CN101077390B - Gymnadenia conopsea extraction and its application in preparing medicine for treating pulmonary fibrosis - Google Patents
Gymnadenia conopsea extraction and its application in preparing medicine for treating pulmonary fibrosis Download PDFInfo
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Abstract
The present invention discloses one kind of conic gymnadenia tuber extract and its application in preparing medicine for treating pulmonary fibrosis. The conic gymnadenia tuber extract is prepared through crushing conic gymnadenia tuber, sieving to obtain conic gymnadenia tuber powder, soaking the conic gymnadenia tuber powder in 50-70 % concentration alcohol water solution of 4-10 time volume for 18-30 hr, heating reflux to extract for 0.5-3 hr, filtering, repeating the extraction step for 1-3 times, merging the filtrate, decompression concentrating to density at 50 deg.c of 1.05-1.2. Experiment proves the treating effect of the conic gymnadenia tuber extract on pulmonary silicosis, and this reveals its use value in treating other pulmonary fibrosis diseases.
Description
Technical field
The invention belongs to technical field of Chinese medicines, relate to a kind of Chinese medicine extract and the application in preparation treatment pulmonary fibrosis medicine thereof.
Background technology
Pulmonary fibrosis is that finally to cause interstitial pulmonary fibrosis with diffusivity alveolitis and alveolar structure disorder be the disease of feature.Multiple factor such as antibacterial, dust, radioactive substance, wound and medicine all can cause pulmonary fibrosis.Because pulmonary fibrosis sickness rate height, cure rate is low, lacks effective medicine at present and becomes the disease of serious harm people ' s health.As: the disease pneumoconiosis that can cause pulmonary fibrosis is a kind of main occupation disease of China, and China's accumulative total is reported pneumoconiosis case people more than 580,000 since the fifties in last century, the people more than 140,000 that dies in heaven, existing patient people more than 440,000.Pneumoconiosis popular presents mass-sending property, becomes younger and new feature that disability rate is high in recent years, and about calendar year 2001-2003 year China annual New Development pneumoconiosis patient 10,000 examples, therefore the direct economic loss that causes reaches more than 90 hundred million, and indirect loss is difficult to counting.And for example: impacted the atypical pneumonia SARS of the unexpected incidents in the whole nation, and also can cause patient's pulmonary fibrosis in 2003.In a word, along with the industrialized process of China, the aggravation of environmental pollution, the sickness rate of the respiratory system disease that a variety of causes causes improves year by year, and majority can lapse to and be pulmonary fibrosis, and Chinese national economy and social development have been caused very big influence.At present, the medicine of the pulmonary fibrosis that is used for the treatment of clinically exists side effect big, shortcomings such as weak curative effect, and the new drug of exploitation treatment pulmonary fibrosis is very urgent and important.
Rhizoma Gymnadeniae belongs to the tuber of the orchid family herbaceos perennial, sweet and slightly bitter taste, and property is flat, goes into lung, spleen channel, and multiple efficacies such as benefiting vital QI and blood, promoting the production of body fluid to quench thirst, astringing to arrest bleeding are arranged.Modern pharmacological research shows that Rhizoma Gymnadeniae contains various active compositions such as polysaccharide, glycoside, luxuriant and rich with fragrance class and stilbene class, has multiple pharmacological effect such as antiinflammatory, antiallergic and immunomodulating.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of Rhizoma Gymnadeniae extract is provided.
Second purpose of the present invention provides the application of a kind of Rhizoma Gymnadeniae extract in preparation treatment pulmonary fibrosis medicine.
Technical scheme of the present invention is summarized as follows:
A kind of Rhizoma Gymnadeniae extract, make with following method:
With Rhizoma Gymnadeniae crude drug grinding and sieving, with described Rhizoma Gymnadeniae crude drug grain weight amount 4-10 concentration of volume percent doubly is after 50～70% ethanol waters soak 18-30 hour, heating and refluxing extraction 0.5-3 hour, filter, described extraction time is 1-3 time, relative density was 1.05-1.2 when merging filtrate, filtrate decompression were concentrated into 50 ℃, promptly made a kind of Rhizoma Gymnadeniae extract.
The addition of described ethanol water is 8 times of Rhizoma Gymnadeniae crude drug grain weight amount, and the concentration of described ethanol water is 60%.
The soak time of described Rhizoma Gymnadeniae crude drug powder is 24 hours.
The Rhizoma Gymnadeniae extract of one of claim 1-3, the application in preparation treatment pulmonary fibrosis medicine.
