Use of an active part of dendrobium swinhoii flowers in preparation of a medicine for treating primary dysmenorrhea

Abstract

The present application relates to Dendrobium officinale flower active part, and particularly to application of Dendrobium officinale flower active part in preparation of a drug for treating primary dysmenorrhea; the present application shows that the Dendrobium officinale flower water extract can be applied to the drug for treating primary dysmenorrhea, and a new use of the Dendrobium officinale flower water extract is developed. DOF-EA treats dysmenorrhea through multi-target point effect, and has smaller side effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the active part of Dendrobium candidum flower, and particularly relates to application of the active part of Dendrobium candidum flower in preparation of a drug for treating primary dysmenorrhea BACKGROUND

[0002] Primary dysmenorrhea is a common gynecological disease, which refers to menstrual pain without pelvic lesions, and is characterized by repeated attacks during menstruation and crampy lower abdominal pain. Globally, 50%-90% of women during menstruation are affected by primary dysmenorrhea [1] . Primary dysmenorrhea affects the mood of patients and significantly reduces the quality of life of patients, and causes social and economic losses due to absenteeism and other problems. In addition, pain is often accompanied by nausea, vomiting, diarrhea, headache, fatigue and insomnia and other systemic symptoms [2] . Primary dysmenorrhea is related to uterine smooth muscle contraction caused by excessive secretion of prostaglandin F 2α (PGF 2α ), and the pain intensity is positively correlated with PGF 2α . Prostaglandins are generated from arachidonic acid under the action of cyclooxygenase (COX), and increased expression of COX-2 causes increased secretion of PGF 2α during menstruation, which is a possible mechanism for inducing primary dysmenorrhea [5] . In addition, increased calcium ion levels in the uterine muscle layer also cause uterine smooth muscle contraction and lead to the occurrence of primary dysmenorrhea. Two sources of increased intracellular calcium ion concentration are: extracellular calcium ions entering the intracellular through calcium ion channels (receptor-operated calcium channels and voltage-dependent calcium channels) or from intracellular sarcoplasmic reticulum calcium release [6] .

[0003] At present, most patients take non-steroidal anti-inflammatory drugs (such as ibuprofen, naproxen sodium, mefenamic acid, celecoxib, etc.) to relieve pain symptoms [3] . Non-steroidal anti-inflammatory drugs have gastrointestinal side effects, affect the cardiovascular system and liver and kidney, and in addition, about 18% of dysmenorrhea patients do not respond to non-steroidal anti-inflammatory drugs [4] . Therefore, there is an urgent need to seek drugs with small side effects and effective in relieving primary dysmenorrhea.

[0004] Dendrobium officinale Kimura et Migo is a kind of dry stem of orchidaceae plant Dendrobium officinale Kimura et Migo. It is praised as the first of the nine Chinese fairy grasses in Taoist Canon, and is called the life-saving fairy grass in folk. Dendrobium officinale Kimura et Migo has been used as traditional Chinese medicine for more than 2000 years, and is used for daily health care and treatment of diabetes and gastrointestinal diseases. Due to the increasing application and demand in the field of medicine and food, large-scale artificial planting has been realized in Zhejiang, Yunnan and Anhui, etc., with an annual output of about 10,000 tons and raw material output value of more than 200 million. Dendrobium officinale Kimura et Migo mainly contains polysaccharides, amino acids and alkaloids and other chemical components. The chemical components of Dendrobium officinale Kimura et Migo flower are similar to those of the stem, and are used to make herbal tea to relieve tension [7,8] It has been reported that the water extract of Dendrobium officinale Kimura et Migo flower has the effects of anti-COX-2, anti-oxidation, anti-glycation and anti-aging, indicating that the water extract of Dendrobium officinale Kimura et Migo flower has the potential of anti-primary dysmenorrhea [9] . SUMMARY

[0005] OBJECTIVE

[0006] The purpose of the present application is to provide a new use of the water extract of Dendrobium officinale Kimura et Migo flower. The present application provides an application of Dendrobium officinale Kimura et Migo flower extract in the preparation of a drug for treating primary dysmenorrhea.

