A Lycium barbarum polysaccharide, its preparation method, and its application in the field of health.

Abstract

This invention discloses a Lycium barbarum polysaccharide, its preparation method, and its application in the field of health and wellness. The invention claims protection for a Lycium barbarum polysaccharide prepared from dried, ripe Lycium barbarum fruit. The method involves steaming, alkaline water extraction, and alcohol precipitation to obtain crude polysaccharide, followed by protein removal, ethanol fractionation precipitation, desalting, and drying. This invention has found that the fractionated polysaccharide prepared from Lycium barbarum using the method described herein significantly enhances the chemosensitivity of sorafenib-resistant liver cancer to sorafenib chemotherapy, an effect not observed in conventionally extracted Lycium barbarum fractionated polysaccharides. Therefore, the Lycium barbarum polysaccharide provided by this invention has the potential to be developed into a drug that enhances the chemosensitivity of sorafenib-resistant liver cancer to sorafenib chemotherapy, and also has the potential to be developed into a functional food that helps enhance the chemosensitivity of sorafenib-resistant liver cancer to sorafenib chemotherapy.

Description

Technical Field

[0001] This invention belongs to the field of chemistry and relates to novel polysaccharides and their preparation and application, specifically to a wolfberry polysaccharide, its preparation method, and its application in the field of health. Background Technology

[0002] Goji berries (Lycium barbarum) are a deciduous shrub belonging to the Solanaceae family. Originating in China, they are mainly distributed in temperate and subtropical regions, and are cultivated in most provinces of northern China, commonly found on hillsides, roadsides, or near villages. Rich in various nutrients and active ingredients, goji berries have become a popular topic in traditional Chinese medicine research and modern health preservation. With the development of science and technology, extensive research and development of the effective components of goji berries have provided theoretical basis and potential drugs for clinical application.

[0003] Modern phytochemical studies have revealed that wolfberry contains over 200 chemical components, primarily including 4 polysaccharides, 15 glycolipids, 20 flavonoids, 21 anthocyanins, 6 anthocyanins, 4 proanthocyanidins, 13 alkaloids, phenolic acids, 5 carotenoids, 7 vitamins, 18 amino acids, and 32 trace elements (Reference: Wang Shixue et al., Research Progress on Chemical Constituents and Pharmacological Effects of Wolfberry, Chinese Journal of Pharmacology and Toxicology, 2023). The main active ingredients in wolfberry are wolfberry polysaccharides, betaine, and wolfberry pigments. Early applications of wolfberry were limited to treating single symptoms; however, research has shown that combining wolfberry with other traditional Chinese medicines can treat more complex diseases. Modern scientists have conducted in-depth research in clinical medicine, basic pharmacy, and biological resources. A large amount of experimental data shows that wolfberry has clinical pharmacological effects such as enhancing immune regulation, anti-oxidation, anti-aging, anti-tumor, lowering blood sugar, regulating blood lipids, anti-inflammation, antibacterial, improving eyesight, protecting nerves, and preventing radiation.

[0004] Polysaccharides are a class of complex macromolecular compounds composed of multiple monosaccharide molecules, widely found in animal cell membranes and plant cell walls. In recent years, research on polysaccharides has mainly focused on the extraction and analysis of their bioactivity. As macromolecules, the bioactivity of polysaccharides is influenced by factors such as their monosaccharide composition, uronic acid content, and spatial configuration. Different extraction methods affect the sugar content, physicochemical properties, and structural characteristics of polysaccharides, thus affecting their bioactivity (Reference: Wang Shenglin et al., Comparative Study on the Bioactivity of Five Lycium barbarum Polysaccharides, China Journal of Traditional Chinese Medicine, 2024).

