CN110496145A - A kind of preparation method and its usage of Sievers wormwood active component - Google Patents
A kind of preparation method and its usage of Sievers wormwood active component Download PDFInfo
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Abstract
The present invention relates to providing a kind of preparation method and its usage of Sievers wormwood active component, the active component be from Sievers wormwood (Artemisia sieversiana) middle using conventional soak extraction, heating and refluxing extraction, cold soaking extracts, after microwave radiation exaraction or ultrasonic wave assisted extraction, solvent is removed under reduced pressure and obtains low polar fraction, n-hexane extraction is used after water dissolution dispersion, raffinate part is dissolved in acetonitrile after being evaporated, after filtering, it is evaporated filtrate and obtains Sievers wormwood active component, the Sievers wormwood active component that the method obtains through the invention confirms that it can be used for preventing and treating by nitric oxide (NO) in vitro, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant factor 1(MCP-1) inflammation caused by excess, and the purposes in the anti-inflammatory drug of its formation.
Description
Technical field
The present invention relates to chemistry and biomedicine fields, and in particular to arrives Sievers wormwood purposes in preparing anti-inflammatory drugs.
Background technique
Inflammation is a kind of common pathologic process, and being body, (including physics, chemistry, biology etc. are more to various destructive stimulus
Aspect) caused by a kind of pathological reaction based on defence.Under normal conditions, inflammation is beneficial, is the automatic anti-of human body
Imperial reaction, but sometimes, inflammation be also it is harmful, modern medicine proves that inflammation takes part in pneumonia, asthma, rheumatoid joint
The occurrence and development of the diseases such as inflammation, Alzheimer's disease, atherosclerosis.In addition, the inflammation that privileged sites or organ are occurred
Serious consequence can be caused, if the inflammation of brain or brain can oppress vital center, vocal cords inflammation obstruction throat causes to suffocate, the serious heart
Myositis can influence cardiac function.Macrophage is broken up by monocyte, is a kind of important phagocyte, widely distributed
In different tissues.Macrophage is in opposing stationary state, hardly secrete cytokines isoreactivity under normal circumstances
Substance, but there is phagocytosis, chemotactic and proliferative capacity, it is the important component of nospecific immunity, composition body is important to be prevented
Line.After pathogen invades body, free macrophage enters the tissue invaded by pathogen under the guidance of chemotactic factor (CF).
The macrophage activated by pathogen, phagocytic activity, secretion capacity are improved, and then it is living to the killing of pathogen to improve it
Property.Once body is wound, macrophage can largely secrete a variety of Macrophage derived biotic factors, including transforming growth factor
(Transforming growth factor, TGF), interleukins (Interleukin, IL), tumor necrosis factor
(Tumor necrosis factor, TNF), the monocyte chemoattractant factor 1 (Monocyte chemoattractant protein-1,
MCP-1) and nitric oxide (Nitric oxide, NO) etc., these bioactive substances directly affect body and repair process.NO
Belong to intracellular messenger, plasma membrane is easily entered and left by disperse, is directly entered adjacent cell, plays a role locally.Many diseases occur
When, NO content increases, and promotes the exudation of blood plasma and the formation of oedema by increasing regional flow, by Drug inhibition LPS
The research for the excessive NO that the inflammatory cell model of induction generates, can disclose the anti-inflammatory activity of drug.In infective inflammation, by removing from office
The bacterial infection inflammation that endotoxic main component lipopolysaccharides (LPS) is induced in Lan Shi negative cell walls is most commonly seen
Inflammatory type, human immune system fight bacterial invasion when, LPS is caught as important antigen molecule by antigen presenting cell
It obtains, starts intracellular signal transfer chain, cause nuclear factor kB activation, a variety of proinflammatory cytokines are expressed and discharged in starting karyogene transcription
The factor (IL-6, TNF-α etc.), pro-inflammatory mediator (NO, prostaglandin E2 (PGE2) etc.) and transforming growth factor β.The generation of inflammation
All along with a large amount of releases of inflammatory factor, so the content of intracellular inflammatory factor directly reflects the level of inflammation of cell.
