Application of hippophae rhamnoides fruit pulp or hippophae rhamnoides fruit oil in preparation of medicine for preventing and treating pharyngitis caused by smoking

Abstract

The invention provides application of hippophae rhamnoides fruit pulp or hippophae rhamnoides fruit oil in preparation of a medicine for preventing and treating pharyngitis caused by smoking, and belongs to the technical field of biological medicine. The sea buckthorn oil is sea buckthorn fruit oil and / or sea buckthorn seed oil. Mouse experiments prove that the hippophae rhamnoides fruit pulp, the hippophae rhamnoides fruit oil and the hippophae rhamnoides fruit seed oil all have the effects of preventing and treating pharyngitis caused by smoking, natural food rich in nutrient substances is used for replacing traditional drugs for treatment, and the traditional Chinese medicine composition is small in side effect, economical, practical and suitable for being taken for a long time and has obvious prevention and treatment effects.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of seabuckthorn pulp or seabuckthorn oil in preparing medicine for preventing and treating pharyngitis caused by smoking. Background Art

[0002] Smoking, as a common bad habit, poses a major threat to global human health. Among the more than 7,000 chemical substances released by tobacco combustion, nicotine, tar, carbon monoxide, etc. have serious impacts on human health. Nicotine is highly addictive, affecting the function of the nervous system, causing vasoconstriction and increased blood pressure; carcinogens and irritants in tar destroy tissue structure, cause inflammation and increase the risk of cancer; carbon monoxide reduces the oxygen-carrying capacity of hemoglobin, leading to systemic hypoxia.

[0003] The damage caused by smoking to the pharynx is mainly manifested in direct stimulation and inflammatory response, immune function damage and increased risk of infection. Nicotine and tar in smoke cause pharyngeal mucosal blood vessel contraction, blood circulation obstruction, triggering the release of inflammatory mediators, causing mucosal congestion, swelling, pain and other symptoms. Long-term smoking can also lead to decreased function of pharyngeal mucosal immune cells, increase the risk of infection, and cause upper respiratory tract infections such as pharyngitis and tonsillitis.

[0004] Existing treatments include medication and physical therapy. Antibiotics in medication can quickly relieve inflammatory symptoms, but long-term or irrational use can lead to drug resistance; gargles and lozenges can temporarily relieve symptoms, but cannot repair mucosal damage caused by smoking. Although physical therapies such as laser therapy and microwave therapy can improve symptoms, there is a risk of damaging normal tissues and large differences in effects.

[0005] As a natural resource with the potential to improve pharyngitis, seabuckthorn contains rich nutrients and antioxidant vitamins, which can promote mucosal repair, scavenge free radicals, and reduce oxidative stress. The flavonoids in seabuckthorn have anti-inflammatory activity, which can inhibit inflammatory responses and alleviate pharyngeal mucosal symptoms. In addition, seabuckthorn also has immunomodulatory effects, which can enhance the activity of immune cells, reduce the risk of infection, and provide a comprehensive and effective solution for the treatment of pharyngitis. Studies have shown that seabuckthorn extracts can reduce mucosal damage in animal models of pharyngitis induced by smoking, and enhance cell repair and regeneration capabilities, showing the potential of seabuckthorn in the treatment of pharyngitis. Summary of the invention

[0006] The purpose of the present invention is to provide the use of seabuckthorn pulp or seabuckthorn oil in the preparation of a medicine for preventing and treating pharyngitis caused by smoking, so as to provide a new safe and effective medicine for pharyngitis caused by smoking.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides application of seabuckthorn pulp or seabuckthorn oil in preparing medicine for preventing and treating pharyngitis caused by smoking.

[0009] Preferably, the sea buckthorn oil is sea buckthorn fruit oil and / or sea buckthorn seed oil.

[0010] Preferably, the seabuckthorn pulp and seabuckthorn fruit oil are obtained by extracting seabuckthorn fruit raw materials through supercritical carbon dioxide extraction;

[0011] The seabuckthorn seed oil is obtained by extracting seabuckthorn seed raw materials through supercritical carbon dioxide extraction method.

