Small-leaf broadleaf holly leaf polysaccharide as well as preparation method and application thereof

Abstract

The invention provides a small-leaf broadleaf holly leaf polysaccharide as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) pretreatment: crushing small-leaf broadleaf holly leaves to obtain small-leaf broadleaf holly leaf powder; (2) degreasing: adding the small-leaf broadleaf holly leaf powder into 95% edible ethanol for extraction to obtain small-leaf broadleaf holly leaf filter residues, and drying the small-leaf broadleaf holly leaf filter residues; (3) polysaccharide extraction: adding distilled water into the dried small-leaf broadleaf holly leaf filter residue, carrying out hot water extraction for multiple times, combining filtrate, concentrating the filtrate to obtain a concentrated solution, then adding 95% edible ethanol, standing and centrifuging to obtain a precipitate; and (4) refining: adding distilled water into the precipitate for dissolving, filtering and carrying out vacuum freeze drying to obtain the small-leaf broadleaf holly leaf polysaccharide. It is proved that the lobular broadleaf holly leaf polysaccharide has potential application value in the aspect of treating chemotherapeutic drug toxicity deafness and senile deafness, and a new direction is provided for development of the lobular broadleaf holly leaf polysaccharide in the field of functional food and biological medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of food and medicine, and in particular relates to a small-leaf Kudingcha tea polysaccharide and a preparation method and application thereof. Background Art

[0002] Hearing loss is one of the world's most significant health issues, affecting approximately 1.5 billion people worldwide, or one-fifth of the total population. Its incidence is increasing significantly, with projections predicting that by 2050, one-quarter of the world's population will experience varying degrees of hearing loss. Environmental factors, such as aging, exposure to high-intensity noise, and the use of ototoxic medications, are all associated with the development of acquired hearing loss. Ototoxicity, caused by drug-induced inner ear damage, is the leading cause of acquired hearing loss worldwide. Cisplatin, primarily used to treat various solid tumors in children and adults, serves as a cornerstone chemotherapy agent in chemotherapy regimens and is widely used in the treatment of ovarian, lung, head and neck, and testicular cancers. However, cisplatin is currently the most ototoxic drug in clinical use. Studies have shown that up to 60% of adults and children treated with cisplatin experience bilateral, progressive, and permanent sensorineural hearing loss. Cisplatin-induced hearing loss (CIHL) often leads to social isolation, speech impairment, depression, or cognitive impairment, severely impacting the normal lives and social interactions of cancer patients and imposing a significant burden on individuals, their families, and society. Despite significant advances in ototoxicity research in recent years, there are currently no effective treatment strategies for cisplatin-induced ototoxicity in adults or children with metastatic cancer. Currently, no FDA-approved treatments are available for the prevention or treatment of CIHL. Therefore, identifying effective approaches to prevent and treat CIHL is of great clinical and socioeconomic importance.

[0003] Presbycusis, also known as age-related hearing loss (ARHL), is one of the most common chronic sensory deficits in the elderly population. It refers to irreversible sensorineural hearing loss characterized by symmetrical, progressive, and high-frequency hearing loss in both ears due to the gradual degeneration of the inner ear structure with aging. ARHL often leads to communication difficulties, social isolation, and cognitive decline, increasing the risk of Alzheimer's disease, significantly reducing the quality of life of the elderly, and even causing a series of adverse effects on the mental, psychological, and physiological aspects of the elderly. The onset of presbycusis involves the interaction of multiple factors, such as genetics, aging, noise exposure, ototoxic drugs, oxidative stress, inflammation, diet, and other factors. The molecular mechanisms of presbycusis are very complex and include oxidative damage, damage to mitochondrial structure and function, and damage to the vascular striae. Recent studies have found that disturbances in intracellular NAD(+) levels are clinically associated with the progression of age-related diseases. It has been reported that nicotinamide adenine dinucleotide (NAD) is increased in the liver, heart, kidney, and lung of elderly animals and in human skin tissue. + The NAD / NADH ratio decreases, + The decrease in the NAD / NADH ratio is attributed to the altered redox mechanism caused by the accumulation of oxidative damage and the subsequent DNA damage-induced overactivation of PARP1. In addition, the enzyme complex α-ketoglutarate dehydrogenase (α-KGDH) of the tricarboxylic acid cycle in mitochondria is activated by NAD + A decrease in the NAD / NADH ratio will promote the generation of reactive oxygen species (ROS), and + A decrease in the NAD / NADH ratio favors the generation of ROS in respiratory chain complex I. Therefore, maintaining sufficient NAD + Levels may be a key factor in delaying cellular aging and may become a useful strategy for treating age-related diseases.

