A Gardenia Polysaccharide, Its Preparation Method and Application

Abstract

This invention provides a gardenia polysaccharide, its preparation method, and its application in the preparation of drugs for treating pulmonary fibrosis, relating to the fields of traditional Chinese medicine extracts and biomedical technology. This invention extracts a novel polysaccharide structure, GP121, from gardenia. The extraction method for GP121 provided by this invention is simple, with high polysaccharide extraction rate and low production cost. This invention further studies the anti-pulmonary fibrosis bioactivity of GP121 at the cellular level. The results show that GP121 provided by this invention can delay the fibrosis process of MRC-5 cells induced by TGF-β1, thereby slowing the fibrosis process by inhibiting the phosphorylation of Smad3 protein and thus reducing the expression of α-SMA, Collagen I, and FN proteins. GP121 provided by this invention can serve as a potential therapeutic drug for pulmonary fibrosis, providing a new application of gardenia polysaccharide in the treatment of pulmonary fibrosis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extracts and biomedical technology, specifically to a gardenia polysaccharide, its preparation method, and its application in the preparation of drugs for treating pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis (PF) is a fibrotic disease caused by stimulation or influence on lung tissue, leading to extensive inflammatory infiltration, excessive deposition of extracellular matrix, and abnormal proliferation of connective tissue. The incidence of this disease is increasing and it is difficult to cure, easily causing organ structural damage and functional decline, and even endangering life. Currently, commonly used drugs for treating pulmonary fibrosis include anti-fibrotic drugs such as nintedanib, pirfenidone, and glucocorticoid immunosuppressants; however, these drugs can only slow the decline in lung function and have high toxicity and side effects. Therefore, the search for new drugs to treat pulmonary fibrosis is of great significance.

[0003] Gardenia (Gardenia jasminoides Ellis) is the dried, ripe fruit of the Gardenia jasminoides plant, belonging to the Rubiaceae family. The fruit is oblong or elliptical in shape; the surface is reddish-yellow or brownish-red; the pericarp is thin and brittle, slightly glossy; the inner surface is lighter in color and glossy; it has a faint odor and a slightly sour and bitter taste. The dried, ripe fruit is used medicinally. It is cold in nature and bitter in taste, and enters the heart, lung, and triple burner meridians. It has the effects of purging fire and relieving irritability, clearing heat and promoting diuresis, cooling blood and detoxifying. Gardenia contains polysaccharides, iridoids, volatile oils, flavonoids, diterpenoids, and other components, possessing neuroprotective, anti-inflammatory, blood sugar-regulating, hepatoprotective, and choleretic effects. Gardenia polysaccharides, as one of the important pharmacologically active substances in gardenia, have anti-inflammatory, antioxidant, immunomodulatory, and bile acid-regulating biological activities.

[0004] The specific pathogenesis of pulmonary fibrosis remains unclear, and there is an urgent need to find therapeutic drugs with definite efficacy and low toxicity. This invention aims to provide a pure polysaccharide extract from gardenia with a novel structure and to investigate its application in the treatment of pulmonary fibrosis. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides a gardenia polysaccharide, a method for preparing the same, and its application in the preparation of drugs for treating pulmonary fibrosis.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a gardenia polysaccharide, the structural formula of which is shown below:

[0010]

[0011] In the above structure, Galp represents galactose, Glcp represents glucose, Rhap represents rhamnose, GalpA represents galacturonic acid, GlcpA represents galacturonic acid, and Ara represents arabinose.

[0012] Furthermore, the molecular weight range of the gardenia polysaccharide is 1-90 kDa.

[0013] Furthermore, the molecular weight of the gardenia polysaccharide ranges from 30 to 77.5 kDa.

