Use of dendrobium nobile in preparation of medicine for treating oral ulcer in combination with pueraria

Abstract

This invention specifically discloses the application of Dendrobium in the preparation of a drug for treating oral ulcers in combination with Pueraria lobata, relating to the field of biomedical technology. The Dendrobium exosomes combined with Pueraria lobata exosomes provided by this invention can simultaneously and effectively reduce inflammation, promote oxidation, and accelerate the proliferation and migration of epithelial cells, thereby fundamentally accelerating the healing process of oral ulcers.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of Dendrobium in the preparation of a drug for the treatment of oral ulcers in combination with Pueraria lobata. Background Technology

[0002] Currently, there are many types of drugs available for the clinical treatment of oral ulcers, but most of them share common drawbacks such as limited efficacy, single action, and poor user experience. These drawbacks are specifically manifested in the following aspects:

[0003] 1. Limited Mechanism of Action, Treating Symptoms but Not the Root Cause: Most medications (such as mouthwashes, sprays, and powders) primarily relieve surface symptoms through antibacterial, anti-inflammatory, analgesic, or physical isolation (such as forming a protective film). They cannot effectively regulate the complex pathophysiological processes behind ulcer healing, such as excessive immune inflammatory responses, oxidative stress damage, and inhibited cell proliferation / migration, resulting in superficial therapeutic effects and a high recurrence rate.

[0004] 2. Unsatisfactory therapeutic effect and slow healing speed: Especially for patients with severe, recurrent, or poorly responded-to hormonal drugs, existing medications often only alleviate pain but cannot significantly shorten the complete healing period of the ulcer (usually still 7-14 days). Patients still have to endure inconvenience and pain for a long time.

[0005] 3. Poor user experience and low adherence to topical medications: Ointments and patches are easily washed away by saliva and cannot remain on the ulcer surface for long, requiring frequent application. Sprays and mouthwashes may cause irritation, pain, or taste discomfort. These unpleasant user experiences reduce patient adherence to treatment, thus affecting the final outcome.

[0006] 4. Limitations of Hormone and Antibiotic Use: To enhance anti-inflammatory and antibacterial effects, some medications contain corticosteroids or antibiotics. Long-term use of hormones carries risks such as local tissue atrophy and secondary infections; while overuse of antibiotics can lead to dysbiosis and antibiotic resistance.

[0007] There is a lack of effective means to promote tissue regeneration: In existing treatment options, drugs that can actively and efficiently promote the regeneration of mucosal epithelial cells and accelerate the formation of granulation tissue are very scarce, which is the key to completely curing ulcers. Summary of the Invention

[0008] (a) Technical problems to be solved

[0009] Therefore, one of the main objectives of this invention is to provide the use of Dendrobium in the preparation of a medicament for the treatment of oral ulcers in combination with Pueraria lobata. It can simultaneously and effectively combat inflammation and oxidation, and promote epithelial cell proliferation and migration, thereby fundamentally accelerating the healing process of oral ulcers.

[0010] (II) Technical Solution

[0011] To achieve the above objectives, the present invention provides the use of Dendrobium exosomes in the preparation of a medicament for the prevention and / or treatment of oral ulcers in combination with Pueraria exosomes.

[0012] In one embodiment, the ratio of Dendrobium exosomes to Pueraria exosomes is (0.5~2):1.

[0013] In one embodiment, the ratio of Dendrobium exosomes to Pueraria exosomes is 2:1.

[0014] In one embodiment, the dosage of the Dendrobium exosomes is 20 μL; the dosage of the Pueraria exosomes is 10 μL.

[0015] In one embodiment, Dendrobium exosomes are applied first, followed by Pueraria exosomes.

[0016] In one embodiment, kudzu exosomes are applied first, followed by dendrobium exosomes.

[0017] In one embodiment, Dendrobium exosomes and Pueraria exosomes are applied simultaneously.

[0018] In one embodiment, the subjects to which the treatment is administered include both mammals and non-mammals.

[0019] Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds, fish, or other non-mammals.

[0020] In one embodiment, the subject is a rat.

[0021] In one embodiment, the subject is healthy.

[0022] In one embodiment, the subject is unhealthy.

[0023] In one embodiment, the subject had oral ulcers.

[0024] In one embodiment, the subject did not have oral ulcers.

[0025] In another aspect, the present invention provides a pharmaceutical composition comprising:

[0026] (1) Therapeutic effective amounts of Dendrobium exosomes and Pueraria exosomes;

[0027] (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0028] In one embodiment, the ratio of Dendrobium exosomes to Pueraria exosomes is (0.5~2):1.

[0029] In one embodiment, the ratio of Dendrobium exosomes to Pueraria exosomes is 2:1.

