CN115227772A

CN115227772A - Traditional Chinese medicine formula for improving fine particulate matter induced lung injury

Info

Publication number: CN115227772A
Application number: CN202210886459.0A
Authority: CN
Inventors: 张媛媛; 潘子倩; 寇俊萍; 张力元; 阎琨
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2022-07-26
Filing date: 2022-07-26
Publication date: 2022-10-25
Anticipated expiration: 2042-07-26
Also published as: CN115227772B

Abstract

The invention discloses a traditional Chinese medicine formula for improving fine particle induced lung injury, and belongs to the field of natural medicines. The traditional Chinese medicine formula consists of honey mulberry leaf, dwarf lilyturf tuber, common anemarrhena rhizome, coastal glehnia root and ginseng. The invention adopts a fine particle induced mouse subacute lung injury model, and the indexes of lung wet-dry ratio measurement, pathological morphology observation, inflammatory factor expression, oxidative stress level and the like prove that the formula has the effect of obviously improving the subacute lung injury induced by the fine particles, and the formula is prompted to be used for preventing and treating the subacute lung injury induced by the fine particles.

Description

Traditional Chinese medicine formula for improving fine particulate matter induced lung injury

Technical Field

The invention belongs to the field of natural medicines, and particularly relates to a traditional Chinese medicine formula for improving lung injury induced by fine particles.

Background

Air pollution is listed as the fifth global risk of death. In 2019, the number of deaths worldwide due to air pollution reaches 667 thousands of people ((1) Munzel T, gori T, al-Kindi S, et Al. Effects of gases and solid constraints of air polarization function European heart J. 2018 (38) 3543-3550. (2) GBD 2019 Risk Factors of Global reactors of Global barders of 87 tissue Factors 204 roads and terrierites, 1990-2019 a systematic analysis for the Global Burden of Disease Std 2019J. Lance.396; 396 (58): 1223-1029.). Fine particles, also known as PM2.5, refer to particles having an aerodynamic diameter of less than or equal to 2.5 μm in ambient air. The characteristics of small particle size and relatively large surface area make it highly adsorptive, and The harmful substance-adsorbed PM2.5 is also greatly harmful to The organism, and its main chemical components are elemental carbon, organic carbon compounds, sulfates, nitrates, ammonium salts, etc. (3) gentle, dingyuan, yanpeng, etc. The compositional characteristics and source analysis of PM _ (2.5) in Beijing area [ J ] applied meteorology, 2007, (05) 645-654. In Beijing in authun [ J ] The Science of The total nutrient, 2018; 630-72. 82. Niu Y, chen R, xia Y, et al. When The fine particulate matter reaches The lungs, a large fraction of The fine particulate matter is deposited in The alveoli ((6) Kulkarni N, pierse N, rushton L, et al carbon in air creatures and lung function in children [ J ]. The New England and Journal of Medicine,2006,355 (1): 21-30.), and The fine particulate matter deposited in The alveoli can penetrate The blood-gas barrier and reach The whole body with The action of The blood circulation, so that in addition to The Respiratory system, the fine particulate matter has a clear correlation with The occurrence of various Diseases such as cardiovascular disease, diabetes, tumor, stroke, etc. (7) Li D, li Y, li G, et al, fluoro receipt on disposition of PM2.5 in and 1 in, PNAS,2019,116 (7): 2488-2493. (8) Schraufnagel DE, balmes JR, cowlct, et al, air accommodation and noise capable Diseases A.A. recovery form of The tissue of The heart, emission of The tissue of The patient, 120. Environment and noise, 2. The. A. Recovery of The tissue of The patient, 120. Environment of The patent of The trade, 2. J..

For respiratory system injury caused by environmental pollution, the currently effective personal protection measures are mainly to use air purifiers, masks and other filter devices, and there are no specific medicines or health care products on the market (9) Rajagopalan S, brauer M, bhatnagar a, et al, personal-level protective actions such as obtained from the American Heart Association [ J ] Circulation,2020,142 (23): e411-e 431.), and the clinical commonly used therapeutic drugs are glucocorticoid drugs such as dexamethasone, but the long-term use of glucocorticoid drugs causes more adverse reactions ((10) 31845pottery, rational use of yellow wave glucocorticoid [ J ] clinical drugs therapy, 2010,8 (01): 23-28). The traditional Chinese medicine compound is a prescription which is composed of two or more medicinal materials and is designed for relatively determined diseases, and has the characteristic of multiple components and multiple target points, so that the traditional Chinese medicine compound has unique advantages in the aspect of preventing and treating lung injury caused by air pollution. In addition, the traditional Chinese medicine composition has the advantages of improving the compliance of patients and the like due to small toxic and side effects, so that the traditional Chinese medicine composition has a considerable market and gradually becomes a research hotspot.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine formula and a preparation method thereof.