Advantage of the present invention is: the experiment proved that Rhizoma Gymnadeniae extract has certain therapeutical effect to experimental silicosis, the prompting Rhizoma Gymnadeniae extract also has use value in other lung fibrosis pathological changes.
Description of drawings
The influence (HE dyeing, * 100) of Fig. 1 Rhizoma Gymnadeniae ethanol extract to dying silicious dust rat different time points lung tissue form;
Fig. 2 Rhizoma Gymnadeniae ethanol extract is to dying silicious dust rat different time points lung tissue I, the synthetic influence of III collagen (day scarlet dyeing of wolf, * 200).
The specific embodiment
Embodiment 1
A kind of Rhizoma Gymnadeniae extract, make with following method:
With Rhizoma Gymnadeniae crude drug grinding and sieving, concentration of volume percent with 8 times of described Rhizoma Gymnadeniae crude drug grain weight amounts is after 60% ethanol water soaks 24 hours, heating and refluxing extraction 2 hours, filter, extract 2 times, relative density was 1.05 when merging filtrate, filtrate decompression were concentrated into 50 ℃, promptly made a kind of Rhizoma Gymnadeniae extract.
Embodiment 2
A kind of Rhizoma Gymnadeniae extract, make with following method:
With Rhizoma Gymnadeniae crude drug grinding and sieving, concentration of volume percent with 4 times of described Rhizoma Gymnadeniae crude drug grain weight amounts is after 70% ethanol water soaks 18 hours, heating and refluxing extraction 3 hours, filter, extract 1 time, relative density was 1.2 when merging filtrate, filtrate decompression were concentrated into 50 ℃, promptly made a kind of Rhizoma Gymnadeniae extract.
Embodiment 3
A kind of Rhizoma Gymnadeniae extract, make with following method:
With Rhizoma Gymnadeniae crude drug grinding and sieving, concentration of volume percent with 10 times of described Rhizoma Gymnadeniae crude drug grain weight amounts is after 50% ethanol water soaks 30 hours, heating and refluxing extraction 0.5 hour, filter, extract 3 times, relative density was 1.1 when merging filtrate, filtrate decompression were concentrated into 50 ℃, promptly made a kind of Rhizoma Gymnadeniae extract.
It is similar that the interstitial pulmonary fibrosis that pulmonary fibrosis due to the various interstitial diseases (as pneumosilicosis), collagenosis causes changes pathological process: from alveolitis, cause pulmonary fibrosis in development and excessive repair process.The present invention adopts rat trachea exposure method to inject silicious dust (50mg/ only) and causes the pneumosilicosis animal model, rat is divided into four groups at random, be matched group, dye silicious dust group, Rhizoma Gymnadeniae treatment group, positive drug control group, rat dye dirt after 24 hours each treatment group begin gastric infusion (the Rhizoma Gymnadeniae group give Rhizoma Gymnadeniae ethanol extract 8g/kg every day, give tetrandrine 50mg/kg next day of the positive drug group), observing time, point was 7 days, 14 days, 21 days, 28 days, 60 days.At each time point, animal is dissected the back abdominal aortic blood and wins lungs under narcotism, make pathology and biochemical analysis.Leading indicator comprises: organ coefficient; The scarlet dyeing observation of it wolf I III collagen is synthetic; Mda content; Superoxide dismutase activity; Glutathione peroxidase activity; Conventional H E dyeing observation lung pathology changes.The result shows: all there are significant difference in pneumosilicosis group and treatment group on These parameters, with traditional anti-pneumosilicosis medicine tetrandrine therapeutic equivalence.Thereby, confirmed that Rhizoma Gymnadeniae extract of the present invention can be effective to the treatment of pulmonary fibrosis resistant.
Utilize a kind of Rhizoma Gymnadeniae extract of the present invention and pharmaceutically acceptable carrier, make various dosage forms, as form oral administrations such as granule, tablet, capsule, pill and liquid preparations.
Embodiment 4
The pharmacodynamics test material and the method for Rhizoma Gymnadeniae extract of the present invention
Material
160 of 1 animal cleaning level Wistar rats, male, body weight 200-240g purchases in Test Animal Centre, Academy of Military Medical Sciences, P.L.A.
2 reagent SiO
2Purchase the Sigma company in the U.S., purity 99%, particle size scope are at 0.5-10 μ m, and 80% particle diameter is made into the 50g/L suspension with normal saline between 1-5 μ m, standby behind high pressure steam sterilization; It wolf is scarlet purchases the chemical reagents corporation in Mobay; MDA, SOD and GHS-Px test kit are purchased and are built up bio-engineering research institute in Nanjing.
