A stigmas of saffron crocus extract enriched in polysaccharides, and a preparation method and application thereof

Abstract

The application discloses a saffron crocus flower part extract rich in polysaccharides and a preparation method and application thereof, and belongs to the field of natural product extraction and medical biotechnology, and solves the technical problems of insufficient utilization of non-medicinal parts of saffron crocus, complex extraction process and insufficient development of related applications in the prior art. The saffron crocus flower part extract rich in polysaccharides is prepared from the saffron crocus flower part remaining after the column head is collected, and the preparation process comprises the following steps: drying and crushing, ethanol pretreatment, hot water extraction, reduced-pressure concentration and alcohol precipitation. In the saffron crocus flower part extract, the total sugar content is greater than or equal to 40% by weight, the sugar acid content is greater than or equal to 40% by weight, and the protein content is less than 1.0% by weight. The saffron crocus flower part extract has the effects of improving intestinal inflammation and protecting intestinal mucosal barrier, and can be used for preparing a medicine for improving or relieving intestinal mucosal barrier damage, and realizes high-value utilization of the non-medicinal parts of saffron crocus.

Description

Technical Field

[0001] This invention relates to the fields of natural product extraction and pharmaceutical biotechnology, specifically to a polysaccharide-enriched saffron flower extract (without stigmas), its preparation method, and its applications. Background Technology

[0002] Saffron ( Crocus sativus Saffron (L.), also known as crocus, is a perennial herb belonging to the genus Crocus in the family Iridaceae. According to the *Pharmacopoeia of the People's Republic of China*, the dried stigma is the medicinal part, possessing properties such as promoting blood circulation, removing blood stasis, cooling the blood, detoxifying, relieving depression, and calming the mind. It can be used for amenorrhea, abdominal masses, postpartum blood stasis, febrile rashes, depression, palpitations, and mania. Besides medicinal uses, saffron can also be used as a food additive, food coloring, and high-grade flavoring.

[0003] In current production processes, only the stigma of the pistil of saffron is used medicinally. Harvesting the stigma results in a large amount of byproducts, including the stigma-free flower parts. These byproducts typically include petals, stamens, and some remaining styles, accounting for approximately 92.6% of the whole flower's weight. Discarding these byproducts directly not only wastes resources but also hinders the high-value utilization of saffron. Existing research indicates that the stigma-free saffron flower parts still contain various active ingredients such as flavonoids, phenolic acids, and polysaccharides, showing potential for further development and utilization.

[0004] Existing technologies have been used to develop non-medicinal parts of saffron. For example, Chinese patent document CN102657747A discloses a method for extracting and purifying flavonoids using saffron by-products as raw materials; Chinese patent document CN114601885A discloses a method for preparing stigma-free saffron flower extract enriched with flavonoids and its application; and Chinese patent document CN109320628A discloses a method for extracting polysaccharides from saffron petals and its application, which involves further separation and purification using various methods after alcohol precipitation to obtain polysaccharide components.

[0005] While the aforementioned existing technologies provide a reference for the development and utilization of active ingredients from saffron by-products, they still have certain shortcomings. On the one hand, some existing technologies use specific tissues such as petals and stamens after separation as raw materials, requiring further separation or screening of the stigma-removed by-products, which increases the raw material processing steps and costs. On the other hand, some technologies introduce multiple separation and purification steps, resulting in a longer process flow and more complex operation, which is not conducive to the large-scale utilization of stigma-removed saffron flower by-products using a simple process.

[0006] In addition, existing technologies are mostly focused on the preparation of flavonoid extracts or petal polysaccharides and their applications in anti-oxidation, hypoglycemia, and lipid reduction. However, there are no systematic reports on technical solutions for preparing polysaccharide-enriched extracts from whole saffron flowers without stigmas.

[0007] Therefore, there is an urgent need to develop a method and application that uses stigma-removed saffron flowers as raw materials, with a relatively simple process, and suitable for resource utilization, so as to realize the high-value utilization of non-medicinal parts of saffron. Summary of the Invention

[0008] This invention addresses the shortcomings and deficiencies of existing technologies by providing a method that utilizes the whole flower of saffron (stigma removed) as raw material, directly using the byproducts after stigma harvesting. This method balances the overall utilization of raw materials, process simplicity, and application value in colitis intervention, thereby improving raw material utilization and reducing raw material processing costs. The polysaccharide-enriched saffron flower extract, along with its preparation method and applications, has the effect of improving colitis-related intestinal mucosal barrier damage.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution: The method for preparing polysaccharide-enriched saffron flower extract provided by the present invention uses the remaining saffron flower parts after harvesting the stigmas as raw materials. After drying and pulverizing, ethanol pretreatment and filtration, the filter residue is collected and dried. The filter residue is then subjected to hot water extraction, vacuum concentration and alcohol precipitation to obtain polysaccharide-enriched saffron flower extract. The extract of saffron flowers without stigmas, by weight fraction, contains ≥40% total sugar, ≥40% uronic acid, and <1.0% protein.