[0005] The incidence and mortality rates of hepatocellular carcinoma (HCC) are on the rise both domestically and internationally, with more than half of the cases occurring in my country. Treatment of HCC is subject to several limitations, including late diagnosis, heterogeneity, and chemotherapy resistance. Sorafenib, a tyrosine kinase inhibitor (TKI), has long been the only approved drug for the treatment of advanced HCC; however, most patients exhibit primary or acquired resistance, limiting its efficacy. Therefore, there is an urgent need to develop drugs that can reverse the sensitivity of sorafenib-resistant HCC to sorafenib chemotherapy.

[0006] CN115192587A discloses the use of Lycium barbarum oligosaccharides in the preparation of pharmaceuticals, foods, and / or health products for the prevention and / or treatment of tumors. This invention found that the Lycium barbarum oligosaccharides can significantly promote apoptosis in HepG2 cells, with a much stronger inhibitory effect on HepG2 cells than on L02 cells. However, this literature studies the killing effect of Lycium barbarum oligosaccharides on ordinary liver cancer cells.

[0007] Currently, no literature discloses the effects of Lycium barbarum polysaccharides on sorafenib-resistant liver cancer cells.

[0008] To overcome the shortcomings of the existing technology, this invention is proposed. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art. The first purpose is to provide a Lycium barbarum polysaccharide, the second purpose is to provide a method for preparing the Lycium barbarum polysaccharide, and the third purpose is to provide the application of the Lycium barbarum polysaccharide in the field of health (including the preparation of drugs to enhance the sensitivity of sorafenib-resistant liver cancer to sorafenib chemotherapy and the preparation of functional foods to help enhance the sensitivity of sorafenib-resistant liver cancer to sorafenib chemotherapy).

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] A type of wolfberry polysaccharide is obtained by first steaming, extracting with alkaline water and precipitating with alcohol to obtain crude polysaccharide, and then removing protein, precipitating with ethanol, desalting and drying.

[0012] In one specific embodiment, the steaming temperature is 110~130℃.

[0013] In one specific embodiment, in the alkaline water heating extraction step, the pH of the alkaline water is 7.5~8.5, and the heating temperature is 80~100℃.

[0014] In one specific embodiment, during the alcohol precipitation step, 3 to 5 times the volume of anhydrous ethanol is added and the mixture is allowed to stand to precipitate.

[0015] In one specific embodiment, in the ethanol fractionation precipitation step, 25-35% volume fraction of ethanol precipitate is first removed, and then 55-65% volume fraction of ethanol precipitate is collected.

[0016] A method for preparing the above-mentioned Lycium barbarum polysaccharide includes the following steps:

[0017] Using dried, ripe wolfberry fruit as raw material, the raw material is first steamed at 110-130℃, extracted by heating in alkaline water with pH=7.5-8.5 at 80-100℃, and precipitated with 3-5 times the volume of anhydrous ethanol to obtain crude polysaccharide. Then, the polysaccharide is obtained by deproteinization, ethanol fractionation precipitation, desalting, and drying. In the ethanol fractionation precipitation step, 25-35% volume fraction of ethanol precipitate is removed first, and then 55-65% volume fraction of ethanol precipitate is collected.

[0018] In one specific embodiment, the Sevage method was used for protein removal.

[0019] In one specific embodiment, dialysis is used for desalination.

[0020] The above-mentioned Lycium barbarum polysaccharide was used in the preparation of a drug to enhance the sensitivity of sorafenib-resistant liver cancer to sorafenib chemotherapy.

[0021] The above-mentioned Lycium barbarum polysaccharide was used in the preparation of functional foods that help enhance the sensitivity of sorafenib-resistant liver cancer to sorafenib chemotherapy.