[0004] Small-leaf Kudingcha (Ilex kudingcha) is a general term for a large group of traditional plant-based tea substitutes in China, second only to tea. With a history of consumption and folk medicine spanning over 2,000 years in southwestern China, it is a health food with both medicinal and edible properties, known as "beauty tea," "slimming tea," and "green gold." It possesses antibacterial and anti-inflammatory properties, clears heat and toxins, and relieves pain and fluid retention. It is widely consumed by the public as a tea for dispelling wind-heat, clearing the head, relieving thirst, lowering blood lipids, and promoting weight loss. Literature reports indicate that small-leaf Kudingcha is rich in saponins, phenylethanoid glycosides, triterpenes, flavonoids, polyphenols, and polysaccharides, demonstrating health and medicinal benefits, including anti-cardiovascular disease, antioxidant properties, lipid-lowering properties, improved lipid metabolism disorders, liver damage alleviation, antiviral, and anti-inflammatory properties. Polysaccharides are one of the main active ingredients in small-leaf Kudingcha, but the structure of these polysaccharides and their therapeutic effects in treating neurological hearing loss are currently unknown. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method and application of small-leaf Kudingcha polysaccharide to solve the problem that the above-mentioned presbycusis can only be treated externally by using hearing aids, cochlear implants and hearing rehabilitation means, and cannot be fundamentally treated from the internal cause, and the small-leaf Kudingcha has a large number of active ingredients that cannot be fully utilized.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides a method for preparing small-leaf Kudingcha polysaccharide, comprising the following steps:

[0008] (1) Pretreatment: crushing the small-leaf Kudingcha tea to obtain small-leaf Kudingcha tea powder;

[0009] (2) defatting: extracting the small-leaf Kudingcha powder in 95% edible ethanol to obtain the small-leaf Kudingcha residue, and drying;

[0010] (3) Polysaccharide extraction: distilled water was added to the dried filtrate of the small-leaf Kudingcha tea, and hot water extraction was performed. The extraction was repeated multiple times, and the filtrates were combined and concentrated to obtain a concentrate. 95% edible ethanol was then added, and the mixture was allowed to stand and centrifuged to obtain a precipitate.

[0011] (4) Purification: Add distilled water to the precipitate to dissolve it, filter it and freeze-dry it in a vacuum to obtain the small-leaf Kudingcha polysaccharide.

[0012] Preferably, in step (1), the fineness of the small-leaf Kudingcha powder is 20-30 mesh.

[0013] Preferably, in step (2), the volume ratio of the small-leaf Kudingcha powder to 95% edible ethanol is 1:(1-3), and the extraction times are more than 2 times.

[0014] Preferably, in step (3), the volume ratio of the filter residue of the small-leaf Kudingcha tea to distilled water is 1:(3-8).

[0015] Preferably, in step (3), the temperature of the hot water is 95° C., and the extraction time is 2 to 5 hours.

[0016] Preferably, in step (3), the volume of the concentrate is 1 / 10 of the volume of the filtrate.

[0017] Preferably, in step (3), 95% edible ethanol is added to make the alcohol content of the concentrate reach 60-70%, and then the concentrate is allowed to stand at 4°C for 10-14 hours, and then centrifuged at 1000-5000 r / min for 5-15 minutes to obtain a precipitate.

[0018] Preferably, in step (4), 0.5 L of distilled water is added to the precipitate, which is filtered and frozen overnight at -20°C, and then vacuum freeze-dried to obtain the small-leaf Kudingcha tea polysaccharide.

[0019] Preferably, in step (4), the relative molecular masses of the polysaccharides from the small-leaf Ilex kudingchai are 13592Da and 1175Da respectively.

[0020] The second aspect of the present invention provides the use of the small-leaf Kudingcha polysaccharide obtained by the above-mentioned preparation method in the preparation of a method for preventing and / or treating chemotherapy-induced deafness and presbycusis.

[0021] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0022] 1. The present invention can ensure the maximum release and extraction of polysaccharide components through multiple hot water extractions.

[0023] 2. The polysaccharide extracted from the small-leaf Kudingcha tea of ​​the present invention has a clear molecular weight distribution, which is closely related to its biological activity, providing support for its subsequent application in the treatment of presbycusis.