[0012] Preferably, the temperature during the extraction of seabuckthorn pulp and seabuckthorn oil is 30-60° C., the pressure is 10-30 MPa, and the CO2 flow rate is 5-20 L / h.

[0013] Preferably, after the extraction of seabuckthorn pulp and seabuckthorn oil is completed, the extraction products are collected and solid-liquid separation is performed to obtain a lower aqueous phase of seabuckthorn pulp and an upper oil phase of seabuckthorn oil.

[0014] Preferably, the solid-liquid separation is performed by centrifugation, and the speed of the centrifugation is 3000-6000 rpm and the time is 15-40 min.

[0015] Preferably, the seabuckthorn seeds are crushed to a particle size of 0.5 to 2 mm before the seabuckthorn seed oil is extracted.

[0016] Preferably, the temperature during seabuckthorn seed oil extraction is 35-60° C., the pressure is 15-35 MPa, and the CO2 flow rate is 8-25 L / h.

[0017] Preferably, after the extraction of seabuckthorn seed oil is completed, the extraction product is collected, and the solid-liquid separation is performed to obtain the upper oil phase which is seabuckthorn seed oil.

[0018] Preferably, the solid-liquid separation is performed by centrifugation, and the speed of the centrifugation is 4000 to 8000 rpm, and the time is 15 to 40 min.

[0019] The invention proves through mouse experiments that seabuckthorn pulp, seabuckthorn fruit oil and seabuckthorn seed oil all have the effects of preventing and treating pharyngitis caused by smoking. The invention replaces traditional drug treatment with natural food rich in nutrients, has little side effects, is economical and affordable, is suitable for long-term use, and has obvious preventive and therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0021] Figure 1 Schematic diagram of the supercritical carbon dioxide extraction device used in Example 1, wherein 1 is an extraction kettle, 2 is a separation column, 3 is a heat exchanger, 4 is a condenser, 5 is a cryogenic refrigerator, 6 is a pump, 7 is a heat exchanger, and 8 is a storage tank;

[0022] Figure 2 HE staining results of pharyngeal tissues of mice in each group in Example 2;

[0023] Figure 3 These are the results of IL-1β and IL-18 staining of the pharyngeal tissues of each group of mice in Example 3. DETAILED DESCRIPTION

[0024] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0025] The invention provides application of seabuckthorn pulp or seabuckthorn oil in preparing medicine for preventing and treating pharyngitis caused by smoking.

[0026] In the present invention, the seabuckthorn oil is seabuckthorn fruit oil and / or seabuckthorn seed oil.

[0027] In the present invention, the seabuckthorn pulp and seabuckthorn fruit oil are obtained by extracting seabuckthorn fruit raw materials through supercritical carbon dioxide extraction;

[0028] The seabuckthorn seed oil is obtained by extracting seabuckthorn seed raw materials through supercritical carbon dioxide extraction method.

[0029] In the present invention, the temperature during extraction of sea buckthorn pulp and sea buckthorn oil is 30-60°C, preferably 40-55°C, further preferably 45-55°C, and more preferably 50°C; the pressure is 10-30MPa, preferably 15-25MPa, further preferably 18-22MPa, and more preferably 20MPa; the CO2 flow rate is 5-20L / h, preferably 8-15L / h, and further preferably 10L / h.

[0030] In the present invention, after the extraction of seabuckthorn pulp and seabuckthorn oil is completed, the extraction products are collected and solid-liquid separation is performed to obtain the lower aqueous phase of seabuckthorn pulp and the upper oil phase of seabuckthorn oil.

[0031] In the present invention, the solid-liquid separation is centrifugation, and the speed of the centrifugation is 3000-6000rpm, preferably 3800-5800rpm, more preferably 4500-5500rpm, more preferably 5000rpm; the time is 15-40min, preferably 20-35min, more preferably 25-32min, more preferably 30min.

[0032] In the present invention, the seabuckthorn seeds are crushed to a particle size of 0.5 to 2 mm, preferably 0.8 to 1.5 mm, and more preferably 1 mm before the seabuckthorn seed oil is extracted.

