CN106588642A - P-hydroxyphenyl propanoic acid extracted from white mulberry root barks as well as preparation method and application thereof - Google Patents
P-hydroxyphenyl propanoic acid extracted from white mulberry root barks as well as preparation method and application thereof Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C59/40—Unsaturated compounds
- C07C59/42—Unsaturated compounds containing hydroxy or O-metal groups
- C07C59/52—Unsaturated compounds containing hydroxy or O-metal groups a hydroxy or O-metal group being bound to a carbon atom of a six-membered aromatic ring
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- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/47—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
Abstract
The invention relates to p-hydroxyphenyl propanoic acid extracted from white mulberry root barks as well as a preparation method and an application thereof to effectively solve the problems of extracting the p-hydroxyphenyl propanoic acid from the white mulberry root barks and applying the p- hydroxyphenyl propanoic acid to preparation of medicines for treating LPS (lipopolysaccharide) induced HPAEC (Human Pulmonary Artery Endothelial Cells) injuries. The preparation method comprises the following steps: performing decoction extraction on the white mulberry root barks; performing vacuum concentration on an extract to obtain a dried product; adding distilled water into the dried product to perform suspension; performing treatment on a resin column; performing gradient elution by using water and ethanol in sequence; concentrating eluant of each gradient until the eluant is dried; dissolving the dried product by using methanol; adding silica gel; adding the mixture to the liquid surface of a chromatographic column; performing gradient elution; performing TLC (thin layer chromatography) analysis and combination on each fraction; performing vacuum concentration to obtain gross samples A1, A2 and A3; and dissolving the gross sample A1 by using methanol, performing treatment on a Toyopearl HW-40 column, performing elution by using methanol, performing thin layer detection and combination to obtain a fraction A1-2, performing vacuum concentration and drying, performing dissolution by using methanol, performing treatment on an ODS (octadecylsilyl) column, performing elution by using methanol, performing thin layer detection, combining parts containing the same fractions, and performing vacuum concentration to obtain the p-hydroxyphenyl propanoic acid. The method provided by the invention is novel and unique, is easy to operate and use, and opens up a new application of the white mulberry root barks.
Description
Technical field
The present invention relates to medical, particularly a kind of para hydroxybenzene propanoic acid extracted from Cortex Mori and preparation method thereof with should
With.
Background technology
Cortex Mori is moraceae plants mulberry (Classification system：Morus alba L.) dry root skin, another name Cortex Mori, Mulberry
Root bark, Cortex Mori, Morus alba skin, autumn Mo fallen leaves excavate root before secondary spring germination, scrape off yellowish-brown rough bark, longitudinally cut open, strip
Root bark, dries.Containing flavones ingredient：Mulberrin (mulberrin), mulberrochromene (mulberrochromene), cyclomulberrin
(cyclomulberrin), cyclomulberrochromene (cyclomulberro-chromene), Morusin (morusin), ring Mulberry
Pi Su (cyclomorusin), oxidation dihydro Morusin (oxydihydromomsin), phellinus ketone (kuwanon) A, B, C, D,
E, F, G (i.e. albanin F, moracenin B), H (i.e. albanin G, moracenin A), I, K, L, Y, Z, moracenin
(moracenin) C, D, sanggenon (sffnggenone) A～P；Again containing Mulberrofuran (mulberrofuran) A, B, C, K, N, 0,
M, P, Q, the acid of birch skin；Coumarinses：5,7- Hydroxycoumarins (5,7-dihydmxycoumarin) umbelliferone
(umbelliferone), the composition such as scopoletin (i.e. scopoletin, scopoletin), hyoscyami lactone.Polysaccharide：Mucus
Element, Mulberry polysaccharide, chitin, shitosan；Also contain α-and β-amyrin, sitoesterol, volatile oil, tannin etc..With blood pressure lowering, diuresis,
The effect of sedation and analgesia, antiinflammatory, antibacterial, suppression platelet aggregation, blood sugar lowering, to the suppression ratio of human cervical carcinoma JTC-26 strains it is
70% or so, act on inducement interferon.
