Gardenia pectic polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine active polysaccharide and preparation thereof, and particularly relates to gardenia pectic polysaccharide as well as a preparation method and application thereof. The gardenia pectic polysaccharide is composed of rhamnose, galactose, glucose and galacturonic acid. The molar ratio of the rhamnose to the galactose to the glucose to the galacturonic acid is 1: 1.07: 1.52: 2.00. The preparation method provided by the invention is simple in process, green and environment-friendly, and relatively high in polysaccharide purity and yield. The pectic polysaccharide prepared by the invention has the effect of promoting and regulating the proliferation of intestinal flora, particularly obviously promotes the growth of intestinal acid-producing and gas-producing bacteria, and has a wide application prospect in the fields of functional foods and health-care products.

Description

Technical Field

[0001] This application belongs to the technical field of active polysaccharides of traditional Chinese medicine and their preparation technology, specifically relating to a gardenia pectin polysaccharide and its preparation method and application. Background Technology

[0002] Prebiotics are a class of bioactive ingredients that selectively promote the proliferation of beneficial bacteria in the gut, playing an important role in maintaining the balance of the gut microbiota. Pectin polysaccharides, as an important natural prebiotic, have bioactivity closely related to their source and structural characteristics (such as monosaccharide composition, molecular weight, and uronic acid content). Gardenia ( Gardenia jasminoides Gardenia (Gardenia jasminoides) is a plant belonging to the Rubiaceae family and the Gardenia genus. It is mainly distributed in southern my country, such as Jiangxi, Hunan, and Hubei provinces, and possesses significant economic and medicinal value. Its dried, mature fruit is a commonly used traditional Chinese medicine, with a bitter and cold nature, and enters the heart, lung, and triple burner meridians. The *Compendium of Materia Medica* records that gardenia "treats hematemesis, epistaxis, bloody dysentery, hematochezia, hematuria, traumatic blood stasis, typhoid fever relapse, feverish headache, hernia, and burns." It has the effects of purging fire and relieving irritability, clearing heat and dampness, cooling blood and detoxifying. Clinically, it is often used to treat symptoms such as feverish irritability, jaundice with dark urine, painful hematuria, hematemesis due to blood heat, and red, swollen, and painful eyes. In modern industry, gardenia is also widely used. Its fruit is rich in natural pigments, especially gardenia yellow pigment, which is safe, non-toxic, brightly colored, and has good stability, and is widely used in food, beverages, and cosmetics. However, a large amount of yellow gardenia shells generated during gardenia processing are usually discarded as waste, resulting in resource waste.

[0003] In recent years, gardenia polysaccharides have attracted attention due to their various biological activities. Existing studies have shown that gardenia polysaccharides possess pharmacological effects such as anti-nonalcoholic steatohepatitis, anti-pulmonary fibrosis, anti-cholestatic liver injury, and anti-liver fibrosis. For example, existing technologies report a gardenia polysaccharide containing rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose, which can effectively alleviate lipid accumulation and liver fibrosis; other studies have isolated a novel polysaccharide GP121 from gardenia, confirming that it can delay the progression of pulmonary fibrosis by inhibiting Smad3 phosphorylation; and patents indicate that gardenia polysaccharides can improve ANIT / DDC-induced cholestatic liver injury and CCl4-induced liver fibrosis, suggesting its potential application in the field of liver protection. Furthermore, existing technologies have achieved the co-production of geniposide, gardenia yellow pigment, and gardenia polysaccharides using a high-pressure ultrasound-assisted method, improving the comprehensive utilization rate of raw materials.

[0004] However, the aforementioned existing technologies share significant limitations: the raw materials used are all whole gardenia fruits or the pulp after shelling, and there is no systematic research on extracting functional polysaccharides using gardenia peel as the sole or primary raw material. Gardenia peel yields a large surplus after pigment and geniposide extraction, and the structural characteristics, physicochemical properties, and biological activities of its polysaccharide components have not been fully explored. Furthermore, currently disclosed methods for preparing gardenia polysaccharides generally suffer from low molecular weight (e.g., average molecular weight of only 3.5–6.5 kDa) and complex separation and purification techniques.