[0010] Preferably, it includes the following steps: (1) Raw material pretreatment: The raw material of saffron flower parts without stigma is dried and then crushed to obtain saffron flower parts without stigma powder; saffron flower parts without stigma are the remaining flower parts after the stigma is harvested, including one or more of the petals, stamens and residual styles; (2) Alcohol pretreatment: Add an aqueous ethanol solution to the stigma-free saffron flower powder and reflux extract, filter, collect the residue and dry it; (3) Water extraction: The filter residue obtained in step (2) is added to water for hot water extraction, followed by solid-liquid separation, and the water extract is collected; (4) Alcohol precipitation: The aqueous extract obtained in step (3) was concentrated under reduced pressure and then ethanol was added for alcohol precipitation. After standing, the precipitate was collected by centrifugation, washed with ethanol, and dried to obtain polysaccharide-enriched saffron flower extract without stigmas.

[0011] Preferably, in step (4), the final volume fraction of ethanol in the mixed system is 70% during alcohol precipitation, and the precipitate is collected by centrifugation after standing at 4°C for 24 h; the precipitate is washed with 75% ethanol ≥ 2 times.

[0012] Preferably, in step (2), the volume fraction of the ethanol aqueous solution used is 60% to 80%, the reflux extraction temperature is 75°C to 85°C, the extraction time is 2h to 4h each time, and the number of reflux extractions is 2 to 3 times.

[0013] Preferably, in step (2), the ratio of stigma-removed saffron flower powder to ethanol aqueous solution is 1 g: (8-15) mL.

[0014] Preferably, in step (3), the filter residue obtained in step (2) is added to water at a material-to-liquid ratio of 1 g:(20-30) mL, and extracted at 95℃-100℃ for 3-4 hours.

[0015] The polysaccharide-enriched saffron flower extract was prepared by the above-mentioned method for preparing the polysaccharide-enriched saffron flower extract. By weight fraction, the total sugar content was ≥40%, the uronic acid content was ≥40%, and the protein content was <1.0%.

[0016] The above-mentioned application of the polysaccharide-enriched saffron flower extract is to use the polysaccharide-enriched saffron flower extract to prepare drugs that improve or alleviate intestinal mucosal barrier damage.

[0017] The above-mentioned application of the polysaccharide-enriched saffron flower extract is to use the polysaccharide-enriched saffron flower extract to prepare a drug for treating colitis, specifically ulcerative colitis or DSS-induced colitis.

[0018] This invention provides a polysaccharide-enriched stigma-free saffron flower extract, its preparation method, and its applications. It possesses the following beneficial effects: (1) The polysaccharide-enriched saffron flower extract without stigma, its preparation method and application, of the present invention use the whole flower of saffron without stigma as raw material, directly utilize the by-products after harvesting the stigma, without the need for further separation of petals, stamens and other tissues, which is conducive to improving the utilization rate of raw materials and reducing the raw material processing cost; and by weight fraction, the total sugar content is ≥40%, the uronic acid content is ≥40%, and the protein content is <1.0%.

[0019] This polysaccharide-enriched saffron flower extract showed good intervention effects in a DSS-induced mouse colitis model, demonstrating the ability to improve or alleviate intestinal mucosal barrier damage, and especially showing promise for improving or alleviating colitis.

[0020] (2) The preparation method of the present invention mainly consists of ethanol pretreatment, hot water extraction and alcohol precipitation. The process is relatively simple and does not require complex purification processes such as dialysis, ion exchange column chromatography and gel column chromatography, which is conducive to the resource utilization of saffron flower by-products without stigma.

[0021] (3) The present invention optimizes the extraction process through single-factor experiments, full-factor experiments, steepest climbing method and face-centered design. The optimal process conditions are clear and the verification results are close to the model prediction values, indicating that the established extraction process has good stability and repeatability. Attached Figure Description

[0022] Figure 1 The figure shows the effect of extraction temperature and material-to-liquid ratio on the yield of polysaccharides enriched from saffron flowers after destigma removal. (1A) is a three-dimensional response surface plot of the effect on the yield of enriched polysaccharides, and (1B) is a contour plot of the effect on the yield of enriched polysaccharides. The horizontal axis represents the extraction temperature (°C), the vertical axis represents the material-to-liquid ratio (g / mL, based on raw material mass / solvent volume), and the Z-axis and color change represent the polysaccharide yield (%).