LPS is endotoxic main component, and endotoxin can cause urgency by inducing excessive inflammatory reaction into after body
Property injury of lung (acute lung injury, ALI), the ALI of LPS inductions be occur after severe trauma or infection earliest, sickness rate
One of highest complication, pulmonary vascular endothelial cell damage are the key links that LPS causes acute lung injury.Therefore study blood vessel
Treatment and defence tool significance of the impaired and its reparation of endothelium to ALI.Can secrete after inner skin cell function imbalance is impaired
Cytokine profiles, inflammatory factor, such as chemotactic factor (MCP-1), adhesion factor (ICAM-1, VCAM-1, E-selectin), inflammation
Inflammation factor (TNF-α, IL-1 β, IL-6) etc..Simultaneously endotheliocyte can also discharge various active substances, coordinate vasodilation because
Balance between son and contraction factor, the destruction of such as vaso-excitor material ET-1 and expansion angiogenic substance NO, ET-1/NO balance is tremulous pulse
The marked feature of vessel endothelium and induced endothelial.INOS is a kind of a kind of material for producing in the case where inflammatory factor stimulates, and its content can be represented
The degree of blood vessel injury, it has recently been demonstrated that the AMPK of activation can be significantly improved in the damage of TNF-α inducing endothelial cell
The core displacement of NF- κ B, 5-aminoimidazole-4-carboxamide-1- β-d-ribofuranoside (AICAR) also can
Enough inflammatory reactions for suppressing LPS inductions by activating AMPK.But so far there are no effective active position pair is extracted from Cortex Mori
Hydroxy phenylpropionic acid (abbreviation SP-6) simultaneously treats the application in the HPAEC damage medicines of LPS inductions for preparation.
The content of the invention
For above-mentioned situation, to overcome the defect of prior art, the purpose of the present invention is just to provide one kind from Cortex Mori
Para hydroxybenzene propanoic acid of extraction and preparation method and application, can effectively solving extract from Cortex Mori para hydroxybenzene propanoic acid (letter
Claim SP-6), and the application problem in the HPAEC damage medicines for preparing treatment LPS inductions.
The technical scheme that the present invention is solved is, described para hydroxybenzene propanoic acid be the effective active that extracts from Cortex Mori into
Point, as shown in Figure 1, the preparation method of the para hydroxybenzene propanoic acid is molecular structural formula：
Cortex Mori is decocted and is extracted, extracting solution concentrating under reduced pressure obtains dried object, and dried object adds distilled water to be suspended, is centrifuged off not
Molten thing, obtains supernatant；Resin column on supernatant, successively with water, 30% ethanol gradient elution of mass concentration, each gradient eluent
It is concentrated to dryness, dry methanol dissolves, and adds silica gel, must be loaded with the silica gel of sample；The silica gel for being loaded with sample is added to into color
On the liquid level of spectrum post, gradient elution is carried out, the stream part collected is analyzed through TLC, merge the stream part containing target component, decompression
Concentration, obtains gross sample A1, A2, A3 containing target component；Gross sample A1 methanol dissolves, and upper Toyopearl HW-40 posts use first
Alcohol eluting, thin layer inspection are known, and merge stream part containing identical, obtain subflow part A1-2, and concentrating under reduced pressure, drying are dissolved with methanol, on
ODS posts, with methanol-eluted fractions, thin layer inspection knowledge, merge stream part containing identical, and concentrating under reduced pressure obtains target component white powder to hydroxyl
Base benzenpropanoic acid (abbreviation SP-6)：
The HPAEC that the para hydroxybenzene propanoic acid is induced to LPS is damaged protective effect, effective for preparing treatment LPS inductions
HPAEC damage medicine, realize para hydroxybenzene propanoic acid prepare treatment LPS induction HPAEC damage medicines in application.
The present invention is a kind of effective active composition para hydroxybenzene propanoic acid (abbreviation SP-6) extracted from Cortex Mori, its preparation
Method novel and unique, easy to operate, the para hydroxybenzene propanoic acid which is prepared are preparing treatment LPS inductions as sole active position
HPAEC damage medicines in application, opened up the new application of Cortex Mori, had good economic and social benefit.
Description of the drawings
Fig. 1 is the molecular structure of the para hydroxybenzene propanoic acid (abbreviation SP-6) of the present invention.
Fig. 2 is impact figures of the SP-6 of the present invention to HPAEC cell injury.
Fig. 3 is impact figures of the AMPK antagonisies Compound C of the present invention to SP-6.
Fig. 4 is impact figures of the SP-6 of the present invention to HPAEC cell TNF-αs.
Fig. 5 is impact figures of the SP-6 of the present invention to HPAEC cell IL-1 β.
Fig. 6 is impact figures of the SP-6 of the present invention to HPAEC cell IL-6.
Fig. 7 is impact figures of the SP-6 of the present invention to HPAEC cell ET-1.
Fig. 8 is impact figures of the SP-6 of the present invention to HPAEC cell NO.
Fig. 9 is impact figures of the SP-6 of the present invention to HPAEC cell iNOS.
Figure 10 is impact figures of the SP-6 of the present invention to HPAEC cell ICAM-1.
Figure 11 is impact figures of the SP-6 of the present invention to HPAEC cell VCAM-1.
Figure 12 is impact figures of the SP-6 of the present invention to HPAEC cell E-selectin.
Figure 13 is impact figures of the SP-6 of the present invention to HPAEC cell MCP-1.