[0033] In the present invention, the temperature during sea buckthorn seed oil extraction is 35-60°C, preferably 45-58°C, and more preferably 55°C; the pressure is 15-35MPa, preferably 18-32MPa, more preferably 20-30MPa, and more preferably 25MPa; the CO2 flow rate is 8-25L / h, preferably 10-20L / h, and more preferably 15L / h.

[0034] In the present invention, after the extraction of seabuckthorn seed oil is completed, the extraction product is collected, and the solid-liquid separation is performed to obtain the upper oil phase which is the seabuckthorn seed oil.

[0035] In the present invention, the solid-liquid separation is centrifugation, and the speed of the centrifugation is 4000-8000rpm, preferably 5000-7000rpm, more preferably 5500-6500rpm, more preferably 6000rpm; the time is 15-40min, preferably 20-35min, more preferably 25-32min, more preferably 30min.

[0036] Example 1

[0037] Sea buckthorn pulp, sea buckthorn fruit oil and sea buckthorn seed oil are prepared by supercritical carbon dioxide extraction. The schematic diagram of the supercritical carbon dioxide extraction device used is as shown in Figure 1 As shown, the specific preparation method of seabuckthorn pulp, seabuckthorn fruit oil and seabuckthorn seed oil is:

[0038] 1. Preparation of sea buckthorn pulp and sea buckthorn oil

[0039] The seabuckthorn fruit raw material is crushed and loaded into the extraction kettle 1, and the extraction temperature required for the experiment is set to 50°C. The CO2 coming out of the storage tank 8 is pressurized by the pump 6, preheated to the set temperature of 50°C in the heat exchanger 7, and the air inlet valve is opened to allow the CO2 fluid to enter the extraction kettle 1 and contact with the crushed seabuckthorn fruit raw material. The CO2 flow rate is controlled to be 10L / h. At the set extraction temperature of 50°C and pressure of 20MPa, the seabuckthorn pulp and seabuckthorn oil contained in the seabuckthorn fruit raw material are fully dissolved in the supercritical CO2, and the material is discharged after a certain residence time of 25min. The seabuckthorn pulp, seabuckthorn oil and CO2 are separated in the separation column 2, and the separated products are discharged from the vent valve and collected. The CO2 gas enters the storage tank 8 for recycling after passing through the heat exchanger 3, the condenser 4, and the deep freezer 5. The circulation is stopped until basically no product is collected at the vent valve. The collected product is centrifuged at 5000rpm for 30min. The lower layer is seabuckthorn pulp and the upper layer is seabuckthorn oil.

[0040] 2. Preparation of sea buckthorn seed oil

[0041] The seabuckthorn seed raw material is crushed to a particle size of 1mm and then loaded into the extraction kettle 1, and the extraction temperature required for the experiment is set to 55°C. The CO2 coming out of the storage tank 8 is pressurized by the pump 6, preheated to the set temperature of 55°C in the heat exchanger 7, and the air inlet valve is opened to allow the CO2 fluid to enter the extraction kettle 1 and contact with the crushed seabuckthorn seed raw material. The CO2 flow rate is controlled to be 15L / h, and the seabuckthorn seed oil contained in the seabuckthorn seed raw material is fully dissolved in the supercritical CO2 at the set extraction temperature of 55°C and the pressure of 25MPa. The material is discharged after a certain residence time of 25min. The seabuckthorn seed oil and CO2 are separated in the separation column 2, and the separated product is discharged from the vent valve and collected. The CO2 gas enters the storage tank 8 for recycling after passing through the heat exchanger 3, the condenser 4, and the deep freezer 5. The circulation is stopped until basically no product is collected at the vent valve. The collected product is centrifuged at 6000rpm for 30min, and the upper layer is the seabuckthorn seed oil.

