CN108743801A - A kind of Pseudobulbus Bletillae (Rhizoma Bletillae) extract and its application - Google Patents
A kind of Pseudobulbus Bletillae (Rhizoma Bletillae) extract and its application Download PDFInfo
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Abstract
The invention discloses a kind of Pseudobulbus Bletillae (Rhizoma Bletillae) extract and its application, the Pseudobulbus Bletillae (Rhizoma Bletillae) extract is prepared as follows：Bletilla dry powder is taken, with volumetric concentration 60-90% ethanol water refluxing extractions, filtrate, as bletilla alcohol extract are collected in filtering；Bletilla alcohol extract is adsorbed through polyamide, D101 macroporous resin columns successively, 35%~45% ethanol eluate of collected volume concentration, and evaporated under reduced pressure obtains Pseudobulbus Bletillae (Rhizoma Bletillae) extract.The present invention confirms the bletilla active component EFB of the present invention by the experiment of internal SD rat model of chronic bronchitis, it can be by inhibiting the inflammatory factors such as TNF-α, IL-1 β, IL-6, with by improving oxidative stress, therapeutic effect is played to chronic bronchitis.
Description
(1) technical field
The present invention relates to a kind of Pseudobulbus Bletillae (Rhizoma Bletillae) extract and applications.
(2) background technology
Bletilla is the dry tuber of orchid bletilla Bletilla striata (Thunb.) Reiehb.f., has and receives
Hemostasis, detumescence and promoting granulation and other effects are held back, for spitting blood, haematemesis, traumatic hemorrhage, sore swollen toxin, the diseases such as chapped skin.《Compendium of Materia Medica》
Calling it " can enter lung hemostasis, myogenic controls sore."《Collected statements on the herbal foundation》It records：" bletilla holds back gas, oozes phlegm, hemostasis, the key medicine ... for the carbuncle that disappears
There is the magical effect for asking old life new "；《The collection of prescriptions with notes》Then clearly record：Bletilla grinds powder, and rice drink is taken after mixing with liquid, and cough, consumptive lung disease, spitting of blood can be controlled
Red phlegm.It can be seen that bletilla accepts the effect of pulmonary diseases such as pulmonary fibrosis already, and modern clinic application further demonstrates that,
It is applied alone bletilla to have good therapeutic effect to pulmonary tuberculosis and silicosis, and coordinates anti-consumptive disease Western medicine isoniazid, Pasid, strepto-
Element etc. can play attenuation, synergy, reduce the curative effects such as drug resistance generation, be greatly facilitated phthistic's rehabilitation, improved its life
Quality.In addition, bletilla also has significant curative effect to burn and scald.But in addition to bletilla polysaccharide, rarely has bletilla correlation effective component report.
We early-stage study have found that 95% ethanol extract of bletilla removes 20% ethanol elution object through polycaprolactam, collects 95% second
Alcohol eluate, being studied through internal Silicotic rat confirms that the bletilla ingredient has significant anti-inflammatory and pulmonary fibrosis is inhibited to live
Property；By further furtheing investigate, we are prepared for one containing known component content up to 50% or more, and with notable anti-inflammatory work
Property bletilla drug activity position, zoopery prove the active site to chronic bronchitis have obvious therapeutic action, mesh
It is preceding that there is not been reported both at home and abroad.
(3) invention content
It is an object of the present invention to provide a kind of Pseudobulbus Bletillae (Rhizoma Bletillae) extract and its applications, solve existing Chinese medicine bletilla preparation effective component
It is unknown, the problems such as quality of the pharmaceutical preparations is unstable, while also specifying the bletilla active component to the effective for the treatment of chronic bronchitis
Using.