3 medicine Rhizoma Gymnadeniaes are purchased in the Tibet drug company, and evaluation meets medicinal through this school crude drug teaching and research room.Method preparation with embodiment 1.
Method
The grouping of 1 laboratory animal and the foundation of model: according to randomization with 160 rats be divided into matched group, dye the silicious dust group, Rhizoma Gymnadeniae extract treatment group, positive drug control group, each 40.After the anesthesia of lumbar injection 0.4% pentobarbital, under aseptic condition, at rat neck skin median line longitudinal cut, expose trachea, inject the 1ml sterile saline in the control rats trachea, dye silicious dust group, Rhizoma Gymnadeniae treatment group and positive drug control group and inject the silicious dust suspension that 1ml prepares.Dye 2d behind the dirt, Rhizoma Gymnadeniae treatment group is pressed 8gkg
-1D
-1Irritate the stomach Rhizoma Gymnadeniae extract of the method preparation of embodiment 1, positive drug control group is pressed 50mgkg
-13d
-1Give tetrandrine, matched group, dye the silicious dust group and give etc. to hold distilled water.
2 plasma malonaldehyde contents (MDA), superoxide dismutase activity (SOD); The mensuration of glutathione peroxidase activity (GHS-Px) dyes behind the dirt the 7th, 14,21,28, each group of 60d is got 8 rats respectively, after weighing, and abdominal aortic blood, centrifugal blood plasma adopts thiobarbituricacid determination of color content; Adopt the nitrite forming method to measure the SOD vigor; Adopt the dinitrobenzoic acid direct method to measure the GHS-Px vigor.Operating procedure is all undertaken by the test kit description.
The processing of 3 lung tissue samples is put to death rat after getting blood, through right heart perfusion normal saline flushing lung inner blood, whole lungs are taken out in the thoracic cavity, reject connective tissues such as trachea, bronchus, rinsing once in normal saline, the pathological change of record naked eyes, electronic balance claims lung heavy, calculates lung coefficient (lung coefficient=lung weight/body weight).It is liquid-solid fixed with 10% neutral buffered formalin to get left lung stage casing, conventional dehydration, and paraffin embedding, serial section is carried out HE dyeing and day scarlet dyeing of wolf.
The formation of Nikon E600POL polarized light microscope observing lung tissue of rats I III Collagen Type VI of 4 image analysises.At least observe 5 animals for every group, section of every animal random observation, making a video recording and deposit computer in every section picked at random upper, middle and lower, left and right 5 visuals field, uses Image-Pro Plus Version 4.5for windows then
TMImage analysis system is analyzed.(Hue, Saturation Intensity) separate positive target from background colour, represent collagen content with the area percentage of positive region with the HIS pattern.
All numerical value of 5 statistical procedures all use x ± s to represent, the LSD method of relatively using one factor analysis of variance between four groups of different time points, and the statistical procedures of total data all adopts SPSS 11.5for windows statistical analysis software.
Result and analysis
1 lungs device coefficient: dye each time point lung tissue organ coefficient of silicious dust group rat and all be higher than matched group, significant difference (P＜0.01) is arranged at 14d, 21d, 28d, 60d.With dye the silicious dust group relatively, Rhizoma Gymnadeniae extract treatment group lung tissue organ coefficient significantly reduces (P＜0.05) when 14d, 21d, 28d, 60d, positive drug control group significantly reduces (P＜0.05) when 14d, 28d, 60d, all do not have significant difference at each time point between two groups.See Table 1.
2I, III collagen content: dye silicious dust group type i collagen area percentage and be the increase of carrying out property, compare with matched group, each time point all has significant difference (P＜0.01); The Rhizoma Gymnadeniae extract treatment is organized each time point type i collagen area percentage and also is higher than matched group, significant difference (P＜0.05) is all arranged. with dye the silicious dust group relatively, each time point type i collagen area percentage of Rhizoma Gymnadeniae extract treatment group and positive drug control group is lower than and dyes the silicious dust group, difference has significance (P＜0.01). show that silicious dust can increase the formation of dying silicious dust rat type i collagen, Rhizoma Gymnadeniae extract is inhibited to the increase of the inductive type i collagen of silicious dust, act on suitable with positive drug. compare with matched group, dye each time point III area of collagen percentage ratio of silicious dust group and all be higher than matched group, significant difference (P＜0.05) is arranged; With dye the silicious dust group relatively, Rhizoma Gymnadeniae extract and positive drug control group treatment are organized each time point III area of collagen percentage ratio and all are lower than and dye the silicious dust group, 14d, 21d have significant difference (P＜0.01). see Table 2-3.