[0024] 3. The small-leaf Kudingcha polysaccharide of the present invention has been shown to have potential application value in treating presbycusis, which provides a new direction for its development in the field of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the appearance of the polysaccharide from the small-leaf Kudingcha tea;

[0026] Figure 2 This is the HPLC spectrum of the polysaccharide from the small-leaf Kudingcha tea;

[0027] Figure 3 Changes in hearing thresholds of mice in different treatment groups; A: C57BL / 6J mouse model and intervention plan; B: ABR follow-up and hearing thresholds of aged model mice; C: ABR follow-up and hearing thresholds of cisplatin model guinea pigs;

[0028] Figure 4 Figure 3. The polysaccharide from the small leaf Kudingcha tea significantly inhibited aging-induced auditory nerve and structural damage (whole cochlear innervation pattern of WT group, 18M group and 18M+small leaf Kudingcha tea polysaccharide group; A: Phalloidin (red) labeled cytoskeleton, Myosin7a (green) labeled HCs, the total length in the figure is 200 μm; B: quantitative analysis of control (****p<0.0001, ***p<0.001, **p<0.01, n=mean not significant); C: Neurofilament (green) labeled auditory nerve fibers, Myosin7a (gray) labeled HCs; D: quantitative analysis of control (****p<0.0001);

[0029] Figure 5:A: CCK8 assay for cell viability modeling after 72h of D-gal concentration gradient treatment; B: quantitative analysis control (****p<0.0001, ***p<0.001, **p<0.01, n.s.mean not significant);

[0030] Figure 6 :A: CCK8 assay of cell viability model after 72h of D-gal concentration gradient treatment; B: quantitative analysis control (***p<0.001, **p<0.01, nsmeant not significant);

[0031] Figure 7 To verify the effect and possible mechanism of Ilex kudingcha polysaccharides in HEI-OC1 aging model by RNAseq. DETAILED DESCRIPTION

[0032] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0033] Example 1

[0034] This embodiment provides a method for preparing small-leaf Kudingcha polysaccharide, comprising the following steps:

[0035] (1) Pretreatment: crush 1000 g of small-leaf Kudingcha to obtain 20-30 mesh small-leaf Kudingcha powder;

[0036] (2) Degreasing: Add the small leaf Kudingcha powder to 2 times the volume of 95% edible ethanol for extraction twice to obtain the small leaf Kudingcha residue, and dry it;

[0037] (3) Polysaccharide extraction: Add 5 times the volume of distilled water to the dried residue of the small-leaf Kudingcha tea, extract in hot water at 95°C for 3 h, extract twice, combine the filtrates, concentrate the filtrates to 1 / 10, and obtain a concentrate. Then, add 95% edible ethanol to make the alcohol content of the concentrate reach 65%, let it stand at 4°C for 12 h, and then centrifuge at 3000 r / min for 10 min to obtain a precipitate.

[0038] (4) Purification: Add 0.5 L of distilled water to the precipitate to dissolve it, filter it, freeze it at -20°C overnight, and freeze-dry it in a vacuum to obtain 30 g of small leaf Kudingcha polysaccharide. The extraction rate of small leaf Kudingcha polysaccharide is 0.3%. The appearance of small leaf Kudingcha polysaccharide is as follows: Figure 1 As shown in the figure, it can be seen that the polysaccharide of small-leaf Kudingcha is yellow.

[0039] Example 2

[0040] The physicochemical properties of the small-leaf Kudingcha polysaccharide prepared in Example 1 were analyzed. Specifically, high performance gel permeation chromatography (HPGPC) was used to analyze and determine the purity and molecular weight distribution of the small-leaf Kudingcha polysaccharide sample.

[0041] First, the retention times of Dextran series dextran standards with molecular weights of 5,000, 25,000, 80,000, 150,000, 420,000, and 670,000 were measured under the same conditions. A molecular weight calibration curve (y = -0.4174x + 9.3434) was generated using Agilent GPC data analysis software. The relative molecular weight (Mw) of the polysaccharide samples was then calculated based on the calibration curve. Test conditions: Agilent 1260 Infinity system; TSK G5000 PWxl (7.8 × 300 mm) column; mobile phase: double-distilled water; injection volume: 50 μL; flow rate: 0.5 mL / min; column temperature: 30°C; evaporative light detector. Figure 2 After testing, the crude polysaccharide of the small leaf Ilex kudingcha obtained in Example 1 mainly has two main peaks (t R 12.4, 15min), and their relative molecular masses are 13592Da and 1175Da respectively.