[0023] Figure 2 Effects of polysaccharide-enriched saffron flower extract (without stigmas) on phenotypic indices in DSS-induced mice; (2A) Body weight change curves of mice in each group; (2B) Disease Activity Index (DAI) score changes; (2C) Representative images of colon morphology in mice in each group; (2D) Statistical analysis of colon length; (2E) Spleen index; (2F) Thymus index. Note: Data are expressed as mean ± standard deviation (n=8); one-way ANOVA was used for comparisons between groups; compared with the normal group, # indicates P<0.05, ## indicates P<0.01; compared with the DSS model group, * indicates P<0.05, ** indicates P<0.01.

[0024] Figure 3 The images show the HE and AB-PAS staining results of DSS-induced mouse colon tissue obtained from the extract of stigma-free saffron flower for enriching polysaccharides; (3A) HE staining results, which are representative images at magnifications of 5×, 10× and 20× respectively; (3B) AB-PAS staining results, which are representative images at magnifications of 5×, 10× and 20× respectively.

[0025] Figure 4 Immunohistochemical staining results of DSS-induced mouse colon tissue using stigma-free saffron flower extract enriched for polysaccharide extraction. (4A) Immunohistochemical staining results of ZO-1 protein; (4B) Immunohistochemical staining results of Occludin protein; (4C) Immunohistochemical staining results of Claudin-1 protein; each figure includes representative images at 5×, 10×, and 20× magnification. Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0027] This invention provides a polysaccharide-enriched saffron flower extract (without stigmas), its preparation method, and its application in the preparation of drugs to improve or alleviate colitis. The method uses the remaining saffron flowers after stigma harvesting as raw material, and obtains a polysaccharide-enriched saffron flower extract through drying and pulverizing, ethanol pretreatment, hot water extraction, vacuum concentration, and alcohol precipitation. By weight fraction, the extract contains ≥40% total sugar, ≥40% uronic acid, and <1.0% protein.

[0028] The total sugar content was determined using the phenol-sulfuric acid method in the examples, as follows: Establishment of the total sugar standard curve and determination of total sugar content The phenol-sulfuric acid method was used, with glucose as a reference, to establish a standard curve and determine the total sugar content.

[0029] (1) Preparation of glucose standard solution: Accurately weigh 10 mg of glucose standard, dissolve it in distilled water and make up to 10 mL to prepare a 1.0 mg / mL glucose standard solution.

[0030] (2) Establishment of standard curve: Take 0 mL, 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL of glucose standard solution respectively, and dilute to 5.0 mL with distilled water and mix well. Take 0.5 mL of each of the above standard solutions into a test tube, add 0.5 mL of 5% phenol solution, mix well, and then quickly add 2.5 mL of concentrated sulfuric acid. Let stand for 10 min, then heat in a boiling water bath for 20 min, cool to room temperature, and measure the absorbance value at a wavelength of 490 nm.

[0031] A standard curve was plotted with glucose concentration on the x-axis and absorbance values ​​on the y-axis. The resulting linear regression equation was: y = 4.291x + 0.0735, with a coefficient of determination of R0. 2 = 0.9999. The results show that within the measured concentration range, there is a good linear relationship between glucose concentration and absorbance value, which can be used for the determination of total sugar content in samples.

[0032] (3) Determination of total sugar content in the sample: Take the enriched polysaccharide extract obtained in Example 1, prepare a 1 mg / mL sample solution, determine it according to the phenol-sulfuric acid method above, and calculate the total sugar content by substituting it into the standard curve.

[0033] Example 1 A method for preparing polysaccharide-enriched stigma-free saffron flower extract includes the following steps: (1) Raw material pretreatment: After drying the stigma-removed saffron flower parts at room temperature, place them in a 50℃ forced-air drying oven to dry to constant weight and then pulverize to obtain stigma-removed saffron flower part powder.

[0034] (2) Alcohol pretreatment: Take 30g of the stigma-free saffron flower powder and add 70% ethanol aqueous solution at a material-to-liquid ratio of 1g:10mL. Reflux and extract twice at 80℃, 3 h each time. After extraction, filter, collect the residue, and dry to constant weight at 35℃.

[0035] (3) Water extraction: Take the filter residue after ethanol pretreatment, crush it, and obtain dried saffron petal powder after ethanol extraction.

[0036] Take 5g of each sample and place them in three similar Erlenmeyer flasks. Add distilled water at a ratio of 1g:24.39mL, stir thoroughly with a glass rod, seal with plastic wrap, and place in a 97.4℃ water bath for 3.5 h. After extraction, cool to room temperature and filter using a Buchner funnel to separate the solid and liquid, collecting the aqueous extract. Combine the filtrates and repeat the filtration process once more.

[0037] (4) Alcohol precipitation: The clarified filtrate was concentrated to the minimum volume under reduced pressure at 55°C in a rotary evaporator, and 95% ethanol was added to make the final volume fraction of ethanol in the mixture 70%. The mixture was stirred evenly and allowed to stand at 4°C for 24 h.