[0042] Example 2

[0043] Twenty-eight 6-week-old ICR male mice were evenly distributed into seven cages, with 4 mice in each cage, and recorded as blank control (NCD) group, model group, levofloxacin group, seabuckthorn oil group, seabuckthorn pulp group and seabuckthorn seed oil group. Except for the NCD group, cigarette insertion devices were installed at the ventilation holes of the cages of the remaining groups, and fans were used to assist combustion above the cages to ensure uniform distribution of smoke. The small ventilation holes of the cages were opened to allow smoke and air to enter the cages. The smoke concentration was controlled to 387 mg / L through smoke collection and delivery equipment, and the smoke was controlled to be evenly distributed. During the experiment, the mice were observed for hypoxic behaviors and physiological states such as difficulty breathing and accelerated breathing to prevent the mice from dying of hypoxia. The mice were ventilated for 10 minutes after smoking for 20 minutes, and repeated 3 times a day. After 3 days of adaptation, starting from the 4th day, the mice in the levofloxacin group were injected with levofloxacin, and the mice in the seabuckthorn oil group, seabuckthorn pulp group and seabuckthorn seed oil group were gavaged with seabuckthorn oil, seabuckthorn pulp and seabuckthorn seed oil prepared in Example 1, respectively, and the injection and gavage volumes were both 200 μL / day; the mice in the NCD group and the Model group were gavaged with an equal amount of normal saline. The injection and gavage treatment lasted for 7 days, during which the model group and the treatment group continued to be given smoking treatment. On the 11th day (the second day after the injection and gavage treatment), the mice in each group were anesthetized with 5% chloral hydrate and sacrificed for sampling. The pharynx was subjected to HE staining for comparative observation.

[0044] The steps of HE staining are as follows:

[0045] (1) Dewaxing: Soak in xylene I for 5 min; soak in xylene II for 5 min;

[0046] (2) Hydration: 100% ethanol I treatment for 3 min → 100% ethanol II treatment for 3 min → 95% ethanol I treatment for 3 min → 95% ethanol II treatment for 3 min → 85% ethanol treatment for 3 min → distilled water rinse for 3 min, rinse 3 times in total;

[0047] (3) Hematoxylin staining: stain with hematoxylin solution for 3 min, wash with running water for 3 min;

[0048] (4) Differentiation: Differentiation with 1% hydrochloric acid alcohol for 10 seconds, followed by sufficient washing with running water;

[0049] (5) Eosin staining: Soak in 0.5% eosin solution for 1 min and rinse thoroughly with running water;

[0050] (6) Dehydration: 75% ethanol treatment for 30 s → 85% ethanol treatment for 30 s + 95% ethanol I treatment for 1 min → 95% ethanol II treatment for 2 min → 100% ethanol I treatment for 2 min → 100% ethanol II treatment for 3 min;

[0051] (7) Transparency: xylene treatment for 3 min, twice in total;

[0052] (8) Sealing: Use neutral gum to seal the slides;

[0053] (9) Filming: Observe the cell morphology by HE staining under a microscope and collect images.

[0054] The results of HE staining of pharyngeal tissues of mice in each group are shown in Figure 2 As shown, the scale bar in the figure is 50μm. It can be seen that the pharyngeal tissue structure of the rats in the blank control group is clear, the mucosal epithelium is smooth, and no thickening is observed. There are scattered lymphoid tissues and glands of normal size in the lamina propria. Some small blood vessels can be seen under the mucosa, no granulation tissue is seen, the muscular layer fibers are arranged tightly, there are no acinar cavities in the glands, and there are no secretions and other foreign bodies in the pharyngeal cavity. Compared with the blank control group, the model group has keratinization and shedding of the membrane epithelium, and the thickness of the pharyngeal mucosa is uneven; the acinus in the gland is obviously cavitated, the gland wall is obviously thickened, the nucleus of the wall cells is significantly enlarged, the muscular layer fibers are loosely arranged, and there are many secretions, red blood cells, and carbon dust particles in the pharyngeal cavity; some capillaries can be seen to be dilated. After the corresponding drug treatment, the pharyngeal tissues have been significantly improved, among which the seabuckthorn seed oil group has the best improvement effect. It can be seen that the uneven thickness of the mucosa has been improved, the acinar cavities in the glands have been reduced, and there are no secretions and other foreign bodies in the pharyngeal cavity. The keratinization and shedding of the membrane epithelium have been improved, and the uneven thickness of the pharyngeal mucosa has been improved. There is no obvious cavity in the glandular alveoli, the glandular wall is slightly thickened, the nucleus is slightly enlarged, the loose arrangement of the muscle layer fibers is improved, and no capillary dilation is observed. This shows that the seabuckthorn fruit oil, seabuckthorn pulp and seabuckthorn seed oil prepared by the present invention all have a good effect of improving pharyngeal inflammation caused by smoking, and the improvement effect of seabuckthorn seed oil is the best.

