Herbal polysaccharide exovesicle compound for maintaining retina thickness and optic nerve repairing function as well as preparation method and application of herbaceous polysaccharide exovesicle compound

Abstract

The invention provides a herbaceous polysaccharide exovesicle compound generated through fermentation, a preparation method thereof and application of the herbaceous polysaccharide exovesicle compound in promoting nerve repair and protecting optic nerves. The herbaceous polysaccharide exovesicle compound disclosed by the invention can maintain the thickness of the retina so as to avoid deterioration of retina injury caused by diabetes mellitus, and has an optic nerve protection function and an effect of promoting nerve repair.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation exovesicle technology. Through microbial fermentation technology, polysaccharide-rich herbal plants, such as Dendrobium, golden fruit, and wolfberry, are fermented to generate herbal polysaccharide exovesicle complexes. Herbal polysaccharide exovesicle complexes can maintain retinal thickness, thereby preventing the deterioration of retinal damage caused by diabetes, and have optic nerve protection function and can promote nerve repair. Background Technology

[0002] Extracellular vesicles (EVs) have been proven to be useful as carriers due to their low immune resistance, easy absorption and utilization by the human body, ability to transport drug components, small size, and high bioavailability. This structure has also been confirmed to exist in microorganisms and plants. EVs are tiny vesicles with a bilayer lipid membrane structure released from the paracrine system of living cells into the surrounding environment. They are mainly classified into four categories: exosomes, microvesicles (microparticles), apoptosis bodies, and oncosomes. However, they can be further subdivided into ectosomes, synaptic vesicles, and matrix vesicles. They can be produced by complex eukaryotes, including Gram-negative and Gram-positive bacteria, mycobacteria, and fungi; almost all cells can release EVs.

[0003] Vision health ingredients currently focus on supplementing visual pigments, such as lutein and zeaxanthin. Their main function is antioxidation, helping visual cells resist light damage and preventing cell damage caused by excessive oxidation. Vision health can be divided into two levels: 1. supplementing visual pigments; 2. protecting and repairing the optic nerve. Currently, ingredients for optic nerve repair can be divided into nerve-protecting ingredients or nerve-repairing ingredients based on their functions. Optic nerve-protecting ingredients are mostly composed of polysaccharides and can be used as food. Optic nerve-repairing ingredients are mainly composed of medicinal substances, such as stem cell components or growth factors, fibroblast growth factors, etc., but are currently only used for medical purposes.

[0004] The health care of the optic nerve needs to take into account both protection and repair. At present, consumers can only choose foods that protect the optic nerve, while the repair of the optic nerve is still mainly done through medical means. The vision care market is large, with more than 90% of the products being visual pigment products and the remaining 10% being optic nerve protection products. If functional raw materials that can take into account both the protection and repair functions of the optic nerve can be developed, then vision care can be more comprehensive. Summary of the Invention

[0005] In view of the above reasons, the present invention generates external vesicles through microbial fermentation technology. These external vesicles have high bioavailability and strong tolerance, and can also cross the vascular barrier, which can improve the absorption of active ingredients.

[0006] The herbal polysaccharide exovesicle complex of the present invention contains both polysaccharides and β-nicotinamide mononucleotide (β-NMN), and can simultaneously have optic nerve protection and repair functions.

[0007] This invention produces exovesicles through a single process, which simultaneously ferments polysaccharide herbs and generates exovesicles in the same process. The exovesicles contain the desired active ingredients without additional processing or the addition of other complex raw materials. Polysaccharides and β-NMN can be extracted during the fermentation process.

[0008] Furthermore, this invention develops raw materials that have both optic nerve protection and repair functions using a food processing method, and uses external vesicles as carriers, which can not only protect the optic nerve but also promote its repair.

[0009] This invention provides a method for preparing a herbal polysaccharide exovesicle complex, comprising:

[0010] The herbal pretreatment step is as follows: three herbal fruits and vegetables rich in polysaccharides, namely dendrobium, golden fruit and wolfberry, are placed in three separate fermentation tanks and the herbal fruits and vegetables are crushed to obtain dendrobium, golden fruit and wolfberry fermented materials respectively.

