides actually extracted. These factors lead to significant variations in the hypoglycemic effect of Gynostemma pentaphyllum polysaccharides. Overall, however, compared to commonly used hypoglycemic drugs such as acarbose, the hypoglycemic effect of these Gynostemma pentaphyllum polysaccharides in current technology is still relatively poor. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a Gynostemma pentaphyllum polysaccharide, its extraction process, and its application. This invention uses Gynostemma pentaphyllum from Sichuan as raw material to extract a Gynostemma pentaphyllum polysaccharide. The obtained Gynostemma pentaphyllum polysaccharide has a better blood sugar lowering effect than Gynostemma pentaphyllum polysaccharide obtained by existing technologies.

[0006] The present invention is specifically implemented through the following technical solutions.

[0007] This invention first provides Gynostemma pentaphyllum polysaccharide, obtained by extraction from Gynostemma pentaphyllum produced in Sichuan. The structural formula of Gynostemma pentaphyllum polysaccharide is as follows: ; The number-average molecular weight of Gynostemma pentaphyllum polysaccharide is 65.8 kDa, the weight-average molecular weight is 77.0 kDa, and the z-average molecular weight is 97.9 kDa.

[0008] This invention extracts Gynostemma pentaphyllum from Sichuan and obtains Gynostemma pentaphyllum polysaccharide with the above-mentioned structure, which has a better blood sugar lowering effect than polysaccharides obtained by existing technologies.

[0009] The present invention also provides a process for extracting Gynostemma pentaphyllum polysaccharides, comprising the following steps: Step 1, Water Extraction: Gynostemma pentaphyllum from Sichuan is crushed and sieved to obtain raw material powder; water is added to the raw material powder, water extraction is performed, and the mixture is filtered to obtain an aqueous solution.

[0010] Step 2, alcohol precipitation: Ethanol was added to the aqueous solution obtained in step 1 to precipitate the polysaccharide from the aqueous solution. The precipitate was dried to obtain a crude polysaccharide extract.

[0011] This invention first uses a water extraction and alcohol precipitation method to obtain crude polysaccharide extract. Water is used as a solvent to fully dissolve water-soluble polysaccharides, resulting in high extraction efficiency. Furthermore, the alcohol precipitation step can effectively precipitate the polysaccharides from the aqueous solution, resulting in a high polysaccharide yield. The extracted polysaccharide solution can be separated by a simple alcohol precipitation step, and subsequent processing is also relatively convenient.

[0012] Step 3: Combined impurity removal system (1) Enzymatic hydrolysis: The crude polysaccharide extract was dissolved in water, and papain was added to the aqueous solution of the crude polysaccharide extract for enzymatic hydrolysis. After enzymatic hydrolysis, an enzymatic hydrolysate was obtained.

[0013] (2) Protein removal: Collect the aqueous phase of the enzymatic hydrolysis mixture, add chloroform and n-butanol to the aqueous phase of the enzymatic hydrolysis mixture. Chloroform and n-butanol are used to remove protein. After mixing evenly, shake and collect the upper aqueous phase.

[0014] (3) Removal of fat-soluble impurities: Petroleum ether is then added to the aqueous phase obtained in step (2) above for extraction and degreasing. After mixing and shaking, the oil and water are separated and the aqueous phase is collected.

[0015] (4) Adsorption of pigments and other small molecule organic impurities: Add macroporous resin AB-8 to the aqueous phase obtained in step (3) above for adsorption and decolorization, and collect the supernatant.

[0016] (5) Dialyze the supernatant after impurity removal in step (4) using a 3000Da dialysis bag, and then dry it. During the dialysis process, small molecules with a molecular weight less than 3000Da are removed to obtain polysaccharides with a molecular weight greater than 3000Da. This method can effectively remove small molecule impurities from the polysaccharide solution and maintain the polysaccharide activity to the maximum extent.

[0017] (6) The polysaccharide sample after dialysis in step (5) is separated and purified, and then the structure and content of the separated and purified product are identified to obtain Gynostemma pentaphyllum polysaccharide.

[0018] Preferably, during water extraction, the material-to-liquid ratio is 1g:10mL~20mL (in traditional processes, when using water for polysaccharide extraction, the ratio is often 1g:30mL~50mL, and this invention reduces solvent consumption by 30%-60%), the water extraction temperature is 60℃±2℃ (in traditional processes, hot water extraction is often used, typically at 80℃~100℃, while this invention uses 60℃±2℃ for extraction, avoiding sugar chain breakage caused by high temperatures and improving polysaccharide extraction efficiency), and the extraction time is 3.5~4h (traditionally, it requires 6h~8h). Combined with intermittent stirring (30min / time), efficiency is improved and energy consumption is reduced by 40%.

[0019] Preferably, a two-step ethanol precipitation is used, with 60% to 80% of the volume of the aqueous solution being added each time. More preferably, 60% of the volume of the aqueous solution is added first, and 80% of the volume of the aqueous solution is added second. The precipitation temperature is 3 to 4°C. Traditional single-step ethanol precipitation is mostly done at room temperature, which introduces more impurities.

[0020] Preferably, during enzymatic hydrolysis, the mass concentration of the aqueous solution of crude polysaccharide extract is 4%~6%, the ratio of papain to the aqueous solution of crude polysaccharide extract is 0.4g~0.6g:600ml~1L, and the enzymatic hydrolysis conditions are: overnight enzymatic hydrolysis at room temperature (>8h) to avoid protein denaturation residue caused by high temperature heating.

[0021] Preferably, when removing proteins, the treatment temperature is 25°C, the ratio of the total volume of chloroform and n-butanol to the volume of the aqueous phase of the enzymatic hydrolysis mixture is 0.2~0.3:1, preferably 0.25:1, the volume ratio of chloroform to n-butanol is 3.5~4.5:1, preferably 4:1, the shaking time is 14~16 min, preferably 15 min, and the shaking is repeated twice.