[0014] The second invention provides a method for preparing gardenia polysaccharide, wherein the gardenia polysaccharide is prepared according to the following steps:

[0015] 1) The dried gardenia fruit was pulverized into powder using a pulverizer, and defatted in industrial ethanol to obtain defatted gardenia fruit;

[0016] 2) Extract defatted gardenia from step 1) using boiling water extraction 4-6 times, then combine the extracts, concentrate to one-fifth of the extract volume, centrifuge to remove the precipitate, and retain the supernatant; dialyze the supernatant with running water for more than 72 hours, then let it stand overnight; centrifuge to obtain polysaccharide precipitate, dry at 60℃ to obtain gardenia crude polysaccharide GP;

[0017] 3) Dissolve the crude gardenia polysaccharide obtained in step 2) in deionized water, stir overnight, centrifuge, and load the supernatant onto a DEAE Sepharose™ Fast Flow anion exchange column. Elute sequentially with deionized water, 0.1M, and 0.2M NaCl solutions at a flow rate of 100 mL / min using an automated collector. Take 20 μL from each tube, develop color using the sulfuric acid-phenol method, and measure the absorbance at 490 nm using a microplate reader. Plot an elution curve using absorbance and elution volume. Collect the corresponding polysaccharide fractions according to the elution curves, then concentrate, dialyze, and freeze-dry. Separately, take the crude gardenia polysaccharide and continue the polysaccharide separation and purification using the above method. Finally, combine the polysaccharide fractions eluted twice with 0.1M NaCl and name them GP1. Dissolve GP1 in deionized water, centrifuge at 3500 r / min for at least 10 min, and load the supernatant onto a Sephacryl S-200HR gel column using 0.2M NaCl. The solution was eluted with NaCl at a flow rate of 0.3 mL / min, and the corresponding components were collected using an automatic collector. Elution curves were plotted using the sulfuric acid-phenol method, and the desired components were collected, concentrated, dialyzed, and freeze-dried to obtain gardenia polysaccharide, which was named GP121.

[0018] Furthermore, in step 1) of the above preparation method, the dialysate used for water dialysis is a mixture of extract and 95% ethanol at a volume ratio of 1:3-5.

[0019] Thirdly, the present invention provides the application of Gardenia polysaccharide GP121 in the preparation of drugs for treating pulmonary fibrosis.

[0020] (III) Beneficial Effects

[0021] This invention extracts a novel polysaccharide structure, GP121, from gardenia. The extraction method for GP121 provided by this invention is simple, yields a high extraction rate, and has low production costs. Furthermore, this invention investigates the anti-pulmonary fibrosis bioactivity of GP121 at the cellular level. The results show that GP121 can delay the progression of TGF-β1-induced fibrosis in MRC-5 cells by inhibiting the phosphorylation of Smad3 protein, thereby reducing the expression of α-SMA, Collagen I, and FN proteins. GP121 provided by this invention can serve as a potential therapeutic agent for pulmonary fibrosis, offering a new application of gardenia polysaccharides in the treatment of pulmonary fibrosis. Attached Figure Description

[0022] Figure 1 The HPGPC spectrum of Gardenia polysaccharide GP121.

[0023] Figure 2 The image shows the one-dimensional NMR spectrum of gardenia polysaccharide GP121.

[0024] Figure 3 This is the NMR spectrum of gardenia polysaccharide GP121, where A represents the NMR spectrum of gardenia polysaccharide GP121. 1 1H NMR spectrum; B represents gardenia polysaccharide GP121. 13 C NMR spectrum.

[0025] Figure 4 This indicates the inhibitory effect of Gardenia polysaccharide GP121 on TGF-β1-induced MRC-5 cell proliferation. Compared with the control group, the model group showed... ## P<0.01; *P<0.05; **P<0.01 compared with the model group.

[0026] Figure 5 The effect of Gardenia polysaccharide GP121 on TGF-β1-induced transcription of α-SMA, Collagen I, and Fibronectin (FN) mRNA in MRC-5 cells was investigated. Compared with the control group, the model group showed... ## P<0.01; *P<0.05; **P<0.01 compared with the model group.

[0027] Figure 6 The effect of Gardenia polysaccharide GP121 on the expression of α-SMA, Collagen I, and Fibronectin (FN) proteins induced by TGF-β1 in MRC-5 cells was investigated. Compared with the control group, the model group showed... ## P<0.01; *P<0.05; **P<0.01 compared with the model group.