[0022] Beneficial effects:

[0023] Those skilled in the art know that polysaccharides, as macromolecules, have biological activities influenced by factors such as their monosaccharide composition, uronic acid content, and spatial configuration. Different extraction methods can affect the sugar content, physicochemical properties, and structural characteristics of polysaccharides, thereby affecting their biological activity (Reference: Wang Shenglin et al., Comparative Study on the Biological Activities of Five Lycium barbarum Polysaccharides, China Journal of Traditional Chinese Medicine, 2024). This invention found that the graded polysaccharides prepared from Lycium barbarum through steaming and alkaline water extraction using the method of this invention significantly enhance the chemosensitivity of sorafenib-resistant liver cancer to sorafenib, while this effect was not observed in conventionally extracted graded Lycium barbarum polysaccharides. Therefore, the Lycium barbarum polysaccharides provided by this invention have the potential to be developed into drugs that enhance the chemosensitivity of sorafenib-resistant liver cancer to sorafenib, and also have the potential to be developed into functional foods that can help enhance the chemosensitivity of sorafenib-resistant liver cancer to sorafenib. Detailed Implementation

[0024] The substantive content of the present invention will be described in detail below with reference to specific embodiments. However, those skilled in the art should know that the scope of protection of the present invention should not be limited to these specific embodiments.

[0025] Example 1: Preparation and determination of polysaccharide content of Lycium barbarum polysaccharides

[0026] I. Experimental Materials

[0027] The goji berries were purchased from Bairuiyuan Goji Berry Co., Ltd., and are the dried, ripe fruits of *Lycium barbarum* L., a plant belonging to the Solanaceae family. All other experimental materials were commercially available and commonly used.

[0028] II. Experimental Methods and Results

[0029] 1. Extraction of crude polysaccharides

[0030] (1) Steaming and alkali extraction method (method of this invention)

[0031] Goji berries were placed in breathable gauze bags and placed in an autoclave. The mixture was heated to 121℃ and 0.1MPa (gauge pressure) and then steamed for 30 minutes. The steamed goji berries were then dried in an oven at 55℃ for 12 hours, pulverized using a pulper, and 500g of the dried powder was weighed out. Distilled water (pH=8.0) was added at a ratio of 1:5 (g / mL) and adjusted with 0.1mol / L NaOH and 0.1mol / L HCl solutions (pH adjusted). Extraction was carried out at 90℃ for 3 hours. The supernatant was collected after filtration. The collected supernatant was then filtered again to remove residue. The solution was concentrated under reduced pressure to 1000mL, and 4000mL of anhydrous ethanol was added. The mixture was allowed to stand at room temperature for 12 hours to precipitate, and then filtered. The residue was the crude polysaccharide.

[0032] (2) Conventional water extraction method (existing technology method)

[0033] After drying the goji berries in an oven at 55℃ for 12 hours, they were pulverized using a pulping machine. 500g of the dried goji berry powder was weighed out and added to distilled water at a ratio of 1:5 (g / mL). The mixture was extracted at 90℃ for 3 hours. After filtration, the supernatant was collected. The collected supernatant was then filtered again to remove the residue. The mixture was concentrated under reduced pressure to 1000mL and 4000mL of anhydrous ethanol was added. After standing at room temperature for 12 hours to precipitate, the mixture was filtered. The residue was the crude polysaccharide.

[0034] 2. Deproteinization of crude polysaccharides

[0035] The crude polysaccharide was deproteinized using the conventional Sevage method. The specific steps were as follows: the crude polysaccharide was reconstituted with distilled water to prepare a 10 mg / mL solution, and 1 / 4 volume of chloroform-n-butanol mixture (4:1, V / V) was added and mixed well. The mixture was then shaken on a shaker (40℃, 200 r / min, 1 h). The mixture was allowed to stand for separation, and the supernatant was collected to remove the denatured protein. This process was repeated multiple times (5 times) until there was no white precipitate at the reagent interface after shaking. The supernatant was then concentrated and freeze-dried to obtain the deproteinized crude polysaccharide.