[0005] Therefore, conducting targeted extraction, structural analysis, and functional evaluation of polysaccharides from gardenia shells—a long-neglected byproduct—not only helps expand the high-value utilization pathways of gardenia resources but also fills the gap in existing technologies for research on polysaccharides derived from fruit shells. Transforming gardenia shells into polysaccharide products with well-defined structures and biological activities aligns with the development concepts of green, low-carbon, and circular economy, and also provides a new material basis and technical support for the development of novel functional foods, pharmaceuticals, or cosmetics. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a gardenia pectin polysaccharide, its preparation method, and its application. Specifically, the following technical solution is adopted: In a first aspect, the present invention provides a gardenia pectin polysaccharide, wherein the gardenia pectin polysaccharide is composed of rhamnose, galactose, glucose and galacturonic acid; wherein the molar ratio of rhamnose, galactose, glucose and galacturonic acid is 1:1.07:1.52:2.00.

[0007] As a further preferred embodiment, the total uronic acid content in the gardenia pectin polysaccharide is 75%~95%.

[0008] As a further preferred embodiment, the weight-average molecular weight of the gardenia pectin polysaccharide is 200 kDa to 1000 kDa.

[0009] Secondly, the present invention provides a method for preparing the above-mentioned gardenia pectin polysaccharide, comprising the following steps: Gardenia fruit shells were crushed into gardenia fruit shell powder, then ethanol solution was added and stirred for 2 h to 3 h. The extraction was repeated 1 to 2 times. The residue was then filtered. The residue was extracted with water, centrifuged, reconstituted, and concentrated by rotary evaporation to obtain a concentrated solution. The concentrate was subjected to alcohol precipitation and dialysis to obtain the gardenia pectin polysaccharide.

[0010] As a further preferred embodiment, the ratio of gardenia fruit shell powder to ethanol solution is 1g:10mL.

[0011] As a further preferred embodiment, the volume ratio of the concentrated solution to the ethanol solution during alcohol precipitation is 1:3.

[0012] As a further preferred embodiment, the alcohol precipitation time is 10 h to 15 h.

[0013] As a further preferred embodiment, the dialysis uses a dialysis bag with a molecular weight cutoff of 8000 Da to 14000 Da.

[0014] Thirdly, the present invention provides the application of the above-mentioned gardenia pectin polysaccharide in the preparation of products that regulate intestinal flora.

[0015] As a further preferred embodiment, the product includes health supplements, food, or medicine.

[0016] The beneficial effects of this invention are as follows: Existing methods for preparing gardenia polysaccharides typically use whole or whole gardenia fruit as raw material. After extraction, purification methods such as ethanol fractionation or column chromatography are required to obtain high-purity components. These processes are cumbersome and costly in terms of time and money. This invention provides a method for extracting high-purity gardenia pectin polysaccharides from gardenia fruit shells. The method is simple to operate, achieves a polysaccharide extraction rate of 2%–7%, and has low production costs. This invention also investigated the activity of the extracted high-purity gardenia pectin polysaccharides in regulating intestinal flora. Experimental results show that the high-purity gardenia pectin polysaccharides provided by this invention can promote intestinal flora growth and lower the pH of the fermentation broth in an in vitro fermentation model. It is gradually decomposed and utilized within 0–48 hours, and can be used as an effective component for regulating intestinal flora in products promoting intestinal health. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The figure shows the high performance liquid chromatography gel elution curve of gardenia pectin polysaccharide.

[0019] Figure 2 The image shows the results of the monosaccharide composition test for gardenia pectin polysaccharide. Detailed Implementation

[0020] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0021] Example 1 A method for preparing high-purity gardenia pectin polysaccharide, specifically including the following steps: (1) Remove the shell from the dried gardenia fruit and separate it into fruit and shell. Crush the fruit and set aside. Weigh 200 g of gardenia shell powder and add 95% ethanol at a ratio of 10:1 (mg / mL). Stir magnetically for 2 h, filter, and rinse the residue with water.