[0028] Figure 7 The effect of Gardenia polysaccharide GP121 on the delayed fibrosis process of Smad3 protein phosphorylation induced by TGF-β1 in MRC-5 cells was investigated. Compared with the control group, the model group showed... ## P<0.01; *P<0.05; **P<0.01 compared with the model group. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1

[0031] Preparation and determination of gardenia polysaccharide GP121

[0032] 1. Preparation of Gardenia Polysaccharide GP121

[0033] 1) The dried gardenia fruit was pulverized into powder using a pulverizer, and defatted in industrial ethanol to obtain defatted gardenia fruit;

[0034] 2) Extract 3.0 kg of defatted gardenia from step 1) four times using boiling water extraction. Then combine the extracts and concentrate them to one-fifth the volume of the extract. Centrifuge to remove the precipitate. Dialyze the supernatant with running water for more than 72 hours, then let it stand overnight. After centrifugation, the polysaccharide precipitate is obtained and dried at 60℃ to obtain crude gardenia polysaccharide GP. The dialysate used for running water dialysis is a mixture of the extract and 95% ethanol at a volume ratio of 1:3-5.

[0035] 3) Dissolve 50g of Gardenia crude polysaccharide GP obtained in step 2) in 500mL of deionized water, stir overnight, centrifuge, and load the supernatant onto a DEAE Sepharose™ Fast Flow anion exchange column. Elute sequentially with deionized water, 0.1M, and 0.2M NaCl solutions at a flow rate of 100mL / min using an automatic collector. Take 20μL from each tube, develop color using the sulfuric acid-phenol method, and measure the absorbance at 490nm using an ELISA reader. Plot an elution curve using absorbance and elution volume. Collect the corresponding polysaccharide fractions according to the elution curves, then concentrate, dialyze, and freeze-dry. Take another 50g of Gardenia crude polysaccharide GP and continue the polysaccharide separation and purification using the above method. Finally, combine the polysaccharide fractions eluted twice with 0.1M NaCl and name it GP1. Dissolve 200mg of crude polysaccharide GP1 in 5mL of deionized water, centrifuge at 3500r / min for 10min, and load the supernatant onto a Sephacrylase column. The S-200HR gel column was used for elution with 0.2M NaCl eluent at a flow rate of 0.3 mL / min. The corresponding components were collected using an automatic collector. Elution curves were plotted using the sulfuric acid-phenol method. The desired components were collected, concentrated, dialyzed, and freeze-dried to obtain pure gardenia polysaccharide, which was named GP121.

[0036] 2. Determination of Gardenia polysaccharide GP121

[0037] 2.1 Experimental Objective

[0038] The molecular weight, monosaccharide composition, sugar residue linkage mode and structure of Gardenia polysaccharide GP121 were determined by HPGPC, HPLC, GC-MS and NMR.

[0039] 2.2 Experimental Materials

[0040] 2.2.1 Instruments

[0041] GL-2M centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd.); TGL-16C centrifuge (Shanghai Antingke Instrument Factory); 1ES-224DS electronic analytical balance (Tianjin De'ant Sensor Technology Co., Ltd.); vortex oscillator (LICHEN Ltd.); Agilent 1260 high performance liquid chromatography system (Agilent Technologies).

[0042] 2.2.2 Reagents

[0043] PMP (1-phenyl-3-methyl-5-pyrazolone), trifluoroacetic acid, and CMC (1-cyclohexyl-3-(2-morpholinylethyl)carbodiimide methyl-p-toluenesulfonate) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Other chemicals and reagents were analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0044] 2.3 Methods

[0045] 2.3.1 Determination of the molecular weight of Gardenia polysaccharide GP121

[0046] Polysaccharide GP121 in series with KS-802 (resistivity limit 1x10) 4 Da) and KS-804 (resistance limit is 4x10) 5 The chromatographic conditions for the characteristic chromatograms on the gel column were as follows: mobile phase: 0.1M NaNO3; flow rate: 0.6mL / min; Agilent 1260 liquid chromatograph; column temperature: 35℃; detectors: differential detector and ultraviolet detector.

[0047] 2.3.2 Determination of Monosaccharide Composition in Gardenia Polysaccharide GP121

[0048] Monosaccharide composition was determined by HPLC. An Agilent 1260 HPLC system was used, with a flow rate of 1 mL / min, a column temperature of 25 °C, a UV detection wavelength of 254 nm, and a sample injection volume of 10 μL / sample.