[0038] After settling, the precipitate was centrifuged at 4℃ and 6000 rpm for 15 min, collected, and washed twice with 75% ethanol. The washed precipitate was freeze-dried and pulverized to obtain the polysaccharide-enriched extract from stigmaless floral saffron. Crocus sativus L.). For ease of description, the "stigma-free saffron flower extract enriched with polysaccharides" will be abbreviated as PFRC below.

[0039] The results showed that the total sugar content in the polysaccharide-enriched saffron flower extract prepared in Example 1 was 46.78%.

[0040] Comparative Example 1 The effect of different extraction methods on the extraction efficiency of polysaccharides from saffron flowers without stigmas.

[0041] To compare the effects of different extraction methods on the extraction efficiency of polysaccharides from stigma-free saffron flowers, water extraction, acid extraction, and alkali extraction were used for extraction, and the total sugar yield of the resulting extracts was determined.

[0042] 1.1 Water Extraction Method: Accurately weigh 0.3 g of the dried saffron powder pretreated with ethanol obtained in step (3) of Example 1 into three clean test tubes. Add distilled water at a material-to-liquid ratio of 1 g: 30 mL and extract in an 80℃ water bath for 3 h. After extraction, cool to room temperature, take the water extract, centrifuge at 10000 rpm for 15 min, and collect the supernatant. Dilute the extract 100 times and determine the total sugar content using the phenol-sulfuric acid method. Calculate the yield and take the average value to obtain a total sugar content of 15.20%.

[0043] 1.2 Acid Extraction Method: Accurately weigh 0.3 g of the powder pretreated with ethanol obtained in step (3) of Example 1, add 0.1 M HCl at a material-to-liquid ratio of 1 g:30 mL, and extract in an 80℃ water bath for 3 h. After extraction, cool to room temperature and neutralize to pH 7 by titration with 1 M NaOH. Then centrifuge at 10000 rpm for 15 min and collect the supernatant. After diluting the extract 100 times, determine the total sugar content using the phenol-sulfuric acid method. The parallel experiment was repeated three times, and the average value was taken to obtain a total sugar content of 12.52%.

[0044] 1.3 Alkaline Extraction Method: Accurately weigh 0.3 g of the powder pretreated with ethanol obtained in step (3) of Example 1, add 1% NaOH at a material-to-liquid ratio of 1 g:30 mL, and extract by stirring at room temperature for 3 h. After extraction, neutralize to pH 7 by titration with 1 M HCl. Then centrifuge at 10000 rpm for 15 min and collect the supernatant. After diluting the extract 100 times, determine the total sugar content using the phenol-sulfuric acid method. The experiment was repeated in parallel 3 times, and the average value was taken to obtain a total sugar content of 14.45%.

[0045] The above measurement results show that the polysaccharide yields obtained by different extraction methods are: acid extraction 12.52%, alkali extraction 14.45%, and water extraction 15.20%. This indicates that under the experimental conditions, the polysaccharide yield obtained by water extraction is higher than that obtained by acid extraction and alkali extraction. Therefore, water extraction is selected as the main extraction method in the subsequent extraction process of this invention.

[0046] Example 2 Single-factor experimental test of polysaccharide extraction process from stigma-free saffron flowers To investigate the effects of different extraction conditions on the yield of polysaccharides from stigma-removed saffron flowers, single-factor experiments were conducted with extraction temperature, extraction time, material-to-liquid ratio, and number of extractions as the factors to be investigated.

[0047] 2.1 Effect of extraction temperature on polysaccharide yield Accurately weigh 0.3 g of the powder pretreated with ethanol obtained in step (3) of Example 1. With a fixed material-to-liquid ratio of 1 g:30 mL, an extraction time of 3 h, and one extraction cycle, gradient extraction was performed at extraction temperatures of 60℃, 70℃, 80℃, 90℃, and 100℃. After extraction, the mixture was cooled to room temperature, the volume was replenished, and the mixture was centrifuged at 8000 rpm for 15 min. The supernatant was collected, and the total sugar content was determined using the phenol-sulfuric acid method. The optimal extraction temperature was screened, and the extraction was repeated three times in parallel, with the average value calculated.

[0048] 2.2 Effect of extraction time on polysaccharide yield Accurately weigh 0.3 g of the powder pretreated with ethanol obtained in step (3) of Example 1, fix the material-liquid ratio at 1 g: 30 mL, the extraction temperature at 90 °C, and the number of extractions at 1 time, and set the extraction times at 1 h, 2 h, 3 h, 4 h and 5 h respectively. Measure the polysaccharide yield under each condition, repeat the process three times in parallel, and calculate the average value.

[0049] 2.3 Effect of material-to-liquid ratio on polysaccharide yield The extraction temperature was fixed at 90℃, the extraction time was 3 h, and the extraction was performed once. The material-to-liquid ratios were set as 1g:10mL, 1g:20mL, 1g:30mL, 1g:40mL, and 1g:50mL. The polysaccharide yield was measured under each condition, and the results were repeated three times in parallel. The average value was calculated.