The traditional Chinese medicine formula comprises honey mulberry leaf, dwarf lilyturf tuber, common anemarrhena rhizome, coastal glehnia root and ginseng.

Further, the traditional Chinese medicine formula comprises the following components in parts by mass: 12 to 14 portions of honey mulberry leaf, 1 to 4 portions of dwarf lilyturf tuber, 7 to 11 portions of common anemarrhena rhizome, 7 to 11 portions of coastal glehnia root and 2 to 6 portions of ginseng.

The preparation method of the traditional Chinese medicine prescription comprises the following steps of mixing traditional Chinese medicine decoction pieces according to a ratio of 1: adding 8 volumes of pure water, decocting twice for one hour each time, combining the filtrates, and concentrating under reduced pressure to obtain the final product. The concentrated solution can be added with pharmaceutically acceptable carrier and/or excipient, and made into clinically acceptable dosage forms such as tablet, pill, capsule, granule, oral liquid, etc. by conventional preparation method.

The invention also aims to provide application of the traditional Chinese medicine formula in preparation of a medicine for treating subacute lung injury induced by fine particles.

The subacute lung injury is non-cardiogenic progressive or hypoxic respiratory insufficiency or decline caused by various internal and external pathogenic factors such as harmful gas inhalation, severe infection, trauma, poisoning, shock and the like, and acute respiratory distress syndrome is caused by severe patients.

In the embodiment 1-3 of the invention, a mouse subacute lung injury model induced by fine particles is adopted, and the compound MAG is proved to have the effect of obviously improving the subacute lung injury induced by the fine particles by measuring indexes such as lung wet-dry ratio, pathological morphological observation, inflammatory factor expression, oxidative stress level and the like, so that the compound MAG can be used for preventing and treating the subacute lung injury induced by the fine particles.

The invention also provides application of the traditional Chinese medicine formula in preparation of medicines for preventing and treating lung diseases, wherein the lung diseases comprise emphysema, chronic obstructive pulmonary disease, pulmonary hypertension and pulmonary fibrosis.

Emphysema refers to the pathological state of hypoelasticity, excessive expansion, gas filling and lung volume enlargement of the airway at the distal end of the terminal bronchiole or the condition accompanied with airway wall destruction; chronic obstructive pulmonary disease refers to chronic bronchitis and/or emphysema which are related to abnormal inflammatory reaction of harmful gas and harmful particles and have the characteristics of airflow obstruction; pulmonary hypertension refers to a hemodynamic and pathophysiological state in which the pulmonary arterial pressure rises above a certain threshold; pulmonary fibrosis is an end-stage change in lung disease characterized by fibroblast proliferation and massive extracellular matrix aggregation with inflammatory injury, destruction of tissue architecture.

The embodiment 4 of the invention proves that the high-dose compound MAG (20 g/kg) has no obvious side effect on male and female mice, no toxicity to liver and kidney and wide application prospect through the weight and the drinking water amount of the mice, the index measurement of each organ, the tissue morphology observation of each organ and the measurement of the contents of glutamic-oxalacetic transaminase and creatinine.

The traditional Chinese medicine formula disclosed by the invention is reasonable in component collocation and interaction, has the effects of improving lung injury and resisting inflammation, does not generate any toxic or side effect, is easy to popularize, simple to prepare and convenient to operate, and can be used for industrial production.

Drawings

FIG. 1 is the result of the experiment of compound MAG in example 1 to improve lung tissue morphology and edema in mice with sub-acute lung injury induced by fine particles (

n = 6). Wherein: ^### P<0.001vs. blank; ^** P<0.01, ^*** P<0.001vs. model set.

FIG. 2 shows the experimental results of the compound MAG in example 1 for improving the pathological structural damage of lung tissue in mice with subacute lung injury induced by fine particles (

n = 6). Wherein: ^### P<0.001vs. blank group; ^** P<0.01, ^*** P<0.001vs. model set.