[0011] The yeast fermentation step is as follows: 0.2-2% (w / w) of Saccharomyces cerevisiae is added at a concentration of 1*10... 6 The bacterial count was inoculated into three independent fermentation tanks at a CFU / mL level, and the fermentation environment was controlled, including a sugar content of 20 Brix, a temperature of 25–28°C, and the addition of 0.01% nicotinic acid as an NMN precursor; and

[0012] The lactic acid bacteria fermentation step involves mixing the contents of three independent fermentation tanks that have undergone yeast fermentation in a 1:1:1 ratio, and simultaneously adding 1*10... 6 The bacteria were inoculated with 0.2-2% (w / w) of Lactobacillus plantarum at a CFU / mL concentration, and fermented under the following conditions: sugar content 20 Brix, fermentation temperature 25-28℃. After fermentation was completed, the herbal polysaccharide exovesicle complex of the present invention could be obtained.

[0013] In some embodiments, the polysaccharide-rich herbal fruits and vegetables are placed in separate fermentation tanks according to the weight ratios of water:dendrone; water:golden fruit; water:goji berries 1:1, 10:1, and 10:1 respectively.

[0014] The present invention also provides a herbal polysaccharide extracellular vesicle complex comprising extracellular vesicles (EVs), wherein the extracellular vesicles further comprise vegetable and fruit fermentation broth, polysaccharides, or nicotinamide mononucleotides.

[0015] In some embodiments, the fermented vegetable and fruit liquid contains a variety of polysaccharide-rich herbal fruits and vegetables, wherein the polysaccharide-rich herbal fruits and vegetables are one or more of Dendrobium, golden fruit, and wolfberry.

[0016] The present invention also provides the use of herbal polysaccharide exovesicle complex in the preparation of medicaments for treating or alleviating nerve damage.

[0017] In some embodiments, the therapeutic or nerve-relief effects include promoting the repair of the optic nerve.

[0018] The present invention also provides the use of herbal polysaccharide exovesicle complex in the preparation of a drug for protecting the optic nerve.

[0019] In some embodiments, the protective effect on the optic nerve includes maintaining retinal thickness or maintaining ganglion function. Attached Figure Description

[0020] Figure 1 The expression of Claudin-5 protein in ganglion cells in each group of the present invention is shown, where the values ​​are expressed as mean ± standard deviation; where: a, P<0.05 indicates a significant difference compared with diabetic retinopathy mice (DR); b, P<0.05 indicates a significant difference compared with polysaccharide extract only; c, P<0.05 indicates a significant difference compared with β-NMN extract only.

[0021] Figure 2 The expression of Occludin protein in ganglion cells in each group of the present invention is shown, where the values ​​are expressed as mean ± standard deviation, where: a, P<0.05 indicates a significant difference compared with diabetic retinopathy mice (DR); b, P<0.05 indicates a significant difference compared with polysaccharide extract only; c, P<0.05 indicates a significant difference compared with β-NMN extract only.

[0022] Figure 3The expression of ZO-1 protein in ganglion cells in each group of the present invention is shown, where the values ​​are expressed as mean ± standard deviation, where: a, P<0.05 indicates a significant difference compared with diabetic retinopathy mice (DR); b, P<0.05 indicates a significant difference compared with polysaccharide extract only; c, P<0.05 indicates a significant difference compared with β-NMN extract only.

[0023] Figure 4 The expression of SREBO2 protein in ganglion cells in each group of the present invention is shown, where the values ​​are expressed as mean ± standard deviation, where: a, P<0.05 indicates a significant difference compared with diabetic retinopathy mice (DR); b, P<0.05 indicates a significant difference compared with polysaccharide extract only; c, P<0.05 indicates a significant difference compared with β-NMN extract only.

