The application discloses application of an active part of Dendrobium candidum flowers in preparation of a medicine for treating cervicitis

Abstract

The present application relates to Dendrobium officinale flower extract, in particular to a Dendrobium officinale flower active part in the preparation of a drug for treating cervicitis; a Dendrobium officinale flower active part in the preparation of a drug for treating cervicitis; characterized in that the Dendrobium officinale flower active part is prepared by the following steps: weighing 4g of Dendrobium officinale flower, adding 200mL of purified water, boiling water bath for 1h, extracting 3 times, then filtering and combining the filtrate, removing most of the water, and then water bath evaporation; the extract is added with 100mL of purified water to prepare a suspension, extracted with ethyl acetate, and extracted 3 to 4 times until complete extraction; the concentrated extract is the ethyl acetate part DOF- EA of the Dendrobium officinale flower boiling water extract. The present application adopts a mouse cervicitis model induced by phenol glue, and DOF- EA is administered by gavage, and the results show that DOF- EA can significantly improve the damage of cervical mucosa tissue, reduce the level of inflammatory factors and oxidative stress, and has a good effect on anti-cervicitis.

Description

TECHNICAL FIELD

[0001] The present application relates to Dendrobium officinale Kimura & Migo flower extract, in particular to the application of Dendrobium officinale Kimura & Migo flower active part in the preparation of a medicine for treating cervicitis BACKGROUND

[0002] Cervicitis is a common gynecological disease, and the general incidence population is women of childbearing age. The incidence rate and recurrence rate are high, and it is a high-risk factor for inducing cervical cancer. Cervicitis mainly manifests as inflammation of the uterine cervix and vaginal part and inflammation of the uterine cervix canal mucosa. Clinically, it is divided into acute and chronic cervicitis. Acute cervicitis often coexists with acute endometritis or acute vaginitis, but chronic cervicitis is more common. Chronic cervicitis has various manifestations such as cervical erosion, cervical hypertrophy, cervical polyps and cervical ectropion. The current drugs for treating cervicitis in clinical practice are mainly antibiotics, such as the first-line drug for treating cervicitis in clinical practice, azithromycin and erythromycin. After long-term use, drug resistance is easily produced, that is, antibiotics are ineffective for cervicitis caused by drug-resistant strains. Therefore, it is urgent to find more effective drugs.

[0003] Dendrobium officinale Kimura & Migo is a plant of Orchidaceae Dendrobium, which grows on semi-shady and humid rocks in mountainous areas and is mainly distributed in Anhui, Zhejiang and Fujian of China. Dendrobium officinale Kimura & Migo is one of traditional Chinese medicines in China and has a long history of medicinal use. Its stems are mainly used as medicines and have the effects of reducing blood sugar, enhancing body immunity, resisting oxidation and resisting aging. Studies have shown that Dendrobium officinale Kimura & Migo flowers contain various amino acids, polysaccharides, total flavonoids and total phenols and have the effects of resisting oxidation, resisting inflammation and resisting tumors (Miao Y X, Liao M X, Sun A H, et al. Study on ultrasonic-ethanol extraction of total flavonoids from Dendrobium officinale Kimura & Migo flowers and its in vitro antioxidant activity. China Brewing, 2019, 38(4): 155-159). SUMMARY

[0004] OBJECTIVE

[0005] The purpose of the present application is to provide the application of Dendrobium officinale Kimura & Migo flower active part in the preparation of a medicine for treating cervicitis.

[0006] TECHNICAL SOLUTION

[0007] The application of Dendrobium officinale Kimura & Migo flower active part in the preparation of a medicine for treating cervicitis is characterized in that the Dendrobium officinale Kimura & Migo flower active part is prepared by the following steps: 4 g of Dendrobium officinale Kimura & Migo flowers are weighed, 200 mL of purified water is added, and boiling water bath is performed for 1 h. After extraction for 3 times, the filtrate is combined after filtration, and after most of the water is removed, water bath evaporation is performed until dryness. The extract is added with 100 mL of purified water to prepare a suspension, and extraction is performed with ethyl acetate. The extraction is performed for 3 to 4 times until complete extraction. The concentrated extract is the ethyl acetate part (DOF-EA) of the boiling water extract of Dendrobium officinale Kimura & Migo flowers.