[0022] Preferably, when removing fat-soluble impurities, the ratio of the volume of petroleum ether added to the volume of the aqueous phase is 0.2~0.3:1, more preferably 0.25:1. The petroleum ether degreasing stage is maintained at room temperature with shaking for 15 min, which significantly shortens the time and lowers the temperature compared to traditional Soxhlet extraction (6-8 h, 60-80 °C).

[0023] Preferably, when adsorbing pigments and other small molecule organic impurities, the volume of AB-8 macroporous resin is 1 / 2 of the volume of the aqueous phase, and the adsorption time at room temperature is >12h, which replaces the traditional high-temperature boiling method of activated carbon (80℃~100℃, 1h~2h) and avoids high-temperature degradation.

[0024] Preferably, dialysis at 4°C for 24-48 hours reduces microbial contamination compared to traditional room temperature dialysis, while retaining small molecule active ingredients (>3kDa).

[0025] Preferably, the polysaccharide separation and purification is carried out by tandem ion exchange purification (DEAE-52 column) and gel chromatography (Sephadex G-100). During ion exchange purification, the dialyzed polysaccharide sample is dissolved in water at a sample-to-water ratio of 1g:50mL, centrifuged, and the supernatant is passed through the ion exchange column with a sample loading volume of 10% to 15% of the exchange capacity. The ion column is equilibrated at pH 6.7 to 6.9, preferably 6.8, and gradient elution is performed using 0 to 0.5 mol / L NaCl aqueous solution. The eluent is collected, dialyzed, and then dried.

[0026] Preferably, when using gel chromatography for purification, the ion-exchange purified sample is added to pure water at a ratio of 1g:20mL, centrifuged, and the supernatant is passed through a gel chromatography column for separation and purification at a flow rate of 1~1.5mL / min. Pure water is used to elute 1.5~2 column volumes, and one tube is collected for every 10mL. All eluent is collected, dialyzed, and then dried.

[0027] For structural identification, molecular weight determination was performed using a gel chromatography column combination: Ohpak SB-805 HQ + SB-803 HQ in series, with a column temperature of 45℃ (traditional single-column methods have large temperature fluctuations and low resolution), and an elution flow rate of 0.6 ml / min (optimized flow rate to balance separation efficiency and time cost). The detection coupling was multi-angle laser light scattering (DAWN HELEOS II) coupled with differential refractive index (Optilab T-rEX). The absolute molecular weight was directly calculated using the Mark-Houwink equation with an error rate of <5% (compared to >15% for traditional single differential methods).

[0028] Multidimensional NMR Spectroscopy Analysis: Scanning parameters: 600MHz superconducting NMR (traditionally 300-400MHz), resolution improved to 0.26Hz (rotating sample).

[0029] Pulse sequence combination: 1H / 13C / COSY / HSQC / HMBC / NOESY full spectrum coverage, with a monosaccharide residue linkage site resolution accuracy of over 95% (traditional methylation-dependent analysis has an accuracy of only 60-70%).

[0030] Sample dissolution: The concentration prepared in D2O is ≥40mg / ml (traditionally <20mg / ml), and the signal-to-noise ratio is improved to 888:1.

[0031] Electron microscopic morphological characterization: Gold sputtering thickness: Nanoscale gold film deposition (10nm) achieves 1nm resolution under Zeiss field emission electron microscope (conventional SEM resolution is 3-5nm), which can clearly observe the irregular particles (particle size 50-200nm) and rough topology on the polysaccharide surface.

[0032] Compared with the prior art, the present invention has the following beneficial effects: This invention uses Gynostemma pentaphyllum from Sichuan as raw material to extract a Gynostemma pentaphyllum polysaccharide. Its structure is different from the Gynostemma pentaphyllum polysaccharides disclosed in the prior art. The types of monosaccharides, the linkage sequence, the linkage mode, and the molecular weight are all different. That is, this invention provides a new Gynostemma pentaphyllum polysaccharide. Furthermore, subsequent tests have found that compared with the Gynostemma pentaphyllum polysaccharides obtained by commonly used methods, the Gynostemma pentaphyllum polysaccharide obtained by this invention exhibits superior hypoglycemic activity.

[0033] Gynostemma pentaphyllum from different origins varies in its active ingredients, and different extraction processes also yield different results. This leads to differences in the composition of the final Gynostemma pentaphyllum polysaccharide. The raw material selected in this invention differs from commonly used raw materials. Currently, the commonly used raw material is Gynostemma pentaphyllum from the Qinling-Bashan region, while this invention uses Gynostemma pentaphyllum from Sichuan as the raw material and provides a new extraction process for Gynostemma pentaphyllum polysaccharide. First, the raw material from this origin is subjected to water extraction and alcohol precipitation to obtain crude polysaccharide extract. Then, the crude polysaccharide extract is subjected to a combined impurity removal system, which includes enzymatic hydrolysis, removal of proteins, removal of fat-soluble impurities, adsorption of pigments and other small molecule organic impurities, dialysis to obtain high molecular weight polysaccharides, and separation and purification of polysaccharides. Through the above steps, impurities are removed and polysaccharide loss is reduced. Finally, Gynostemma pentaphyllum polysaccharide is extracted from Gynostemma pentaphyllum from Sichuan. Compared with Gynostemma pentaphyllum polysaccharide from the Qinling-Bashan region, the polysaccharide of this invention has higher hypoglycemic activity, providing a new source of Gynostemma pentaphyllum polysaccharide for further development of pharmaceutical-grade polysaccharide preparations and laying the foundation for subsequent research. Attached Figure Description

[0034] Figure 1 This is a graph showing the raw data for determining the molecular weight of Gynostemma pentaphyllum polysaccharides.

[0035] Figure 2 This is a one-dimensional hydrogen spectrum.

[0036] Figure 3 This is a one-dimensional carbon spectrum.

[0037] Figure 4 This is the COSY spectrum.

[0038] Figure 5 This is the HMBC spectrum.

[0039] Figure 6 This is an HSQC spectrum.

[0040] Figure 7 This is the NOESY spectrum.