[0024] Figure 5 The figures show the expression of HMGCR protein in ganglion cells in each group of the present invention, where the values ​​are expressed as mean ± standard deviation, where: a, P<0.05 indicates a significant difference compared with diabetic retinopathy mice (DR); b, P<0.05 indicates a significant difference compared with polysaccharide extract only; c, P<0.05 indicates a significant difference compared with β-NMN extract only.

[0025] Figure 6 This invention demonstrates the expression of SIRT1 protein in ganglion cells in each group.

[0026] Figure 7 This invention shows the staining results of ocular sections from different experimental groups.

[0027] Figure 8 This invention demonstrates the effect of different experimental groups of herbal polysaccharide extravesicular complex (EVs-NMN) on the concentration of superoxide dismutase (SOD) enzyme in serum.

[0028] Figure 9 This invention demonstrates the effect of different experimental groups of herbal polysaccharide extravesicular complex (EVs-NMN) on serum glutathione (GSH) concentration.

[0029] Figure 10 This is a diagram illustrating the mechanism of action of the herbal polysaccharide exovesicle complex of the present invention. Detailed Implementation

[0030] To make the description of the present invention more detailed and complete, illustrative descriptions of the embodiments and specific examples of the present invention are provided below; however, these are not the only forms of implementing or utilizing the specific examples of the present invention. The embodiments cover the features of multiple specific examples and the methods and uses for operating these specific examples; however, other specific examples may also be used to achieve the same or equivalent effects. It should be understood that these embodiments are for illustrative purposes only and not for limiting the scope of the present invention.

[0031] Example 1: Preparation of Herbal Polysaccharide Exovesicle Complex

[0032] The herbal polysaccharide exovesicle complex of the present invention is prepared by the following steps:

[0033] (A) Herbal Pretreatment

[0034] Golden fruit, dendrobium stems, and goji berries were prepared separately in individual fermentation tanks according to water:vegetable / fruit weight ratios of 1:1, 10:1, and 10:1, respectively. Then, the herbs and fruits in each fermentation tank were ground using a grinding device to obtain fermented materials for golden fruit, dendrobium, and goji berries, respectively, for later use.

[0035] (B) Yeast fermentation

[0036] Three independent fermentation tanks were each inoculated with 0.5% (w / w) of the fermentation strain Saccharomyces cerevisiae, with a bacterial count of 1*10⁻⁶. 6 The concentration of CFU / mL was adjusted to 20 Brix, the fermentation temperature was controlled at 25-28℃, and finally 0.01% nicotinic acid was added as a precursor of NMN for yeast fermentation.

[0037] After 14 days of fermentation, Saccharomyces cerevisiae can produce 0.5%–3% β-NMN.

[0038] (C) Lactic acid bacteria fermentation

[0039] Next, the contents of the three fermentation tanks after yeast fermentation were mixed in a 1:1:1 (v / v / v) ratio, and 0.5% (w / w) of the fermentation strain Lactobacillus plantarum was introduced into the mixture for lactic acid fermentation, with a bacterial count of 1*10⁻⁶. 6 The concentration of CFU / mL was then adjusted to 20 Brix, and the fermentation temperature was controlled at 25–28°C.

[0040] After 30 days of lactic acid bacteria fermentation, Lactobacillus plantarum undergoes cell division during fermentation, forming a large number of exovesicles.

[0041] Example 2: Establishment and Group Description of the Animal Model of the Present Invention

[0042] After establishing an animal model of diabetic retinopathy (DR), diabetic mice were divided into 5 groups of male mice, including:

[0043] Control group (WT group): No diabetes was induced;

[0044] Control group (DR group): Diabetic retinopathy control group;

[0045] Low-dose group (LD group): Diabetes was induced and mice were given a low dose (0.195 mg per kg of body weight) of fermentation complex or polysaccharide extract or β-NMN (LD);

[0046] Recommended Dosage Group (MD Group): Diabetes was induced and mice were given the recommended dose (0.39 mg per kg of body weight) of fermentation complex or polysaccharide extract or β-NMN; and

[0047] High-dose group (HD group): Diabetes was induced and the mice were given a high dose (1.17 mg per kg of body weight) of fermentation complex or polysaccharide extract or β-NMN.