[0055] Example 3

[0056] 28 6-week-old ICR male mice were randomly divided into 4 groups, 7 mice in each group, and recorded as blank control (NCD) group, model group, seabuckthorn oil group, seabuckthorn pulp group, seabuckthorn seed oil group and levofloxacin group. Except for the blank control group, the mice in the other groups were treated with smoking, and the treatment method was the same as in Example 2, and the treatment was carried out for 7 days. Starting from the 8th day of treatment, except for the blank control group, the mice in the other groups were gavaged or intraperitoneally injected with the corresponding drugs before the smoking treatment, wherein the seabuckthorn oil group, seabuckthorn pulp group, and seabuckthorn seed oil group mice were gavaged with seabuckthorn oil, seabuckthorn pulp, and seabuckthorn seed oil prepared in Example 1, respectively, and the levofloxacin group mice were intraperitoneally injected with levofloxacin, and the dosage of gavage and intraperitoneal injection was 200 μL / day; the mice in the NCD group and the Model group were gavaged with the same amount of normal saline. Injection and gavage treatment for 10 days. 24 hours after the treatment, the mice were anesthetized with 5% chloral hydrate, and the samples were collected after being killed. The pharyngeal tissues of the mice in each group were observed by immunohistochemical staining.

[0057] Place the pre-cut slides in a 65°C oven for 30 minutes and then perform immunohistochemical staining at room temperature. The staining steps are as follows:

[0058] (1) Xylene dewaxing: Soak in xylene I for 10 min; soak in xylene II for 10 min;

[0059] (2) Alcohol gradient immersion: 100% ethanol I soak for 5 min; 100% ethanol II soak for 5 min; 90% ethanol soak for 5 min; 80% ethanol soak for 5 min; 70% ethanol soak for 5 min; slowly rinse with distilled water for 3 min;

[0060] (3) Citric acid repair: Place the slide in a beaker containing citrate repair solution and heat the beaker in a microwave oven. Start timing when the citrate repair solution boils and continue for 10 minutes (antigen repair treatment). After completion, cool the slide at room temperature (2 hours). After cooling, wash with PBS three times, 5 minutes each time.

[0061] (4) Blocking: Add hydrogen peroxide to the slides repaired with citric acid to block endogenous peroxidase activity for 20 min; then wash three times with PBS, each time for 5 min;

[0062] (5) Blocking: Add goat serum to the blocked slide for 1 h;

[0063] (6) Primary antibody: add the primary antibody purchased from Abcam to the blocked slide and store at 4°C after overnight treatment;

[0064] (7) Rewarming: Place the slides treated with primary antibodies at 20°C for 30 min. Wash three times with PBS, 5 min each time.

[0065] (8) Add the reaction enhancement solution (DAB + nickel enhancer) purchased from Abcam to the rewarmed slide and treat for 20 minutes; then wash three times with PBS, each time for 5 minutes;

[0066] (9) Secondary antibody: Add the secondary antibody purchased from Affinity onto the slide and treat for 20 min. Wash with PBS three times, 5 min each time.

[0067] (10) Color development: Add DAB color developing solution to the slide and observe the color change under a microscope. Stop the color development when a distinct yellow color appears. Wash with PBS three times, 5 min each time.