[0022] (2) Add 2L of distilled water to the residue in step (1), stir magnetically at room temperature for 3 h, extract twice, filter and combine the extracts, concentrate by rotary evaporation to 1000 mL, add 3 times the volume of anhydrous ethanol solution, precipitate with alcohol for 12 h, centrifuge at 4000 rpm for 20 min, reconstitute the precipitate with distilled water, concentrate by rotary evaporation, dialyze for 48 h (molecular weight cutoff: 8000~14000 Da), freeze dry to obtain gardenia pectin polysaccharide, with a yield of 2.3%.

[0023] Example 2 A method for preparing high-purity gardenia pectin polysaccharide, specifically including the following steps: (1) Remove the shell from the dried gardenia fruit and separate it into fruit and shell. Crush the fruit and set aside. Weigh 199.2 g of gardenia shell powder and add 95% ethanol at a material-to-liquid ratio of 10:1 (mg / mL). Stir magnetically (600 rpm) for 2 h and filter. Repeat the extraction on the residue under the same conditions and rinse with water for later use.

[0024] (2) Add 2.2 L of distilled water to the residue in step (1), stir magnetically at room temperature for 2 h, extract twice, filter and combine the extracts, concentrate by rotary evaporation to 1000 mL, add 3 times the volume of anhydrous ethanol solution, precipitate with alcohol for 12 h, centrifuge at 4000 rpm for 20 min, reconstitute the precipitate with distilled water, concentrate by rotary evaporation, dialyze for 48 h (molecular weight cutoff: 8000~14000 Da), freeze dry to obtain gardenia pectin polysaccharide, with a yield of 6.4%.

[0025] Example 3 A method for preparing high-purity gardenia pectin polysaccharide, specifically including the following steps: (1) Remove the shell from the dried gardenia fruit and separate it into fruit and shell. Crush the fruit and set aside. Weigh 100.0 g of gardenia shell powder and add 95% ethanol at a material-to-liquid ratio of 10:1 (mg / mL). Stir magnetically (600 rpm) for 2 h and filter. Repeat the extraction on the residue under the same conditions and rinse with water for later use.

[0026] (2) Add 1.0 L of distilled water to the residue in step (1), stir magnetically at room temperature for 2 h, extract twice, filter and combine the extracts, concentrate by rotary evaporation to 1000 mL, add 3 times the volume of anhydrous ethanol solution, precipitate with alcohol for 12 h, centrifuge at 4000 rpm for 20 min, reconstitute the precipitate with distilled water, concentrate by rotary evaporation, dialyze for 48 h (molecular weight cutoff: 8000~14000 Da), freeze dry to obtain gardenia pectin polysaccharide, with a yield of 4.8%.

[0027] Example 4 The primary structure of the high-purity gardenia pectin polysaccharide prepared in Example 1 was characterized. The uronic acid content, molecular weight, and monosaccharide composition of gardenia pectin polysaccharide were determined by colorimetry, high performance liquid chromatography-permeation gel chromatography, and ion chromatography.

[0028] Experimental materials: Dextran series standards of different molecular weights (Dextran T-10, Dextran T-40, Dextran T-50, Dextran T-70, Dextran T-500, and blue dextran) were from Pharmacia, USA; 8 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, and fructose) and 2 uronic acid standards (galacturonic acid and glucuronic acid) were from Sigma, USA; phenol, sulfuric acid, etc., were all domestically produced analytical grade.

[0029] Main instruments and equipment: TU-1900 UV-Vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd.), Model 5450 high-performance liquid chromatograph (Hitachi, Japan); ICS 6000 ion chromatograph (Thermo Fisher Scientific, USA). Determination of the content of gardenia pectin polysaccharide uronic acid: The uronic acid content of gardenia polysaccharides was determined using the sulfuric acid-carbazole method with galacturonic acid as the standard. The samples were measured in triplicate, and the average value was taken.