[0041] Figure 8 In the image, (a), (b), (c), and (d) are scanning electron microscope images of Gynostemma pentaphyllum obtained in Example 1 at different magnifications. Detailed Implementation

[0042] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention. Unless otherwise specified, the experimental methods and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials described are commercially available.

[0043] Gynostemma pentaphyllum from different origins exhibits differences in its active ingredients, and different extraction processes also yield varying extraction results, leading to variations in the composition of the final Gynostemma pentaphyllum polysaccharides. To obtain higher-activity Gynostemma pentaphyllum polysaccharides, this invention has conducted long-term research and ultimately selected Gynostemma pentaphyllum from Sichuan Province as the extraction target. Sichuan has a diverse climate, belonging to the subtropical humid climate, characterized by warmth, humidity, abundant rainfall, frequent fog, and limited sunshine. Furthermore, its moderate altitude, rich vegetation, and favorable ecological environment contribute to its superior growth environment compared to the commonly found Gynostemma pentaphyllum from the Qinling-Bashan Mountains. Sichuan-grown Gynostemma pentaphyllum has larger, darker green leaves, thicker stems, and overall better quality with higher content of active ingredients. This invention extracts polysaccharides from Sichuan-grown Gynostemma pentaphyllum and analyzes the monosaccharide composition of the polysaccharides. The structural formula of the obtained Gynostemma pentaphyllum polysaccharide is as follows:

[0044] ; The number-average molecular weight of Gynostemma pentaphyllum polysaccharide is 65.8 kDa, the weight-average molecular weight is 77.0 kDa, and the z-average molecular weight is 97.9 kDa.

[0045] The following provides the above-mentioned extraction process for Gynostemma pentaphyllum polysaccharides, and details the factors affecting the extraction, separation, and purification of polysaccharides. To highlight the advantages of the extraction process of this invention, the traditional Qinba Gynostemma pentaphyllum extraction process is used as a comparison. For the traditional Qinba Gynostemma pentaphyllum extraction process, please refer to the published technology: "Research Progress on Extraction, Separation, Chemical Structure and Bioactivity of Gynostemma pentaphyllum Polysaccharides," Shang Xiaoya et al., Natural Product Research and Development. Authorization Announcement No.: CN 101717452 B, Invention Title: A Method for Preparing Neutral Polysaccharides from Gynostemma pentaphyllum.

[0046] Compared with current extraction processes for Gynostemma pentaphyllum polysaccharides, the extraction process of this invention differs from current processes in both the overall extraction sequence and the specific extraction details. This invention first involves water extraction and alcohol precipitation of the Gynostemma pentaphyllum raw material to obtain a crude polysaccharide extract. Then, the crude polysaccharide extract undergoes a combined impurity removal system, sequentially including enzymatic hydrolysis, protein removal, removal of lipid-soluble impurities, adsorption of pigments and other small-molecule organic impurities, dialysis to obtain high-molecular-weight polysaccharides, and separation and purification of the polysaccharides, ultimately yielding Gynostemma pentaphyllum polysaccharides. Through precise parameter control, this process achieves efficient and low-loss extraction of Gynostemma pentaphyllum polysaccharides. It is important to emphasize that the extraction steps are closely related, and their order cannot be arbitrarily adjusted or replaced. Specifically, the Gynostemma pentaphyllum polysaccharide extraction process of this invention includes the following steps:

[0047] Step 1, Water Extraction: Gynostemma pentaphyllum from Sichuan is crushed and sieved to obtain raw material powder; water is added to the raw material powder, water extraction is performed, and the mixture is filtered to obtain an aqueous solution.

[0048] In a preferred embodiment of the present invention, during water extraction, the material-to-liquid ratio is 1g:10mL~20mL (in traditional processes, when using water for polysaccharide extraction, the ratio is usually 1g:30mL~50mL, and the present invention reduces solvent consumption by 30%-60%), the water extraction temperature is 60℃±2℃ (in traditional processes, hot water extraction is used, usually at 80℃~100℃, and the present invention uses water at 60℃±2℃, which avoids sugar chain breakage caused by high temperature and improves polysaccharide extraction effect), and the extraction time is 3.5~4h (traditionally it takes 6h~8h). Combined with intermittent stirring (30min / time), the efficiency is improved and the energy consumption is reduced by 40%.

[0049] Step 2, alcohol precipitation: Ethanol is added to the aqueous solution to precipitate the polysaccharide, which is then dried to obtain a crude polysaccharide extract. In a preferred embodiment of the present invention, stepwise ethanol precipitation is employed, with 60% to 80% of the volume of the aqueous solution being added to ethanol each time. More preferably, 60% of the volume of the aqueous solution is added to ethanol in the first step, and 80% of the volume of the aqueous solution is added in the second step. The precipitation temperature is 3 to 4°C. Traditional single-step ethanol precipitation is mostly carried out at room temperature, which introduces more impurities.

[0050] It should be noted that, compared to the ethanol extraction method used for extracting Gynostemma pentaphyllum from Qinba, this invention first sieves the pulverized raw material and then uses a water extraction and alcohol precipitation method to extract Gynostemma pentaphyllum from Sichuan. The water extraction and alcohol precipitation method utilizes water as a solvent, which can fully dissolve water-soluble polysaccharides, resulting in high extraction efficiency. Furthermore, the alcohol precipitation step effectively precipitates the polysaccharides from the aqueous solution, leading to a high polysaccharide yield. The extracted polysaccharide solution can be separated through a simple alcohol precipitation step, and subsequent processing is also relatively convenient. In contrast, the solution extracted by the traditional ethanol reflux extraction method requires multiple filtrations and concentrations, making subsequent steps more cumbersome.

[0051] Step 3: Combined impurity removal system (1) Enzymatic hydrolysis to degrade proteins: Dissolve crude polysaccharide extract in water, add papain to the aqueous solution of crude polysaccharide extract for enzymatic hydrolysis, and obtain an enzymatic hydrolysate mixture after hydrolysis.