[0048] Each group and each test sample consisted of 12 mice. In the groups that received the fermentation complex, the fermentation complex, polysaccharide extract, or β-NMN were administered via tube feeding for a total of 28 days.

[0049] Example 3: Expression of growth-promoting proteins in optic ganglion cells

[0050] This experiment used the animal model established in Example 2. Subsequently, retinal cells were sampled from mice to analyze the expression of SIRT1, SREBP2, and HMGCR proteins.

[0051] The results are as follows Figures 1-6 As shown, the DR group will cause retinal oxidative stress damage. The inflammatory factor TNF-α caused by oxidative damage increases, which promotes the adhesion of white blood cells to the blood vessel wall, thereby blocking the retinal-vascular barrier (blood-retinal barrier, BRB). When nutrients and blood cannot pass smoothly into the retina, retinal cell damage is caused.

[0052] Repair of the blood-retinal barrier (BRB) relies on the formation of tight junction proteins. Increased levels of Claudin-5, Occludin, and ZO-1 proteins promote BRB repair and maintain the normal morphology of optic ganglion cells. Experimental results show that the herbal polysaccharide extravesicular complex (EVs-NMN) significantly increased protein expression at both the recommended and high-dose groups, demonstrating a significant difference compared to the use of polysaccharide extracts or β-NMN alone. These results indicate that EVs-NMN promotes optic ganglion repair.

[0053] Furthermore, according to the retinal ganglion repair pathway, studies have shown that increased SIRT1 protein expression can promote increased cholesterol concentration in ganglion cells, which is beneficial for cellular DNA repair. It can also increase the expression levels of transcription factor SREBP2 (Sterol Regulatoryelement Binding Protein-2) and HMGCR protein in ganglion cells. This pathway can increase the number of retinal ganglion cells (RGCs). Analysis results show that the use of EVs-NMN can significantly increase SIRT1 protein expression in retinal cells, while simultaneously increasing the expression levels of SREBP2 and HMGCR. Therefore, it can help increase the number of RGCs, achieving the effect of repairing visual nerve cells.

[0054] Example 4: Eyeball Section Staining Experiment

[0055] This experiment used the animal model established in Example 2. Mice were sacrificed after being fed for 28 days, and mouse eyeballs were sampled and stained.

[0056] The results are as follows Figure 7 As shown, Part B represents the control group mice, whose ganglion cell layer showed significant vacuolation and angiogenesis; the inner plexiform layer also showed vacuolation and thinning; the inner nuclear layer had a scattered and irregular structure and was significantly thinned; and the outer nuclear layer also showed thinning. This indicates that long-term hyperglycemia can lead to retinal thinning, damage to optic nerve cells, and ultimately, a risk of blindness. Part C represents the recommended dose group mice. The results show that after using the recommended dose of EVs-NMN, the vacuolation of ganglion cells was significantly improved, the ganglion cells gradually became more orderly arranged, and the inner plexiform layer, inner nuclear layer, and outer nuclear layer also showed a tendency to thicken, with a more orderly structure. This indicates that EVs-NMN can improve retinal thinning, protect retinal layer thickness, restore damaged ganglion cells, and inhibit retinal angiogenesis.

[0057] Example 5: Analysis of the antioxidant protective function of the optic nerve / retina

[0058] This experiment used the animal model established in Example 2. Serum samples were collected from mice in the control group, control group, low-dose group, recommended dose group, and high-dose group to analyze the concentration of superoxide dismutase (SOD) and glutathione (GSH) in the serum.

[0059] The results are as follows Figures 8-9 As shown, research indicates that retinopathy caused by light damage or high blood sugar is accompanied by a decrease in the concentration of SOD or antioxidants in the body after its onset, which in turn damages retinal cells, causing the retina to thin and impairing vision. In diabetic retinopathy mice treated with the EVs-NMN of this invention, the concentrations of SOD and GSH in the blood significantly increased, indicating that EVs-NMN can improve the antioxidant capacity of the retina, thereby protecting the function of the retina and nerve cells.