[0030] Determination of molecular weight of gardenia pectin polysaccharide: A 1.0 mg / mL Gardenia pectin polysaccharide GHP solution was prepared using the mobile phase, and its purity was identified by high-performance gel permeation chromatography (HPLC). Chromatographic conditions: Guard column: Ultrahydrogel.TM Guard Column (6×40mm), Ultrahydrogel Analytical Column TM 500 column (7.8 × 300 mm, 10 μm); column temperature: 35℃; mobile phase: aqueous solution (with 0.02% NaNO3 added); flow rate: 0.6 mL / min; injection volume: 20 μL. Detection was performed using a G1362A differential detector at 35℃; data were acquired and analyzed using a ChemStation chromatography workstation.

[0031] Determination of monosaccharide composition of gardenia pectin polysaccharide: Monosaccharide composition was determined using ion chromatography. Three 5.0 mg aliquots of GHP were accurately weighed and placed into sample hydrolysis tubes. 0.5 mL of 12 M H₂SO₄ solution was transferred and stirred in an ice bath for 30 min. Then, 2.5 mL of water was added, and the solution was sealed and hydrolyzed at 100 °C for 2 h. The hydrolysate was brought to a final volume of 50 mL, then diluted a certain factor. The sample hydrolysate was filtered through a 0.22 μm filter membrane before injection for analysis.

[0032] The total uronic acid content of Gardenia pectin polysaccharide GHP-1 was determined to be 86.3 ± 3.3% by the sulfuric acid-carbazole method.

[0033] like Figure 1 As shown, the HPGPC elution curve of Gardenia pectin polysaccharide GHP-1 shows a single symmetrical peak, and its relative molecular mass is calculated to be 832 kDa.

[0034] like Figure 2 As shown, the results indicate that Gardenia pectin polysaccharide GHP-1 is mainly composed of rhamnose (Rha), galactose (Gal), glucose (Glc), and galacturonic acid (GalA), with the corresponding monosaccharide molar ratio being: Rha:Gal:Glc:GalA = 1:1.07:1.52:2.00.

[0035] Example 5 Investigating the effects of gardenia pectin polysaccharide on gut microbiota growth This embodiment uses the effect of gardenia pectin polysaccharide on intestinal microbial growth provided in Example 1 to evaluate the application prospects of gardenia pectin polysaccharide in the preparation of prebiotics and other health products.

[0036] 1. Experimental Materials Peptone; yeast extract; NaCl; KH2PO4; K2HPO4; MgSO4•7H2O; CaCl2•6H2O; NaHCO3; L-cysteine; taurine; vitamin K1; azurite; heme; PBS buffer.

[0037] 2. Experimental Methods 2.1 In vitro fermentation experiment of GHP Each 1L of basal nutrient medium contained 2 g peptone, 2 g yeast extract, 2 g NaHCO3, 5 mg CaCl2, 0.1 g NaCl, 0.04 g KH2PO4, 0.04 g K2HPO4, 0.01 g MgSO4·7H2O, 0.5 g L-cysteine ​​hydrochloride, 0.5 g bile salts, 2 mL Tween 80, and 1 mg resazurin. GHP was added to the basal nutrient medium to a concentration of 10 mg / mL. Fresh fecal samples were obtained from three healthy volunteers who had not experienced digestive system diseases or received antibiotic treatment in the past 90 days. Fresh fecal samples were mixed and a 10% (w / v) fecal suspension was prepared by adding PBS buffer containing 0.1% L-cysteine ​​hydrochloride. The suspension was transferred to an anaerobic glove box and filtered through sterile double-layered 200-mesh gauze; the filtrate was used as fecal inoculum. Fecal filtrate was mixed with GHP-containing culture medium at a ratio of 1:9 and then incubated in an anaerobic incubator at 37°C for 48 h. The basal culture medium served as a control group. Samples were taken at 0, 6, 12, 24, and 48 h, and OD values ​​were measured. 600 Value, gas production and pH.

[0038] 2.2 Determination of gas production, absorbance, and pH At specified time points during fermentation, the anaerobic tube was removed, and the amount of gas produced during fermentation was measured using a 10 mL disposable syringe. The syringe needle was inserted into the tube through the rubber stopper at the top; the height to which the syringe plunger rose represents the gas production during fermentation. The absorbance of the fermentation broth diluted twice with sterile PBS was measured at 600 nm using a microplate reader to monitor the bacterial community in the fermentation medium. The pH of the fermentation broth was measured using a pH meter.