[0050] 2.4 Effect of extraction times on polysaccharide yield With a fixed material-to-liquid ratio of 1 g:30 mL, an extraction temperature of 90℃, and an extraction time of 3 h, the number of extractions was set to 1, 2, 3, 4, and 5 times. The polysaccharide yield under each condition was measured, and the results were repeated three times in parallel to calculate the average value.

[0051] Table 1 Summary of Single-Factor Experimental Data

[0052] The results showed that changes in extraction temperature, extraction time, and material-liquid ratio all affected the yield of polysaccharides from saffron flowers without stigmas. Among these, extraction temperature had a more significant impact on extraction efficiency, while the number of extractions had a limited effect on improving the overall yield. Therefore, the number of extractions was no longer considered as a primary factor in subsequent optimizations.

[0053] Example 3 Optimization Experiment of Polysaccharide Extraction Process from Saffron Flower Parts without Stigmas Based on single-factor experiments, extraction temperature, extraction time, and solid-liquid ratio were selected as further optimization factors to significantly affect the yield of polysaccharides from stigma-removed saffron flowers. The results showed that extraction temperature, extraction time, and solid-liquid ratio all affected the polysaccharide yield, with extraction temperature having the most significant impact. The number of extractions had a limited effect on improving the yield; therefore, the number of extractions was no longer considered a primary factor in subsequent optimizations.

[0054] To further investigate the main effects and interactions of each factor, a full factorial experiment was conducted to analyze the extraction process. Based on the results of the single-factor experiments, a three-factor, two-level full factorial experiment was performed, setting extraction temperatures of 60℃ and 100℃, extraction times of 1 h and 4 h, and solid-liquid ratios of 1:10 g / mL and 1:30 g / mL, respectively. The results showed that extraction temperature had the most significant impact on polysaccharide yield, while the solid-liquid ratio and extraction time also had some influence. Further supplementary experiments indicated that under conditions of higher temperature, suitable solid-liquid ratio, and longer extraction time, the polysaccharide yield could be further improved, suggesting that the optimal extraction conditions lie in the mid-to-high range of the aforementioned investigation.

[0055] Based on this, the steepest climbing method was used to further approach the optimal extraction region. Starting with the combination with higher yield in the supplementary experiment, the extraction time was fixed at 3.5 h, and the extraction temperature and solid-liquid ratio were gradually adjusted. The results showed that as the temperature gradually increased from 90℃ and the solid-liquid ratio was gradually adjusted from 1:30 g / mL to around 1:24 g / mL, the polysaccharide yield showed a trend of first increasing and then slightly decreasing. Under the conditions of 97.5℃, solid-liquid ratio of 1:24 g / mL, and extraction time of 3.5 h, the yield reached a relatively high level, indicating that this region was close to the optimal extraction range.

[0056] Based on the results of the steepest ascent experiment, a face-centered central composite design (FCCCD) was further employed to optimize the extraction process of polysaccharides from stigma-removed saffron flowers. Extraction temperature and solid-liquid ratio were used as the factors of consideration, with a fixed extraction time of 3.5 h. A response surface model was established using Design-Expert 13 software to optimize the polysaccharide extraction yield. The results showed that both extraction temperature and solid-liquid ratio significantly affected the polysaccharide yield, and there was a certain interaction between the two. Response surface analysis indicated that the optimal extraction region was located around a temperature of approximately 97.5℃ and a solid-liquid ratio of approximately 1:24 g / mL.

[0057] Based on the response surface methodology (RSM) optimization results, the optimal extraction conditions for polysaccharides from stigma-free saffron flowers were determined to be: extraction temperature 97.4℃, solid-liquid ratio 1:24.39 g / mL, and extraction time 3.5 h. Under these conditions, the model predicted a yield of 22.77%, and the validation experiment yielded 22.79%, which is close to the predicted value, indicating that the established optimization model has good reliability and repeatability. Compared with the optimal yield under single-factor conditions, the extraction efficiency increased by approximately 28.4%, demonstrating that the extraction process established through full-factor experiments, the steepest ascent method, and RSM optimization can more effectively improve the extraction efficiency of polysaccharides from stigma-free saffron flowers.

[0058] Example 4 Determination of reducing sugar content The reducing sugar content of the polysaccharide-enriched saffron flower extract (PFRC) obtained in Example 1 was determined using the DNS method. The reagent used was the DNS assay kit from Beijing Solarbio Science & Technology Co., Ltd., and the procedure was strictly followed according to the kit instructions. To ensure that the sample values ​​fell within the linear range of the standard curve, the samples were diluted 10-fold before measurement.