[0007] TECHNICAL SCHEME

[0008] In the present application, Dendrobium officinale Kimura et Migo flower is used as raw material to prepare Dendrobium officinale Kimura et Migo flower active part, i.e. Dendrobium officinale Kimura et Migo flower water extract ethyl acetate part extract (DOF-EA), and the effect of the active part in treating primary dysmenorrhea is studied.

[0009] An application of Dendrobium officinale Kimura et Migo flower active part in the preparation of a drug for treating primary dysmenorrhea.

[0010] The Dendrobium officinale Kimura et Migo flower active part is prepared by the following steps: 4g of Dendrobium officinale Kimura et Migo flower is weighed, 200mL of pure water is added, and boiling water bath is performed for 1h; after extraction for 3 times, the filtrate is combined and filtered, and after most of the water is removed, water bath is performed for drying; the extract is added with 100mL of pure water to prepare a suspension, and ethyl acetate is used for extraction, and the extraction is performed for 3 to 4 times until the extraction is complete; the concentrated extract is the ethyl acetate part extract DOF-EA.

[0011] Further,

[0012] The application adopts estradiol benzoate combined with oxytocin to induce a primary dysmenorrhea model of mice, and the time when the mice start to show writhing reaction and the number of writhing reactions of the mice within 30 minutes after injection of oxytocin are observed. After administration of DOF-EA, the writhing reaction time of the primary dysmenorrhea mice is significantly prolonged and the number of writhing reactions is significantly reduced, indicating that DOF-EA can play a role in analgesia. Hematoxylin-eosin staining is used to observe the morphology of the uterine tissue of the mice, and after administration of DOF-EA, the edema of the lamina propria of the uterine tissue is improved and the inflammatory cell infiltration is reduced, that is, DOF-EA can improve the pathological state of the uterine tissue of the primary mice. The expression level of the inflammatory factor TNF-α in the uterine tissue is detected by an enzyme-linked immunosorbent assay, and after administration of DOF-EA, the expression level of TNF-α is significantly reduced, indicating that DOF-EA can play an anti-inflammatory role. PGF 2α Excessive secretion can cause uterine smooth muscle contraction and uterine spasm, and the expression level of PGF 2α in the uterine tissue is detected by an enzyme-linked immunosorbent assay, and after administration of DOF-EA, the expression level of PGF 2α is significantly reduced, indicating that DOF-EA can relax the uterine smooth muscle. In vitro experiments are used to observe the spontaneous contraction and oxytocin and KCl induced secondary contraction of the uterine tissue strip by a Power Lab 8 / 35 isolated tissue perfusion system, and the calcium ion influx caused by oxytocin and high potassium depolarization can promote uterine contraction. The results show that after administration of DOF-EA, the spontaneous contraction and oxytocin and KCl induced secondary contraction of the isolated uterine tissue are reduced in a dose-dependent manner, indicating that DOF-EA can play a role in spasmolysis. Therefore, DOF-EA can play a role in treating primary dysmenorrhea by spasmolysis, anti-inflammation and analgesia.

[0013] The technical key points of the application are:

[0014] 1. DOF-EA can improve the symptoms of primary dysmenorrhea mice, including reducing the number of writhing and prolonging the writhing reaction time, reducing the TNF-α level and PGF 2α level in the uterine tissue, and improving the pathological changes of the uterine tissue.

[0015] 2. DOF-EA can reduce the spontaneous and secondary contractions of the isolated uterine tissue.