[0030] In one embodiment, the dosage of the Dendrobium exosomes is 20 μL; the dosage of the Pueraria exosomes is 10 μL.

[0031] In one embodiment, the pharmaceutical composition comprises:

[0032] (1) Dendrobium exosomes and Pueraria exosomes with a therapeutic effective ratio of (0.5~2):1;

[0033] (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0034] In one embodiment, the pharmaceutical composition comprises:

[0035] (1) The ratio of Dendrobium exosomes and Pueraria exosomes with a therapeutic effective dose of 2:1;

[0036] (2) Pharmaceutically or immunologically acceptable carriers or excipients.

[0037] In another aspect, the present invention provides a pharmaceutical preparation comprising the above-described pharmaceutical composition.

[0038] In one embodiment, the dosage form of the pharmaceutical preparation includes granules, lotions, liniments, aerosols, sprays, elixirs, ointments, suspensions, infusions, tablets, suppositories, injections, capsules, creams, lozenges, tinctures, and / or pastes.

[0039] In one embodiment, the dosage form of the pharmaceutical preparation is a liniment.

[0040] In another aspect, the present invention also provides a product comprising the above-described pharmaceutical composition and / or pharmaceutical preparation.

[0041] In another aspect, the present invention also provides the use of the above-described pharmaceutical compositions and / or pharmaceutical preparations and / or products in the preparation of medicaments for the prevention and / or treatment of oral ulcers.

[0042] (III) Beneficial Effects

[0043] This invention provides the application of Dendrobium in the preparation of a drug for treating oral ulcers in combination with Pueraria lobata. Compared with the prior art, it has the following beneficial effects:

[0044] 1. Significantly accelerates ulcer healing: Through multi-target action, it significantly shortens (significantly reduces) the complete healing time of oral ulcers, and is expected to effectively shorten the healing cycle from the usual 7-14 days.

[0045] 2. Synergistic regulation of the healing microenvironment: potent anti-inflammatory effect: significantly inhibits the infiltration of inflammatory cells, reduces the level of pro-inflammatory factors, and effectively relieves acute symptoms such as redness, swelling and pain; highly effective antioxidant effect: significantly reduces the level of local oxidative stress in ulcers (such as significantly reducing MDA content), enhances the body's antioxidant capacity (such as increasing GSH level), and protects cells from free radical damage. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 This is an electron microscope image of kudzu exosomes.

[0048] Figure 2 This is an electron microscope image of Dendrobium exosomes.

[0049] Figure 3 This is a graph showing the NTA detection results of kudzu exosomes.

[0050] Figure 4 This is a graph showing the NTA detection results of Dendrobium exosomes.

[0051] Figure 5 This is a standard curve for the quantitative analysis of exosomal proteins.

[0052] Figure 6 This is a graph showing the improvement in the area of ​​oral ulcers.

[0053] Figure 7 This is a bar chart showing the area of ​​oral ulcers.

[0054] Figure 8 This is a picture of the HE staining results.

[0055] Figure 9 This is a bar chart of malondialdehyde (MDA) concentration.

[0056] Figure 10 This is a graph showing glutathione concentration.

[0057] Figure 11 This is a graph of serum iron concentration. Detailed Implementation

[0058] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0059] Terms and Definitions

[0060] As used in this article, the term "exosomes" refers to lipid-bound, cell-secreting vesicles that are released from the cell via fusion with the plasma membrane through multivesicular endosomes (MVEs) and mediate intercellular communication through the intercellular transport of proteins and RNA. Typically, exosomes range in size from about 30 nm to about 200 nm, and sometimes from 30–150 nm or 30–100 nm, and have a double-membrane structure and a saucer-like morphology.

[0061] As used herein, the term "plant material" has the meaning commonly understood in the art. Plant materials can be fresh or dried.

[0062] As used herein, the term "pharmaceutical composition" refers to a composition comprising Dendrobium exosomes and Pueraria exosomes formulated together with one or more pharmaceutically acceptable carriers.

[0063] The formulation of the pharmaceutical composition can be adjusted according to the application. In particular, the pharmaceutical composition can be formulated using methods known in the art to provide rapid, continuous, or delayed release of the active ingredient upon administration to mammals. For example, the formulation can be selected from any of the following: granules, lotions, liniments, aerosols, sprays, elixirs, ointments, suspensions, infusions, tablets, suppositories, injections, capsules, creams, lozenges, tinctures, and / or pastes.