[0059] The standard curve was prepared as follows: 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of 1 mg / mL glucose standard solution were taken, and each volume was brought to 1.0 mL with distilled water. 2.0 mL of DNS reagent was added to each volume, and the mixture was boiled in a water bath for 5 minutes. After cooling to room temperature, 9.0 mL of distilled water was added, and the absorbance was measured at 540 nm to plot the standard curve. For sample determination, 20 mg of PFER was added to distilled water to make a 20 mg / mL sample stock solution. After a 10-fold dilution, a sample dilution was obtained. 1.0 mL of the sample dilution was taken, and 2.0 mL of DNS reagent was added. The remaining procedures were the same as for the standard curve determination. The absorbance was measured at 540 nm, and the reducing sugar content was calculated based on the standard curve. The experiment was repeated three times in parallel, and the average value was taken. The results showed that the reducing sugar content of the polysaccharide-enriched saffron flower extract (without stigmas) was 9.04%.

[0060] Example 5 Protein content determination The protein content of the polysaccharide-enriched, stigma-free saffron flower extract (PFRC) obtained in Example 1 was determined using the Coomassie Brilliant Blue assay. The reagent used was the Nanjing Jiancheng Protein Quantitative Reagent Kit, and the procedure was performed according to the kit instructions. For sample determination, 20 mg of PFRC was added to distilled water to a final volume of 20 mg / ml. Blank wells, standard wells, and assay wells were prepared. 5 μL of distilled water and 250 μL of Coomassie Brilliant Blue reagent were added to the blank wells; 5 μL of protein standard solution and 250 μL of Coomassie Brilliant Blue reagent were added to the standard wells; and 5 μL of sample solution and 240 μL of Coomassie Brilliant Blue reagent were added to the assay wells. After mixing, the mixture was allowed to stand at room temperature for 10 min, and the absorbance of each well was measured at 595 nm. The protein content of the sample was calculated using the formula provided in the kit instructions and converted to a mass fraction. The experiment was repeated three times in parallel, and the average value was taken. The results showed that the protein content of the polysaccharide-enriched, stigma-free saffron flower extract was 0.47%.

[0061] Example 6 Determination of uronic acid content The polysaccharide-enriched saffron flower extract (PFRC) obtained in Example 1 was analyzed using the polysaccharide uronic acid content determination kit (microplate method) from Suzhou Greens Biotechnology Co., Ltd. The specific operation was strictly performed according to the kit instructions. To ensure the sample values ​​were within the linear range of the standard curve, a 20 mg / mL stock solution was prepared and further diluted 5-fold to obtain the test solution.

[0062] Prepare uronic acid standard solutions of different concentrations according to the kit requirements, and measure the absorbance values ​​at the specified wavelength. Plot a standard curve with the standard concentration on the x-axis and the absorbance values ​​on the y-axis. The linear regression equation is: y = 1.9826x + 0.0459, R0 2 = 0.9978. The results indicate that within the investigated concentration range, there is a good linear relationship between uronic acid concentration and absorbance value, which can be used for the quantitative analysis of uronic acid content in samples. The diluted sample solution was subjected to a colorimetric reaction according to the kit instructions, and the absorbance value was measured. The experiment was repeated three times in parallel, and the average value was taken. The uronic acid content in the sample was calculated based on the regression equation of the standard curve, and converted using the dilution factor. The results showed that the uronic acid contents obtained in the three parallel experiments were 43.32%, 45.03%, and 41.18%, respectively, with an average of 43.18%. The results indicate that the polysaccharide-enriched saffron flower extract contains a certain proportion of uronic acid components and exhibits certain acidic characteristics.

[0063] Example 7 Efficacy evaluation of DSS-induced mouse colitis model Fifty-six male SPF-grade C57BL / 6 mice were randomly divided into seven groups of eight mice each after 7 days of acclimatization: a normal control group, a DSS model group, a low-dose DSS+PFRC group, a medium-dose DSS+PFRC group, a high-dose DSS+PFRC group, a 5-ASA positive control group, and a high-dose DSS alone group. PFRC was dissolved in ultrapure water to prepare solutions of corresponding concentrations, with low, medium, and high doses of 100 mg / kg, 200 mg / kg, and 400 mg / kg, respectively. The 5-ASA positive control group received 100 mg / kg of PFRC. All groups were pretreated with 2.5% DSS via gavage for 14 days. Subsequently, except for the normal control group and the high-dose DSS alone group, the other groups were induced with 2.5% DSS for 7 days while continuing gavage administration. Mice were sacrificed on day 8, and disease activity index (DAI), final body weight, colon length, spleen weight, and thymus weight were recorded. Spleen index and thymus index were calculated. Colon tissue was also harvested for subsequent molecular biological analysis and histological observation. All data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA was used for comparisons between groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely significant.