[0049] 2.3.3 Determination of GP121 Methylation Linkage Mode of Gardenia Polysaccharide

[0050] Monosaccharide composition analysis revealed a small amount of acidic monosaccharides in the polysaccharide; therefore, the Needs method was used for methylation experiments. First, 10 mg of the dried sample was dissolved in 2 mL of dimethyl sulfoxide. Next, 100 mg of sodium hydroxide powder was rapidly added, and the mixture was stirred at room temperature for 2.5 hours. Then, 1 mL of iodomethane was slowly added dropwise in an ice bath to induce methylation. The reaction mixture was stirred at room temperature for another 2.5 hours, followed by quenching with 2 mL of distilled water. After extraction, the methylated polysaccharide was hydrolyzed at 110 °C with 2 M trifluoroacetic acid for 4 hours. Finally, acetylation was performed at 100 °C for 1.5 hours to obtain partially methylated acetylated aldosterones (PMAAs), which were then analyzed by gas chromatography-mass spectrometry.

[0051] 2.3.4 NMR determination of Gardenia polysaccharide GP121

[0052] Take 30 mg of gardenia polysaccharide GP121, dissolve it in 0.5 mL of D2O, add 5 μL of acetone as an internal standard (δ = 2.29 ppm, δ = 31.5 ppm), and measure the one-dimensional nuclear magnetic resonance spectrum on a Bruker AVANCE III 500M NMR spectrometer.

[0053] 3 Results

[0054] like Figure 1 The HPGPC spectrum of GP121 shown is a single symmetrical peak, and its molecular weight is calculated to be 77.5 kDa.

[0055] like Figure 2 The results showed that, based on the total amount of sugar, Gardenia polysaccharide GP121 mainly contains galactose (35-42.88%), arabinose (35-44.45%), rhamnose (3-6.24%), galacturonic acid (2-4.61%), and glucose (1-1.81%).

[0056] The methylation results are shown in Table 1. The GP121 sugar residue linkage modes include fourteen modes: T-Ara, 1,3-Ara, 1,5-Ara, T-Rha, 1,2-Rha, T-Gal, 1,3,5-Ara, 1,2,4-Rha, 1,4-Gal, 1,4-GlcpA, 1,3-Gal, 1,6-Glc, 1,6-Gal, and 1,3,6-Gal.

[0057] Table 1. Methylation results of GP121

[0058] Linkages Molar Rato% T-Ara 11.24 1,3-Ara 9.79 1,5-Ara 18.41 T-Rha 0.99 1,2-Rha 2 T-Gal 10.43 1,3,5-Ara 3.26 1,2,4-Rha 3.38 1,4-Gal 19.09 1,4-GlcpA - 1,3-Gal 5.46 1,6-Glc 2.01 1,6-Gal 4.69 1,3,6-Gal 9.25

[0059] Figure 3 A represents polysaccharide GP121. 1 The 1,6-H NMR spectra show that the chemical shifts of H1-H6 in the 1,6-linked galactan backbone are 4.55 ppm, 3.62 ppm, 3.71 ppm, 3.95 ppm, 3.84 ppm, and 3.85 ppm, respectively. Figure 3 B represents polysaccharide GP121. 13 The CNMR spectrum shows C1-C6 chemical shifts of 104.41 ppm, 72.05 ppm, 73.57 ppm, 69.89 ppm, 74.01 ppm and 70.19 ppm.

[0060] The structure of gardenia polysaccharide GP121 prepared in this embodiment is shown below:

[0061]

[0062] Example 2

[0063] This embodiment provides the effect of Gardenia polysaccharide GP121 on TGF-β1-induced fibrosis in MRC-5 human embryonic lung fibroblasts, and is used to evaluate the application prospects of Gardenia polysaccharide GP121 in the preparation of drugs for treating pulmonary fibrosis.

[0064] 1. Experimental Materials

[0065] The BCA protein kit was purchased from Shanghai Solarbio Biotechnology Co., Ltd. (Shanghai, China); the high-sensitivity enhanced chemiluminescence assay (ECL) was purchased from Shanghai Yisheng Biotechnology Co., Ltd. (Shanghai, China); all primary and secondary antibodies were purchased from Beijing Bio-Sen Biotechnology Co., Ltd. (Beijing, China); all reagents used in the experiments were analytical or chromatographic grade.