Specific embodiment
The specific embodiment of the present invention is elaborated below in conjunction with concrete condition.
In being embodied as, described para hydroxybenzene propanoic acid is the effective active composition extracted from Cortex Mori to the present invention,
As shown in Figure 1, the preparation method of the para hydroxybenzene propanoic acid is molecular structural formula：
By Cortex Mori 2.4kg every time with the distillation water boiling and extraction 3 times of its weight 8-12 times, each 60min, united extraction
Liquid, concentrating under reduced pressure obtain dried object, plus the distilled water of dried object bulking value 2-3 times is suspended, and bulking value refers to solidss with g
Meter, in terms of mL, 6000r/min is centrifuged off insoluble matter to liquid, obtains supernatant；Take 7.0L Diaion HP-20 macroporous absorption trees
Fat filler, with 95% ethanol of mass concentration immersion 24h dress posts, is washed to without alcohol taste with distillation, and supernatant concentration is relative to 50 DEG C
Density 1.2-1.4, upper resin column, successively with water, 30% ethanol elution of mass concentration, each gradient eluent consumption is 10 posts
Volume, flow velocity are 2BVh-1, and each gradient eluent is concentrated to dryness, and obtain 30% ethanol elution component and crude extract, are added
50mL methanol dissolves, and adds the silica gel 40g of 160-200 mesh sieves, and 45 DEG C of recovered under reduced pressure methanol obtain being loaded with the silicon of sample
Glue；The silica gel 500g of 200-300 mesh sieves was taken, dichloromethane 800mL is added, is transferred to the chromatographic column of 80cm length × 8cm internal diameters
In, post bed is enriched with dichloromethane, liquid level is down at the 5cm of silica gel face, then the above-mentioned silica gel for being loaded with sample is added to into color
On the liquid level of spectrum post, volume ratio is sequentially added for 100:3、100:5、100:The methylene chloride-methanol of 10 gradients, each gradient
4L, carries out gradient elution, and flow velocity is 10mLmin-1, per 100mL as 1 stream part, the stream part collected is analyzed through TLC,
Merge the stream part containing target component, concentrating under reduced pressure obtains gross sample A1, A2, A3 containing target component, described TLC analyses
Used in adsorbent be GF254, developing solvent be volume ratio 20:1-5:1 methylene chloride-methanol；Gross sample A1 mass concentrations
30% methanol dissolves, upper Toyopearl HW-40C posts, and with 30% methanol-eluted fractions of mass concentration, flow velocity is 1mLmin-1, often
2mL is a stream part, and thin layer inspection knowledge, merging stream part containing identical obtain subflow part A1-1, A1-2；Gross sample A2 mass concentrations
35% methanol dissolves, upper Toyopearl HW-40 posts, and with 35% methanol-eluted fractions of mass concentration, flow velocity is 1mLmin-1, per 2mL
For a stream part, thin layer inspection knowledge, merge stream part containing identical, obtain subflow part A2-1, A2-2；Gross sample A3 mass concentrations 50%
Methanol dissolves, upper Toyopearl HW-40 posts, and with 50% methanol-eluted fractions of mass concentration, flow velocity is 1mLmin-1, it is one per 2mL
Individual stream part, thin layer inspection are known, and merge stream part containing identical, obtain subflow part A3-1；Gained 45 DEG C of concentrating under reduced pressure of each subflow part, obtain
To the sample containing target component；Subflow part A1-2 31% methanol of mass concentration dissolves, upper ODS posts, with mass concentration 31%
Methanol-eluted fractions, flow velocity are 5mLmin-1, it is a stream part per 10mL, thin layer inspection is known, and merges stream part containing identical, 45 DEG C of decompressions
Concentration, obtains target component white powder para hydroxybenzene propanoic acid (SP-6)：
The HPAEC that the para hydroxybenzene propanoic acid is induced to LPS is damaged protective effect, effective for preparing treatment LPS inductions
HPAEC damage medicine, realize para hydroxybenzene propanoic acid prepare treatment LPS induction HPAEC damage medicines in application.
The effective active composition that the inventive method is extracted from Cortex Mori, Jing liquid chromatography for measuring, its structural formula such as Fig. 1
It is shown, para hydroxybenzene propanoic acid (abbreviation SP-6) is named as, damaging with the HPAEC induced to LPS has protective effect, and Jing experiments
Obtain fully proving, relevant information is as follows：
Materials and methods
1 experiment material
1.1 cell
HPAEC cell strains are purchased from Chinese Academy of Sciences's cell bank.
1.2 cell culture
HPAEC cell culture (is contained into penicillin and chain in being 1640 culture medium of 10% hyclone containing volume fraction
Mycin 100kUL-1), it is placed in 37 DEG C, 5%CO2Cultivate in saturated humidity incubator, trophophase cell of taking the logarithm is used for real
Test.