[0008] The active part of Dendrobium officinale flower is the ethyl acetate fraction (DOF-EA) of the boiling water extract of Dendrobium officinale flower prepared by the above method. This invention studies the drug use of DOF-EA in anti-cervicitis. The specific method is as follows: A mouse cervicitis model was established by phenol gel method (Wei Yun, Wu Aiping, Yang Shiping, et al. Effect of Jindan Fuyankang capsules on phenol gel-induced chronic pelvic inflammatory disease model in rats. Medical Frontier, 2013(30):27-29). After successful modeling, the mice were treated by gavage, and the pathological damage changes in the cervical tissue and the changes in the levels of inflammation and oxidative stress in the cervical tissue were observed. The experimental results showed that DOF-EA can significantly improve the damage of cervical mucosa tissue in mice with cervicitis, reduce the levels of inflammatory factors (TNF-α, IL-6) and oxidative stress (SOD, MDA), and show an effective anti-cervicitis effect. It can be expected to be developed into a drug for the prevention and treatment of cervicitis.

[0009] The key technical point of this invention is:

[0010] 1. A method for preparing the active components of Dendrobium officinale flowers;

[0011] 2. An active component of Dendrobium officinale flower can significantly improve cervical mucosal tissue damage, reduce inflammation and oxidative stress levels, and exert a therapeutic effect on mice with cervicitis.

[0012] Beneficial effects

[0013] Cervicitis is a common gynecological disease, and antibiotics are generally used to treat it. However, antibiotic overuse leads to drug resistance and other side effects. Therefore, finding a safe and effective drug is a pressing technical problem in this field. This invention is the first to discover that the ethyl acetate fraction of the water extract of Dendrobium officinale flowers has a therapeutic effect on cervicitis. Its efficacy is not significantly different from that of the commonly used clinical drug, Fuke Qianjin tablets. Furthermore, the dosage of Fuke Qianjin tablets is 1g / kg by gavage, while this invention uses 100mg / kg by gavage; from a dosage comparison, this invention is actually more advantageous. At the same time, Dendrobium officinale flowers themselves have no side effects and can be used to treat cervicitis by steeping in water or adding to beverages, thus possessing better clinical and promotional value and increasing the application of Dendrobium officinale flowers.

[0014] Specifically, this invention utilizes a phenol-gel-induced mouse cervicitis model. DOF-EA was administered via gavage. Results showed that DOF-EA significantly improved cervical mucosal tissue damage, reduced inflammatory factors and oxidative stress levels, and demonstrated good anti-cervicitis effects. Furthermore, DOF-EA showed no significant difference from the positive control drug, Fuke Qianjin tablets, meaning that this invention has the same efficacy as Fuke Qianjin tablets. However, DOF-EA, being an extract of Dendrobium officinale flowers, avoids antibiotic resistance. Attached Figure Description

[0015] Figure 1 HPLC chromatogram of the ethyl acetate fraction of the water extract of Dendrobium officinale flowers: Peak 1—caffeic acid glucoside; Peak 2—coumaryl glucoside; Peak 3—caffeic acid; Peak 4—p-coumaric acid; Peak 5—rutin; Peak 6—isoquercetin; Peak 7—quercetin-3-O-malonyl glucoside; Peak 8—kaempferol rutin; Peak 9—astragaloside;

[0016] Figure 2 Results of HE staining of mouse sections;

[0017] Figure 3 Changes in the levels of TNF-α(A) and IL-6(B) in the cervical tissue of mice in each group;

[0018] Figure 4 Changes in the levels of MDA(A) and SOD(B) in the cervical tissue of mice in each group. Detailed Implementation

[0019] Example 1

[0020] 1. Experimental Materials

[0021] (1) Reagents

[0022] 25% phenol gel (5g phenol, added to 20mL with 1% CMC-Na solution), Fuke Qianjin tablets (Zhuzhou Qianjin Pharmaceutical Co., Ltd.), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) enzyme-linked adsorption reaction (ELISA) kit (Wuhan Yilairuit Biotechnology Co., Ltd.), malondialdehyde (MDA) and superoxide dismutase (SOD) detection kit (Beyotime Biotechnology Co., Ltd.).