[0036] 3. Fractionation and purification of crude polysaccharides

[0037] The deproteinized crude polysaccharide was reconstituted with distilled water to prepare a 25% (w / w) solution. Anhydrous ethanol was added to bring the ethanol volume fraction in the solution to 30%. The solution was allowed to stand at 4°C for 12 hours to precipitate, and then centrifuged at 5000 rpm for 10 minutes. The supernatant was collected. Anhydrous ethanol was added to the supernatant to bring the ethanol volume fraction in the solution to 60%. The solution was allowed to stand at 4°C for 12 hours to precipitate, and then centrifuged at 5000 rpm for 10 minutes. The precipitate was collected to obtain a 60% ethanol precipitate.

[0038] 4. Dialysis desalination

[0039] The 60% ethanol precipitate was redissolved in distilled water and placed in a dialysis bag with a molecular weight cutoff of 3500 Da. Dialysis was performed for 48 hours with continuous stirring (the ultrapure water was replaced every 8 hours). The dialysate was then freeze-dried under vacuum to obtain the target Lycium barbarum polysaccharide. The target Lycium barbarum polysaccharide obtained by the alkali extraction method was named ZZJT-P, and the target Lycium barbarum polysaccharide obtained by the conventional water extraction method was named CGST-P.

[0040] 5. Content determination

[0041] The polysaccharide content was determined using the conventional phenol-sulfuric acid method. The polysaccharide contents of ZZJT-P and CGST-P were 85.2% and 88.7%, respectively, with the polysaccharide content of the conventional water extraction method being slightly higher.

[0042] Example 2: Activity determination of Lycium barbarum polysaccharides prepared in Example 1

[0043] I. Experimental Materials

[0044] Polysaccharides ZZJT-P and CGST-P were prepared according to the method in Example 1.

[0045] All other test materials were standard commercially available materials.

[0046] II. Test Methods

[0047] HepG2 / Sorafenib cells (a sorafenib-resistant strain of human hepatocellular carcinoma) were cultured in DMEM high-glucose medium (complete medium) containing 10% FBS, 100 U / mL penicillin, and 100 mg / mL streptomycin at 37 °C in an incubator containing 5% CO2. Cells in the logarithmic growth phase were collected and diluted to 5 × 10⁻⁶ cells / mL. 4Cells were seeded at a density of 100 μL / mL in 96-well plates. After 6 h, the culture medium was changed and cultured for another 48 h according to the groupings shown in Table 1 (3 replicates per group). At the end of the culture, 10 μL of CCK-8 solution was added to each well, and the plates were cultured for another 2 h. The absorbance (A) value was measured at 450 nm. Blank wells were also set up with only culture medium and no cell suspension. Cell viability was calculated using the formula: Cell viability (%) = (A value of drug group - A value of blank group) / (A value of control group - A value of blank group) × 100%.

[0048] Table 1 Grouping and Culture Protocol

[0049] 1 control group Complete culture medium 2 Sorafenib-L group Complete culture medium containing 1 μM sorafenib 3 Sorafenib-M group Complete culture medium containing 2 μM sorafenib 4 Sorafenib-H group Complete culture medium containing 5 μM sorafenib 5 ZZJT-PL Group Complete culture medium containing 100 μg / mL ZZJT-P 6 ZZJT-P-L+Sorafenib-L group Complete culture medium containing 100 μg / mL ZZJT-P and 1 μM sorafenib 7 ZZJT-P-L+Sorafenib-M group Complete culture medium containing 100 μg / mL ZZJT-P and 2 μM sorafenib 8 ZZJT-P-L+Sorafenib-H group Complete culture medium containing 100 μg / mL ZZJT-P and 5 μM sorafenib 9 ZZJT-PH Group Complete culture medium containing 200 μg / mL ZZJT-P 10 ZZJT-P-H+Sorafenib-L group Complete culture medium containing 200 μg / mL ZZJT-P and 1 μM sorafenib 11 ZZJT-P-H+Sorafenib-M group Complete culture medium containing 200 μg / mL ZZJT-P and 2 μM sorafenib 12 ZZJT-P-H+Sorafenib-H group Complete culture medium containing 200 μg / mL ZZJT-P and 5 μM sorafenib 13 CGST-PL Group Complete culture medium containing 100 μg / mL CGST-P 14 CGST-P-L+Sorafenib-L group Complete culture medium containing 100 μg / mL CGST-P and 1 μM sorafenib 15 CGST-P-L+Sorafenib-M group Complete culture medium containing 100 μg / mL CGST-P and 2 μM sorafenib 16 CGST-P-L+Sorafenib-H group Complete culture medium containing 100 μg / mL CGST-P and 5 μM sorafenib 17 CGST-PH group Complete culture medium containing 200 μg / mL CGST-P 18 CGST-P-H+Sorafenib-L group Complete culture medium containing 200 μg / mL CGST-P and 1 μM sorafenib 19 CGST-P-H+Sorafenib-M group Complete culture medium containing 200 μg / mL CGST-P and 2 μM sorafenib 20 CGST-P-H+Sorafenib-H group Complete culture medium containing 200 μg / mL CGST-P and 5 μM sorafenib

