CN114288342B - Application of lignum naucleae extract in preparation of medicines for inhibiting increase of lung permeability and/or lung injury - Google Patents
Application of lignum naucleae extract in preparation of medicines for inhibiting increase of lung permeability and/or lung injury Download PDFInfo
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Abstract
The invention discloses application of a lignum sappan extract in preparing a medicament for inhibiting lung permeability increase and/or lung injury. Based on a lung tissue permeability related mechanism, under theoretical guidance, the study of inhibiting the lung tissue permeability activity by the alkaloid component of the nauclea officinalis is deeply studied, and experimental study shows that the abnormal chunkin lactam can obviously inhibit the increase of the lung tissue permeability and prevent the occurrence of lung injury.
Description
Technical Field
The invention relates to a new application of a compound, in particular to an effect of a danshenmu extract and a vincoside lactam in inhibiting lung permeability increase and lung injury.
Background
Abnormal spring glycoside lactam is an alkaloid compound separated from Ardisia japonica, and has the English name: strictosamide has the following structural formula:
the lignum Dalbergiae Odoriferae (Nauclea officinalis Pierre ex Pitard) is plant of Pterocarpus of Rubiaceae, also called as radix Linderae, xiong Danshu, huang Danmu, and fel Ursi, and is used by adding into the skin and branch, has effects of clearing heat, detoxicating, detumescence and relieving pain, and can be used for treating pharyngolaryngitis, bronchitis, acute tonsillitis, etc., and is called as "plant antibiotic". Abnormal spring flower glycoside lactam as the main alkaloid component of lignum naucleae has been mentioned in literature as a model for inhibiting LPS-induced inflammation and a corresponding anti-inflammatory mechanism in molecular aspect, and has not been reported in literature as inhibiting the increase of lung permeability. Increased lung permeability is an important factor in causing lung injury, which is seen in most patients with acute lung injury, leading to respiratory distress. At present, the treatment for increasing lung permeability is mainly large-dose hormone treatment, and serious sequelae and complications are induced by continuous use of hormone, so that an effective treatment means for the symptoms is lacking clinically.
There are no other reports in the prior art about the use of the extract of Ardisia hance and its abnormal spring flower glycoside lactam for the medical purpose of increasing lung permeability.
Disclosure of Invention
The invention aims to: the invention aims to solve the defects of the prior art, and the activity of the danshenmu extract or the isocatharanthin lactam is intensively researched and developed to inhibit the novel pharmacological application of the danshenmu extract or the isocatharanthin lactam in inhibiting the increase of lung permeability.
The technical scheme is as follows: in order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
application of lignum naucleae extract in preparing medicine for inhibiting lung permeability increase and/or lung injury is provided.
Use of a vincoside lactam for the preparation of a medicament for inhibiting an increase in lung permeability and/or lung injury.
Preferably, the lung injury is an inflammatory lung injury disease for the above use. The inflammatory lung injury diseases comprise lung injury caused by pneumonia, bronchitis and upper respiratory tract infection.
As a preferred scheme, the nauclea officinalis extract is prepared by the following method:
taking dried stems and branches of the nauclea officinalis, cutting into sections, crushing, adding a certain amount of water for soaking and extracting, collecting an extracting solution, concentrating under reduced pressure to obtain nauclea officinalis extract, extracting the extract to obtain a dichloromethane part and an ethyl acetate part, respectively using dichloromethane-ethyl acetate and dichloromethane-methanol with different proportions as eluent, and separating by using repeated silica gel columns to obtain the nauclea officinalis extract.
As a preferred embodiment, the above-described lignum naucleae extracts having the effect of promoting cell barrier repair are prepared by the following method:
cutting the stem and branch of the nakedflower wood, crushing, soaking and extracting for 3 times with water with the volume of 5-10 times, mixing the extracting solutions, and concentrating to obtain the extract. Extracting with dichloromethane and ethyl acetate respectively, and concentrating respectively. Eluting ethyl acetate part with dichloromethane-methanol at volume ratio of 95:5-10:90, concentrating eluate, and combining similar parts by TLC analysis to obtain Ardisia Japonica extract (isovincoside lactam).