[0064] As used herein, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or animals without excessive adverse effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0065] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to a carrier used for the administration of therapeutic agents, encompassing a variety of excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0066] Pharmaceutically acceptable carriers in a composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption-delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, flow aids, pH adjusters, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc., compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may contain other active compounds that provide complementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin, are possible. Other examples of carriers include culture media, such as DMEM or RPMI; and cryogenic storage media containing components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates, and ion concentrations to balance intracellular states at low temperatures; and mixtures of organic solvents with water.

[0067] The pharmaceutical compositions of the present invention can be administered using any known method. One of a variety of methods known to those skilled in the art can be used to administer the substance, compound, or agent to a subject using the terms "give" or "apply".

[0068] For example, compounds or agents can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). Compounds or agents can also be suitably introduced via rechargeable or biodegradable polymeric devices or other devices (e.g., patches and pumps or formulations) that provide prolonged, slowed, or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more prolonged periods.

[0069] As used herein, the term “therapeutic effective dose” refers to a dose sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level includes subject type and severity, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors known in the medical field.

[0070] As used herein, the term “treatment” for a symptom or patient refers to steps taken to achieve a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, eliminating, substantially inhibiting, slowing, or reversing the progression of a disease, symptom, or condition; substantially improving or alleviating the clinical or aesthetic symptoms of a symptom; substantially preventing the clinical or aesthetic symptoms of a disease, symptom, or condition; and avoiding harmful or unpleasant symptoms. Treatment also refers to achieving one or more of the following: (a) reducing the severity of the symptom; (b) limiting the development of characteristic symptoms of the symptom being treated; (c) limiting the exacerbation of characteristic symptoms of the symptom being treated; (d) limiting the recurrence of the symptom in patients who previously had the symptom; and / or (e) limiting the recurrence of symptoms in patients who previously did not have symptoms of the symptom.

[0071] The term "prevention" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms.

[0072] As used herein, the phrases “significantly reduced” or “significantly different” indicate a sufficiently high difference between two values ​​(typically one value associated with a molecule and the other with a reference / comparison molecule) such that a person skilled in the art would consider the difference between the two values ​​to be statistically significant in the context of the biological characteristic measured by the value (e.g., the KD value). Depending on the value of the reference / comparison molecule, for example, the difference between the two values ​​is greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and / or greater than about 50%.

[0073] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0074] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0075] Example 1: Preparation of exosomes:

[0076] Fresh plant materials (Dendrobium and Kudzu) were surface-sterilized with 75% alcohol and placed in a biosafety cabinet for 20 minutes. Unusable parts were removed. The peeled parts of the fresh Dendrobium stems and the roots of Kudzu were cut into small pieces for juicing. The juice was extracted using a sterilized juicer and collected. PBS buffer was added to dilute the juice and stirred well. The juice was then centrifuged to remove impurities and the supernatant was collected.

[0077] Add the purified plant juice to a well-mixed Solution A reagent and incubate at 37°C for 16 hours (overnight). Adjust the pH of the incubated mixture to 7.0 using 1M NaOH and 1% citric acid under a biosafety cabinet. Centrifuge the pH-adjusted mixture at 4°C and 10,000 g for 20 min, and collect the supernatant. Further purify the supernatant by passing it through 1 μm and 0.45 μm filter membranes.

[0078] Then add Solution B reagent, vortex for 1 min to mix, let stand at 4°C for 4 hours, centrifuge again at 5000 g for 10 min, aspirate the supernatant, and gently pipette the precipitate with an appropriate amount of PBS until dissolved. Transfer the precipitate to a new 1.5 ml EP tube, centrifuge at 10000 g for 10 min, and retain the supernatant, which contains plant vesicle particles (exosomes).

[0079] The collected crude plant vesicle particles were transferred to an EPF column (Exosome Purification Filter) for final purification. The column was centrifuged at 3000 g for 10 minutes, and the filtrate was collected. The purified plant vesicles were aliquoted and stored at -80℃ to avoid repeated freeze-thaw cycles.

[0080] Example 2: Confirmation of exosomes:

[0081] The particle size and concentration of exosomes prepared in Example 1 were detected using NTA, vesicle morphology was observed by electron microscopy, particle size distribution was analyzed using a Malvern particle size analyzer, and protein concentration was determined by protein quantification (standard curve as shown). Figure 5 As shown in Table 1, the test results are as follows.

[0082] The results are as follows Figures 1-4 As shown, the exosomes were successfully prepared. The concentration of kudzu root exosomes was 7.512 mg / ml, and the particle size was 100-200 nm. The concentration of dendrobium exosomes was 1.02 mg / ml, and the particle size was 100-200 nm.

[0083] Table 1. Results of exosome protein quantification

[0084]

[0085] Example 3: Construction of an animal model:

[0086] glacial acetic acid etching method:

[0087] 1. Anesthesia: 2% sodium pentobarbital (40 mg / kg, intraperitoneal injection).