[0016] Beneficial effects

[0017] The application shows that the water extract of Dendrobium candidum flowers can be applied to a medicine for treating primary dysmenorrhea, and develops a new use of the water extract of Dendrobium candidum flowers. DOF-EA treats dysmenorrhea through multi-target action, and has smaller side effects. Specifically:

[0018] DOF-EA administration can reduce the PGF 2αThe DOF-EA and ibuprofen can significantly reduce the number of writhing of mice, and produce analgesic effect. The analgesic and anti-inflammatory effects are equivalent to the effect of ibuprofen, and there is no significant difference. The water extract of D. candidum flower has no obvious side effects. The treatment of dysmenorrhea by DOF-EA is through multi-target effect. Therefore, the DOF-EA has more market development value. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 , HPLC chart of the ethyl acetate fraction of the water extract of D. candidum flower; wherein: peak 1-coffee acid glucoside; peak 2-coumaroyl glucoside; peak 3-coffee acid; peak 4-p-coumaric acid; peak 5-rutin; peak 6-isorhamnetin; peak 7-quercetin-3-O-malonyl glucoside; peak 8-kampferol rutinoside; peak 9-astrin;

[0020] Figure 2 , Effect of DOF-EA on the number of writhing (A) and the duration of writhing (B) of primary dysmenorrhea mice;

[0021] Figure 3 , Effect of DOF-EA on the histopathology of uterine tissue of primary dysmenorrhea mice;

[0022] Figure 4 , Effect of DOF-EA on the TNF-α level of uterine tissue of primary dysmenorrhea mice;

[0023] Figure 5 , Effect of DOF-EA on the PGF2α level of uterine tissue of primary dysmenorrhea mice;

[0024] Figure 6 , Effect of DOF-EA on the spontaneous contraction of isolated uterine tissue, wherein A is the experimental result, and B is the statistical result;

[0025] Figure 7 , Effect of DOF-EA on the oxytocin-induced secondary contraction of isolated uterine tissue, wherein A is the experimental result, and B is the statistical result;

[0026] Figure 8 , Effect of DOF-EA on the KCl-induced secondary contraction of isolated uterine tissue, wherein A is the experimental result, and B is the statistical result. DETAILED DESCRIPTION

[0027] Example 1

[0028] Preparation and analysis of the ethyl acetate fraction (DOF-EA) of the water extract of D. candidum flower

[0029] (1) Preparation of the ethyl acetate fraction of the aqueous extract of Dendrobium candidum flowers

[0030] Weigh 4g of Dendrobium candidum flowers, add 200mL of purified water, and boil in a water bath for 1h. Extract 3 times, then filter the combined filtrate. After removing most of the water, evaporate the filtrate to dryness in a water bath. Add 100mL of purified water to the extract to make a suspension, and extract with ethyl acetate. Extract 3 to 4 times until complete extraction. Concentrate the extract to obtain the ethyl acetate fraction extract DOF-EA.

[0031] (2) HPLC analysis of the ethyl acetate fraction of the aqueous extract of Dendrobium candidum flowers

[0032] Preparation of the test sample solution: Weigh 6mg of the ethyl acetate fraction extract of Dendrobium candidum flowers, and dissolve in 1mL of 70% methanol. Filter through a 0.22μm organic filter membrane to obtain the test sample solution.

[0033] Preparation of the reference solution: Weigh an appropriate amount of rutin, isoquercitrin, and other standard chemicals, and add to 1mL of 70% methanol. Ultrasonically shake and mix to obtain a single reference solution stock solution of a certain concentration. Before injection, filter through a 0.22μm filter membrane.

[0034] HPLC conditions: Use a ThermoAcclaim C18(4.6×250mm, 5μm) column, and a diode array detector for analysis. The mobile phase is 0.1% formic acid water (A) - acetonitrile (B), and the elution gradient is 0-12min, 9% B; 12-13min, 9%-11% B; 13-27min, 11% B; 27-28min, 11%-12% B; 28-46min, 12%-13% B; 46-47min, 13%-14% B; 47-66min, 14%-14.5% B; 66-80min, 14.5%-16.5% B; 80-90min, 16.5%-19% B; 90-110min, 19%-60% B. The injection volume is 10μL, the column temperature is 30℃, and the detection wavelength is 340nm.