[0042] Example 3

[0043] The polysaccharide from the small leaf Kudingcha tea prepared in Example 1 was used in a mouse experiment to study the effect of the polysaccharide from the small leaf Kudingcha tea on improving presbycusis and cisplatin-induced deafness.

[0044] (1) Construction of mouse model

[0045] 1.1 To investigate the effect of Ilex kudingcha polysaccharides on the presbycusis model in vivo, this experiment was conducted using normal aging C57BL / 6J mice. 55 12-month-old mice were randomly divided into two groups, and 6-8-week-old C57BL / 6J mice were used as controls.

[0046] ①Aging control group: no intervention;

[0047] ② Experimental group: 100 mg / kg of the small-leaf Kudingcha polysaccharide prepared in Example 1 was administered by gavage three times a week;

[0048] ③Young control group: no intervention.

[0049] like Figure 3As shown in Figure B, auditory brainstem evoked potential (ABR) tests were performed 3 and 6 months after treatment. The results showed that with age, the hearing threshold of the aging control group significantly increased, while the hearing threshold of the experimental group was significantly lower than that of the aging control group of the same age. In particular, after 6M administration, the hearing threshold of the 100mg / kg experimental group was lower than that of the aging control group at click, 4KHz, 8KHz and 16KHz frequencies by 8.26dB, 10.65dB, 18.36dB and 12.46dB, respectively.

[0050] 1.2 Similarly, the polysaccharide of small-leaf Kudingcha tea has a certain therapeutic effect in the guinea pig model of cisplatin-induced ototoxicity:

[0051] ①Control group: no intervention;

[0052] ② Model group: cisplatin ototoxicity model (guinea pig, 10 mg / kg, subcutaneous injection);

[0053] ③Administration group: 1 mg / ml was administered one day in advance;

[0054] like Figure 3 As shown in C, the ototoxicity model induced by cisplatin was stably constructed. After administration of 1 mg / ml, the hearing thresholds of the drug-treated groups were significantly lower than those of the modeling group in the click, 4-32 kHz frequency range, with significant statistical differences (p<0.05).

[0055] (2) The polysaccharide from the small-leaf Kudingcha tea prepared in Example 1 inhibited the auditory pathway structure and nerve damage induced by aging in C57BL / 6J mice under in vivo conditions.

[0056] Hair cells are epithelial cells that sense auditory stimulation. They can convert sound signals into electrical signals through the movement of the cilia at the top, and then transmit them to the auditory center through the auditory nerve, thereby producing hearing. Myosin7a was used to label inner and outer hair cells to observe the protective effect of Ipomoea australis polysaccharide on hair cells. Figure 4 As shown, three months after treatment, the aged control group experienced severe inner and outer hair cell loss compared to the young control group, with the loss being most pronounced in the basal region, followed by the apical and mid-regions. The loss of outer hair cells was more severe than that of inner hair cells. The number of both inner and outer hair cells in the experimental group was greater than that in the aged control group, regardless of the apical, mid-region, or basal region (p<0.01). This suggests that polysaccharides from Kudingcha spp. can inhibit hair cell damage caused by aging.

[0057] Neuofilament was used to label the cochlear auditory nerve fibers to observe the protective effect of Kudingcha polysaccharides on the auditory nerve. The results showed that compared with the young control group, the auditory nerve fibers of the aging control group were disordered and significantly reduced in number, while the auditory nerve fibers of the experimental group were more neatly arranged and more complete in morphology, especially in the apical and middle turns. Kudingcha polysaccharides can inhibit the damage of auditory nerve fibers caused by aging.

[0058] (3) Inhibitory effect of Ilex kudingcha polysaccharides on aging-induced damage in an in vitro model

[0059] In order to construct a senescence-induced HEI-OC1 cell senescence model, different concentrations of D-galactose (D-gal) were used to induce HEI-OC1 cells for 72 h ( Figure 5 ) (5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 40 mg / mL, 60 mg / mL, and 80 mg / mL). CCK8 results showed that when the concentration was higher than 20 mg / mL, cell viability was highly significantly different (P < 0.0001). Therefore, 20 mg / mL D-gal was used to induce cell senescence and establish a HEI-OC1 cell senescence model.