1.3 Experimental agents：Para hydroxybenzene propanoic acid (abbreviation SP-6) prepared by the inventive method.
1.4 reagent
Lipopolysaccharide (sigma)；Dexamethasone acetate tablets, Chinese medicines quasi-word H12020686, product batch number 13050102, (Tianjin
Tian Yao Pharmaceuticaies limited company)；(Wuhan Yi Lairuite biotechnologies are limited for humanTNF-α, IL-1 β and IL-6ELISA test kits
Company)；ICAM-1, VCAM-1, E-selectin, MCP-1ELISA (Wuhan Yi Lairuite bio tech ltd)；NO,
INOS test kits (Nanjing is built up)；1640 culture medium (Gibco companies of the U.S.)；Gibco companies of the trypsin U.S.)；Tire Sanguis Bovis seu Bubali
Clearly (Gibco companies of the U.S.)；Water is ultra-pure water；PBS etc. is autogamy；Total protein extraction test kit, BCA method protein quantifications
Test kit (Beijing Puli comes gene technology company limited)；It is pure that other various reagents are commercially available analysis.
1.5 instrument
Bio Mate 3S ultraviolet spectrophotometers (Thermo Fisher Scientific)；AB204-N a ten thousandths essence
Close analytical balance (METTLER TOLEDO)；(Eppendorf is public for Centrifuge-5804R miniature high-speeds freezing centrifuge
Department)；IMARK type microplate reader (U.S. BIO-RAD).CO2 gas incubator (Shanghai STIK)；Superclean bench (Su Jing groups)；
IMARK type microplate reader (BIO-RAD companies of the U.S.)；The super combined Superpure water machines (SARTORIUS) of Arium 611VF；It is inverted
Microscope (Nikon)；PB-10 acidometers (German Sai Duolisi groups), culture dish, 96 well culture plates, cell cryopreservation tube
(Corning companies).
2 experimental techniques
2.1LPS induces the foundation of HPAEC cell injury models
By HPAEC cell culture in 1640 culture medium containing 10% hyclone, by former culture medium after cell attachment
Remove, washed one time with PBS, change the blank cultures starvation 12h without serum, then change containing 1 μ g/mL LPS's
1640 culture medium continue culture.HPAEC cell injury model is formed after 24h.
2.2SP-6 the impact to HPAEC cell injury
By HPAEC cells with 1640 culture medium inoculateds containing 10% hyclone in 96 orifice plates, after cell is completely adherent
Former culture medium is removed, is washed one time with PBS, normal group is changed after changing the 1640 culture medium starvation 12h without serum
1640 new culture medium, model group change the culture medium that LPS concentration is 1 μ g/mL, and SP-6 groups change 1 μ g/mL LPS+SP-6's
Culture medium, SP-6+Compound C groups change the culture medium of 1 μ g/mL LPS+SP-6+Compound C (10 μM).Culture 24h
Afterwards, MTT solution (5mg/mL) 20 μ L are added per hole, 37 DEG C are continued culture 4 hours, exhaust culture fluid, 150 μ L is added per hole
DMSO, shakes 10 minutes, is completely dissolved crystal.Returned to zero with DMSO, the absorbance in each hole is determined under 490nm with microplate reader
A。
2.3 experiment packet
Experiment is divided into 5 groups, Normal group (NC)；Model group (M) is (with the LPS culture medium culturing cells containing 1 μ g/mL
24h)；Dexamethasone group (Dex) (dexamethasone, 0.5 μM)；SP-6 groups (1 μ g/mL LPS+3 μM SP-6)；SP-6+C groups (1 μ
g/mL LPS+3μM SP-6+10μM Compound C)。
2.4SP-6 is to HPAEC cell TNF-αs, the impact of IL-1 β, IL-6
The medium supernatant of each group cell is taken, standard substance, sample to be tested is added in the microwell plate of advance coated antibody,
Bathe through constant temperature temperature, after adding biotin antibody working solution, constant temperature temperature bath temperature and washing, add enzyme to combine working solution, constant temperature temperature
Substrate solution (TMB) is added after bath temperature and washing, after temperature bath, adds terminate liquid terminating reaction.Examined under 450nm wavelength with microplate reader
Absorbance (OD) value is surveyed, the activity of these inflammatory factors in sample is calculated by standard curve.
Impacts of the 2.5SP-6 to HPAEC cell ICAM-1, VCAM-1, E-selectin
Each group cell extraction total protein of cell, after being dissolved with appropriate medium, takes the total protein of cell solution for preparing,
Its protein concentration is determined with BCA methods, protein concentration is adjusted, then in the microwell plate of advance coated antibody add standard substance,
Sample to be tested, bathes through constant temperature temperature, after adding biotin antibody working solution, constant temperature temperature bath temperature and washing, adds enzyme to combine work
Substrate solution (TMB) is added after liquid, constant temperature temperature bath temperature and washing, after temperature bath, adds terminate liquid terminating reaction.With microplate reader in 450nm
Absorbance (OD) value is detected under wavelength, the expression of albumen in each group is calculated by standard curve.