[0050] Data analysis was performed using GraphPad Prism 8 software. The experimental results are expressed as mean ± standard deviation, and P < 0.05 indicates that the data are statistically significant.

[0051] III. Test Results

[0052] Table 2. A value and cell viability of each group

[0053] 1 control group 1.473±0.005 100 2 Sorafenib-L group 1.457±0.007 98.84 3 Sorafenib-M group 1.455±0.007 98.70 4 Sorafenib-H group 1.432±0.009 97.03 5 ZZJT-PL Group 1.437±0.009 97.40 6 ZZJT-P-L+Sorafenib-L group 1.322±0.008 89.07 7 ZZJT-P-L+Sorafenib-M group 1.165±0.009 77.71 8 ZZJT-P-L+Sorafenib-H group 0.845±0.007 54.56 9 ZZJT-PH Group 1.409±0.008 95.37 10 ZZJT-P-H+Sorafenib-L group 1.214±0.006 81.26 11 ZZJT-P-H+Sorafenib-M group 0.968±0.009 63.46 12 ZZJT-P-H+Sorafenib-H group 0.540±0.006 32.49 13 CGST-PL Group 1.452±0.007 98.48 14 CGST-P-L+Sorafenib-L group 1.417±0.009 95.95 15 CGST-P-L+Sorafenib-M group 1.402±0.006 94.86 16 CGST-P-L+Sorafenib-H group 1.413±0.012 95.66 17 CGST-PH group 1.415±0.009 95.80 18 CGST-P-H+Sorafenib-L group 1.392±0.006 94.14 19 CGST-P-H+Sorafenib-M group 1.389±0.006 93.92 20 CGST-P-H+Sorafenib-H group 1.384±0.008 93.56 21 Blank group 0.091±0.005 /

[0054] The above experimental results found that:

[0055] 1. Compared with the control group, there was no significant difference in cell survival rate among the ZZJT-PL group, ZZJT-PH group, CGST-PL group and CGST-PH group, indicating that polysaccharides ZZJT-P and CGST-P alone did not significantly inhibit the activity of HepG2 / Sorafenib cells within the tested concentration range.

[0056] 2. Compared with the Sorafenib-L group, Sorafenib-M group, and Sorafenib-H group, the cell survival rate of the ZZJT-P-L+Sorafenib-L group, ZZJT-P-L+Sorafenib-M group, ZZJT-P-L+Sorafenib-H group, ZZJT-P-H+Sorafenib-L group, ZZJT-P-H+Sorafenib-M group, and ZZJT-P-H+Sorafenib-H group was significantly reduced (P<0.05), indicating that polysaccharide ZZJT-P can enhance the chemosensitivity of HepG2 / Sorafenib cells to sorafenib, and the effect of high concentration polysaccharide ZZJT-P is more obvious.