[0035] The analysis results are shown in Table Figure 1 , which shows that the chemical components with higher content in DOF-EA are the ethyl acetate fraction of the aqueous extract of Dendrobium candidum flowers. The HPLC chart shows that: Peak 1 is caffeic acid glucoside; Peak 2 is coumaroyl glucoside; Peak 3 is caffeic acid; Peak 4 is p-coumaric acid; Peak 5 is rutin; Peak 6 is isoquercitrin; Peak 7 is quercetin-3-O-malonyl glucoside; Peak 8 is kaempferol rutinoside; Peak 9 is astrin;

[0036] It has been reported that quercetin can reduce the contraction of isolated uterine

[10] Among them, the content of isoquercitrin in DOF-EA is the highest, so isoquercitrin, a quercetin analogue, is used as the control group, accounting for about 7% (mass percentage), so the isoquercitrin group is added in the following experiments, and the dosage is set to 7% of the DOF-EA dosage, i.e. 7 mg / kg.

[0037] Example 2 Study on the effect of DOF-EA on the primary dysmenorrhea mouse model

[0038] Experimental materials: Female ICR mice weighing 20-22 g were purchased from Jiangsu Huacheng Xinnuo Pharmaceutical Technology Co., Ltd. (License No. SCXK (Su) 2020-0009, Taizhou, China); Ibuprofen (Item No. MB1271) was purchased from Dalian Melun Biotechnology Co., Ltd. (Dalian, China); Isoquercitrin (Item No. BP0793) was purchased from Chengdu Pureway Technology Development Co., Ltd. (Chengdu, China); Estradiol benzoate (Veterinary Drug No. 110202511) and oxytocin (Veterinary Drug No. 110201571) were purchased from Hangzhou Animal Pharmaceutical Factory (Hangzhou, China); Mouse TNF-ɑ ELISA detection kit (KE10002) was purchased from Proteintech Company (Chicago, USA); Mouse PGF 2α ELISA detection kit (E-EL-M1360c) was purchased from Elabscience Company (Wuhan, China).

[0039] Experimental steps:

[0040] SPF female non-pregnant ICR mice (weighting 20-22 g) were randomly divided into 5 groups: control group (Control), model group (Model), DOF-EA group (100 mg / kg, i.g.), ibuprofen group (Ibuprofen 100 mg / kg, i.g.), and isoquercitrin group (Isoquercitrin, 7 mg / kg i.g.), n = 6. Except for the control group (subcutaneous injection of the same volume of normal saline), the rest of the mice were subcutaneously injected with estradiol benzoate for 11 consecutive days, with a high dose of estradiol benzoate (10 mg / kg) on the first and eleventh days, and a low dose of estradiol benzoate (2 mg / kg) on the second to tenth days. Gavage was started on the fourth day, and continuous administration was given for 8 days until the end of the experiment on the eleventh day. The control group and the model group were given the same volume of 0.5% CMC-Na solution by gavage. Within 1 hour after the injection of estradiol benzoate on the eleventh day, oxytocin (100 U / kg) was injected intraperitoneally to establish the primary dysmenorrhea model, and the mouse writhing response within 30 minutes was recorded by video. After the observation, the mice were sacrificed, and the uterine tissue was taken for subsequent analysis.

[0041] (1) Observation of mouse writhing response

[0042] The writhing response in mice is characterized by an inward-curving abdomen, a twisted and raised buttocks, and extended hind limbs. The time from the injection of oxytocin to the onset of the writhing response is recorded as the writhing response delay duration, and the number of writhing responses in mice within 30 minutes after oxytocin injection is counted.

[0043] (2) Observation of uterine tissue morphology by hematoxylin-eosin (HE) staining.