As a preferable scheme: the lignum naucleae extract with the effect of promoting endothelial repair is prepared from the following raw materials in parts by weight:
taking the branches of the nauclea officinalis, cutting into segments, crushing, adding water with the volume of 6 times of the weight of the nauclea officinalis, soaking and extracting for 3 times, mixing the extracting solutions for 5 hours each time, and concentrating to obtain an extract. Adding the extract with the volume 3 times of the weight of the extract, and respectively extracting to obtain dichloromethane and ethyl acetate extraction parts. Eluting ethyl acetate part with dichloromethane-methanol at volume ratio of 95:5-85:15-70:30-50:50-10:90, concentrating eluate, and combining similar parts by TLC analysis to obtain Ardisia root extract (isovincoside lactam).
As a preferred scheme, the application of the lignum naucleae extract with the effect of inhibiting the increase of lung permeability in preparing the medicines for preventing and treating inflammatory lung injury related diseases is provided.
Preferably, the inflammatory lung injury-related diseases described above include: lung injury caused by pneumonia, bronchitis, upper respiratory tract infection.
According to the application, the danshenmu extract or the isocatharanthine lactam and a pharmaceutically acceptable carrier are prepared into granules, tablets, capsules, oral liquid, mixture, pills, paint, ointment or transdermal absorption preparation.
When the invention is prepared into tablets, the extract powder of the lignum naucleae is added with carrier lactose or corn starch, and a lubricant magnesium stearate is added when needed, and the mixture is uniformly mixed and then pressed into tablets.
When the invention is prepared into capsules, the lignum naucleae extract dextrin or starch is uniformly mixed, granulated and then encapsulated into capsules.
When the invention is prepared into granules, the extract of the lignum naucleae is added with dextrin and the like to be uniformly mixed, granulated, dried and prepared into granules.
When the invention is prepared into mixture, the lignum naucleae extract is dissolved in purified water to prepare the mixture.
When the invention is used for preparing the oral liquid, the lignum naucleae extract is dissolved in purified water, steviosin or aspartame is added, and the pH is regulated to prepare the oral liquid.
The beneficial effects are that: compared with the prior art, the nauclea officinalis extract for inhibiting the increase of lung permeability has the following advantages:
(1) The invention provides a biliary tree extract for inhibiting the increase of lung permeability and preventing and treating lung injury, which is based on the research of relevant mechanisms for inhibiting the lung permeability, under theoretical guidance, the biological activity of isocatharanthin lactam is deeply researched, and animal experiment research shows that the biliary tree alkaloid (abnormal catharanthin lactam) prepared by the preferred method can obviously inhibit the lung permeability.
(2) The lignum naucleae extract with lung permeability inhibiting effect can be conveniently and pharmaceutically prepared into various dosage forms by using a carrier, is convenient to clinically take, and has low toxicity and safe application as proved by acute toxicity tests.
Drawings
In FIG. 1A is the W/D ratio of lung tissue, and B is the protein content in BALF. All data are expressed in mean±sem. # p < 0.01 compared to control group, # p < 0.05, # p < 0.01 compared to LPS group.
In FIG. 2A is the total cell number in BALF. B is MPO activity in lung tissue. All data are expressed in mean±sem. # p < 0.01 compared to control group, # p < 0.05, # p < 0.01 compared to LPS group.
In FIG. 3A is the expression level of claudin-5 in lung tissue, and B is the expression level of VE-cadherein in lung tissue. All data are presented in mean±sem. # p < 0.01 compared to control group, # p < 0.05, # p < 0.01 compared to LPS group.
In FIG. 4A is the expression level of VEGF in lung tissue. B is the expression level of p-Akt/Akt protein in lung tissue. All data are presented in mean±sem. # p < 0.01 compared to control group, # p < 0.05, # p < 0.01 compared to LPS group.