[0060] Example 6: Main Functional Mechanism of Action of the Herbal Polysaccharide Exovesicle Complex of the Present Invention The mechanism of action of the EVs-NMN of the present invention is as follows: Figure 10 As shown.

[0061] 1. EVs-NMN can increase the concentration of SOD and GSH, thereby controlling the decrease in TNF-α expression. This leads to an increase in the expression of retinal ganglion cell repair factors Claudin-5, Occludin, and ZO-1, thus maintaining retinal thickness and retinal ganglion function.

[0062] 2. EVs-NMN can increase the expression of SIRT protein, thereby increasing the expression of retinal cell differentiation factor SREBP2 and HMGCR, achieving the effect of optic ganglion repair.

[0063] The above embodiments of the present invention are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope thereof, but all such modifications and substitutions fall within the protection scope of the present invention.

Claims

1. A method for preparing a herbal polysaccharide exovesicle complex, characterized in that, Include: The herbal pretreatment step is as follows: three kinds of herbal fruits and vegetables rich in polysaccharides are placed in separate fermentation tanks and the fruits and vegetables are crushed to obtain three kinds of herbal fruit and vegetable fermentation materials. The yeast fermentation step is as follows: 0.2-2% (w / w) of Saccharomyces cerevisiae is added at a concentration of 1*10... 6 The bacteria were inoculated into separate fermentation tanks with a CFU / mL count, and the fermentation environment was controlled at a sugar content of 20 Brix and a temperature of 25-28℃. 0.01% nicotinic acid was added as an NMN precursor. and The lactic acid bacteria fermentation step is as follows: the contents of three independent fermentation tanks after yeast fermentation are mixed in a 1:1:1 ratio, and 1*10 are added to the mixture. 6 The bacteria were inoculated with 0.2–2% (w / w) of *Lactobacillus plantarum* at a concentration of CFU / mL, and fermentation was carried out at a sugar concentration of 20 Brix and a fermentation temperature of 25–28°C. Once fermentation is complete, the herbal polysaccharide exovesicle complex can be obtained.

2. The preparation method according to claim 1, characterized in that, The polysaccharide-rich herbs and vegetables include dendrobium, golden fruit, and goji berries.

3. The preparation method according to claim 2, characterized in that, The polysaccharide-rich herbal fruits and vegetables were placed in their respective independent fermentation tanks according to the following weight ratios: water: dendrobium; water: golden fruit; water: goji berries, respectively, at 1:1, 10:1, and 10:

1.

4. A herbal polysaccharide exovesicle complex, characterized in that, It contains extracellular vesicles (EVs), which further contain fruit and vegetable fermentation broth, polysaccharides, or nicotinamide mononucleotides.

5. The herbal polysaccharide exovesicle complex as described in claim 4, characterized in that, The fermented vegetable and fruit liquid is a fermented liquid containing herbal fruits and vegetables rich in polysaccharides, wherein the herbal fruits and vegetables rich in polysaccharides include dendrobium, golden fruit and goji berries.

6. Use of the herbal polysaccharide exovesicle complex as described in claim 4 in the preparation of a medicament for treating or alleviating nerve damage.

7. The use of the herbal polysaccharide exovesicle complex as described in claim 6 for the preparation of a medicament for treating or alleviating nerve damage, characterized in that, The treatment or relief of nerve damage includes promoting the repair of the optic nerve.

8. Use of the herbal polysaccharide exovesicle complex as described in claim 4 in the preparation of a medicament for protecting the optic nerve.

9. The use of the herbal polysaccharide exovesicle complex as described in claim 8 in the preparation of a medicament for protecting the optic nerve, characterized in that, The protection of the optic nerve includes maintaining retinal thickness or maintaining ganglion function.

Images