There are two main classes for inflammation treatment medicine used at present: steroidal anti-inflammatory medical instrument has powerful anti-inflammatory and immunosupress to make
With, but long-time service can cause many adverse reactions such as abnormal carbohydrate metabolism and osteoporosis, and clinical application is limited big.Non-steroidal is anti-
It is most extensive that scorching medicine (NSAIDs) treats use in many diseases associated with inflammation, but NSAIDs and stomach and intestine lesion is used for a long time, goes out
Blood, peptic ulcer and renal insufficiency are related.Now traditional anti-inflammatory drug has been difficult to satisfy social needs, from natural resources
It is middle to find efficient, low toxicity, the hot spot that more boots point anti-inflammatory drugs have become pharmacy and functional food is studied.In traditional medicine,
Sievers wormwood is a kind of Chinese medicine, is the wild herb in nature, and complete stool can be used as medicine, medical value with higher.
Sievers wormwood has effects that reduce swelling (washout), stop blooding, clearing away heat and removing summer；Control hot summer weather uncomfortable in chest, dysentery, knife wound etc.；External application decocting, which is washed, to be controlled
Scabies, rheumatism etc.；Highlands is also used to burn caused by treating solar ultraviolet radiation.Chinese medicine is then used as medicine with its bud, tool
There is anti-inflammatory analgetic；Carbuncle, swelling and furuncle can be treated by being decocted in water for oral dose, and affected part is washed in external application decocting can treat impetigo, skin eczema, palace
Neck is rotten to the corn.Up to the present, about the research of Sievers wormwood be only limitted to Wang Jinlan, Zhao Yanlei, Zhang Qi, Xu Fuchun etc. to Sievers wormwood into
Capable chemical composition analysis, and there is antiviral and antitumor activity from wherein isolated several compounds, but anti-to it
There is not been reported for scorching active research.Sievers wormwood (Artemisia sieversiana) active component of the present invention can press down
Cell pro-inflammatory cytokine IL-6 processed, IL-1 β, TNF-α, the monocyte chemoattractant factor 1 (MCP-1) and inflammatory mediator NO, and be in certain agent
Measure dependence, it is seen that it is with good anti-inflammatory activity.
Summary of the invention
Present invention aims at provide a kind of preparation method and its usage of Sievers wormwood active component, which is
It is middle using conventional soak extraction, heating and refluxing extraction, cold soaking extraction, microwave from Sievers wormwood (Artemisia sieversiana)
After assisted extraction or ultrasonic wave assisted extraction, solvent is removed under reduced pressure and obtains low polar fraction, uses n-hexane after water dissolution dispersion
Extraction, raffinate part is dissolved in acetonitrile after being evaporated, after filtering, be evaporated filtrate and obtain Sievers wormwood active component, through the invention institute
State method acquisition Sievers wormwood active component confirm in vitro its can be used for prevent and treat it is bad by nitric oxide (NO), tumour
Inflammation and its shape caused by necrosis factor-α (TNF-α), interleukin-6 (IL-6), the monocyte chemoattractant factor 1 (MCP-1) excess
At anti-inflammatory drug in purposes.
A kind of preparation method of Sievers wormwood active component of the present invention, follows these steps to carry out:
A, it by Sievers wormwood small polar organic solvent petroleum ether, methylene chloride, chloroform, acetone, ethyl acetate or ether, adopts
With directly impregnating, being heated to reflux solvent extraction or the extraction of ultrasonic wave secondary solvent, then vacuum distillation removes solvent, obtains big seed
The low polar fraction of wormwood artemisia；
Or Sievers wormwood is used into big polar solvent concentration 50%- anhydrous methanol or concentration 50%- dehydrated alcohol, temperature 20-
It extracted under the conditions of 82 DEG C using cold soaking, be heated to reflux solvent extraction or Microwave-assisted solvent extraction, extract is obtained, by extract
It is dissolved and is dispersed with water after concentration, then extracted with petroleum ether, methylene chloride or chloroform, obtain the low polar fraction of Sievers wormwood；
B, the low polar fraction of Sievers wormwood that step a is obtained is dissolved in water, then with n-hexane extraction, water position extracted is steamed
It after dry, is dissolved in acetonitrile, filters, be evaporated filtrate to get Sievers wormwood active component.