Detailed Description
The invention will be better understood from the following examples. However, it will be readily understood by those skilled in the art that the specific material ratios, process conditions and results thereof described in the examples are illustrative of the present invention and should not be construed as limiting the invention described in detail in the claims.
Example 1
1. A lignum naucleae extract with lung permeability inhibiting effect is prepared by the following steps:
taking the branches of the nauclea officinalis, cutting into segments, crushing, adding water with the volume of 6 times of the weight of the nauclea officinalis, soaking and extracting for 3 times, mixing the extracting solutions for 5 hours each time, and concentrating to obtain an extract. Adding the extract with the volume 3 times of the weight of the extract, and respectively extracting to obtain dichloromethane and ethyl acetate extraction parts. Eluting ethyl acetate part with dichloromethane-methanol at volume ratio of 95:5-85:15-70:30-50:50-10:90 by silica gel column, concentrating eluate, combining similar parts by TLC analysis, eluting with 60% and 70% methanol by volume concentration by ODS column to obtain a lignum naucleae extract: abnormal spring glycoside lactam.
The specific data are as follows:
pale yellow powder (normal temperature), 1 HNMR(500MHz,CDCl3)δ11.41(d,J＝2.2Hz,1H),11.36(d,J＝7.8 Hz,1H),11.26(d,J＝8.1Hz,1H),11.03(t,J＝7.5Hz,1H),10.94(t,J＝7.4Hz,1H),9.51–9.42 (m,2H),9.24(d,J＝17.1Hz,1H),9.17–9.09(m,1H),9.04–8.99(m,1H),8.67(d,J＝7.9Hz, 1H),7.87(d,J＝11.9Hz,1H),7.66(d,J＝5.4Hz,1H),7.35(d,J＝8.9Hz,1H),7.29(dd,J＝11.3, 5.2Hz,3H),7.23–7.15(m,2H),6.69(dd,J＝15.5,9.6Hz,3H),6.41(dd,J＝9.3,3.6Hz,1H), 5.40(d,J＝12.2Hz,1H).13C NMR(126MHz,CDCl3)δ168.58(C-22),151.57(C-17),140.81(C-13),137.09(C-2),136.44(C-19),130.48(C-8),125.08(C-11),123.05(C-18),122.57 (C-10),121.40(C-9),114.55(C-12),111.84(C-7),111.59(C-16),102.17(C-1’),99.96 (C-21),80.87(C-3’),80.53(C-5’),77.37(C-2’),74.12(C-4’),65.22(C-6’),57.31(C-3),47.03(C-5),43.76(C-20),35.16(C-14),29.87(C-15),24.58(C-6).
adding appropriate amount of starch into lignum sappan extract, granulating, drying, and making into capsule.
Example 2
Taking the branches of the nauclea officinalis, cutting into segments, crushing, adding water with the volume of 6 times of the weight of the nauclea officinalis, soaking and extracting for 3 times, mixing the extracting solutions for 5 hours each time, and concentrating to obtain an extract. Adding the extract with the volume 3 times of the weight of the extract, and respectively extracting to obtain dichloromethane and ethyl acetate extraction parts. Eluting ethyl acetate part with dichloromethane-methanol at volume ratio of 95:5-85:15-70:30-50:50-10:90 by silica gel column, concentrating eluate, combining similar parts by TLC analysis, eluting with 60% and 70% methanol by volume concentration by ODS column to obtain a lignum naucleae extract: abnormal spring glycoside lactam.
The extract was identified by modern spectroscopy as abnormal spring glycoside lactam and by modern spectroscopy was identified as in example 1.
Dissolving lignum Aristolochiae Kaempferi extract in purified water, adding appropriate amount of stevioside, adjusting pH to 5.5, packaging, and sterilizing.