[0023] (2) Preparation of the ethyl acetate fraction of Dendrobium officinale flower water extract

[0024] Weigh 4g of Dendrobium officinale flowers, add 200mL of purified water, boil in a water bath for 1 hour, extract 3 times, filter and combine the filtrates, remove most of the water by vortexing and evaporate to dryness in a water bath; add 100mL of purified water to the extract to make a suspension, extract with ethyl acetate, extract 3 to 4 times until extraction is complete; concentrate the extract to obtain the ethyl acetate fraction extract.

[0025] 2. Experimental Methods

[0026] (1) HPLC analysis of the ethyl acetate fraction of the water extract of Dendrobium officinale flowers

[0027] Preparation of the test solution: Weigh 6 mg of the ethyl acetate extract of Dendrobium officinale flowers and dissolve it in 1 mL of 70% methanol; filter it through a 0.22 μm organic filter membrane to obtain the test sample solution.

[0028] Preparation of reference solutions: Accurately weigh appropriate amounts of several standard chemicals such as rutin and isoquercitrin, add 1 mL of 70% methanol, sonicate and shake well to obtain a single reference stock solution of a certain concentration, and filter it through a 0.22 μm microporous membrane before injection.

[0029] HPLC conditions: A Thermo Fisher Scientific UltiMate 3000 HPLC system with a diode array detector was used for analysis. The chromatographic conditions were as follows: Thermo Acclaim C18 (4.6 × 250 mm, 5 μm) column; mobile phase: 0.1% formic acid water (A) - acetonitrile (B); elution gradient: 0–12 min, 9% B; 12–13 min, 9%–11% B; 13–27 min, 11% B; 27–28 min, 11%–12% B; 28–46 min, 12%–13% B; 46–47 min, 13%–14% B; 47–66 min, 14%–14.5% B; 66–80 min, 14.5%–16.5% B; 80–90 min, 16.5%–19% B; 90–110 min, 19%–60% B. The injection volume was 10 μL; the column temperature was 30℃; and the detection wavelength was 340 nm.

[0030] (2) Animal grouping and modeling

[0031] Thirty SPF-grade, female ICR mice, weighing 20-22g, were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd., certificate number SCXK(Su)2020-0009. They were randomly divided into a normal control group, a model group, a DOF-EA (100mg / kg) group, a Fuke Qianjin tablet (1g / kg) group, and an isoquercetin (7mg / kg) group, n=6. Phenol gel (1.44mL / kg) was injected into the vaginal opening using a pipette once daily for 3 consecutive days. After 3 days, redness and swelling or purulent discharge from the vaginal opening indicated successful model establishment.

[0032] Note: Isoquercetin is one of the main components and the largest component of DOF-EA. The calculated content of isoquercetin is 7% (mass percentage) of DOF-EA, so we use a dosage of 7 mg / kg.

[0033] (3) Animal administration

[0034] After successful model establishment, administration of medication via gavage began the following day, once daily for 7 consecutive days. Normal control group and model group: administered physiological saline via gavage; DOF-EA group: administered 100 mg / kg of DOF-EA via gavage; Fuke Qianjin tablet group: administered 1 g / kg of Fuke Qianjin tablets via gavage; Isoquercetin group: administered 7 mg / kg of isoquercetin via gavage.