[0044] A segment of uterine tissue was fixed with 4% paraformaldehyde for more than 24 hours, then dehydrated, embedded in paraffin, sectioned (5μm thick), stained with hematoxylin and eosin (HE), and the morphology of the uterine tissue was observed under an Olympus BX53 optical microscope.

[0045] (3) Enzyme-linked immunosorbent assay (ELISA) was used to detect TNF-α and PGF levels in uterine tissue. 2α level

[0046] 20 mg of uterine tissue was added to 100 μL of PBS for tissue homogenization. The homogenate was centrifuged at 10000g for 10 min, and the supernatant was collected to obtain the sample for testing. 5 μL of the sample was used for BCA protein quantification. The levels of TNF-α and PGF2α in the sample were detected according to the ELISA kit instructions.

[0047] Data analysis was performed using GraphPad Prism 8.0.1 software. One-way ANOVA was used to test the differences between multiple groups. A p-value < 0.05 was considered statistically significant. The results are expressed as mean ± SD.

[0048] Experimental results:

[0049] (1) As Figure 2 As shown in Figures AB, compared with the control group, the number of writhing movements in the model group mice was significantly increased (22.25±4.272 times, P<0.001), and the writhing response time was significantly shortened (2.275±0.229 min, P<0.001). DOF-EA and ibuprofen administration significantly reduced the number of writhing movements in mice (13±1.414 times, P<0.01; 14.25±2.986 times, P<0.01) and increased the writhing response time (4.865±0.9705 min, P<0.001; 4.245±1.225 min, P<0.01). Isoquercetin administration had no significant effect on the number of writhing movements (24.75±4.113 times) or the writhing response time (2.365±0.1865 min) in mice with primary dysmenorrhea. Compared with the control group, ###P<0.001; compared with the model group, **P<0.01, ***P<0.001.

[0050] There was no significant difference between DOF-EA and ibuprofen.

[0051] (2) The control group mice uterine tissue lamina propria tight, epithelial cells arranged in order epithelial structure intact, no obvious inflammatory cell infiltration. Model group mice uterine tissue lamina propria destruction, appear obvious edema (yellow frame), inflammatory cell infiltration (blue frame black arrow), part of the cytoplasm of epithelial cells vacuolization. DOF-EA and ibuprofen administration, improve mouse uterine tissue lamina propria edema, interstitial is more compact, and reduce inflammatory cell infiltration. Isoquercitrin administration lamina propria edema and inflammatory cell infiltration was not significantly improved. Figure 3

[0052] (3) Compared with the control group (234.6±125.4 pg / mg prot.), the model group uterine tissue TNF-α level was significantly increased (730.8±291.6 pg / mg prot., P<0.001), DOF-EA and ibuprofen administration uterine tissue TNF-α level was significantly reduced (321.7±49.9 pg / mg prot., P<0.01; 277.4±80.93 pg / mg prot., P<0.001), and isoquercitrin administration did not significantly reduce the level of TNF-α (553.4±101.6 pg / mg prot.) Figure 4 ) Compared with the control group (659.4±213 pg / mg prot.), the model group uterine tissue PGF 2α expression was significantly increased (1468±199.9 pg / mg prot., P<0.001), DOF-EA and ibuprofen administration can significantly reduce uterine tissue PGF 2α expression (890.3±139.5 pg / mg prot., P<0.001; 948.5±47.14 pg / mg prot., P<0.001), isoquercitrin administration did not significantly affect uterine tissue PGF 2α expression (1318±200 pg / mg prot.) Figure 5 ) Compared with the control group, ###P<0.001; compared with the model group, **P<0.01, ***P<0.001.

[0053] Wherein: DOF-EA and ibuprofen no significant difference.