[0066] 2. Experimental Grouping and Treatment

[0067] MRC-5 cells were seeded in 96-well or 6-well plates. Control group, model group, and groups treated with different doses of Gardenia polysaccharide GP121 (0 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, 1000 μg / mL) were set up. After overnight incubation and cell adhesion, the culture medium was replaced with a medium containing 1% fetal bovine serum for 6 hours of starvation. The medium was then replaced with fresh complete culture medium. Except for the control group, TGF-β1 (10 ng / mL) was added to all other groups. The Gardenia polysaccharide GP121 treatment groups were treated with different concentrations of polysaccharide solution to investigate the effect of Gardenia polysaccharide GP121 on TGF-β1-induced fibrosis in MRC-5 human embryonic lung fibroblasts.

[0068] 3. Effect of Gardenia polysaccharide GP121 on TGF-β1-induced proliferation rate of MRC-5 human embryonic lung fibroblasts.

[0069] MRC-5 cells were seeded in 96 blanks and subjected to experiments according to the experimental groups and treatments in step 2. MTT (0.5 mg / mL) was added to each well and incubated for 4 h. The culture medium was then discarded, and the cells were washed three times with PBS. 150 μL of DMSO was added to each well and the cells were shaken for 15 min. The absorbance was then measured at 570 nm.

[0070] 4. Detection of fibrosis marker mRNA by real-time quantitative PCR

[0071] Cell experiments were performed using the previous method. RNA was extracted using TRIZO, and then reverse transcribed into cDNA using a reverse transcription kit for qRT-PCR detection. The program consisted of 40 cycles: 95℃ pretreatment for 20 seconds, followed by 95℃ for 15 seconds and 58℃ for 20 seconds. Primer sequences are shown in Listing 2.

[0072] Table 2 Primer sequences

[0073]

[0074] 5. Detect the expression of relevant proteins in lung tissue.

[0075] Cellular proteins were lysed using RIP protein lysis buffer, and protein concentrations were determined using a BCA protein kit. Loading buffers were adjusted to the same concentration. 20 μg of protein was loaded onto each lane, and proteins were separated by SDS-PAGE electrophoresis. The protein was transferred to a PVDF membrane, blocked with 5% skim milk, and incubated with primary antibody overnight at 4°C. After washing, the membrane was incubated with secondary antibody for 1 hour, and protein expression was detected using enhanced chemiluminescence (ECL). All data were quantified and normalized using Image software.

[0076] 6. Statistical Analysis

[0077] All statistical analyses were performed using GraphPadPrism8 software (GraphPad software, San Diego, CA, USA). All data are presented as mean ± standard deviation (SD). Comparisons were performed using the Student's test, and one-way ANOVA was used to measure p-values ​​for differences between groups.

[0078] 7 Results

[0079] like Figure 4 As shown, compared with the control group, TGF-β1 significantly induced fibrosis proliferation in MRC-5 human embryonic lung fibroblasts, with a highly significant difference (p<0.01). Treatment with different concentrations of Gardenia polysaccharide GP121 significantly inhibited TGF-β1-induced fibrosis proliferation in MRC-5 human embryonic lung fibroblasts in a dose-dependent manner. These results indicate that the Gardenia polysaccharide GP121 provided in this invention has a significant inhibitory effect on TGF-β1-induced abnormal proliferation of MRC-5 cells.

[0080] α-SMA, FN, and Collagen I are three factors associated with cell fibrosis in MRC-5 cells. We used qRT-PCR to verify the effects of the gardenia polysaccharide GP121 provided in this invention on these three factors. The results are as follows: Figure 5-6 As shown, compared with the blank group, TGF-β1 significantly increased the mRMA and protein expression levels of α-SMA, FN, and Collagen I (p<0.01); compared with the model group, the mRMA and protein expression levels of α-SMA, FN, and Collagen I in the Gardenia polysaccharide GP121 treatment group were significantly decreased (p<0.01). These results indicate that the Gardenia polysaccharide GP121 provided by this invention can significantly reduce the expression of α-SMA, FN, Collagen I, and proteins related to fibrosis, thereby inhibiting cell fibrosis.