[0088] 2. Model making:

[0089] Soak a cotton swab in 50% glacial acetic acid and apply it to the left buccal mucosa (3×3mm).

[0090] Duration of action: 40 seconds;

[0091] Rinse three times with saline solution.

[0092] 3. Success Criteria:

[0093] One day later, visual observation revealed a central grayish-white necrotic area surrounded by a ring of congestion.

[0094] Example 4: Treatment of oral ulcers:

[0095] We treated exosomes of Dendrobium and Pueraria alone, or in different proportions, and observed their effects on ulcer healing.

[0096] The oral ulcer animal model constructed in Example 3 was divided into 7 groups, and each group was given the following treatment:

[0097] Model group: Apply saline solution twice daily;

[0098] Positive drug group: Guilin watermelon frost 1g / kg, prepared with physiological saline, 30μl was administered once and evenly applied to the ulcer surface;

[0099] Dendrobium group: 30ul of Dendrobium exosomes were evenly applied to the ulcer surface;

[0100] Kudzu root group: 30ul of kudzu root exosomes were evenly applied to the ulcer surface;

[0101] Combined medication group (Dendrobium:Pueraria lobata ratio of 1:2): 10ul of Pueraria lobata + 20ul of Dendrobium, applied evenly to the ulcer surface;

[0102] Combined medication group (Dendrobium:Pueraria lobata ratio of 1:1): 15ul Pueraria lobata + 15ul Dendrobium, applied evenly to the ulcer surface;

[0103] Combined medication group (Dendrobium:Pueraria lobata ratio of 2:1): 20ul Pueraria lobata + 10ul Dendrobium, applied evenly to the ulcer surface;

[0104] Meanwhile, a normal group of rats without oral ulcers was set up and treated with saline solution.

[0105] Example 5: Verification of therapeutic effect:

[0106] Photographs of the ulcerated area were taken daily to record the healing process; the degree of tissue inflammation and epithelial repair were observed using HE staining; simultaneously, serum tests were performed on oxidative stress-related biochemical indicators, including serum iron, MDA (malondialdehyde), and glutathione (GSH / GSSG). Results were as follows: Figures 6-11 As shown in Tables 2 to 4.

[0107] Table 2 Malondialdehyde Concentration

[0108]

[0109] Table 3 Glutathione Concentration

[0110]

[0111] Table 4 Serum iron concentration

[0112]

[0113] Experimental results showed that both Dendrobium and Pueraria exosomes could significantly promote the healing of oral ulcers, with the Dendrobium + Pueraria (2:1) combination showing particularly significant effects; pathological sections showed that the infiltration of inflammatory cells was reduced and epithelial repair was accelerated in the treatment group; biochemical indicators showed that the MDA level decreased and the GSH level increased, indicating that the oxidative stress was relieved.

[0114] In conclusion, the plant exosome extraction method provided by this invention is efficient and stable, and the extracted exosomes have good biological activity, which can effectively promote the healing of oral ulcers and has broad clinical application prospects.

[0115] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0116] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of Dendrobium exosomes in the preparation of a drug for the prevention and / or treatment of oral ulcers in combination with Pueraria exosomes, characterized in that, The ratio of Dendrobium exosomes to Pueraria exosomes is 2:

1.

2. The application according to claim 1, characterized in that, The dosage of Dendrobium exosomes is 20 μL; the dosage of Pueraria exosomes is 10 μL.

3. A pharmaceutical composition, characterized in that, include: (1) A therapeutically effective amount of Dendrobium exosomes and Pueraria exosomes, wherein the ratio of Dendrobium exosomes to Pueraria exosomes is 2:1; (2) Pharmaceutically or immunologically acceptable carriers or excipients.

4. A pharmaceutical preparation, characterized in that, Includes the pharmaceutical composition according to claim 3.

5. The pharmaceutical preparation according to claim 4, characterized in that, The dosage forms of the pharmaceutical preparations include granules, lotions, liniments, aerosols, sprays, elixirs, ointments, suspensions, tablets, suppositories, injections, capsules, creams, lozenges, tinctures, and / or pastes.

6. The pharmaceutical preparation according to claim 5, characterized in that, The dosage form of the pharmaceutical preparation is a liniment.

7. A pharmaceutical product, characterized in that, This includes the pharmaceutical composition of claim 3 and / or the pharmaceutical formulation of any one of claims 4-6.

8. The use of the pharmaceutical composition of claim 3 and / or the pharmaceutical preparation of any one of claims 4-6 and / or the pharmaceutical product of claim 7 in the preparation of a medicament for the prevention and / or treatment of oral ulcers.

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