Impacts of the 2.6SP-6 to HPAEC cell chemotactic factor MCP-1
Each group cell extraction total protein of cell, after being dissolved with appropriate medium, takes the total protein of cell solution for preparing,
Its protein concentration is determined with BCA methods, protein concentration is adjusted, then in the microwell plate of advance coated antibody add standard substance,
Sample to be tested, bathes through constant temperature temperature, after adding biotin antibody working solution, constant temperature temperature bath temperature and washing, adds enzyme to combine work
Substrate solution (TMB) is added after liquid, constant temperature temperature bath temperature and washing, after temperature bath, adds terminate liquid terminating reaction.With microplate reader in 450nm
Absorbance (OD) value is detected under wavelength, the expression of albumen in each group is calculated by standard curve.
Impacts of the 2.7SP-6 to HPAEC cell ET-1
The medium supernatant of each group cell is taken, standard substance, sample to be tested is added in the microwell plate of advance coated antibody,
Bathe through constant temperature temperature, after adding biotin antibody working solution, constant temperature temperature bath temperature and washing, add enzyme to combine working solution, constant temperature temperature
Substrate solution (TMB) is added after bath temperature and washing, after temperature bath, adds terminate liquid terminating reaction.Examined under 450nm wavelength with microplate reader
Absorbance (OD) value is surveyed, the expression of ET-1 albumen in each group is calculated by standard curve.
The content of NO in 2.8 nitrate reductase method detection cell supernatant
The medium supernatant of each group cell is taken, according to the content of the operation detection NO of test kit.
The measure of iNOS contents in 2.9 cell supernatants
The medium supernatant of each group cell is taken, according to the content of the operation detection iNOS of test kit.
Note：Colour generation thing mole twilight coefficient is 38.3 × 106
2.10 statistical method
Experimental data mean ± standard deviationRepresent, with SPSS18.0 statistical softwares processing data, compare between each group
Using one factor analysis of variance (One-Way ANOVA), P<0.05 indicates significant, P<0.01 indicates pole significance
Meaning.
3 results
3.1SP-6 the impact to HPAEC cell injury
Compared with blank control group, LPS can cause the damage (P of HPAEC cells<0.05)；Compared with model group, SP-6
Cell injury situation (the P of HPAEC can be improved<0.05), the results are shown in Table 1, Fig. 2.
Impacts of the table 1SP-6 to HPAEC cell injury
Note：Compared with normal group, * represents P<0.05, * * represents P<0.01；Compared with model group, # represents P<0.05, ##
Represent P<0.01.
Impacts of the 3.2AMPK antagonist Compound C to SP-6
Compared with blank control group, LPS can significantly damage HPAEC cell (P<0.05)；Compared with model group, SP-6 can change
Cell injury situation (the P of kind HPAEC<0.05), SP-6+C groups improvement result disappearance (P<0.05), the results are shown in Table 2, Fig. 3.
Impacts of the table 2AMPK antagonist Compound C to SP-6
Note：Compared with normal group, * represents P<0.05, * * represents P<0.01；Compared with model group, # represents P<0.05, ##
Represent P<0.01.
3.3SP-6 is to HPAEC cell injury TNF-αs, the impact of IL-1 β, IL-6
Compared with blank control group, model group TNF-α, IL-1 β, IL-6 levels pole significance raise (P<0.01)；With mould
Type group is compared, and SP-6 can reduce the level (P of TNF-α, IL-1 β, IL-6<0.01), SP-6+C groups TNF-α, IL-1 β, IL-6 water
Mean pole significance raises (P<0.01), the results are shown in Table 3, Fig. 4-6.
Table 3SP-6 is to HPAEC cell TNF-αs, the impact of IL-1 β, IL-6
Note：Compared with normal group, * represents P<0.05, * * represents P<0.01；Compared with model group, # represents P<0.05, ##
Represent P<0.01.
3.4SP-6 is to HPAEC cell ET-1, the impact of NO, iNOS
Compared with blank control group, model group ET-1, iNOS levels pole significance raise (P<0.01), NO levels significantly drop
Low (P<0.01)；Compared with model group, SP-6 groups can reduce ET-1, the level (P of iNOS<0.01), the level (P of elevation of NO<
0.01) the results are shown in Table 4, Fig. 7-9.
Table 4SP-6 is to HPAEC cell ET-1, the impact of NO, iNOS
Note：Compared with normal group, * represents P<0.05, * * represents P<0.01；Compared with model group, # represents P<0.05, ##
Represent P<0.01.