FIG. 3 is the result of experiment of compound MAG in example 2 to improve the expression of inflammatory factors in serum of mice with sub-acute lung injury induced by fine particles (

n = 6). Wherein: ^## P<0.01, ^### P<0.001vs. blank; ^* P<0.05， ^** P<0.01 ^*** P<0.001vs. model set.

FIG. 4 shows the experimental results of the compound MAG of example 3 for improving the malondialdehyde content in lung tissue of mice with sub-acute lung injury induced by fine particles (

n = 6). Wherein: ^### P<0.001vs. blank group; ^* P<0.05， ^** P<0.01vs. model set.

FIG. 5 shows the effect of high-dose MAG on the body weight and dietary intake of male and female mice in example 4 (

n＝10，ns:no significance)。

FIG. 6 shows the effect of high-dose compound MAG on the morphology of each organ of male and female mice in example 4.

FIG. 7 shows the effect of high-dose compound MAG on organ index of male and female mice in example 4 (

n＝10,ns:no significance)。

FIG. 8 shows the effect of high-dose MAG on glutamic-oxaloacetic transaminase and creatinine in serum of male and female patients in example 4 (

n＝10，ns:no significance)。

Detailed Description

The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, steps or conditions of the present invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.

The novel medicine composition MAG in the embodiment of the invention comprises 10 parts of honey mulberry leaf, 1.3 parts of dwarf lilyturf tuber, 9.3 parts of common anemarrhena rhizome, 9.3 parts of coastal glehnia root and 4 parts of ginseng, and decoction pieces are purchased from Shangyitang medicine Limited company of Nanjing and are prepared according to the proportion of 1:8, decocting the mixture for 2 times with water for 1 hour each time, combining the filtrates, and concentrating under reduced pressure.

Example 1

Improvement effect of compound MAG on lung pathological injury of mice with subacute lung injury induced by fine particles

1. Experimental method

a) Determination of the Wet-to-Dry-weight ratio of Lung tissue

A total of 36 male ICR mice were acclimatized for 3 days and randomized into 6 groups: blank group, model + low-dose compound MAG group (1.25 g/kg), model + medium-dose compound MAG group (5 g/kg), model + high-dose compound MAG group (20 g/kg), and model + dexamethasone group (Dex, 1 mg/kg). Model mice were modeled by oropharyngeal inhalation of 40 μ L (10 mg/kg) PM suspension on

days

1,3,5,7,9, 11, 13, and oropharyngeal inhalation of the same volume of PBS on blank groups. The specific operation method for oropharynx inhalation comprises the following steps: the mouse is placed in an isoflurane anesthesia box, and is taken out when the righting reflex is lost and the cough is severe, and the nose of the mouse is pinched by the left hand to make the body of the mouse naturally droop. Holding the pipette with the right hand, pressing down and fixing the tongue with the pipette head, immediately injecting PM suspension from the tongue root of the mouse, keeping the tongue fixed, and after the mouse breathes vigorously for 5 times, putting the mouse back into the cage in a supine position to enable the mouse to revive naturally. The mice of each administration group are administered with the corresponding drugs by intragastric administration every day from 1 to 14 days, and the mice of the blank group and the model group are intragastric administered with normal saline with the same volume. The administration time for odd days was 1h before PM inhalation. And (3) killing the mice on the 15 th day, taking down the left lung, accurately measuring the weight of the left lung, recording the weight as the wet weight of the lung tissue, and drying the lung tissue in an oven at the temperature of 60 ℃ for 48 hours until the constant weight is recorded as the dry weight of the lung tissue. The lung tissue wet weight/lung tissue dry weight ratio was calculated and recorded as lung wet weight/dry weight.

b) Pathological observation of lung tissue

The upper right lung lobe of the mouse is taken, fixed by 10% formalin, embedded by paraffin, sliced to be about 4 mu m thick, stained by HE, and read and scored by a pathology professional. The scoring standard is as follows: the scores were 0.5 (mild), 1 (mild), 2 (moderate), 3 (severe), 4 (extremely severe) and no obvious lesion was 0 according to the scores of the severity of lesions. All scores were accumulated and the average score for each animal in each group was calculated