[0035] (4) Detection indicators

[0036] Four hours after the last administration, the animals were sacrificed, the intact uterus of the mice was dissected, a portion of the cervical tissue was taken and fixed in 4% paraformaldehyde for 48 hours, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and the pathological changes of the cervical mucosa were observed under a microscope; the remaining cervical tissue was preserved in liquid nitrogen, and the contents of TNF-α, IL-6, MDA and SOD in the cervical tissue were subsequently measured.

[0037] 3. Experimental Results

[0038] (1) Component identification of DOF-EA

[0039] Under these liquid chromatography conditions, the main chromatographic peaks of each fraction of Dendrobium officinale flower extract achieved good separation (see...). Figure 1 And identified the compounds corresponding to 9 chromatographic peaks (see...). Figure 1 Among them, peaks 1, 2, 3, and 4 belong to phenylpropanoid compounds, while peaks 5, 6, 7, 8, and 9 are flavonoid glycoside compounds.

[0040] (2) Pathological staining results of mouse cervical tissue

[0041] HE staining results showed that in the normal control group, the cervical tissue surface was covered with intact epithelium, without degeneration, necrosis, or ulceration. Loose connective tissue, smooth muscle, and skeletal muscle were observed in the lamina propria, without edema or significant inflammatory cell infiltration. In the model group, the cervical tissue surface was covered with necrotic and sloughed epithelium, ulceration occurred, and significant inflammatory cell infiltration was visible on the free surface of the epithelium. Vascular congestion and inflammatory cell infiltration were significant in the lamina propria, and the myometrium was disordered. After administration of DOF-EA or Fuke Qianjin tablets, cervical tissue damage improved, inflammatory cell infiltration in the lamina propria decreased, and the myometrium became more orderly. There was no significant difference between DOF-EA and Fuke Qianjin tablets. However, the cervical tissue changes in the isoquercitrin group were similar to those in the model group, showing no significant therapeutic effect. (See details...) Figure 2 See Table 1.

[0042] Table 1. Comparison of the degree of cervical tissue lesions in mice of different groups

[0043]

[0044] Note: Compared with the model group, a: P < 0.01; b: P > 0.05. Compared with the Fuke Qianjin Pian group, c: P > 0.05.

[0045] (3) Changes in the levels of inflammatory factors in mouse cervical tissue

[0046] The levels of TNF-α and IL-6 in the cervical tissue of mice were detected using an ELISA kit. The results showed that, compared with the normal control group, the levels of TNF-α and IL-6 in the cervical tissue of the model group mice were significantly increased. After treatment with DOF-EA or Fuke Qianjin tablets, the inflammation level was significantly reduced, and there was no significant difference between DOF-EA and Fuke Qianjin tablets. However, there was no significant change in the isoquercetin group, indicating no therapeutic effect. (See attached figures). Figure 3 .

[0047] (4) Changes in oxidative stress levels in mouse cervical tissue

[0048] The kit detected the levels of MDA and SOD in the cervical tissue of mice. The results showed that, compared with the normal control group, the model group mice had increased MDA levels and decreased SOD levels in their cervical tissue, indicating a high level of oxidative stress. After treatment with DOF-EA or Fuke Qianjin tablets, the oxidative stress level significantly decreased, and there was no significant difference between DOF-EA and Fuke Qianjin tablets; however, isoquercetin did not exert the same effect, as shown in the results below. Figure 4 .

Claims

1. The use of the ethyl acetate fraction of a boiling water extract of Dendrobium officinale flowers in the preparation of a medicament for treating cervicitis, characterized in that... The ethyl acetate fraction of the boiling water extract of Dendrobium officinale flowers was prepared by the following steps: 4 g of Dendrobium officinale flowers were weighed, 200 mL of purified water was added, and the mixture was boiled in a water bath for 1 h. After extraction three times, the filtrates were combined and most of the water was removed by vortexing. The extract was then evaporated to dryness in a water bath. The extract was added to 100 mL of purified water to make a suspension, and then extracted with ethyl acetate three to four times until extraction was complete. The concentrated extract was the ethyl acetate fraction of the boiling water extract of Dendrobium officinale flowers.

Images