Impacts of the 3.5SP-6 to HPAEC cell ICAM-1, VCAM-1, E-selectin
Compared with blank control group, model group ICAM-1, VCAM-1, the expression pole significance of E-selectin albumen are raised
(P<0.01)；Compared with model group, SP-6 can reduce the expression (P of ICAM-1, VCAM-1, E-selectin albumen<0.01), tie
Fruit is shown in Table 5, Figure 10-12.
Impacts of the table 5SP-6 to HPAEC cell ICAM-1, VCAM-1, E-selectin
Note：Compared with normal group, * represents P<0.05, * * represents P<0.01；Compared with model group, # represents P<0.05, ##
Represent P<0.01.
Impacts of the 3.6SP-6 to HPAEC cell MCP-1
Compared with blank control group, the expression pole significance of model group CCL2 raises (P<0.01)；With model group phase
Than SP-6 groups can reduce the expression (P of CCL2<0.01), the results are shown in Table 6, Figure 13.
Impacts of the table 6SP-6 to HPAEC cell MCP-1
Note：Compared with normal group, * represents P<0.05, * * represents P<0.01；Compared with model group, # represents P<0.05, ##
Represent P<0.01.
Conclusion
During acute lung injury, vascular endothelial cell is both impaired first target organs, and active effector lymphocyte, is ground
The change for showing pulmonary vascular endothelial cell (pulmonary vasculary endothelial cell, PVEC) is studied carefully in acute lung
It is particularly significant in damage.In the presence of LPS, cytokine, oxygen-derived free radicals etc., its permeability increases pulmonary vascular endothelial cell,
Secrete and discharge various inflammatory mediators and cytokine so that proinflammatory and Anti-inflammatory mediator is unbalance.There is inflammation during ALI in body
The factor (such as TNF-α, IL-1 β, IL-6), adhesion molecule (ICAM-1, VCAM-1, E-selectin), chemotactic factor (MCP-1)
Expression is significantly raised.In these medium expression regulations, the activation of Nuclear Factor kappa B and core displacement are key factors,
The vasoactive endotheliocyte such as endotoxin makes NF- κ B in endochylema be discharged by its inhibitive factor I κ B, becomes its activity form fast
There is core displacement in speed, the kB site of searching particular target gene promoter area is simultaneously in combination, instructs the expression regulation of downstream gene.
Many researchs show that blocking acute lung injury of the NF- κ B paths to LPS inducing mouses has protective effect, while AMPK is also scorching
A key factor during disease regulation.Although the AMPK of activation is proved to antiinflammatory action, whether AMPK can
Improve the HPAEC cell injury of LPS inductionsEndotheliocyte can also discharge vaso-excitor material ET-1 and expand angiogenic substance simultaneously
The destruction of NO, ET-1/NO balance is the impaired marked feature of arterial endothelium.The content of iNOS can represent the journey of blood vessel injury
Degree, therefore this experiment is by detecting cytokine (TNF-α, IL-1 β, IL-6), adhesion molecule (ICAM-1, VCAM-1, E-
Selectin), the expression of chemotactic factor (MCP-1), ET-1, the content of NO, iNOS, while detecting AMPK antagonist Compound
Impacts of the C to which, inquires into the impact of the HPAEC cell injury that compound SP-6 is induced to LPS, tentatively discloses its mechanism of action,
Lay the foundation for its further development and application.
LPS is the main component of bacterial endotoxin, and its toxic action is mainly too strong anti-by exciting host cell to produce
Should be embodying.We can see that LPS can result in the damage of HPAEC cells from experimental result, and after giving SP-6 can under
Note improves the cell injury of LPS inductions.Cell injury is improved to SP-6 in order to explain AMPK antagonist Compound C simultaneously
Affect, Setup Experiments SP-6+C groups, experimental result show, after addition AMPK antagonist Compound C, SP-6 improves cell
Damage activity disappearance, this also preliminary proof SP-6 improve LPS induction cell injury may be relevant with AMPK/NF- κ B paths.
The HPAEC cell injury essence that LPS causes is excessive inflammatory response, we can see that model group from experimental result
TNF-α, the level of IL-1 β, IL-6 are significantly raised, and after giving SP-6, TNF-α, the level of IL-1 β, IL-6 are remarkably decreased, and SP-
6+C group TNF-αs, the level of IL-1 β, IL-6 are significantly raised, and show that SP-6 reduces the event resolves of inflammatory factor.