The technical solution adopted by the present invention is：
The present invention provides a kind of Pseudobulbus Bletillae (Rhizoma Bletillae) extract, and the extract is prepared as follows：1) it takes bletilla stem tuber slice, do
It is dry, beat powder, cross 50 mesh sieve, obtain bletilla dry powder；Bletilla dry powder is weighed, is carried with the reflux of volumetric concentration 60-90% ethanol waters
It takes and (preferably extracts 3 times), filtrate, as bletilla alcohol extract are collected in filtering；The volumetric concentration 60-90% ethanol water bodies
Product dosage is calculated as 5-10L/kg with bletilla dry powder weight；
2) 60-100 mesh polyamide and distilled water are added into step 1) filtrate, depressurizes and is steamed to no alcohol taste after mixing, obtained
Adsorb the polyamide suspended matter of bletilla alcohol extract；
3) in chromatographic column be added distilled water be suspended 60-100 mesh polyamide, after sedimentation (ensure on cylinder retain 5cm with
Upper distilled water), then the polyamide suspended matter of absorption bletilla alcohol extract in step 2) is fitted into column；
4) distilled water, 15%~25% ethanol water of volumetric concentration (preferably 20%) and volumetric concentration 35% are used successively
~45% ethanol water (preferably 40%) elution, flow velocity is 2mL/min, and collected volume concentration 35%~45% is (excellent
Select the eluent of 40%) ethanol water；
5) distilled water is added in the ethanol eluate collected into step 4), uses D101 macroporous resin columns to flow after mixing
Fast 2ml/min carries out Dynamic Adsorption；
6) 15%~25% ethanol water of volumetric concentration (preferably 20%), 35%~45% second of volumetric concentration are used successively
Alcohol solution (preferably 40%) elution, flow velocity is 2mL/min, collects 35%~45% (preferably 40%) ethanol eluate,
Evaporated under reduced pressure obtains Pseudobulbus Bletillae (Rhizoma Bletillae) extract.
Further, step 1) the ethanol water volumetric concentration 80%, volumetric usage are calculated as 7L/ with bletilla dry powder weight
kg。
Further, the step 2) polyamide and bletilla dry powder quality ratio are 0.15~0.25:1, preferably 0.2:1, institute
It states distilled water volume dosage and 1mL/g is calculated as with bletilla dry powder weight.
Further, the step 3) polyamide and step 1) bletilla dry powder weight ratio are 0.04:1, the distilled water volume
Dosage is calculated as 10ml/g with polyamide weight.
Further, the step 4) distilled water, 15%~25% ethanol water of volumetric concentration and volumetric concentration 35%~
45% ethanol water volumetric usage is respectively 2 times of column volumes, 2 times of column volumes and 4 times of column volumes.
Further, the step 5) distilled water and ethanol eluate volume ratio are 2~4：1.
Further, 15%~25% ethanol water of step 6) volumetric concentration, 35%~45% ethyl alcohol of volumetric concentration are water-soluble
Liquid elutes 2 times of column volumes and 4 times of column volumes respectively.
The present invention also provides a kind of application of Pseudobulbus Bletillae (Rhizoma Bletillae) extract in preparing anti-inflammatory drug.
The present invention also provides a kind of Pseudobulbus Bletillae (Rhizoma Bletillae) extracts to prepare the application in treating chronic bronchitis drug, this hair
Bright Pseudobulbus Bletillae (Rhizoma Bletillae) extract, i.e. active component EFB (Effect Fraction of Bletilla striata, EFB) are to lipopolysaccharides
The reaction of RAW264.7 cellular inflammations caused by (LPS, Sigma company) significantly inhibits effect.
Compared with prior art, advantageous effect of the present invention is mainly reflected in：
It being experimentally confirmed, Pseudobulbus Bletillae (Rhizoma Bletillae) extract active ingredient of the invention includes mainly luxuriant and rich with fragrance class and Bibenzyl compound,
In, 2,7- dihydroxy -4- methoxyl groups -9,10- dihydro phenanthrenes (punctatelip coelogyne herb is peaceful) >=15.85%, 3', 3 "-dihydroxy -5'- methoxyl groups
Bibenzyl (batatasins III) >=29.65%, 3'-O- methyl batatasins III >=6.32%, 3- hydroxy-5-methyl oxygroups bibenzyl >=
2.95%, the peaceful content of punctatelip coelogyne herb, batatasins III, 3'-O- methyl batatasins III, 3- hydroxy-5-methyl oxygroup bibenzyl total amounts are more than
50%.
The present invention confirms the bletilla active component of the present invention by the experiment of internal SD rat model of chronic bronchitis
EFB, can be by inhibiting TNF-α, IL-1 β, the inflammatory factors such as IL-6, and by improving oxidative stress, to chronic branch gas
Pipe inflammation plays therapeutic effect.