[0060] To further confirm the inhibitory effect of Ilex kudingcha polysaccharides on aging-induced damage in an in vitro model, the subjects were divided into four groups according to the intervention method:

[0061] ① Control group: cells were cultured for 84 hours after attachment, with daily medium changes and no intervention;

[0062] ②D-gal group: cells were cultured in 20 mg / mL D-gal medium for 72 h after attachment;

[0063] ③ Group of Ilex kudingcha polysaccharide + D-gal: After cells adhered to the wall, they were pre-protected with 200 μg / mL of Ilex kudingcha polysaccharide culture medium. After that, they were intervened with 20 mg / mL D-gal and 200 μg / mL of Ilex kudingcha polysaccharide simultaneously for 72 hours.

[0064] like Figure 6 As shown, the CCK8 results showed that the cell viability of the small-leaf Kudingcha polysaccharide+D-gal group was significantly higher than that of the D-gal group (P<0.001).

[0065] In order to further explore the mechanism of the protective effect of small leaf Kudingcha polysaccharide on the aging-induced HEI-OC1 cell model, the differentially expressed genes in the D-gal group and the D-gal + small leaf Kudingcha polysaccharide group were detected by RNAseq, and GO analysis was used to detect that the differentially expressed genes were mostly enriched in NAD(+) metabolism-related pathways, and the NAD(+) expression-related genes in the D-gal + small leaf Kudingcha polysaccharide group were significantly upregulated. Figure 7 As shown in the results, it is suggested that the protective effect of Ilex kudingcha polysaccharide on aging model is mediated by NAD(+)-related pathway.

[0066] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A polysaccharide from small-leaf Kudingcha tea and a preparation method thereof, characterized in that: The following steps are involved: (1) Pretreatment: crushing the small-leaf Kudingcha tea to obtain small-leaf Kudingcha tea powder; (2) defatting: extracting the small-leaf Kudingcha powder in 95% edible ethanol to obtain the small-leaf Kudingcha residue, and drying; (3) Polysaccharide extraction: distilled water was added to the dried filtrate of the small-leaf Kudingcha tea, and hot water extraction was performed. The extraction was repeated multiple times, and the filtrates were combined and concentrated to obtain a concentrate. 95% edible ethanol was then added, and the mixture was allowed to stand and centrifuged to obtain a precipitate. (4) Purification: Add distilled water to the precipitate to dissolve it, filter it and freeze-dry it in a vacuum to obtain the small-leaf Kudingcha polysaccharide.

2. The method for preparing small-leaf Kudingcha polysaccharide according to claim 1, characterized in that: In step (1), the fineness of the small-leaf Kudingcha powder is 20-30 mesh.

3. The method for preparing small-leaf Kudingcha polysaccharide according to claim 1, characterized in that: In step (2), the volume ratio of the small-leaf kudingcha powder to 95% edible ethanol is 1:(1-3), and the extraction times are more than 2 times.

4. The method for preparing small-leaf Kudingcha polysaccharide according to claim 1, characterized in that: In step (3), the volume ratio of the filter residue of small-leaf Kudingcha tea to distilled water is 1:(3-8).

5. The method for preparing small-leaf Kudingcha polysaccharide according to claim 1, characterized in that: In step (3), the temperature of the hot water is 95° C., and the extraction time is 2 to 5 hours.

6. The method for preparing small-leaf Kudingcha polysaccharide according to claim 1, characterized in that: In step (3), the volume of the concentrate is 1 / 10 of the volume of the filtrate.

7. The method for preparing small-leaf Kudingcha polysaccharide according to claim 1, characterized in that: In step (3), 95% edible alcohol is added to make the alcohol content of the concentrated solution reach 60-70%, and then the concentrated solution is allowed to stand at 4° C. for 10-14 hours, and then centrifuged at 1000-5000 r / min for 5-15 minutes to obtain a precipitate.

8. The method for preparing small-leaf Kudingcha polysaccharide according to claim 1, characterized in that: In step (4), 0.5 L of distilled water is added to the precipitate, which is filtered and frozen at -20°C overnight, and then vacuum freeze-dried to obtain the small-leaf Kudingcha tea polysaccharide.

9. The method for preparing small-leaf Kudingcha polysaccharide according to claim 1, characterized in that: In step (4), the relative molecular masses of the polysaccharides from the small-leaf Ilex kudingchai are 13592Da and 1175Da respectively.

10. Use of the small-leaf Kudingcha polysaccharide obtained by the preparation method according to any one of claims 1 to 9 in the preparation of a method for preventing and / or treating chemotherapy-induced deafness and presbycusis.

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