2. Results of the experiment

a) Compound MAG (active gas) for improving lung tissue morphology and edema of mice with subacute lung injury induced by fine particles

As shown in A in FIG. 1, the lung of the blank group had a powdery color, smooth surface and good elasticity, and no obvious abnormal damage was observed in appearance, while the lung tissue of the model group had a large amount of black fine particles, dark color, and diffused outward from the bronchi. After different doses of compound MAG (1.25,5, 20 g/kg) and dexamethasone (1 mg/kg) are administrated, lung tissue damage is reduced to different degrees, and the compound MAG has dose dependence, and the improvement effect of a high-dose group is most obvious. As shown in figure 1B, the medium-high dose compound MAG has a remarkable improvement effect on the pulmonary edema of the mice with the subacute lung injury induced by the fine particles.

b) Compound MAG (magnesium active ingredient) for improving pathological structure damage of lung tissue of mouse with acute lung injury induced by fine particles

As shown in FIG. 2, the blank group had a clear structure, no congestion in the alveolar wall, no inflammatory cell infiltration, no increase in the goblet cells in the epithelial cells, and no inflammatory exudate in the lumen. The lung tissue structure of the model group disappears and becomes solid, and a large amount of exudates mainly comprising neutrophils are filled in the alveolar cavity. Slight or mild fibrous tissue hyperplasia is seen in the bronchi and perivascular spaces of the lung tissue, and fine particulate matter is deposited in the lung interstitium. After different doses of compound MAG (1.25,5, 20 g/kg) and dexamethasone (1 mg/kg) are given, the damage is obviously reduced. As shown in B in figure 2, the pathological scoring result of lung tissue shows that different doses of compound MAG (1.25, 5, 20 g/kg) and dexamethasone have obvious inhibition effect on pathological injury under a model, and the high-dose improvement effect is optimal.

Example 2

Improvement effect of compound MAG on lung inflammation of mice with subacute lung injury induced by fine particles

1. Experimental methods

Detection of inflammatory factors in serum

The mouse orbit of example 1 was bled in a 1.5mL EP tube, left to stand at room temperature for 30min, centrifuged at 3500rpm for 10min, and the supernatant, i.e., serum, was collected and stored at-70 ℃. And detecting the expression of the serum inflammatory factor according to an ELISA kit.

2. Results of the experiment

Compound MAG (Metal active ingredient) for reducing expression of inflammatory factors in serum of mice with sub-acute lung injury induced by fine particles

As shown in FIG. 3, compared with the blank group, the levels of inflammatory factors IL-1 beta, IL-6 and TNF-alpha in the alveolar lavage fluid of the model group are increased, the inflammatory factors are inhibited after different doses of compound MAG (1.25, 5, 20 g/kg) and dexamethasone (1 mg/kg) are administered, and the effect of the high dose group (20 g/kg) is the most significant.

Example 3

Improving effect of compound MAG on oxidative stress level of mice with subacute lung injury induced by fine particles

1. Experimental methods

Detection of malondialdehyde content in mouse lung tissue

The lung tissue of the mouse of example 1 was accurately weighed, and the weight (g): volume (mL) =1, adding 9 times volume of physiological saline, mechanically homogenizing, taking homogenate to be tested, and determining the content of malondialdehyde in lung tissue according to the malondialdehyde kit instruction.

2. Results of the experiment

Compound MAG (active gas) for reducing malondialdehyde content in lung tissue of mouse with subacute lung injury induced by fine particles

As shown in FIG. 4, compared with the blank group, the malondialdehyde content in the lung tissue of the model group is significantly increased, and the malondialdehyde content in the lung tissue can be reduced after the administration of the high-dose compound MAG (20 g/kg) and dexamethasone (1 mg/kg).