(4) it illustrates
Fig. 1 lung tissue sections HE coloration results, a-f is respectively normal group, model group, Dexamethasone group, EFB low dosages
80% ethanol extract group of group, EFB high dose groups and bletilla.
(5) specific implementation mode
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Bletilla of the present invention according to《Pharmacopoea Chinensis》It is prepared by 2010 editions acquisitions.
1 Pseudobulbus Bletillae (Rhizoma Bletillae) extract of embodiment
1, the preparation of Pseudobulbus Bletillae (Rhizoma Bletillae) extract：
1) bletilla stem tuber is sliced, is dry, beating powder, crosses 50 mesh sieve, bletilla dry powder 500g is weighed, with 3.5L volumetric concentrations 80%
Filtrate is collected in ethanol water refluxing extraction 1h, filtering, repeats extraction 3 times altogether, and merging filtrate, as bletilla alcohol extract are (dry
It is dry, it is total to be denoted as E)；
2) 100g 60-100 mesh polyamide (City of Taizhou road and bridge tetramethyl biochemistry plastics are added into step 1) filtrate
Factory, lot number 2015617), 500mL distilled water, after mixing decompression steam to no alcohol taste, the polyamide for obtaining absorption bletilla alcohol extract mixes
Outstanding object；
3) the 60-100 mesh polyamide dry powder 20g that 200mL distilled water is suspended are added in the chromatographic column of diameter 5cm, high 20cm,
After sedimentation, the polyamide suspended matter of absorption bletilla alcohol extract in step 2) is fitted into column；
4) successively with the volumetric concentration of the distilled water of 2 times of column volumes (evaporated under reduced pressure obtained by position be denoted as E0), 2 times of column volumes
20% ethanol water (evaporated under reduced pressure obtained by position be denoted as E20), 40% ethanol water of volumetric concentration of 4 times of column volumes, 3 times
The volumetric concentration of column volume is that 60% ethanol water (position obtained by evaporated under reduced pressure is denoted as E60) and the volume of 2 times of column volumes are dense
Degree is 80% ethanol water elution (position obtained by evaporated under reduced pressure is denoted as E80), and flow velocity is 2mL/min, collected volume concentration
The eluent of 40% ethanol water；
5) 2.4L distilled water is added in the 40% ethanol eluate 0.8L collected into step 4), D101 macropores are gone up after mixing
Resin column (diameter 4cm, pillar height 20cm), flow velocity 2mL/min carry out Dynamic Adsorption；
6) it is eluted with 2mL/min with 20% ethanol water of 500mL volumetric concentrations, then uses 40% ethyl alcohol of 1L volumetric concentrations
Aqueous solution elutes, and collects 40% ethanol eluate, and evaporated under reduced pressure obtains Pseudobulbus Bletillae (Rhizoma Bletillae) extract (being denoted as bletilla active component EFB)
2.78g。
2, Pseudobulbus Bletillae (Rhizoma Bletillae) extract Contents of Main Components measures：
Using high performance liquid chromatography, to four main monomer components of Pseudobulbus Bletillae (Rhizoma Bletillae) extract, (punctatelip coelogyne herb is peaceful, batatasins III, 3'-
O- methyl batatasins III, 3- hydroxy-5-methyl oxygroups bibenzyl) assay is carried out, chromatographic condition is：Wear peace UltiMate3000 high
Effect liquid phase chromatogram system, Dai An120C18 chromatographic columns (4.6 × 250mm, 5 μm, 120 apertures, lot018-01-
080), Detection wavelength 274.5nm and 193.0nm, mobile phase are -0.1% formic acid (B) of acetonitrile (A)；Gradient elution：0-5min, A
From 10% graded to 35%；5-12min maintains A 35%；12-16min, A are from 35% graded to 45%；16-
22min, A maintain 45%；22-25min, A are from 45% graded to 52%；25-37min, A maintain 52%；Last A is returned
To 10% balance 10min.Flow velocity is 1mL/min；Column temperature is 30 DEG C.