3 plasma malonaldehyde contents: with matched group relatively, dye each time point contents of mda of silicious dust group rat and significantly raise, P＜0.01 when 7d, 14d, 21d, 28d wherein, P during 60d＜0.05.Rhizoma Gymnadeniae extract treatment group and each time point contents of mda of positive drug group rat all are lower than dyes the silicious dust group, and significant difference (P＜0.05) is wherein arranged when 7d, 14d, 21d, 28d.See Table 4.
4 blood plasma superoxide dismutase activity and glutathione peroxidase activities: with matched group relatively, different time polishes silicious dust group rat plasma SOD and the GSH-PX vigor changes similar: except that when the 7d slightly the height, all the other each time points all reduce.SOD significantly reduces (P＜0.05) at 14d, 21d, 28d, 60d, GSH-PX reduces significantly at 14d, 21d, 28d that ((P＜0.05) wherein suddenly drops to minimum (P＜0.01) during 14d and begins slow increase then.With dye the silicious dust group relatively, Rhizoma Gymnadeniae treatment group and positive drug group rat plasma SOD and GSH-PX vigor begin all to increase to some extent from 14d, and significant difference (P＜0.05) is arranged when 14d, 21d, 28d.See Table 5-6.
5 pathological examinations:
5.1HE coloration result: matched group, each time point lung tissue structure is normal substantially.Dye the silicious dust group, inject 7d behind the silicious dust, the alveolar septum edema also obviously thickens, in have lymphocyte, mononuclear phagocyte to invade profit, the little granuloma of alveolar space is filled the air existence, and is fused into bigger granuloma, but the generative center necrosis.Form and the hyaloid variation with fibroblast proliferation, collagen fiber in the granuloma, collagen tuberosity and fibrosis occur and change, the intralesional alveolar space diminishes.During 14d, alveolar septum still has more lymphocyte, macrophage and minority neutrophilic granulocyte to invade profit, with more fibroblast and smooth muscle cell proliferation and be arranged in parallel, alveolar wall thickens, as seen collagen fiber gather, and alveolar structure destroys, and patch shape fibrosis occurs.Cell tuberosity quantity is many, and volume is big.During 21d, the alveolar septum phenomenon of rupture is more serious, and the granuloma large-area fibreization has the kitchen range emphysema to form between tuberosity.During 28d, alveolar wall significantly thickens, and the big portion of alveolar structure is destroyed, and the part alveolar space disappears, and is occupied the extensive fibrosis of lung tissue, visible fibrous tubercle by collagen fiber and fibroblast.The obvious hypertrophy of fibrous tissue during 60d forms huge fibroid silicon tuberosity.The Rhizoma Gymnadeniae extract treatment organize corresponding time point HE coloration result between and silicious dust group and matched group between, alveolitis and fibrosis have alleviating in various degree.See Fig. 1.Among Fig. 1 A:7 days, matched group; B:7 days, the pneumosilicosis group; C:7 days, Rhizoma Gymnadeniae extract treatment group; D:14 days, matched group; E:14 days, the pneumosilicosis group; F:14 days, Rhizoma Gymnadeniae extract treatment group; G:21 days, matched group; H:21 days, the pneumosilicosis group; I:21d days, Rhizoma Gymnadeniae extract treatment group; J:28 days, matched group; K:28 days, the pneumosilicosis group; L:28 days, Rhizoma Gymnadeniae extract treatment group; M:60 days, matched group; N:60 days, the pneumosilicosis group; O:60 days, Rhizoma Gymnadeniae extract treatment group.