NO is the stable key of endothelial function, with vasodilator, suppresses hematoblastic adhesion, suppresses smooth muscle cell to increase
Grow, assemble and the effect such as adhesion from mononuclear cell to endotheliocyte.And ET-1 is the most strong long-acting blood vessel of current known effect
Contracting agent, can promote propagation and macrophage accumulation, the adhesion of smooth muscle cell.INOS is a kind of in the case where inflammatory factor stimulates
A kind of material for producing, its content number can represent the degree of blood vessel injury.Experimental result shows that LPS can be induced
The damage of HPAEC cells, while causing the reduction of NO contents, promotes the generation of ET-1, iNOS, and after giving SP-6, NO contents are notable
Raise, ET-1, iNOS content is reduced, and SP-6+C groups NO content is reduced, ET-1, iNOS content is raised, SP-6 improvement results disappear
Lose.
ICAM-1, VCAM-1, E-selectin, MCP-1 that inflammatory reaction is produced etc. is in the generation development of the diseases such as ALI
Very important effect is played, the vascular endothelial cell of damage may promote by increasing adhesion factor, the secretion of chemotactic factor
Inflammatory cell adheres to blood vessel endothelium tube wall in a large number and assembles, and accelerates inflammatory reaction, causes vascular endothelium dysfunction, from experiment
As a result we can see that model group ICAM-1, VCAM-1, the expression of E-selectin, MCP-1 is significantly raised.After giving SP-6
ICAM-1, VCAM-1, E-selectin, MCP-1 expression is significantly reduced, and adds Compound C, SP-6 after AMPK inhibitor
Reduce the event resolves of adhesion factor and chemotactic factor.
From the above, it is seen that the present invention sets up the HPAEC cell injury moulds of LPS inductions on the basis of previous experiments
Type, is screened SP-6 pharmaceutically actives, and the water of TNF-α, IL-1 β, IL-6 in each group cell supernatant is detected by ELISA method
It is flat, the content of ET-1, NO, iNOS, the expression of the level and MCP-1 of VCAM-1, ICAM-1, E-selectin in each group cell,
Impacts of the AMPK antagonist Compound C to SP-6 is detected simultaneously, studies protection mechanisms of the SP-6 to HPAEC cell injury, it is special
It is not people's lung lipopolysaccharide (lipopolysaccharide, LPS) induced by development test, Cortex Mori effective monomer SP-6
Protective effect that arterial endothelial cell (Human pulmonary artery endothelial cells, HPAEC) is damaged and
Mechanism of action.The HPAEC cell injury models of LPS inductions are set up, protective effects of the SP-6 to which is detected, while detecting that each group is thin
Tumor necrosis factor-alpha (tumor necrosis factor- α, TNF-α), interleukin-1 ' beta ' in born of the same parents' supernatant
The level of (interleukin-1 β, IL-1 β) and interleukin-6 (interleukin-6, IL-6), Endothelin (ET-1),
The content of nitric oxide (NO), nitricoxide synthase (iNOS), intercellular adhesive moleculer-1 (the vascular cell of each group cell
Adhesion molecule-1, VCAM-1), intercellular adhesion molecule-1 (intercellular adhesion molecule-
1, ICAM-1), the level and monocyte chemoattractant protein-1 (monocyte of E-Selectin (E-selectin)
Chemoattractant protein-1, MCP-1) expression, while detect shadows of the AMPK antagonist Compound C to SP-6
Ring, inquire into the protection mechanism that SP-6 is damaged to HPAEC.Test result indicate that, LPS can result in the damage (P of HPAEC cells<
0.01), compared with normal group, model group cell TNF-α, the level of IL-1 β, IL-6 significantly raise (P<0.05 or P<0.01),
The content of the level and iNOS of ET-1 raises (P<0.01), the content of NO reduces (P<0.01), adhesivemoleculeICAM1, VCAM-
1st, the level of E-selectin and chemotactic factor MCP-1 significantly raises (P<0.01).Compared with model group, SP-6 significantly can change
Cell injury situation (the P of kind HPAEC<0.01) TNF-α, the level (P of IL-1 β, IL-6 are reduced,<0.05 or P<0.01), reduce
Content (the P of the level and iNOS of ET-1<0.01), the content (P of elevation of NO<0.01) adhesivemoleculeICAM1, VCAM- are reduced,
1st, the level (P of E-selectin and chemotactic factor MCP-1<0.01).The HPAEC that SP-6 is induced to LPS is damaged and is made with protection
With, and its mechanism of action may be relevant with AMPK/NF- κ B paths.
