CN110669055A

CN110669055A - Compound for treating pneumonia

Info

Publication number: CN110669055A
Application number: CN201910956742.4A
Authority: CN
Inventors: 杜娟
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-10-10
Filing date: 2019-10-10
Publication date: 2020-01-10

Abstract

The invention belongs to the technical field of medicine preparation, and relates to a compound for treating pneumonia, which is named as mulberry leaf flavone A. It is prepared by refluxing folium Mori with 70-90% ethanol, extracting with petroleum ether and ethyl acetate, separating ethyl acetate part by silica gel column chromatography and Sephadex LH-20 column chromatography, and recrystallizing with methanol. The compound for treating pneumonia is separated from traditional clinical traditional Chinese medicine mulberry leaves, is easy to obtain raw materials, novel in structure, strong in streptococcus pneumoniae-resisting activity, small in dosage, good in curative effect on pneumonia models and wide in application prospect.

Description

Compound for treating pneumonia

Technical Field

The invention belongs to the technical field of medicine preparation, and particularly relates to a compound for treating pneumonia.

Background

The main cause of lung disease is pneumonia, which is most commonly bacterial pneumonia with the common symptoms of cough, expectoration, or exacerbation of the original respiratory symptoms, with purulent or bloody sputum with or without chest pain. The bacterial pneumonia mainly comprises pneumonia such as streptococcus pneumoniae, staphylococcus aureus, klebsiella pneumoniae, haemophilus influenzae and pseudomonas aeruginosa, and has great threat to the health of children and old people. More typical sputum of staphylococcus aureus pneumonia is purulent yellow or bloody purulent sputum, streptococcus pneumoniae is rust color sputum, klebsiella pneumoniae is brick red mucus-like sputum, pseudomonas aeruginosa is light green sputum, and anaerobic bacteria infected sputum is often accompanied by odor. Antibacterial therapy is the key to determining the prognosis of bacterial pneumonia.

Mulberry leaves, which are dry leaves of Moraceae plant mulberry, are daily food of silkworm, also known as mulberry, wattle, mulberry, Chinese wampee leaves and the like, are widely planted in various places in the south and north of China, and have abundant yield. Silkworm is a major food. The whole leaf is in the shape of wide or oval, the base of the leaf is heart-shaped, the top is slightly sharp, the edge is provided with saw teeth, and the veins are dense, white and soft. Old leaves are thick and dark green. Thin tender leaves, yellow green. Crisp and easy to hold. Light smell, slightly bitter and astringent taste. According to the report of the literature, the mulberry leaf juice has good growth inhibition effect on most gram-positive bacteria, gram-negative bacteria and part of yeasts, and has low required inhibitory concentration, wide pH value range (4-9) and strong heat stability. Rutin in folium Mori can remarkably inhibit traumatic edema of rat, and can prevent development of conjunctivitis, auricle inflammation, and pulmonary edema. The mulberry leaves have stronger anti-inflammatory effect and accord with the effects of dispelling wind and clearing heat.

Disclosure of Invention

In the research process, a compound for treating pneumonia is obtained through separation, and pharmacological research finds that the compound can effectively inhibit the growth of bacteria and reduce various inflammatory indexes in the blood of rats with pneumonia, and is the basis of medicinal materials for treating pneumonia by mulberry leaves.

A compound for treating pneumonia is named as mulberry leaf flavone A, and the specific structure of the mulberry leaf flavone A is shown as follows:

the preparation method of the mulberry leaf flavone A comprises the following steps: pulverizing dried folium Mori, extracting with 10 times of 70-90% ethanol under reflux for 1-3 hr for 2-3 times, mixing extractive solutions, and concentrating under reduced pressure to obtain total extract; dispersing the extract with 2-4 times of water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol at equal volume for three times, and mixing extractive solutions; concentrating ethyl acetate part under reduced pressure to obtain extract, separating with silica gel column chromatography with 20-30 times of filler, eluting with chloroform-methanol at ratio of 10:0, 9:1, 5:1, 1:3, 1:6, 0:10 respectively for 2-3 times of column volume, and eluting with 0.1-0.2 times of column volume, detecting eluate with thin layer chromatography, and mixing; and (3) carrying out Sephadex LH-20 column chromatography separation on 7-13 parts of eluent with 15-25 times of filler, isocratically eluting trichloromethane-methanol at a ratio of 10: 1, collecting according to a color band, drying the eluent with 9-16 times of volume, and recrystallizing with methanol to obtain the mulberry leaf flavonoid A.