5.2 the scarlet dyeing mirror of it wolf is seen down: matched group, alveolar septum is normal. dye the silicious dust group, during 7d, the normal lung bubble structure is destroyed, and the substitute is two Collagen Type VIs that interweave and exist: type i collagen is thicker, is orange, peony; The III Collagen Type VI is thinner, when being light green .14d, as seen diffusivity, the collagen of light green bending is net and knits the shape existence, when being mingled with the orange colour fiber of small number of coarse and silicious dust granule .21d therebetween, light green collagen further increases, salmon pink collagen also increases, but during less relatively .28d, thick salmon pink collagen showed increased, be the netted existence that interweaves with very thin light green collagen, be covered with whole visual field, when being mingled with silicious dust granule .60d therebetween, thick salmon pink collagen further increases, very thin light green collagen is less. and between dying between silicious dust group and the matched group, the quantity of two kinds of collagens has minimizing in various degree to Rhizoma Gymnadeniae ethanol extract treatment group, when 28d and 60d at the collagen staining of corresponding time point, particularly evident. see Fig. 2. among Fig. 2 A:7 days, matched group; B:7 days, the pneumosilicosis group; C:7 days, Rhizoma Gymnadeniae extract treatment group; D:14 days, matched group; E:14 days, the pneumosilicosis group; F:14 days, Rhizoma Gymnadeniae extract treatment group; G:21 days, matched group; H:21 days, the pneumosilicosis group; I:21d days, Rhizoma Gymnadeniae extract treatment group; J:28 days, matched group; K:28 days, the pneumosilicosis group; L:28 days, Rhizoma Gymnadeniae extract treatment group; M:60 days, matched group; N:60 days, the pneumosilicosis group; O:60 days, Rhizoma Gymnadeniae extract treatment group.
Show from above result: Rhizoma Gymnadeniae extract can improve and dyes silicious dust rat antioxidant ability of organism, reduces the oxidative damage that silicious dust causes, and suppresses early stage inflammatory reaction of pneumosilicosis and middle and late stage I, the synthetic increase of III collagen.Thereby, confirmed that Rhizoma Gymnadeniae extract of the present invention can be effective to the treatment of pulmonary fibrosis resistant.
Table 1 Rhizoma Gymnadeniae is to dying the influence of silicious dust rat different time points lungs device coefficient
(n＝8，x±S，mg/g)
* p＜0.05, * * p＜0.01 and matched group are relatively;
#P＜0.05,
##P＜0.01vs and pneumosilicosis group are relatively
Table 2 Rhizoma Gymnadeniae is to dying the influence of silicious dust rat different time points type i collagen area percentage
(n＝8，x±S，％)
P＜0.05, compare with matched group * * p＜0.01;
#P＜0.05,
##P＜0.01vs and pneumosilicosis group are relatively
Table 3 Rhizoma Gymnadeniae is to dying the influence of silicious dust rat different time points III Collagen Type VI area percentage
(n＝8，x±S，％)
* p＜0.05, * * p＜0.01 and matched group are relatively;
#P＜0.05,
##P＜0.01vs and pneumosilicosis group are relatively
Table 4 Rhizoma Gymnadeniae is to dying the influence of silicious dust rat different time points contents of mda
(n＝8，x±S，nmol/ml)
P＜0.05, compare with matched group * * p＜0.01;
#P＜0.05,
##P＜0.01vs and pneumosilicosis group are relatively
Table 5 Rhizoma Gymnadeniae is to dying the influence of silicious dust rat different time points lungs device coefficient
(n＝8，x±S，U/ml)
* p＜0.05, * * p＜0.01 and matched group are relatively;
#P＜0.05,
##P＜0.01vs and pneumosilicosis group are relatively
Table 6 Rhizoma Gymnadeniae is to dying the influence of silicious dust rat different time points blood plasma GHS-PX vigor
(n＝8，x±S，U)
* p＜0.05, * * p＜0.01 and matched group are relatively;
#P＜0.05,
##P＜0.01vs and pneumosilicosis group are relatively
Claims (1)
1. the application of Rhizoma Gymnadeniae extract in preparation treatment pulmonary fibrosis medicine, it is characterized in that described Rhizoma Gymnadeniae extract is to make with following method: with Rhizoma Gymnadeniae crude drug grinding and sieving, with described Rhizoma Gymnadeniae crude drug grain weight amount 4-10 concentration of volume percent doubly is after 50～70% ethanol waters soak 18-30 hour, heating and refluxing extraction 0.5-3 hour, filter, described extraction time is 1-3 time, merging filtrate, relative density was 1.05-1.2 when filtrate decompression was concentrated into 50 ℃, promptly made a kind of Rhizoma Gymnadeniae extract.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1436839A (en) * | 2002-02-05 | 2003-08-20 | 那木太苏荣 | Conic gymnadenia tuber wine and its making process |
| JP2005041788A (en) * | 2003-07-23 | 2005-02-17 | Kurohime Wakanyaku Kenkyusho:Kk | Component of orchidaceous plant and its application |
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| CN1436839A (en) * | 2002-02-05 | 2003-08-20 | 那木太苏荣 | Conic gymnadenia tuber wine and its making process |
| JP2005041788A (en) * | 2003-07-23 | 2005-02-17 | Kurohime Wakanyaku Kenkyusho:Kk | Component of orchidaceous plant and its application |
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