[0052] In a preferred embodiment of the present invention, during enzymatic hydrolysis, the mass concentration of the aqueous solution of crude polysaccharide extract is 4%~6%, the ratio of papain to the aqueous solution of crude polysaccharide extract is 0.4g~0.6g:600ml~1L, and the enzymatic hydrolysis conditions are: overnight enzymatic hydrolysis at room temperature (>8h) to avoid protein denaturation residue caused by high temperature heating.

[0053] It should be noted that this invention uses papain for overnight enzymatic hydrolysis in the crude polysaccharide purification step. In existing extraction processes, Qinba Gynostemma pentaphyllum is purified using 0.5g of papain in 1mol / L HCl. While the addition of HCl enhances the activity of papain under acidic conditions, its degradation effect on certain proteins is limited, especially those with complex structures (rich in disulfide bonds). This invention uses papain for enzymatic hydrolysis, maintaining the stability of the glycosidic bonds of the polysaccharide under mild conditions, avoiding hydrolysis or degradation that may occur under acidic conditions. In contrast, the addition of HCl to papain in traditional methods may disrupt the glycosidic bonds of the polysaccharide under strongly acidic conditions, leading to polysaccharide degradation or structural changes. Furthermore, from an environmental perspective, the enzymatic hydrolysis process of papain mainly relies on biological enzymes, eliminating the need for large amounts of chemical reagents, making it more environmentally friendly and safer than using strong acids like HCl.

[0054] (2) Protein removal: Collect the aqueous phase of the enzymatic hydrolysis mixture, add chloroform and n-butanol to the aqueous phase of the enzymatic hydrolysis mixture. Chloroform and n-butanol are used to remove proteins. After mixing evenly, shake and collect the upper aqueous phase. In a preferred embodiment of the present invention, the treatment temperature for protein removal is 25°C, the ratio of the total volume of chloroform and n-butanol to the volume of the aqueous phase of the enzymatic hydrolysis mixture is 0.2~0.3:1, preferably 0.25:1, the volume ratio of chloroform to n-butanol is 4:1, the shaking time is 15 min, and the shaking is performed twice.

[0055] (3) Removal of fat-soluble impurities: Petroleum ether is then added to the aqueous phase obtained in step (2) above for extraction and defatting. The mixture is stirred and then separated into oil and water, and the aqueous phase is collected. In a preferred embodiment of the present invention, when removing fat-soluble impurities, the ratio of the volume of petroleum ether added to the volume of the aqueous phase is 0.2~0.3:1, preferably 0.25:1. The petroleum ether defatting stage is maintained at room temperature and shaken for 15 min, which significantly shortens the time and lowers the temperature compared to traditional Soxhlet extraction (6-8 h, 60-80 °C).

[0056] (4) Adsorption of pigments and other small molecule organic impurities: Add macroporous resin AB-8 to the aqueous phase obtained in step (3) above for adsorption and decolorization, and collect the supernatant. In a preferred embodiment of the present invention, the volume of AB-8 macroporous resin is 1 / 2 of the volume of the aqueous phase, and the adsorption at room temperature is >12h, which replaces the traditional activated carbon high-temperature boiling method (80℃~100℃, 1h~2h) and avoids high-temperature degradation.

[0057] (5) Dialyze the supernatant obtained after impurity removal in step (4) using a 3000 Da dialysis bag, and then dry it. During the dialysis process, small molecules with a molecular weight less than 3000 Da are removed to obtain polysaccharides with a molecular weight greater than 3000 Da. This effectively removes small molecule impurities from the polysaccharide solution and maximizes the preservation of polysaccharide activity. In a preferred embodiment of the present invention, dialysis is performed at 4°C for 24-48 hours.

[0058] (6) The solution after dialysis in step (5) is subjected to polysaccharide separation and purification. Then, the structure and content of the purified product are identified to obtain Gynostemma pentaphyllum polysaccharide. In a preferred embodiment of the present invention, the polysaccharide separation and purification is carried out by ion exchange purification (DEAE-52 column) and gel chromatography (Sephadex G-100) in series. During ion exchange purification, the polysaccharide sample after dialysis is dissolved in water at a ratio of 1g:50mL. After centrifugation, the supernatant is passed through an ion exchange column with a sample loading amount of 10%~15% of the exchange capacity. The preferred ion column equilibrium pH is 6.8. Gradient elution is performed using 0~0.5mol / L NaCl aqueous solution. The eluent is collected, dialyzed, and then dried. When purifying using gel chromatography, add pure water to the ion-exchange purified sample at a ratio of 1g:20mL, centrifuge, and take the supernatant for separation and purification through a gel chromatography column at a flow rate of 1~1.5mL / min. Elute with pure water for 1.5~2 column volumes, collecting one tube for every 10mL. Collect all eluent, dialyze, and then dry.

[0059] It should be noted that in the combined purification process, this invention uses chloroform and n-butanol to remove proteins, petroleum ether to remove fat-soluble impurities, and macroporous resin AB-8 to adsorb pigments and other small-molecule organic impurities. In contrast, the purification process for Qinba Gynostemma pentaphyllum uses concentrated ammonia to adjust pH, H2O2 for decolorization, and Sevag reagent to remove proteins, resulting in significant differences in reagents and procedures. The amount of concentrated ammonia added needs precise control; otherwise, the pH value may become too high or too low. Polysaccharides may degrade or undergo structural changes under strongly alkaline conditions (pH > 9), especially for some heat-sensitive polysaccharides, affecting their stability. Impure ammonia may introduce additional impurities, increasing the difficulty of subsequent purification. H2O2 has poor decolorization effects on some pigments such as carotenoids and chlorophyll, and may need to be combined with other decolorization methods to achieve the desired effect. For some complex pigment systems, H2O2 decolorization may require a longer time or a higher concentration, increasing the operational difficulty. In this invention, chloroform, n-butanol, petroleum ether, and macroporous resin AB-8 are used in sequence. Chloroform and n-butanol remove proteins, petroleum ether effectively removes fat-soluble impurities such as fats and waxes from Gynostemma pentaphyllum, and macroporous resin AB-8 adsorbs pigments and other small molecule organic impurities. The combined use of these three agents significantly improves the impurity removal efficiency of Gynostemma pentaphyllum and results in purer polysaccharides.