Application of mulberry leaf flavone A, preparation of a medicament for treating pneumonia, in particular preparation of a medicament for treating streptococcus pneumoniae infection.

The compound for treating pneumonia provided by the invention is prepared from traditional clinical traditional Chinese medicines, is novel in structure, strong in streptococcus pneumoniae-resisting activity and small in dosage, shows a good curative effect on a pneumonia model, and has a wide application prospect.

Drawings

FIG. 1: the invention relates to a new compound mulberry leaf flavone A structure. FIG. 2: the inhibition rate of the mulberry leaf flavone on the streptococcus pneumoniae is different.

Detailed Description

The compounds for treating pneumonia according to the present invention are further illustrated below with reference to specific examples, but the scope of the present invention is not limited thereto.

Example 1

Crushing 10kg of dried mulberry leaves, heating and refluxing the crushed mulberry leaves for 3 times by 10 times of 80% ethanol, extracting for 2 hours each time, combining extracting solutions, and concentrating under reduced pressure to obtain about 680g of total extract. Dispersing the extract with 2L water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol at equal volume for three times, and mixing the extractive solutions. Concentrating the ethyl acetate part under reduced pressure to obtain extract 74g, separating with silica gel column chromatography with 25 times of filler, eluting with chloroform-methanol (10:0, 9:1, 5:1, 1:3, 1:6, 0:10) in gradient of 3000ml and 200ml, detecting the eluates with thin layer chromatography, and mixing. And (3) carrying out Sephadex LH-20 column chromatography separation on 7-13 parts of eluent with 20 times of amount of filler, carrying out isocratic elution on chloroform-methanol (10: 1), collecting according to a color band, drying the solvent in 9-16 times of volume of eluent, and recrystallizing the methanol to obtain about 16mg of the mulberry leaf flavonoid A. Structural identification of mulberry leaf flavone A: yellow crystals, HRMS: 388.1311([ M-H) } M/z]^-，calc.for C₂₄H₁₉O₅387.1231)；¹HNMR(150MHz，CDCl₃)δ_ppm：6.85(1H，s，C₃-H)，6.54(1H，s，C₈-H)，7.42(2H，dd，J＝8.1Hz、2.0Hz，C_2’-H、C_6’-H)，6.75(2H，dd，J＝8.1Hz、2.0Hz，C_3’-H、C_5’-H)，6.63(1H，d，J＝10.2Hz，C_1”-H)，5.74(2H，d，J＝10.2Hz，C_2”-H)，1.41(3H，s，C_4”-H)，2.39(2H，d，J＝7.2Hz，C_5”-H)，5.54(1H，m，C_6”-H)，5.84(1H，J＝15.0Hz，C_7”-H)，4.88(2H，s,C_9”-H)，1.82(3H，s,C_10”-H)，13.41(s，5-OH)，10.43(s，4’-OH)。¹³C-NMR(600MHz，CDCl₃)δ_ppm：118.27(C₂)，113.06(C₃)，182.15(C₄)，162.19(C₅)，104.07(C₆)，164.41(C₇)，104.29(C₈)，157.86(C₉)，107.29(C₁₀)，123.14(C_1’)，127.85(C_2’、C_6’)，115.87(C_3’、C_5’)，157.45(C_4’)，119.54(C_1”)，128.76(C_2”)，86.34(C_3”)，21.35(C_4”)，54.12(C_5”)，129.38(C_6”)，134.75(C_7”)，141.57(C_8”)，111.35(C_9”)，24.57(C_10”). Repeating the above process to enrich folium Mori flavone A164 mg.