[0064] (1) Evaluation of apparent indicators The DSS-induced mouse colitis model is a commonly used experimental model for evaluating the effects of candidate drugs on colitis. The disease activity index (DAI) typically scores a combination of weight loss, fecal characteristics, and the presence of occult or gross blood in the stool, providing a relatively comprehensive reflection of changes in colitis symptoms. Results showed that, as Figure 2 As shown in results A and 2B, compared with the normal group, the DSS model group showed a significant decrease in weight and an increase in DAI score; Figure 2 As shown in C, this is accompanied by shortening of the colonic mass, congestion, and edema; as... Figure 2 As shown in D, the length of the colon is significantly shortened; as... Figure 2 As shown in E and 2F, the increased spleen index and decreased thymus index indicate that 2.5% DSS successfully induced a mouse colitis model. These results suggest that DSS can cause significant local inflammatory damage to the colon and exacerbate overall systemic inflammation, inducing abnormal changes in immune organs. Specifically, the increased DAI score, decreased body weight, and shortened colon corroborate each other, reflecting increased disease activity and significant colonic damage in the model group mice; the increased spleen index and decreased thymus index suggest that the increased local inflammation was accompanied by a certain degree of systemic stress and immune imbalance.

[0065] Compared with the DSS model group, PFRC intervention improved the above-mentioned abnormal changes to varying degrees. Specifically, the medium-dose group (DSS+PFRC200) and the high-dose group (DSS+PFRC400) showed significant effects in reducing DAI scores and mitigating weight loss; the medium-dose group (DSS+PFRC200) showed a particularly significant improvement in colonic shortening; the low-dose group (DSS+PFRC100) performed well in reducing the spleen index; and the medium-dose group (DSS+PFRC200) showed a significant effect in increasing the thymus index. Figure 2 The colonic image shown in Figure C also indicates that colonic shortening, congestion, and edema were all reduced after PFRC intervention. These results suggest that PFRC not only alleviates the superficial damage of DSS-induced colitis and reduces disease activity, but also has a certain regulatory effect on inflammation-related abnormal changes in immune organs, thus demonstrating good overall efficacy. The 5-ASA-positive control group also showed good improvement, while the high-dose administration group alone did not show a significant trend of abnormal deterioration compared to the normal group, suggesting that PFRC did not have significant adverse effects under non-DSS conditions.

[0066] (2) Histological and histochemical observation To further investigate the effects of PFRC on DSS-induced pathological damage and changes in the intestinal mucosal barrier in mouse colon tissue, HE staining, AB-PAS staining, and immunohistochemical observation were performed on colon tissues from each group of mice. HE staining results showed that, as Figure 3 As shown in Figure A, compared with the normal group, the DSS model group showed damaged mucosal structure, poor epithelial continuity, disordered crypt arrangement, incomplete crypt structure in some areas, increased inflammatory cell infiltration in the lamina propria, and varying degrees of tissue edema and damage. These pathological changes indicate that the colonic tissue underwent significant inflammatory damage after DSS stimulation. After PFRC intervention, the integrity of the colonic mucosal structure improved, the crypt arrangement became more regular, and the degree of inflammatory damage decreased, suggesting that PFRC has a certain alleviating effect on DSS-induced pathological damage to colonic tissue.

[0067] AB-PAS staining results showed that, Figure 3 As shown in Figure B, compared with the normal group, the DSS model group showed a decrease in the number of goblet cells and a weakening of AB-PAS positive staining, indicating reduced mucus secretion and decreased protective function of the mucus layer. A decrease in goblet cells and reduced mucus secretion usually reflects damage to the intestinal mucosal chemical barrier and also suggests a decreased ability of colonic tissue to defend against intraluminal stimuli. After PFRC intervention, the number and staining intensity of goblet cells in the colonic tissue showed a recovery trend, indicating that PFRC plays a role in maintaining the mucus barrier on the intestinal mucosa while improving colonic inflammatory damage.

[0068] Further immunohistochemical methods were used to observe the expression of relevant barrier proteins in the colon tissue of each group. For example... Figure 4 The results showed that, compared with the normal group, the DSS model group ZO-1 ( Figure 4 A), Occludin ( Figure 4 B) and Claudin-1 Figure 4 C) Decreased positive expression and poor linear staining continuity at epithelial cell junctions, along with unclear local localization, indicate damage to tight junction structures and disruption of the intestinal mucosal mechanical barrier. After PFRC intervention, the positive expression of the above proteins increased, and the linear distribution at intercellular junctions became more continuous and clear, suggesting that PFRC can, to some extent, improve the abnormal expression and disordered localization of tight junction proteins induced by DSS, thereby playing a certain protective role in the intestinal mucosal mechanical barrier.

[0069] (3) Results Analysis In summary, PFRC intervention improved the overall pathological state of DSS-induced colitis in mice to varying degrees. It not only alleviated apparent abnormalities such as weight loss, elevated DAI, and colonic shortening, but also histologically demonstrated an ameliorative effect on colonic mucosal damage and mucus barrier impairment. These results suggest that PFRC has a good overall intervention effect on DSS-induced colitis in mice, and its efficacy may be related to reducing local inflammatory responses and improving intestinal mucosal barrier function.