[0060] It should be noted that the final step in processing Qinba Gynostemma pentaphyllum involves dialysis of the filtered supernatant with distilled water for 48 hours, collecting the solution from the dialysis bag, adding 4 times the volume of ethanol, incubating at 4°C for 10-12 hours, centrifuging at 5000 rpm for 10 minutes, collecting the precipitate, washing it sequentially with ethanol, acetone, and ether, and freeze-drying for 12 hours to obtain white purified Gynostemma pentaphyllum polysaccharides. The process of this invention utilizes a more convenient and efficient 3000 Da dialysis bag. The 3000 Da dialysis bag has a molecular weight cutoff of 3000 Da, meaning that small molecules with a molecular weight less than 3000 Da (such as salts, monosaccharides, oligosaccharides, organic solvents, etc.) can freely pass through the dialysis bag, while polysaccharides with a molecular weight greater than 3000 Da are retained within the bag. This method effectively removes small molecule impurities from the polysaccharide solution, maximizing the preservation of polysaccharide activity. Furthermore, the dialysis operation is relatively simple, requiring no complex equipment, making it suitable for both laboratory and small-scale production. Both acidic and neutral polysaccharides can effectively remove small molecule impurities.

[0061] It should be noted that, in the separation and purification of polysaccharides, compared with the ion exchange purification of Qinba Gynostemma pentaphyllum using a DEAE-52 cellulose column, this invention utilizes a gel chromatography column for purification. Gel chromatography separates polysaccharides based on their molecular size; larger molecular weight polysaccharides are eluted first, followed by smaller molecular weight polysaccharides. This separation mechanism is suitable for separating polysaccharides of different molecular weights, especially for samples with a wide molecular weight distribution. The DEAE-52 cellulose column separates polysaccharides based on their charge characteristics; negatively charged polysaccharides (such as acidic polysaccharides) bind to the anion exchange groups on the column, while neutral polysaccharides elute directly. This separation mechanism is suitable for separating charged polysaccharides, but its separation effect on neutral polysaccharides is poor. Gel chromatography is suitable for all polysaccharides of different molecular weights, whether acidic, neutral, or other types; as long as there is a difference in molecular weight, they can be separated using gel chromatography.

[0062] Compared with previous methods for extracting Gynostemma pentaphyllum polysaccharides, the new technology provided by this invention exhibits significant innovations in several aspects. Firstly, in terms of impurity removal, this method employs a multi-step process, including enzymatic hydrolysis, organic solvent extraction, and macroporous resin adsorption, which can more thoroughly remove impurities such as proteins, fats, and pigments, significantly improving the purity of the polysaccharides. In particular, the use of macroporous resin AB-8 effectively adsorbs pigments and small molecule impurities, further enhancing the impurity removal efficiency. Secondly, in terms of purification technology, this method introduces gel chromatography for the separation and purification of polysaccharides, enabling precise separation based on molecular weight to obtain polysaccharide components with higher purity. Compared with traditional alcohol precipitation or ion exchange methods, gel chromatography offers higher separation precision and controllability.

[0063] In summary, this new Gynostemma pentaphyllum polysaccharide extraction technology is significantly innovative in terms of impurity removal efficiency and purification precision. It can extract and identify Gynostemma pentaphyllum polysaccharides more efficiently and accurately, providing more reliable technical support for their application in food, medicine and other fields.

[0064] Molecular weight determination was performed using a gel chromatography column combination: Ohpak SB-805 HQ + SB-803 HQ in series, column temperature 45℃ (traditional single-column methods have large temperature fluctuations and low resolution), elution flow rate: 0.6 ml / min (optimized flow rate to balance separation efficiency and time cost), detection coupling: multi-angle laser light scattering (DAWN HELEOS II) and differential refractive index (Optilab T-rEX), absolute molecular weight was directly calculated using the Mark-Houwink equation, with an error rate of <5% (traditional single differential method error >15%).

[0065] Multidimensional NMR Spectroscopy Analysis: Scanning parameters: 600MHz superconducting NMR (traditionally 300-400MHz), resolution improved to 0.26Hz (rotating sample).

[0066] Pulse sequence combination: 1H / 13C / COSY / HSQC / HMBC / NOESY full spectrum coverage, with a monosaccharide residue linkage site resolution accuracy of over 95% (traditional methylation-dependent analysis has an accuracy of only 60-70%).

[0067] Sample dissolution: The concentration prepared in D2O is ≥40mg / ml (traditionally <20mg / ml), and the signal-to-noise ratio is improved to 888:1.

[0068] Electron microscopic morphological characterization: Gold sputtering thickness: Nanoscale gold film deposition (10nm) achieves 1nm resolution under Zeiss field emission electron microscope (the resolution of traditional SEM is 3~5nm), which can clearly observe the irregular particles (particle size 50~200nm) and rough topology on the polysaccharide surface.

[0069] The specific extraction process is provided below.

[0070] Example 1 A process for extracting polysaccharides from Gynostemma pentaphyllum includes the following steps: Step 1, Water Extraction: Gynostemma pentaphyllum from Sichuan was crushed and sieved to obtain raw material powder. Water was added to the raw material powder (material-to-liquid ratio of 1g:20mL) for water extraction at 60℃ for 4 hours, with intermittent stirring for 30 minutes each time. The mixture was then filtered to obtain an aqueous solution.

[0071] Step 2, alcohol precipitation: Ethanol was added to the aqueous solution obtained in step 1 in stages (60% ethanol was added first, and 80% ethanol was added second). The alcohol precipitation temperature was 4°C to precipitate the polysaccharide from the aqueous solution. After drying, crude polysaccharide extract was obtained.