[0081] To further investigate the mechanism by which gardenia polysaccharide GP121 reduces the expression of fibrosis proteins, we examined the phosphorylation expression of Smad3 protein in MRC-5 cells after treatment with gardenia polysaccharide GP121. The results are as follows: Figure 7 As shown in Figures AB, compared with the control group, the phosphorylation expression of Smad3 protein and the expression level of the fibrosis marker α-SMA in MRC-5 cells of the model group were significantly increased (p<0.01); compared with the model group, the phosphorylation expression of Smad3 protein and the expression level of the fibrosis marker α-SMA in MRC-5 cells of the Gardenia polysaccharide GP121 treatment group were significantly decreased (p<0.01). These results indicate that the Gardenia polysaccharide GP121 provided by this invention can inhibit the phosphorylation of Smad3 protein, thereby reducing the expression of the fibrosis marker α-SMA. The Gardenia polysaccharide GP121 may exert its anti-pulmonary fibrosis effect by inhibiting the phosphorylation of Smad3 protein.

[0082] 8 Conclusions

[0083] The gardenia polysaccharide GP121 provided by this invention has the effect of delaying the TGF-β1-induced fibrosis process in MRC-5 cells. It slows the fibrosis process by inhibiting the phosphorylation of Smad3 protein, thereby reducing the expression of α-SMA, Collagen I, and FN proteins. The gardenia polysaccharide GP121 provided by this invention can serve as a potential therapeutic agent for pulmonary fibrosis, providing a new application of gardenia polysaccharide in the treatment of pulmonary fibrosis.

[0084] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gardenia polysaccharide, characterized in that, The structural formula of the gardenia polysaccharide is shown below: R2=T-Ara-(1"[3)-Ara-(1]3" R3=T-Rha-(1"4)-GlcpA-(1′ n＝5 In the above structure, Galp represents galactose, Glcp represents glucose, Rhap represents rhamnose, GalpA represents galacturonic acid, GlcpA represents galacturonic acid, and Ara represents arabinose.

2. The gardenia polysaccharide according to claim 1, characterized in that, The molecular weight range of the gardenia polysaccharide is 1-90 kDa.

3. The gardenia polysaccharide according to claim 1, characterized in that, The molecular weight range of the gardenia polysaccharide is 30-77.5 kDa.

4. The method for preparing gardenia polysaccharide according to any one of claims 1 to 3, characterized in that, The gardenia polysaccharide was prepared according to the following steps: 1) The dried gardenia fruit was pulverized into powder using a pulverizer, and defatted in industrial ethanol to obtain defatted gardenia fruit; 2) Extract defatted gardenia from step 1) using boiling water extraction 4-6 times, then combine the extracts, concentrate to one-fifth of the extract volume, centrifuge to remove the precipitate, and retain the supernatant; dialyze the supernatant with running water for more than 72 hours, then let it stand overnight; centrifuge to obtain polysaccharide precipitate, dry at 60℃ to obtain gardenia crude polysaccharide GP; 3) Dissolve the crude gardenia polysaccharide obtained in step 2) in deionized water, stir overnight, centrifuge, and load the supernatant onto a DEAE Sepharose™ Fast Flow anion exchange column. Elute sequentially with deionized water, 0.1M, and 0.2M NaCl solutions at a flow rate of 100 mL / min using an automated collector. Take 20 μL from each tube, develop color using the sulfuric acid-phenol method, and measure the absorbance at 490 nm using a microplate reader. Plot an elution curve using absorbance and elution volume. Collect the corresponding polysaccharide fractions according to the elution curves, then concentrate, dialyze, and freeze-dry. Separately, take the crude gardenia polysaccharide and continue the polysaccharide separation and purification using the above method. Finally, combine the polysaccharide fractions eluted twice with 0.1M NaCl and name them GP1. Dissolve GP1 in deionized water, centrifuge at 3500 r / min for at least 10 min, and load the supernatant onto a Sephacryl S-200HR gel column using 0.2M NaCl. The solution was eluted with NaCl at a flow rate of 0.3 mL / min, and the corresponding components were collected using an automatic collector. Elution curves were plotted using the sulfuric acid-phenol method, and the desired components were collected, concentrated, dialyzed, and freeze-dried to obtain gardenia polysaccharide, which was named GP121.

5. The method for preparing gardenia polysaccharide according to claim 4, characterized in that, In step 1), the dialysate used for water dialysis is a mixture of extract and 95% ethanol at a volume ratio of 1:3-5.

6. The use of gardenia polysaccharide as described in claim 1 in the preparation of a drug for delaying the progression of TGF-β1-induced fibrosis in MRC-5 cells.

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