In a word, SP-6 can significantly improve the HPAEC cell injury that LPS causes, reduce inflammatory factor TNF-α, IL-1 β,
The level of IL-6, elevation of NO content reduce ET-1, iNOS content, reduce ICAM-1, VCAM-1, E-selectin, MCP-1 table
Reach, and after adding AMPK inhibitor Compound C, SP-6 improves the event resolves of HPAEC damages, points out SP-6 to induce LPS
HPAEC cell injury there is protective effect, and its mechanism of action may be relevant with AMPK/NF- κ B.Effective for preparing treatment
The medicine that the HPAEC of LPS inductions is damaged, realizes that para hydroxybenzene propanoic acid (abbreviation SP-6) is damaged in the HPAEC for preparing treatment LPS inductions
Application in vulnerary thing, has opened up the medical value and new application of Cortex Mori, is on the HPAEC damage medicines for treat LPS inductions
Innovation, have good economic and social benefit.
Claims (3)
1. a kind of para hydroxybenzene propanoic acid extracted from Cortex Mori, is the effective active position extracted from Cortex Mori, molecule knot
Structure formula is：
2. the preparation method of the para hydroxybenzene propanoic acid extracted from Cortex Mori described in claim 1, it is characterised in that by Cortex Mori
Every time with the distillation water boiling and extraction 3 times of its weight 8-12 times, each 60min, united extraction liquid, concentrating under reduced pressure are obtained skin 2.4kg
Dried object, plus the distilled water of dried object bulking value 2-3 times is suspended, bulking value refers to solidss in terms of g, liquid in terms of mL,
6000r/min is centrifuged off insoluble matter, obtains supernatant；7.0L Diaion HP-20 macroporous adsorbent resin fillers are taken, it is dense with quality
95% ethanol immersion 24h dress posts are spent, is washed to without alcohol taste with distillation, supernatant concentration is to 50 DEG C of relative densities 1.2-1.4, upper tree
Fat post, successively with water, 30% ethanol elution of mass concentration, each gradient eluent consumption is 10 column volumes, and flow velocity is 2BVh-
1, each gradient eluent is concentrated to dryness, and obtains 30% ethanol elution component and crude extract, adds the dissolving of 50mL methanol, adds
The silica gel 40g of 160-200 mesh sieves is crossed, 45 DEG C of recovered under reduced pressure methanol obtain being loaded with the silica gel of sample；Took 200-300 mesh sieves
Silica gel 500g, adds dichloromethane 800mL, is transferred in the chromatographic column of 80cm length × 8cm internal diameters, enriches post with dichloromethane
Bed, liquid level is down at the 5cm of silica gel face, then the above-mentioned silica gel for being loaded with sample is added on the liquid level of chromatographic column, successively plus
Enter volume ratio for 100:3、100:5、100:The methylene chloride-methanol of 10 gradients, each gradient 4L carry out gradient elution, flow velocity
For 10mLmin-1, per 100mL as 1 stream part, the stream part collected is analyzed through TLC, merges the stream containing target component
Part, concentrating under reduced pressure obtains gross sample A1, A2, A3 containing target component, and the adsorbent used in described TLC analyses is
GF254, developing solvent are volume ratio 20:1-5:1 methylene chloride-methanol；Gross sample A1 30% methanol of mass concentration dissolves, on
Toyopearl HW-40C posts, with 30% methanol-eluted fractions of mass concentration, flow velocity is 1mLmin-1, it is a stream part per 2mL, it is thin
Layer inspection is known, and merges stream part containing identical, obtains subflow part A1-1, A1-2；Gross sample A2 35% methanol of mass concentration dissolves, on
Toyopearl HW-40 posts, with 35% methanol-eluted fractions of mass concentration, flow velocity is 1mLmin-1, it is a stream part per 2mL, thin layer
Inspection is known, and merges stream part containing identical, obtains subflow part A2-1, A2-2；Gross sample A3 50% methanol of mass concentration dissolves, on
Toyopearl HW-40 posts, with 50% methanol-eluted fractions of mass concentration, flow velocity is 1mLmin-1, it is a stream part per 2mL, thin layer
Inspection is known, and merges stream part containing identical, obtains subflow part A3-1；Gained 45 DEG C of concentrating under reduced pressure of each subflow part, obtain containing target
The sample of composition；Subflow part A1-2 31% methanol of mass concentration dissolves, upper ODS posts, with 31% methanol-eluted fractions of mass concentration, flows
Speed is 5mLmin-1, it is a stream part per 10mL, thin layer inspection is known, and merges stream part containing identical, and 45 DEG C of concentrating under reduced pressure obtain mesh
Mark composition white powder para hydroxybenzene propanoic acid (SP-6).
3. the hydroxy phenylpropionic acid that prepared by claim 2 methods described is preparing treatment LPS inductions as sole active position
Application in HPAEC damage medicines.
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| CN110934855A (en) * | 2020-01-31 | 2020-03-31 | 中山大学 | Application of p-hydroxyphenylpropionic acid in preparing medicine for preventing and treating respiratory tract infection |
| CN115252598A (en) * | 2022-06-13 | 2022-11-01 | 贵州大学 | Application of cyclomousin in preparation of anti-inflammatory drugs |
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