Example 4

Compound MAG safety assessment

1. Experimental methods

a) Changes in body weight and drinking amount of mice

After adaptive breeding for 3 days, 40 SPF-grade ICR mice with half male and female are randomly divided into four groups, namely a male blank group, a male administration group, a female blank group and a female administration group, wherein each group comprises 10 mice. The administration group continuously administers high-dose compound MAG (20 g/kg) for 14 days, the blank group administers normal saline with corresponding volume, and after the gastric lavage is finished, the mice are normally raised, the death and growth activity conditions of the mice within 14 days are observed and recorded, and the weight, food intake and water drinking conditions of the mice are recorded.

b) Organ index measurement

Each group of mice was sacrificed and dissected after blood was taken from the eyeballs, and their hearts, livers, spleens, lungs, kidneys, brains were taken, weighed and recorded using an electronic balance, and the relevant visceral organ index was calculated.

c) Morphological changes of organ tissue

Each group of mice was sacrificed and dissected after blood was taken from the eyeballs, and the heart, liver, spleen, lung, kidney and brain were photographed.

d) Liver and kidney injury determination

And (3) detecting indexes such as glutamic-oxalacetic transaminase and creatinine by using the separated serum by using a kit, wherein the specific operation steps are carried out according to relevant kit specifications.

2. Results of the experiment

a) The high-dose compound MAG has no obvious influence on the weight and the drinking amount of the mice

Body weight gain is one of the important indicators for evaluating the growth of mice. The results are shown in fig. 5, a, where the mice in the compound MAG group showed the same tendency of increasing the intrabody weight in 14 days as the blank group, and the mice in both the male and female groups grew normally. Statistical results show that the weight gain of mice in the administration group and the blank group has no significant difference. As shown in B and C in figure 5, the water and food intake of the mice in the administration group has no significant change compared with the administration group within 14 days, and the result shows that the high-dose compound MAG has no significant influence on the weight gain and the water and food intake of the mice.

b) The high-dose compound MAG has no obvious influence on the tissue morphology of each organ of the mouse

After the experiment, the animals were sacrificed and observed by naked eyes, as shown in fig. 6, no obvious morphological differences were observed in the major organs such as heart, liver, spleen, lung, kidney, brain, etc.

c) The high-dose compound MAG has no obvious influence on the organ index of each organ of the mouse

The results of the organ index calculation after weighing the major organs such as the heart, the liver, the spleen, the lung, the kidney, the brain and the like are shown in fig. 7, and the major organ indexes of the high-dose compound MAG administration group are not significantly changed compared with the blank group. Experimental results show that the high-dose compound MAG has no significant influence on the index of the main organs of the mouse, and the compound MAG can be preliminarily considered to have no obvious targeted toxicity on the main organs of the mouse.

d) The high-dose compound MAG has no obvious influence on the liver and kidney injury of mice

The results of the determination of glutamic-oxaloacetic transaminase and creatinine in serum show that the contents of glutamic-oxaloacetic transaminase and creatinine in the serum of the male and female mice in the administration group and the blank group have no obvious change, and show that the high-dose compound MAG has no obvious hepatorenal toxicity to the male and female mice.

Claims

1. A Chinese medicinal composition comprises folium Mori preparata, radix Ophiopogonis, rhizoma anemarrhenae, radix Glehniae, and radix Ginseng.

2. The traditional Chinese medicine composition according to claim 1, characterized in that: the medicine comprises, by mass, 12-14 parts of honey mulberry leaves, 1-4 parts of radix ophiopogonis, 7-11 parts of rhizoma anemarrhenae, 7-11 parts of radix glehniae and 2-6 parts of ginseng.

3. The method for preparing the traditional Chinese medicine composition of claim 1, which is characterized in that: mixing honey mulberry leaves, dwarf lilyturf tuber, common anemarrhena rhizome, coastal glehnia root and ginseng according to a volume ratio of 1:8 adding pure water, decocting twice for one hour each time, mixing filtrates, and concentrating under reduced pressure to obtain the final product.

4. The use of the Chinese medicinal composition of claim 1 in the preparation of a medicament for the prevention and treatment of subacute lung injury induced by fine particulate matter.

5. A medicine for preventing and treating subacute lung injury, which comprises the traditional Chinese medicine composition of claim 1 as an active ingredient.

6. The subacute lung injury control drug according to claim 5, characterized in that: also comprises a pharmaceutically acceptable carrier and/or excipient.

7. The use of the Chinese medicinal composition of claim 1 in the preparation of a medicament for the prevention and treatment of pulmonary diseases, including emphysema, chronic obstructive pulmonary disease, pulmonary hypertension, and pulmonary fibrosis.

8. A medicine for preventing and treating lung diseases, which comprises the Chinese medicinal composition of claim 1 as an active ingredient.

9. The pulmonary disease control agent according to claim 8, wherein: also comprises a pharmaceutically acceptable carrier and/or excipient.