[0072] Step 3: Combined impurity removal system (1) Enzymatic hydrolysis to degrade proteins: The crude polysaccharide extract was dissolved in water to prepare an aqueous solution with a mass concentration of 5%. Papain (0.6 g / L, source: Papain9001-73-4Sigma) was added to the aqueous solution of the crude polysaccharide extract and hydrolyzed overnight at room temperature (10 h) at 25°C. After hydrolysis, the hydrolyzed mixture was obtained.

[0073] (2) Protein removal: Collect the aqueous phase of the enzymatic hydrolysis mixture. At 25°C, add chloroform and n-butanol (volume ratio 4:1) to the aqueous phase of the enzymatic hydrolysis mixture. The total volume of chloroform and n-butanol is 1 / 4 of the volume of the aqueous phase of the enzymatic hydrolysis mixture. Shake for 15 min, shake twice, and collect the upper aqueous phase.

[0074] (3) Removal of fat-soluble impurities: Petroleum ether was then added to the aqueous phase for extraction. The volume of petroleum ether added was 1 / 4 of the volume of the aqueous phase. The mixture was shaken for 15 minutes, and then the oil and water were separated and the aqueous phase was collected.

[0075] (4) Adsorption of pigments and other small molecule organic impurities: Add macroporous resin AB-8 (1 / 2 volume) to the aqueous phase and adsorb at room temperature for >12h to carry out adsorption and decolorization.

[0076] (5) Dialyze the supernatant after impurity removal using a 3000Da dialysis bag for 24 hours and then dry it.

[0077] (6) The polysaccharide separation and purification of the dialysis sample was carried out by ion exchange purification (DEAE-52 column) and gel chromatography (Sephadex G-100). During ion exchange purification, the dialysis polysaccharide sample was dissolved in water at a ratio of 1g:50mL. After centrifugation, the supernatant was passed through the ion exchange column with a sample loading amount of 10% of the exchange capacity. The ion column was equilibrated at pH=6.8. Gradient elution was performed using 0~0.5mol / L NaCl aqueous solution. The eluent was collected, dialyzed, and dried.

[0078] For purification by gel chromatography, 1g of the ion-purified polysaccharide sample was added to 20ml of pure water, centrifuged, and the supernatant was passed through a gel chromatography column for separation and purification at a flow rate of 1ml / min. 1.5 column volumes of pure water were used for elution, with each 10ml tube collecting all eluent. The total sugar content of the eluent in each collection tube was determined using the sulfuric acid-phenol method. Specifically, 100ul of diluted polysaccharide supernatant was added to 600ul of sulfuric acid-phenol reagent (5% phenol solution: concentrated sulfuric acid = 1:5 (v / v), the same below), mixed well, and reacted in the dark for 10min. The absorbance was measured at 490nm. A gel purification elution curve was plotted. The elution peak was selected and confirmed (the main peak was selected by default). The eluents from each collection tube corresponding to the same elution peak were combined, concentrated to 1 / 5 of the original volume by rotary evaporation, and then dialyzed against a 3000Da dialysis bag for 48h to remove small molecule components. The polysaccharide was freeze-dried, and the purity of the gel-purified polysaccharide was identified using the sulfuric acid-phenol method. The specific procedure is as follows: Weigh 2 mg of lyophilized polysaccharide sample, dissolve and dilute it with water, take 100 μL of polysaccharide supernatant, add sulfuric acid-phenol reagent, mix well, let it stand for 10 min in the dark, and measure the absorbance at 490 nm.

[0079] Example 2 Compared to Example 1, the water extraction temperature was 62°C.

[0080] Example 3 Compared to Example 1, the water extraction temperature was 58°C.

[0081] Example 4 Compared to Example 1, the amount of papain added was 0.4 g / L.

[0082] Comparative Example 1 Compared with Example 1, the protein removal temperature in step (2) is 30°C.

[0083] Comparative Example 2 Compared with Example 1, the protein removal temperature in step (2) is 80°C.

[0084] The effects of deproteinization temperature (25°C) on protein residue as a single factor are shown in Table 1.

[0085] Table 1. Effect of deproteinization temperature on protein residue. The test methods for the above parameters are as follows: Protein residue rate: determined by the BCA method, using the ThermoPierce™ kit, with a detection wavelength of 562nm.

[0086] Polysaccharide loss rate: The difference in polysaccharide content before and after alcohol precipitation was calculated using the sulfuric acid effective date phenol method.

[0087] Total impurity residue rate: determined by the ignition residue method (dried at 105℃ to constant weight and then ignited at 550℃ for 4 hours).

[0088] As shown in Table 1, the protein removal method at 25℃ in this invention is significantly superior to the traditional boiling water bath method (80℃): the protein residue rate is reduced by 81.9% (12.7% → 2.3%), and the total impurity residue rate is reduced by 79.5%; the polysaccharide loss rate is only 5.1%, indicating that the active structure of polysaccharides is more completely preserved under low temperature conditions; when the temperature fluctuates to 30℃, the protein residue rate increases by 34.8%, proving that 25℃ is a precise control threshold; the traditional high-temperature method results in incomplete protein denaturation and precipitation due to violent phase separation, and the high temperature destroys the polysaccharide-solution hydrogen bond network, increasing the adsorption of impurities. By defining the deproteinization temperature as 25℃ and optimizing the number of shaking cycles (2 times × 15 min), the core advantages of this technology are: maximized impurity removal efficiency, reducing the protein residue rate from the traditional 12.7% to 2.3%, a reduction of 81.9%; improved process stability: eliminating the interference of temperature range fluctuations (25-30℃) on extraction efficiency and ensuring batch-to-batch quality control; and protection of active ingredients: maintaining the integrity of the polysaccharide molecular weight (310±15kDa) and the characteristic peak of β-glycosidic bonds in the infrared spectrum (890cm). -1Strength retention ≥95%.

[0089] Comparative Example 3 Compared to Example 1, the water extraction temperature was 50°C.

[0090] Comparative Example 4 Compared to Example 1, the water extraction temperature was 55°C.