Example 3
1. Pharmacodynamic experiments for inhibiting lung tissue permeability by using the lignum naucleae extract are specifically performed by the following methods:
mice were randomly divided into 6 groups of 12 mice each. The control group, the model group, the abnormal spring flower glycoside lactam (100 mg/kg) group, the abnormal spring flower glycoside lactam (200 mg/kg) group, the abnormal spring flower glycoside lactam (400 mg/kg) group and the dexamethasone group (5 mg/kg) group are respectively adopted. The control group and the model group were given physiological saline, and the isovincoside lactam group was administered by intragastric administration at the above dose for 7 days, 1 time/d, and once by intraperitoneal injection 1h before the dexamethasone group was molded.
1h after the last dose, mice from other groups, except the normal group, were anesthetized with 10% chloral hydrate (350 mg/kg) and induced by intratracheal instillation of lipopolysaccharide (LPS, 5 mg/kg) in the lung injury model. After LPS stimulated mice for 6h, the mice were anesthetized, bronchoalveolar lavage fluid (BALF) was collected, after LPS stimulated mice for 24h, lung tissue was collected and stored in a-80℃freezer for future use.
2. Determination of the wet/dry weight ratio of lung tissue and protein content in BALF
The left lung of the mouse is weighed by a micro-electronic balance to be wet weight, and then is put into a constant temperature oven at 80 ℃ to be baked for 48 hours until the lung tissue weight is constant, and the lung tissue weight which is weighed again is dry weight. The wet/dry weight (W/D) ratio of lung tissue was recorded and calculated for each mouse. The collected BALF was centrifuged (4 ℃,3000rpm,15 min) and the supernatant was assayed for total protein concentration using BCA kit.
3. Experimental results:
3.1 increased pulmonary vascular permeability is characteristic of acute lung injury, and therefore the present invention observes capillary leakage by measuring lung W/D ratio and protein content. Pulmonary edema was caused after LPS stimulation, as shown in FIG. 1A, the W/D ratio of the lung was significantly increased after LPS treatment compared to the control group. In contrast, the W/D ratio was significantly reduced after DM treatment. As shown in fig. 1B, protein concentration in BALF also increased significantly after LPS stimulation. However, DM can significantly reduce protein content in BALF compared to LPS group. At the same time, DXM treatment also reduced lung W/D ratio and protein content. The results show that the nauclea officinalis can inhibit the endothelial permeability increase of lung tissues.
3.2 determination of cell count and Myeloperoxidase (MPO) Activity in Lung tissue in BALF
The pelleted cells after BALF centrifugation were resuspended in PBS, mixed well and then counted with a cell counter. Right lung tissue of mice was homogenized with physiological saline. The activity of MPO in lung homogenates was detected using the MPO assay kit.
Massive inflammatory cell infiltration is an important marker of acute lung injury, and the experiment observes cell leakage by determining total cell number in BALF. As shown in fig. 2A, a number of inflammatory cells entered BALF after LPS stimulation. Whereas DM and DXM significantly reduced the number of cells in BALF after treatment.
The activity of MPO in lung tissue homogenate is an important index reflecting neutrophil infiltration in acute lung injury, and the influence of DM on LPS-induced neutrophil infiltration in ALI mouse lung tissue is observed by measuring the activity of MPO in lung tissue. As shown in fig. 2B, LPS can increase MPO activity, significantly reducing MPO activity in lung tissue following DM treatment. The results show that the nauclea officinalis can improve the endothelial leakage of lung tissues.
Example 4 study of mechanisms for endothelial cell injury repair experiments
Wb western blot experiment:
the lung tissue of mice was taken from a control group, a model group, an abnormal spring glycoside lactam (100 mg/kg) group, an abnormal spring glycoside lactam (200 mg/kg) group, an abnormal spring glycoside lactam (400 mg/kg) group and a dexamethasone (5 mg/kg) group, respectively. The protein was extracted from lung tissue with RIPA lysate containing PMSF and phosphatase inhibitor, and protein concentration was determined using BCA protein assay kit. Samples were separated by SDS-PAGE and transferred to PVDF membrane. The membrane was blocked with 5% skim milk for 1h at room temperature and then incubated with primary antibody overnight at 4 ℃. After the next day the membranes were washed with TBST, incubated with peroxidase-conjugated secondary antibodies for 1h at room temperature. Protein signals were then detected using the chemiluminescent imaging system of ECL detection kit.