[0068] (11) Hematoxylin staining for 2 min (depending on the thickness of the slice); rinse with tap water for 3 min;

[0069] (12) Differentiation with 1% hydrochloric acid alcohol for 2 seconds; rinse with tap water until the nucleus turns blue and the cytoplasm is no longer blue;

[0070] (13) Alcohol gradient dehydration: soak in 70% ethanol for 1 min; soak in 80% ethanol for 1 min; soak in 90% ethanol for 1 min; soak in 100% ethanol I for 2 min; soak in 100% ethanol II for 2 min.

[0071] (14) Xylene transparency: Soak in xylene I for 10 min; soak in xylene II for 10 min.

[0072] (15) Seal the slide with transparent resin and place it in a fume hood for 24 hours. After completion, observe the changes in pharyngeal morphology and cells under a microscope.

[0073] The results are as follows Figure 3 As shown, the scale bar in the figure is 50μm. It can be seen that the pharynx of mice in each group was damaged after smoking. The results of IL-1β and IL-18 immunohistochemical staining showed that the glands of the NCD group showed relatively few stained cells, and the staining was light, indicating that the expression of IL-1β and IL-18 was low under normal conditions; the staining area of ​​the Model group was large and the staining was darker, indicating that the expression of IL-1β and IL-18 increased significantly in the acute pharyngitis model. The staining degree of the seabuckthorn fruit oil and seabuckthorn seed oil groups was between the NCD group and the Model group, and the staining area was reduced, indicating that seabuckthorn seed oil and seabuckthorn fruit oil may have certain anti-inflammatory effects and can reduce the expression of IL-1β and IL-18. The seabuckthorn pulp group still had a large area of ​​staining, and the improvement effect was not obvious; the levofloxacin group, as the positive control group, did not improve the staining area and degree significantly.

[0074] It can be seen from the above embodiments that the seabuckthorn pulp, seabuckthorn fruit oil and seabuckthorn seed oil prepared by the present invention have good improvement and treatment effects on pharyngitis caused by smoking, among which the seabuckthorn fruit oil and seabuckthorn seed oil have better improvement and treatment effects, and are expected to be used as drugs for preventing and treating pharyngitis caused by smoking, and have good application prospects.

[0075] The above is only 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of seabuckthorn pulp or seabuckthorn oil in the preparation of medicines for preventing and treating pharyngitis caused by smoking.

2. The use according to claim 1, characterized in that The seabuckthorn oil is seabuckthorn fruit oil and / or seabuckthorn seed oil.

3. The use according to claim 2, characterized in that The seabuckthorn pulp and seabuckthorn fruit oil are obtained by extracting seabuckthorn fruit raw materials through supercritical carbon dioxide extraction; The seabuckthorn seed oil is obtained by extracting seabuckthorn seed raw materials through supercritical carbon dioxide extraction method.

4. The use according to claim 3, characterized in that The temperature for extracting seabuckthorn pulp and seabuckthorn oil is 30-60°C, the pressure is 10-30MPa, and the CO2 flow rate is 5-20L / h.

5. The use according to claim 4, characterized in that After the extraction of seabuckthorn pulp and seabuckthorn oil is completed, the extraction products are collected and solid-liquid separation is performed to obtain the lower aqueous phase of seabuckthorn pulp and the upper oil phase of seabuckthorn oil.

6. The use according to claim 5, characterized in that The solid-liquid separation is performed by centrifugation, the speed of the centrifugation is 3000-6000 rpm, and the time is 15-40 minutes.

7. The use according to claim 3, characterized in that Before extracting the seabuckthorn seed oil, the seabuckthorn seeds are crushed to a particle size of 0.5 to 2 mm.

8. The use according to claim 3, characterized in that The temperature during seabuckthorn seed oil extraction is 35-60°C, the pressure is 15-35MPa, and the CO2 flow rate is 8-25L / h.

9. The use according to claim 8, characterized in that After the seabuckthorn seed oil extraction is completed, the extraction product is collected, and the solid-liquid separation is performed to obtain the upper oil phase which is the seabuckthorn seed oil.

10. The use according to claim 9, characterized in that The solid-liquid separation is performed by centrifugation, the speed of the centrifugation is 4000-8000 rpm, and the time is 15-40 minutes.

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