Pharmacological Activity test

In vitro bacteriostatic experiment: dissolving standard Streptococcus pneumoniae strain in liquid culture medium, recovering, transferring for three generations, centrifuging the culture broth with bacteria, centrifuging at 3000r/min for 15min, discarding supernatant, collecting bacterial precipitate, dissolving bacterial precipitate with small amount of physiological saline, making the bacterial solution into original bacterial solution, and accurately preparing into 1.0 × 10 with sterile physiological saline⁹CFU/m L. Adding 190 μ L of bacterial liquid into each sterile 48-well plate, respectively adding 10 μ L of folium Mori flavone A liquid medicine with mass concentration of 2.0, 1, 0.5, 0.25, 0.1mg/mL, each with mass concentrationRepeating for 3 times; taking 190 mu L of bacterial liquid and 10 mu L of 5% DMSO as negative control holes; mu.L of the culture medium and 10. mu.L of sterile purified water were used as blank control wells and incubated in an incubator at 37 ℃ for 24 hours. The lowest concentration corresponding to the clear culture solution observed by naked eyes is the MIC of the tested drug solution, namely 0.25 mg/mL. And measuring the absorbance value of each hole at 600nm by using a spectrophotometer, and calculating the bacteriostasis rate of the tested liquid medicine with each concentration, wherein the result is shown in figure 1.

Effect on pneumonia model rats: randomly dividing 24 SD male rats with SPF grade weight of about 200g into blank group, model group, positive group and administration group, each group comprises 6 rats, adaptively feeding for 7 days, lightly anesthetizing the rats by ether inhalation, and respectively administering to the model group and administration group at a concentration of 1.0 × 10⁹0.5mL/kg of CFU/mL bacterial liquid, slowly dripping the bacterial liquid into the nasal cavity of a rat by using an injector with a No. 4 half needle head, wherein the bacterium feeding speed is 0.05 mL/min; equal volume of saline was given in the same manner as the blank group. At 12h and 24h after molding, cefuroxime axetil tablets (45mg/kg) were administered to the positive group, and mulberry flavone A (50mg/kg) was administered to the administration group. 2h after the last administration, rats were dosed ip10 mL/kg of 0.4% sodium pentobarbital solution and blood was routinely tested by abdominal aorta bleeding. The results are shown in Table 1.

Table 1: pneumonia model rat blood regular fruit (n ═ 6)

Compared to the model set:^**P<0.01。

Claims

1. a compound for treating pneumonia is named as mulberry leaf flavone A, and is characterized in that the specific structure of the mulberry leaf flavone A is as follows:

2. the mulberry leaf flavonoid I according to claim 1, which is prepared by the following steps: pulverizing dried folium Mori, extracting with 10 times of 70-90% ethanol under reflux for 1-3 hr for 2-3 times, mixing extractive solutions, and concentrating under reduced pressure to obtain total extract; dispersing the extract with 2-4 times of water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol at equal volume for three times, and mixing extractive solutions; concentrating ethyl acetate part under reduced pressure to obtain extract, separating with silica gel column chromatography with 20-30 times of filler, eluting with chloroform-methanol at ratio of 10:0, 9:1, 5:1, 1:3, 1:6, 0:10 respectively for 2-3 times of column volume, and eluting with 0.1-0.2 times of column volume, detecting eluate with thin layer chromatography, and mixing; and (3) carrying out Sephadex LH-20 column chromatography separation on 7-13 parts of eluent with 15-25 times of filler, isocratically eluting trichloromethane-methanol at a ratio of 10: 1, collecting according to a color band, drying the eluent with 9-16 times of volume, and recrystallizing with methanol to obtain the mulberry leaf flavonoid A.

3. The use of mulberry leaf flavonoid A according to claim 1, characterized in that it is used for the preparation of medicaments for the treatment of pneumonia.

4. The use of mulberry leaf flavonoid A according to claim 1, characterized by the preparation of a medicament for the treatment of Streptococcus pneumoniae infection.