It is prepared by standard curve：By standard items, (punctatelip coelogyne herb is peaceful, batatasins III, 3'-O- methyl batatasins III, 3- hydroxy-5-methyls
Oxygroup bibenzyl) 10,20,30,40,50 and 60 μ g/mL concentration gradients are made into distilled water respectively, according to above-mentioned high performance liquid chromatography
Testing conditions detect the characteristic peaks of various concentration, and with a concentration of abscissa, it is bent to make standard using characteristic peaks as ordinate
Line, the results are shown in Table 1.
1 four standard items calibration curve equations of table
According to the characteristic peaks of Pseudobulbus Bletillae (Rhizoma Bletillae) extract and respective standard curve, four main monomer components contain in Pseudobulbus Bletillae (Rhizoma Bletillae) extract
Amount is respectively：The peaceful content of punctatelip coelogyne herb is up to 17.84%, batatasins III up to 32.75%, 3'-O- methyl batatasins III up to 8.26%, 3-
Hydroxy-5-methyl oxygroup bibenzyl is up to 4.21%, it is known that object total amount is up to 63.06%.
3, Pseudobulbus Bletillae (Rhizoma Bletillae) extract extracorporeal anti-inflammatory is tested
Exponential phase RAW264.7 cells (being purchased from cell institute of the Chinese Academy of Sciences) are collected, DMEM culture mediums is used in combination to be adjusted to density
It is 5 × 105The single cell suspension of a/mL is inoculated in 96 orifice plates, per 100 μ L of hole, i.e., 5 × 104A/hole is placed in 37 DEG C, 5%CO2
It is cultivated for 24 hours in incubator.Culture solution supernatant is abandoned, the DMEM trainings of 200 μ L Pseudobulbus Bletillae (Rhizoma Bletillae) extracts containing various concentration (being shown in Table 2) are added per hole
Nutrient solution is placed in 37 DEG C, 5%CO21h is cultivated in incubator, and LPS (lipopolysaccharides, the Sigma of final concentration of 200ng/mL is then added
Company) culture is stimulated, ATP (atriphos, Sigma companies) the stimulation 15min of final concentration of 2mM are added after 12h per hole, receive
Collect supernatant, is passed through according to specification using BD companies Cytometric Bead Array (CBA) cytokine detection kits
Flow cytometer measures IL-1 β, IL-6, MCP, TNF-a level, and the results are shown in Table 2.From the results, it was seen that bletilla extracts
Object EFB can each inflammatory factor of dose-dependent inhibition it is horizontal, wherein IL-1 β, IL-6 and MCP are especially pronounced.In addition, being all 20 μ
When the processing of g/mL drug concentrations, E0, E20, E60, E80, E are always substantially less than the inhibitory activity of each inflammatory factor prepared
EFB components, it is seen that EFB is a main anti-inflammatory drug effect active site in bletilla.
2 Pseudobulbus Bletillae (Rhizoma Bletillae) extract of table, which secretes LPS stimulation RAW264.7 inflammatory factors, influences (mean ± SD, n=3)
Note：Compared with model group#P < 0.05,*P < 0.01；With the EFB processing group ratio of 20 μ g/mL,△P < 0.01；"E0,
E20, E60, E80 " are respectively distilled water, 20% ethyl alcohol, 60% ethyl alcohol, the 80% ethanol elution object in step 1 4), E chief representative
It is directly concentrated and dried after step 1) bletilla powder 80% ethanol water refluxing extraction of volumetric concentration extract obtained.
4, Pseudobulbus Bletillae (Rhizoma Bletillae) extract (i.e. active component EFB) is to rat chronic bronchitis therapeutic effect
Chronic bronchitis SD rat models are replicated using fumigation, i.e., SD rats are placed in 1m3In smoke-box, with wood shavings,
Sawdust and each 50g of tobacco leaf, daily early, evening each sootiness 1 time, each 30min, continue modeling one month；Normal group SD rats are placed in
It is raised in no smoke environment.It is divided into four groups after modeling, the normal group of (physiology of Tween-80 of the gavage containing volumetric concentration 0.1%
Brine), model group (Tween-80 physiological saline of the gavage containing volumetric concentration 0.1%), positive drug group (gavage dexamethasone
6mg/kg, with the Tween-80 physiological saline solution containing volumetric concentration 0.1%), EFB low dose groups (gavage EFB 15mg/kg, use
Tween-80 physiological saline solution containing volumetric concentration 0.1%), EFB high dose groups (gavage EFB 30mg/kg, with dense containing volume
The Tween-80 physiological saline solution of degree 0.1%) and total processing group (the 80% ethanol extract 30mg/kg of gavage bletilla, with containing body of E
The Tween-80 physiological saline solution of product concentration 0.1%), daily gastric infusion is primary, successive administration two weeks；Rat is put to death, lung is done
Lavation is steeped, streaming CBA methods measure IL-1 β in irrigating solution, IL-6, TNF-α level, SOD and MDA kits measure alveolar filling respectively
SOD and MDA is horizontal in washing lotion；It is another to take left mainstem bronchus, fixed paraffin section HE dyeing microscopic examinations.