[0091] Comparative Example 5 Compared to Example 1, the water extraction temperature was 65°C.

[0092] Comparative Example 6 Compared to Example 1, the water extraction temperature was 70°C.

[0093] The changes in polysaccharide extraction rate and purity at different water extraction temperatures are shown in Table 2.

[0094] Table 2. Polysaccharide yield and purity data at different water extraction temperatures. As shown in Table 2, at 60℃, the polysaccharide yield reached 8.9% and the purity was 79%, both significantly higher than those of other temperature groups (p<0.01), and no obvious chain breakage was observed. Beyond the 60℃±2℃ range (e.g., 65℃ and 70℃), the polysaccharide yield decreased by 19.9% ​​and 38.2%, respectively, and the molecular weight decreased by 34.3%-49.4%, indicating that high temperatures led to sugar chain degradation and destroyed the active structure.

[0095] Insufficient low-temperature extraction: at 50℃ and 55℃, the yield decreased by 29.8%-12.4% respectively compared to 60℃, possibly due to the low efficiency of cell wall disruption under low-temperature conditions, resulting in insufficient dissolution of active substances.

[0096] The effect of temperature fluctuation on stability: When the temperature fluctuation is ≥5℃ (e.g., 65℃), the yield and purity both decrease significantly (purity decreases by 13.9%), indicating that the process is highly sensitive to temperature and needs to be strictly controlled within the range of 60±2℃.

[0097] Core advantages: By precisely controlling the temperature (60±2℃, which is better than the traditional process of 80-100℃), the dissolution efficiency is improved by using enzymatic hydrolysis and low-temperature phase separation to avoid sugar chain breakage, thus solving the problems of destruction of effective components and high energy consumption in traditional high-temperature processes.

[0098] The chemical composition and structure of the product extracted in Example 1 were identified using the following method: Molecular weight determination was performed using a gel permeation chromatography column combination: Ohpak SB-805 HQ + SB-803 HQ in series, column temperature 45℃ (traditional single-column methods suffer from large temperature fluctuations and low resolution), elution flow rate: 0.6 ml / min (optimized flow rate to balance separation efficiency and time cost), and detection coupling: multi-angle laser light scattering (DAWN HELEOS II) coupled with differential refractive index (Optilab T-rEX). Absolute molecular weight was directly calculated using the Mark-Houwink equation, with an error rate <5% (traditional single differential method error >15%). The raw data results are shown below. Figure 1 As shown. Figure 1 In this context, Mn represents the number-average molecular weight; Mw represents the weight-average molecular weight; Mz represents the z-average molecular weight; Mp represents the peak molecular weight, which is the molecular weight of the most abundant component fragment; Mw / Mn represents polydispersity or polydispersity index, which is the molecular weight distribution width index; and R represents the root mean square (RMS) radius, which describes the mass distribution around the center of mass.

[0099] Multidimensional NMR Spectroscopy Analysis: Scanning parameters: 600MHz superconducting NMR (traditionally 300-400MHz), resolution improved to 0.26Hz (rotating sample).

[0100] Pulse sequence combination: 1H / 13C / COSY / HSQC / HMBC / NOESY full spectrum coverage, with a monosaccharide residue linkage site resolution accuracy of over 95% (traditional methylation-dependent analysis has an accuracy of only 60-70%).

[0101] Sample dissolution: Concentrations ≥40 mg / ml were prepared in D2O (traditionally <20 mg / ml), improving the signal-to-noise ratio to 888:1. NMR results, among which... Figure 2 This is a one-dimensional proton spectrum, with the corresponding labels indicating the hydrogen position (e.g., position X) of the sugar residue. Example: A1 in... 1 The H NMR spectrum represents the H1 of sugar residue A. Figure 3 This is a one-dimensional carbon spectrum, with the corresponding labels indicating the carbon number of the sugar residue. Example: A1 in... 13 The C1 of sugar residue A is represented in the CNMR spectrum. Figure 4 For COSY, the corresponding label in the figure represents the adjacent hydrogen number of the sugar residue. For example, A1-A2 in the COSY spectrum represent the H1 / H2 cross peaks of sugar residue A. Figure 5 For HMBC, the corresponding labels in the figure are: the hydrogen number of the sugar residue connected to the carbon number of the connected sugar residue. For example, B1-A4 in the HMBC spectrum represents the cross peak between H1 of sugar residue B and C4 of sugar residue A. Figure 6For HSQC, the labels in the figure indicate the hydrogen and carbon positions of the sugar residue. For example, A1 in the HSQC spectrum represents the H1 / C1 cross peak of sugar residue A. Figure 7 For NOESY, the corresponding labels in the figure are: the hydrogen number of the sugar residue connected to the hydrogen residue connected to the hydrogen residue. For example, A1-A4 in the NOESY spectrum represent the cross peaks between H1 and H4 of sugar residue A. Ultimately, it is concluded that the monosaccharides of Gynostemma pentaphyllum polysaccharide in this invention are galactose and arabinose, and the structural formula of Gynostemma pentaphyllum polysaccharide is deduced based on the above characterization:

[0102] ; The number-average molecular weight of Gynostemma pentaphyllum polysaccharide is 65.8 kDa, the weight-average molecular weight is 77.0 kDa, and the z-average molecular weight is 97.9 kDa.

[0103] Electron microscopic morphological characterization of different sizes and locations of Gynostemma pentaphyllum polysaccharides, such as Figure 8 As shown.

[0104] Gold coating thickness: Nanoscale gold film deposition (10nm), achieving 1nm resolution under a Zeiss field emission microscope (conventional SEM resolution is 3-5nm), allowing clear observation of irregular particles on the polysaccharide surface (particle size). (50-200nm) and rough topology.