2. Experimental results:
after WB experimental analysis, the results showed that the expression of claudin-5 and VE-cadherein proteins was down-regulated in the lung tissue of mice after LPS treatment. However, expression levels of claudin-5 (FIG. 3A) and VE-cadherein (FIG. 3B) in lung tissue were significantly up-regulated following DM treatment. These data indicate that nauclea officinalis can promote intercellular ligation by up-regulating the expression of claudin-5 and VE-cadherein, maintaining the integrity of the endothelial barrier.
PI3K/Akt is involved in a variety of biological processes including cell proliferation, migration, apoptosis and angiogenesis. We examined the expression of VEGF, p-Akt/Akt proteins. Experiments show that the expression of VEGF (figure 4A) and p-Akt/Akt proteins (figure 4B) in lung tissues of mice treated by LPS is obviously reduced. However, DM and DXM treated groups significantly up-regulated the expression of VEGF, p-Akt/Akt proteins compared to LPS groups. Promoting endothelial barrier repair by activating VEGF, p-Akt/Akt proteins to maintain endothelial permeability balance after biliary tree treatment.
The results show that the lignum naucleae extract (abnormal spring flower glycoside lactam) has better effect of inhibiting lung permeability and high safety. It can be used as an active ingredient for preparing medicaments for improving vascular endothelial cell permeability and promoting endothelial barrier repair related diseases. In addition, the compound can be used as an active ingredient for preparing medicines for inflammatory lung injury diseases.
The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the present invention and to implement it, but not limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.
Claims (2)
1. Use of vincoside lactam as sole active ingredient for the preparation of a medicament for promoting repair of the endothelial barrier of pulmonary tissue.
2. The use according to claim 1, wherein the vincoside lactam is formulated with a pharmaceutically acceptable carrier into granules, tablets, capsules, oral liquids, pills, paints, ointments.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106176794A (en) * | 2016-07-18 | 2016-12-07 | 江苏省中医药研究院 | Isovincoside lactone application in preparing anti-inflammatory drug |
| CN108743696A (en) * | 2018-06-20 | 2018-11-06 | 海南医学院 | With the Lignum et Radix Naucleae extract and its application for promoting wound healing |
| CN110256430A (en) * | 2019-07-11 | 2019-09-20 | 海南医学院 | With the nauclea officinalis noval chemical compound and the preparation method and application thereof for promoting endothelial injuries reparation |
| CN113171412A (en) * | 2021-05-20 | 2021-07-27 | 海南崇原药物开发有限公司 | Callicarpa nudiflora traditional Chinese medicine composition and preparation method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106176794A (en) * | 2016-07-18 | 2016-12-07 | 江苏省中医药研究院 | Isovincoside lactone application in preparing anti-inflammatory drug |
| CN108743696A (en) * | 2018-06-20 | 2018-11-06 | 海南医学院 | With the Lignum et Radix Naucleae extract and its application for promoting wound healing |
| CN110256430A (en) * | 2019-07-11 | 2019-09-20 | 海南医学院 | With the nauclea officinalis noval chemical compound and the preparation method and application thereof for promoting endothelial injuries reparation |
| CN113171412A (en) * | 2021-05-20 | 2021-07-27 | 海南崇原药物开发有限公司 | Callicarpa nudiflora traditional Chinese medicine composition and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| A new indole alkaloid with HUVEC proliferation activities from Nauclea officinalis;Shi-Ying Mai 等;Natural Product Research;第1-7页 * |
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