After modeling one month, model mouse expiratory dyspnea, cough, burnout, hair is matt, and feed is reduced, mouth and nose secretion
More, after two weeks, model group rats, which have no, to be clearly better, apathetic, rolls up and moves less, but each administration group rats eating amount is bright
Aobvious to increase, mouth and nose secretion substantially reduces, and cough mitigates, more active.HE is sliced the result shows that (see Fig. 1), normal rat gas
Road epithelial structure is complete, epithelial cell marshalling, no inflammation cell invasion, and alveolar structure is clear；Model group rats tracheae is viscous
Film epithelial cell fall into disarray, some necrosis that fall off have a large amount of inflammatory cells to invade profit in tube wall, and smooth muscle layer thickens obviously,
Inflammatory exudate is more in bronchial lumen, and inflammatory cell infiltration is serious in alveolar space, while alveolar structure significantly changes；Respectively give
Medicine group tracheae luminal sectetion is reduced, and airway epithelia arrangement is obviously improved, and inflammatory cell is invaded profit and substantially reduced, wherein EFB
High dose is particularly evident；And 80% alcohol extract of bletilla (E is total) 30mg/kg processing group is suitable with EFB low dosages 15mg/kg.
(it is shown in Table 3) in terms of each factor level of bronchoalveolar lavage fluid, EFB can dose dependent reduction inflammatory factor IL-1 β, IL-
6, TNF-α is horizontal, while can remarkably promote the recovery of SOD levels, and effectively reduces MDA contents；This explanation, EFB may can lead to
Body oxidative stress and inflammatory reaction system are overregulated, to play the therapeutic effect to chronic bronchitis.In addition, comparison is white
And 80% each inflammatory factor of alcohol extract (E is total) 30mg/kg processing groups it is horizontal and HE slice results is combined to see that EFB high doses are aobvious
It writes and is better than bletilla alcohol extract group, it is seen that bletilla composition and effectiveness EFB anti-inflammatory activities and chronic bronchitis is controlled after enrichment
Treatment effect is significantly improved, and is an important composition and effectiveness in bletilla.
3 each group rat blood serum items factor level (mean ± SD, n=6) of table
Note：Compared with model group*P < 0.01.
Claims (9)
1. a kind of Pseudobulbus Bletillae (Rhizoma Bletillae) extract, it is characterised in that the Pseudobulbus Bletillae (Rhizoma Bletillae) extract is prepared as follows：1) take bletilla stem tuber to be sliced,
Powder is beaten in drying, is crossed 50 mesh sieve, is obtained bletilla dry powder；Bletilla dry powder is weighed, is flowed back with volumetric concentration 60-90% ethanol waters
Filtrate, as bletilla alcohol extract are collected in extraction, filtering；The volumetric concentration 60-90% ethanol water volumetric usages are with bletilla
Dry powder weight is calculated as 5-10L/kg；
2) 60-100 mesh polyamide and distilled water are added into step 1) filtrate, depressurizes and is steamed to no alcohol taste after mixing, adsorbed
The polyamide suspended matter of bletilla alcohol extract；
3) the 60-100 mesh polyamide that distilled water is suspended is added in chromatographic column, after sedimentation, bletilla alcohol extract will be adsorbed in step 2)
Polyamide suspended matter be fitted into column；
4) 35%~45% ethanol water of distilled water, 15%~25% ethanol water of volumetric concentration and volumetric concentration is used successively
Elution, flow velocity is 2ml/min, the eluent of 35%~45% ethanol water of collected volume concentration；
5) distilled water is added in the ethanol eluate collected into step 4), uses D101 macroporous resin columns with flow velocity after mixing
2ml/min carries out Dynamic Adsorption；
6) 15%~25% ethanol water of volumetric concentration, 35%~45% ethanol water of volumetric concentration is used to elute successively, stream
Speed is 2ml/min, 35%~45% ethanol eluate of collected volume concentration, evaporated under reduced pressure, acquisition Pseudobulbus Bletillae (Rhizoma Bletillae) extract.