[0039] 3. Experimental Results As shown in Table 1, compared with the blank control group, the gas production of the GHP group increased significantly from 0 to 6 mL after 48 h of fermentation, the absorbance increased significantly at 12 h, and the pH value decreased significantly, indicating that GHP can promote the growth of intestinal probiotics.

[0040] Table 1. OD values ​​of fermentation broth at different culture times in the in vitro fermentation experiment of Gardenia pectin polysaccharide 600 Gas production and pH value As described above, this invention successfully developed a green preparation method for the directional extraction of high-purity pectin polysaccharides from gardenia processing by-products—gardenia fruit shells. This method is simple, requiring only water extraction, alcohol precipitation, and dialysis purification to efficiently obtain gardenia pectin polysaccharides with uniform structure and no protein residue, providing a feasible path for the high-value utilization of gardenia shells. Structural characterization results show that the obtained gardenia pectin polysaccharide GHP has unique structural characteristics: its total uronic acid content is as high as 86.3±3.3%, its weight-average molecular weight is 832 kDa, and it is mainly composed of rhamnose (Rha), galactose (Gal), glucose (Glc), and galacturonic acid (GalA), with a molar ratio of Rha:Gal:Glc:GalA = 1:1.07:1.52:2.00. High-performance gel permeation chromatography shows a single symmetrical peak, indicating excellent product homogeneity.

[0041] The gardenia pectin polysaccharide provided by this invention has the effect of promoting the proliferation of intestinal bacteria. As shown in Table 1, compared with the blank control group, GHP showed significant probiotic properties during the 48-hour fermentation process: gas production increased significantly from 0 mL to 6.0 ± 0.0 mL, and the OD of the fermentation broth increased significantly. 600 The pH value was significantly higher than that of the control group, and the pH value was significantly reduced to 5.07±0.10. These data fully demonstrate that GHP can be effectively utilized by gut microbiota, significantly promote the proliferation of beneficial bacteria, and regulate the acid-base balance of the gut microenvironment.

[0042] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A gardenia pectin polysaccharide, characterized in that, The gardenia pectin polysaccharide is composed of rhamnose, galactose, glucose and galacturonic acid; the molar ratio of rhamnose, galactose, glucose and galacturonic acid is 1:1.07:1.52:2.

00.

2. The gardenia pectin polysaccharide according to claim 1, characterized in that, The total uronic acid content in the gardenia pectin polysaccharide is 75%~95%.

3. The gardenia pectin polysaccharide according to claim 1, characterized in that, The weight-average molecular weight of the gardenia pectin polysaccharide is 200 kDa to 1000 kDa.

4. The method for preparing gardenia pectin polysaccharide according to any one of claims 1-3, characterized in that, Includes the following steps: Gardenia fruit shells were crushed into gardenia fruit shell powder, then ethanol solution was added and stirred for 2 h to 3 h. The extraction was repeated 1 to 2 times. The residue was then filtered. The residue was extracted with water, centrifuged, reconstituted, and concentrated by rotary evaporation to obtain a concentrated solution. The concentrate was subjected to alcohol precipitation and dialysis to obtain the gardenia pectin polysaccharide.

5. The preparation method according to claim 4, characterized in that, The ratio of gardenia fruit shell powder to ethanol solution is 10 mg: 1 mL.

6. The preparation method according to claim 4, characterized in that, The volume ratio of the concentrated solution to the ethanol solution during alcohol precipitation is 1:3, and the precipitation time is 10 h to 15 h.

7. The preparation method according to claim 4, characterized in that, The dialysis was performed using dialysis bags with a molecular weight cutoff of 8000 Da to 14000 Da.

8. The use of gardenia pectin polysaccharide according to any one of claims 1-3 in the preparation of products that regulate intestinal flora.

9. The application according to claim 8, characterized in that, The products include health supplements, food, or medicine.

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