[0070] This invention relates to a polysaccharide-enriched saffron flower extract (stigma-free), its preparation method, and its applications. The method uses the remaining saffron flowers after stigma harvesting as raw material. The extract is obtained through drying and pulverizing, ethanol pretreatment, hot water extraction, vacuum concentration, and alcohol precipitation. By weight fraction, the total sugar content is ≥40%, the uronic acid content is ≥40%, and the protein content is <1.0%. Under optimal water extraction conditions, the obtained extract has a total sugar content of 46.78%, a reducing sugar content of 9.04%, a protein content of 0.47%, and a uronic acid content of 43.18%. This extract has the effect of improving intestinal inflammation and protecting the intestinal mucosal barrier. This invention realizes the high-value utilization of non-medicinal parts of saffron and has good application prospects for the intervention of colitis.

[0071] The above are merely embodiments of the present invention. For example, the stigma-free saffron flower part refers to the remaining flower part after harvesting the stigma, including one or more of the petals, stamens, and residual styles. All of these can realize the polysaccharide-enriched stigma-free saffron flower part extract, its preparation method, and its application in the present invention.

[0072] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a polysaccharide-enriched saffron flower extract without stigmas, characterized in that, Using the remaining saffron flower parts after harvesting the stigmas as raw materials, the stigmas were dried, pulverized, pretreated with ethanol, filtered, and the filter residue was collected and dried. The filter residue was then extracted with hot water, concentrated under reduced pressure, and precipitated with alcohol to obtain a polysaccharide-enriched saffron flower part extract. The stigma-free saffron flower extract, by weight fraction, has a total sugar content ≥40%, a uronic acid content ≥40%, and a protein content <1.0%.

2. The method for preparing a polysaccharide-enriched saffron flower extract (without stigmas) according to claim 1, characterized in that, Includes the following steps: (1) Raw material pretreatment: The stigma-removed saffron flower parts are dried and then pulverized to obtain stigma-removed saffron flower part powder; the stigma-removed saffron flower parts are the remaining flower parts after the stigma is harvested, including one or more of the petals, stamens and residual styles; (2) Alcohol pretreatment: Add an aqueous ethanol solution to the stigma-free saffron flower powder and reflux extract, filter, collect the residue and dry it; (3) Water extraction: The filter residue obtained in step (2) is added to water for hot water extraction, followed by solid-liquid separation, and the water extract is collected; (4) Alcohol precipitation: The aqueous extract obtained in step (3) is concentrated under reduced pressure and then ethanol is added for alcohol precipitation. After standing, the precipitate is collected by centrifugation, washed with ethanol, and dried to obtain the polysaccharide-enriched saffron flower extract.

3. The method for preparing a polysaccharide-enriched saffron flower extract without stigmas according to claim 2, characterized in that, In step (4), the final volume fraction of ethanol in the mixed system is 70% during alcohol precipitation, and the precipitate is collected by centrifugation after standing at 4°C for 24 h; the precipitate is washed with 75% ethanol ≥ 2 times.

4. The method for preparing a polysaccharide-enriched saffron flower extract (without stigmas) according to claim 2, characterized in that, In step (2), the volume fraction of the ethanol aqueous solution used is 60% to 80%, the reflux extraction temperature is 75℃ to 85℃, the extraction time is 2h to 4h each time, and the reflux extraction is performed 2 to 3 times.

5. The method for preparing a polysaccharide-enriched saffron flower extract (without stigmas) according to claim 4, characterized in that, In step (2), the ratio of stigma-removed saffron flower powder to ethanol aqueous solution is 1 g: (8-15) mL.

6. The method for preparing a polysaccharide-enriched saffron flower extract (without stigmas) according to claim 2, characterized in that, In step (3), the filter residue obtained in step (2) is added to water at a material-to-liquid ratio of 1 g: (20-30) mL, and extracted at 95℃-100℃ for 3-4 hours.

7. A polysaccharide-enriched extract from the stigma-free saffron flower, characterized in that, The extract is prepared by any one of the polysaccharide-enriched saffron flower extracts according to any one of claims 1-5, and by weight fraction, the total sugar content is ≥40%, the uronic acid content is ≥40%, and the protein content is <1.0%.

8. The application of the polysaccharide-enriched saffron flower extract with destigma as described in claim 7, characterized in that, The stigma-free saffron flower extract enriched with polysaccharides was used to prepare drugs that improve or alleviate damage to the intestinal mucosal barrier.

9. The application of the polysaccharide-enriched saffron flower extract with destigma as described in claim 7, characterized in that, The polysaccharide-enriched stigma-free saffron flower extract was used to prepare a drug for treating colitis, specifically ulcerative colitis or DSS-induced colitis.

Images