2. Pseudobulbus Bletillae (Rhizoma Bletillae) extract as described in claim 1, it is characterised in that step 1) the ethanol water volumetric concentration 80%, body
Product dosage is calculated as 7L/kg with bletilla dry powder weight.
3. Pseudobulbus Bletillae (Rhizoma Bletillae) extract as described in claim 1, it is characterised in that the step 2) polyamide is with bletilla dry powder quality ratio
0.15~0.25:1, the distilled water volume dosage is calculated as 1ml/g with bletilla dry powder weight.
4. Pseudobulbus Bletillae (Rhizoma Bletillae) extract as described in claim 1, it is characterised in that the step 3) polyamide and step 1) bletilla dry powder weight
Amount is than being 0.04:1, the distilled water volume dosage is calculated as 10ml/g with polyamide weight.
5. Pseudobulbus Bletillae (Rhizoma Bletillae) extract as described in claim 1, it is characterised in that the step 4) distilled water, volumetric concentration 15%~25%
Ethanol water and 35%~45% ethanol water volumetric usage of volumetric concentration are respectively 2 times of column volumes, 2 times of column volumes and 4
Times column volume.
6. Pseudobulbus Bletillae (Rhizoma Bletillae) extract as described in claim 1, it is characterised in that the step 5) distilled water and ethanol elution liquid
Product is than being 2~4：1.
7. Pseudobulbus Bletillae (Rhizoma Bletillae) extract as described in claim 1, it is characterised in that 15%~25% ethanol water of step 6) volumetric concentration,
35%~45% ethanol water of volumetric concentration elutes 2 times of column volumes and 4 times of column volumes respectively.
8. application of the Pseudobulbus Bletillae (Rhizoma Bletillae) extract in preparing anti-inflammatory drug described in a kind of claim 1.
9. Pseudobulbus Bletillae (Rhizoma Bletillae) extract described in a kind of claim 1 is preparing the application in treating chronic bronchitis drug.
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| CN109498523A (en) * | 2018-12-25 | 2019-03-22 | 广东润洁日化有限公司 | The anti-chap skin care item of one kind and its preparation |
| CN109568494A (en) * | 2019-01-22 | 2019-04-05 | 浙江中医药大学 | A kind of application of bletilla alcohol extract in preparation polymyxins Trimethoprim |
| CN109568494B (en) * | 2019-01-22 | 2021-11-19 | 浙江中医药大学 | Application of bletilla striata alcohol extract in preparation of polymyxin antibacterial synergist |
| CN109678680A (en) * | 2019-01-31 | 2019-04-26 | 遵义医学院 | A method of extracting 2,7- dihydroxy -4- methoxyl group -9,10- dihydro phenanthrene from bletilla |
| CN112675354A (en) * | 2020-12-23 | 2021-04-20 | 浙江中医药大学 | Multifunctional rhizoma bletillae medical material and preparation method and application thereof |
| CN114557985A (en) * | 2022-03-15 | 2022-05-31 | 浙江中医药大学 | Fritillaria-lanning composition for treating osteoarthritis and application thereof |
| CN114557985B (en) * | 2022-03-15 | 2023-06-27 | 浙江中医药大学 | A pharmaceutical composition for treating osteoarthritis and its application |
| CN114902920A (en) * | 2022-04-27 | 2022-08-16 | 贵州省农作物品种资源研究所 | Bletilla striata cultivation method, bletilla striata extract, preparation method of bletilla striata extract and oxidation resistance detection method |
| CN114902920B (en) * | 2022-04-27 | 2024-04-09 | 贵州省农作物品种资源研究所 | Bletilla striata cultivation method, bletilla striata extract, preparation method thereof and oxidation resistance detection method |
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