[0054] Example 3 Effect of DOF-EA on uterine tissue contraction in vitro

[0055] ​Experimental materials: Female ICR mice weighing 20-22 g were purchased from Jiangsu Huacheng Xinnuo Pharmaceutical Technology Co., Ltd. (License No. SCXK (Su) 2020-0009, Taizhou, China); Potassium chloride, sodium chloride, sodium bicarbonate, calcium chloride, glucose were purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). Locke's buffer: 5.6 mM potassium chloride, 154 mM sodium chloride, 3.6 mM sodium bicarbonate, 2.2 mM calcium chloride, 11.1 mM glucose, pH = 7.3, prepared fresh.

[0056] Experimental steps:

[0057] SPF female non-pregnant ICR mice (body weight 20-22 g) were injected intraperitoneally with estradiol benzoate (10 mg / kg) for two consecutive days. After the mice were sacrificed, the uterine tissue was quickly removed and placed in Locke's buffer. A piece of uterine tissue (one side of the Y-shaped, i.e. below the ovary to the uterine body) was cut off and one end was placed on a hook at the bottom of the bath, and the other end was placed on a force sensor, so that the uterine tissue was in a straight state (without additional tension). The constant temperature (37.0 ± 0.1℃) bath contained 5 mL of preheated Locke's buffer, and 95% O2, 5% CO2 gas was uniformly introduced into the bath at a rate of 1-2 small bubbles per second. The uterine tissue strip was given a 1 g load, and after the contraction was stable, the tension and frequency of uterine tissue contraction were observed and recorded by Power Lab 8 / 35 isolated tissue perfusion system, n = 3.

[0058] (1) Observation of the effect of DOF-EA on spontaneous contraction of isolated uterine tissue

[0059] A 50 mg / mL DOF-EA stock solution was prepared. After the uterine tissue strip contracted stably, 25 μL, 25 μL, and 50 μL of the DOF-EA stock solution were added to the bath in sequence using a pipette, so that the concentration of DOF-EA in the bath was 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL, respectively. After each administration, 10 min was observed before the next administration.

[0060] (2) Observation of the effect of DOF-EA on secondary contraction of isolated uterine tissue

[0061] In the oxytocin (10 -6 M) and KCl (60 mM) induced secondary contraction of isolated uterine tissue experiment, after adding oxytocin or KCl to the bath, the uterine tissue strip contracted stably, and DOF-EA was added according to the above method, so that the concentration of DOF-EA in the bath was 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL, respectively.

[0062] Data analysis was performed using GraphPad Prism 8.0.1 software. One-way ANOVA was used to test the differences between multiple groups. A p-value < 0.05 was considered statistically significant. The results are expressed as mean ± SD.

[0063] Experimental results:

[0064] (1) As Figure 6 As shown in AB, compared with the blank control, DOF-EA administration reduced spontaneous contractions of isolated uterine tissue in a dose-dependent manner (81±3%, P<0.001; 60±3.606%, P<0.001; 33.33±2.082%, P<0.001). ***P<0.001 compared with the blank control.

[0065] (2) Figure 7 As shown in AB, DOF-EA administration dose-dependently reduced oxytocin-induced secondary contractions in isolated uterine tissue (85.33±1.528%, P<0.001; 61.67±1.155%, P<0.001; 31±1.732%, P<0.001). DOF-EA administration also dose-dependently reduced KCl-induced secondary contractions in isolated uterine tissue (86.33±0.5774%, P<0.001; 60±1%, P<0.001; 26.33±0.5774%, P<0.001). Figure 8 (AB). Compared with oxytocin, ***P<0.001; compared with KCl, ***P<0.001.

[0066] References

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Claims

1. The application of an ethyl acetate extract of Dendrobium officinale flowers in the preparation of a drug for treating primary dysmenorrhea, characterized in that, The ethyl acetate extract of Dendrobium officinale flowers was prepared by the following steps: Weigh 4 g of Dendrobium officinale flowers, add 200 mL of purified water, boil in a water bath for 1 h, extract 3 times, filter and combine the filtrates, remove most of the water by vortexing and evaporate to dryness in a water bath; add 100 mL 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 extract of Dendrobium officinale flowers.

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