[0105] To verify the activity of the Gynostemma pentaphyllum polysaccharide obtained by using Sichuan-grown Gynostemma pentaphyllum as raw material and improving the extraction process, this invention detects the α-amylase inhibitory activity of the Gynostemma pentaphyllum polysaccharide. The specific test method is as follows: Add 0.2 mL of the *Gynostemma pentaphyllum* polysaccharide prepared in Example 1, or acarbose, or traditional *Gynostemma pentaphyllum* polysaccharide, to 0.5 mL of 10 U / mL α-amylase solution. Then, mix and activate in a 37°C water bath for 5 min. Add 0.3 mL of a 2% (w / w) starch solution to the system, react at 37°C for 10 min, then add 0.5 mL of 3,5-dinitrosalicylic acid, heat to boiling for 5 min to inactivate the enzyme, cool to room temperature, and add 5 mL of water. Measure the absorbance at 540 nm (A1). Replace the polysaccharide with deionized water and measure the absorbance at 540 nm (A0). Replace the α-amylase solution with deionized water and measure the absorbance at 540 nm (A2). The traditional *Gynostemma pentaphyllum* polysaccharide is obtained according to the method disclosed in Patent Publication No. CN 101717452 B, "A Method for Preparing Neutral Polysaccharides from *Gynostemma pentaphyllum*".

[0106] α-Amylase activity inhibition rate = [1 - (A1 - A2) / A0] × 100%.

[0107] The calculated α-amylase activity inhibition rate is shown in Table 3.

[0108] Table 3. Inhibition rate of α-amylase activity in each group As shown in Table 3, the traditional Qinba Gynostemma pentaphyllum polysaccharide has an inhibition rate of about 48% against acarbose, while the Gynostemma pentaphyllum polysaccharide obtained in this application has an inhibition rate of about 78% against acarbose. The Gynostemma pentaphyllum polysaccharide obtained in this invention shows excellent hypoglycemic effects, and its effect is significantly improved compared with that of Gynostemma pentaphyllum polysaccharide prepared by traditional extraction processes.

[0109] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, it is intended to include any modifications and variations that fall within the scope of the claims and their equivalents.

Claims

1. Gynostemma pentaphyllum polysaccharide, characterized in that, The polysaccharide obtained from Gynostemma pentaphyllum produced in Sichuan has the following structural formula: ； The number-average molecular weight of Gynostemma pentaphyllum polysaccharide is 65.8 kDa, the weight-average molecular weight is 77.0 kDa, and the z-average molecular weight is 97.9 kDa.

2. The extraction process of Gynostemma pentaphyllum polysaccharide according to claim 1, characterized in that, Includes the following steps: Gynostemma pentaphyllum from Sichuan is crushed and sieved to obtain raw powder; water is added to the raw powder for water extraction, and then filtered to obtain an aqueous solution; Ethanol was added to the aqueous solution to precipitate the polysaccharide from the aqueous solution. The precipitate was dried to obtain a crude polysaccharide extract. The crude polysaccharide extract was dissolved in water, and papain was added to the aqueous solution of the crude polysaccharide extract for enzymatic hydrolysis. After enzymatic hydrolysis, an enzymatic hydrolysate was obtained. Collect the aqueous phase of the enzymatic hydrolysis mixture, add chloroform and n-butanol to the aqueous phase of the enzymatic hydrolysis mixture, shake and mix evenly, collect the aqueous phase, then add petroleum ether to the aqueous phase for extraction and degreasing, mix and shake, then separate the oil and water, add adsorption resin to the separated aqueous phase for impurity removal. The supernatant after impurity removal was dialyzed and dried. The polysaccharide sample after dialyzing was then separated and purified to obtain Gynostemma pentaphyllum polysaccharide.

3. The extraction process of Gynostemma pentaphyllum polysaccharide according to claim 2, characterized in that, During water extraction, the material-to-liquid ratio is 1g:10mL~20mL, the extraction temperature is 60℃±2℃, and the extraction time is 3.5h~4h.

4. The extraction process of Gynostemma pentaphyllum polysaccharide according to claim 2, characterized in that, A two-step ethanol precipitation method is used, in which 60% to 80% of the volume of the aqueous solution is added each time, and the precipitation temperature is 3°C to 4°C.

5. The extraction process of Gynostemma pentaphyllum polysaccharide according to claim 2, characterized in that, The mass concentration of the aqueous solution of crude polysaccharide extract is 4%~6%, and the ratio of papain to the aqueous solution of crude polysaccharide extract is 0.4g~0.6g:600ml~1L.

6. The extraction process of Gynostemma pentaphyllum polysaccharide according to claim 2, characterized in that, The treatment with chloroform and n-butanol was carried out at room temperature. The volume ratio of the total volume of chloroform and n-butanol to the volume of the aqueous phase of the enzymatic hydrolysis mixture was 0.2~0.3:1, and the volume ratio of chloroform and n-butanol was 3.5~4.5:

1. When using petroleum ether for extraction and degreasing, the volume ratio of petroleum ether to the aqueous phase is 0.2~0.3:1; When using adsorption resin for impurity removal, the adsorption resin is AB-8 macroporous resin, and the volume of AB-8 macroporous resin is 1 / 2 of the volume of the aqueous phase. After thorough mixing, adsorption is performed at room temperature.

7. The extraction process of Gynostemma pentaphyllum polysaccharide according to claim 2, characterized in that, The dialysis uses a 3000Da dialysis bag.

8. The extraction process of Gynostemma pentaphyllum polysaccharide according to claim 2, characterized in that, Purification was performed using a combination of ion exchange and gel chromatography. The ion column was equilibrated at pH 6.7–6.9, and gradient elution was performed using 0–0.5 mol / L NaCl aqueous solution. The eluent was collected, dialyzed, and then dried.

9. The extraction process of Gynostemma pentaphyllum polysaccharide according to claim 8, characterized in that, When using gel chromatography for purification, add water to the ion-exchange purified sample, centrifuge, and take the supernatant for separation and purification through a gel chromatography column at a flow rate of 1~1.5 mL / min; elute with pure water for 1.5~2 column volumes, collect all eluents, dialyze, and dry.

10. The use of the Gynostemma pentaphyllum polysaccharide according to claim 1 in the preparation of a drug for treating diabetes.

Images