[0057] 3. Compared with the Sorafenib-L group, Sorafenib-M group, and Sorafenib-H group, there was no significant difference in cell survival rate among the CGST-P-L+Sorafenib-L group, CGST-P-L+Sorafenib-M group, CGST-P-L+Sorafenib-H group, CGST-P-H+Sorafenib-L group, CGST-P-H+Sorafenib-M group, and CGST-P-H+Sorafenib-H group, indicating that polysaccharide CGST-P did not enhance the sensitivity of HepG2 / Sorafenib cells to sorafenib chemotherapy.

[0058] Therefore, polysaccharide ZZJT-P has the potential to be developed into a drug that enhances the sensitivity of sorafenib-resistant liver cancer to sorafenib chemotherapy, and also has the potential to be developed into a functional food that helps enhance the sensitivity of sorafenib-resistant liver cancer to sorafenib chemotherapy.

[0059] Example 3: Drug Example

[0060] A drug that enhances the sensitivity of sorafenib-resistant liver cancer to sorafenib chemotherapy, the active ingredient being the polysaccharide ZZJT-P prepared in Example 1, and formulated into a pharmaceutically acceptable dosage form, including but not limited to tablets, capsules and injections, with pharmaceutically acceptable excipients.

[0061] Example 4: Functional Food Example

[0062] A functional food for enhancing the sensitivity of sorafenib-resistant liver cancer to sorafenib chemotherapy, the functional ingredient being the polysaccharide ZZJT-P prepared in Example 1, and formulated into a food-acceptable dosage form with food-acceptable excipients, including but not limited to solid beverages, meal replacement powders and gel candies, can be used as a dietary supplement during sorafenib intervention and complies with relevant food safety standards and regulations.

[0063] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. A Lycium barbarum polysaccharide, characterized in that: Using dried, ripe wolfberry fruit as raw material, crude polysaccharide is obtained by steaming, heating with alkaline water for extraction, and precipitating with alcohol. Then, it is obtained by deproteinization, fractionation and precipitation with ethanol, desalting, and drying.

2. The Lycium barbarum polysaccharide according to claim 1, characterized in that: The steaming temperature is 110~130℃.

3. The Lycium barbarum polysaccharide according to claim 1, characterized in that: In the alkaline water heating extraction step, the pH of the alkaline water is 7.5~8.5, and the heating temperature is 80~100℃.

4. The Lycium barbarum polysaccharide according to claim 1, characterized in that: In the alcohol precipitation step, add 3 to 5 times the volume of anhydrous ethanol and let it stand to precipitate.

5. The Lycium barbarum polysaccharide according to claim 1, characterized in that: In the ethanol fractionation precipitation step, 25-35% volume fraction of ethanol precipitate is removed first, and then 55-65% volume fraction of ethanol precipitate is collected.

6. A method for preparing the Lycium barbarum polysaccharide according to any one of claims 1 to 5, characterized in that, The process includes the following steps: using dried, ripe wolfberry fruit as raw material, the raw material is first steamed at 110-130℃, extracted by heating in alkaline water with pH=7.5-8.5 at 80-100℃, and precipitated with 3-5 times the volume of anhydrous ethanol to obtain crude polysaccharide, followed by deproteinization, ethanol fractionation precipitation, desalting, and drying; in the ethanol fractionation precipitation step, 25-35% volume fraction of ethanol precipitate is removed first, and then 55-65% volume fraction of ethanol precipitate is collected.

7. The preparation method according to claim 6, characterized in that: The Sevage method was used for protein removal.

8. The preparation method according to claim 6, characterized in that: Desalination was performed using dialysis.

9. The use of the Lycium barbarum polysaccharide according to any one of claims 1 to 5 in the preparation of a drug for enhancing the sensitivity of sorafenib-resistant liver cancer to sorafenib chemotherapy.

10. The use of the Lycium barbarum polysaccharide according to any one of claims 1 to 5 in the preparation of functional foods that help enhance the sensitivity of sorafenib-resistant liver cancer to sorafenib chemotherapy.