The Sievers wormwood active component that the method obtains is preparing the purposes in anti-inflammatory drug.
Detailed description of the invention
Sievers wormwood active component survives to lipopolysaccharides LPS stimulation RAW264.7 macrophage in Fig. 1 embodiment of the present invention 12
The influence schematic diagram of rate, wherein+indicate lipopolysaccharides ,-indicate no lipopolysaccharides；
Sievers wormwood active component generates NO inhibiting effect to LPS stimulation RAW264.7 cell in Fig. 2 embodiment of the present invention 13
Schematic diagram, wherein+indicate lipopolysaccharides ,-indicate no lipopolysaccharides；
Sievers wormwood active component secretes the influence of IL-6 to LPS stimulation RAW264.7 cell in Fig. 3 embodiment of the present invention 14
Schematic diagram, wherein+indicate lipopolysaccharides ,-indicate no lipopolysaccharides；
Sievers wormwood active component secretes the influence of IL-1 β to LPS stimulation RAW264.7 cell in Fig. 4 embodiment of the present invention 15
Schematic diagram, wherein+indicate lipopolysaccharides ,-indicate no lipopolysaccharides；
Influence of the Sievers wormwood active component to LPS stimulation RAW264.7 cell TNF secretion-α in Fig. 5 embodiment of the present invention 16
Schematic diagram, wherein+indicate lipopolysaccharides ,-indicate no lipopolysaccharides；
Sievers wormwood active component secretes the influence of MCP-1 to LPS stimulation RAW264.7 cell in Fig. 6 embodiment of the present invention 17
Schematic diagram, wherein+indicate lipopolysaccharides ,-indicate no lipopolysaccharides.
Specific embodiment
In order to further illustrate the present invention, detailed retouch is carried out to technical solution provided by the invention below with reference to embodiment
It states, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
A, dry Sievers wormwood herb 3.5Kg is taken, after crushing, with polar organic solvent petroleum ether soak at room temperature 7 days small, filtering
The dregs of a decoction are removed, filtrate utilizes Rotary Evaporators, removes petroleum ether and obtains the low polar fraction of Sievers wormwood；
B, the low polar fraction 4g of the dried Sievers wormwood obtained step a adds the dissolution dispersion of 40mL water, is extracted with n-hexane
3 times, each 40mL, the water position of n-hexane part and raffinate is evaporated respectively, the water position after being evaporated is dissolved with acetonitrile, filtering
Afterwards, filtrate is evaporated up to Sievers wormwood active component.
Embodiment 2
A, dry Sievers wormwood herb 100g is taken, is crushed, 2L tool plug flask is placed in, flask is set into ultrasonic wave extraction instrument, use is small
Polar organic solvent chloroform room temperature ultrasonic extraction 3 times, each 1.5L, each extraction time is 0.5h, and combined extract filters,
In being concentrated to dryness in Rotary Evaporators, the low polar fraction of Sievers wormwood is obtained；
B, the low polar fraction 4g of the dried Sievers wormwood obtained step a adds the dissolution dispersion of 40mL water, is extracted with n-hexane
3 times, each 40mL, the water position of n-hexane part and raffinate is evaporated respectively, the water position after being evaporated is dissolved with acetonitrile, filtering
Afterwards, filtrate is evaporated up to Sievers wormwood active component.
Embodiment 3
A, dry Sievers wormwood herb 100g is taken, is crushed, 2L tool plug flask is placed in, flask is set into ultrasonic wave extraction instrument, use is small
Polar organic solvent acetone room temperature ultrasonic extraction 3 times, each 1.5L, each extraction time is 0.5h, and combined extract filters,
Filtrate decompression is distilled to recover solvent, is collected simultaneously concentrate, obtains the low polar fraction of Sievers wormwood；
B, the low polar fraction 4g of the dried Sievers wormwood obtained step a adds the dissolution dispersion of 40mL water, is extracted with n-hexane
3 times, each 40mL, the water position of n-hexane part and raffinate is evaporated respectively, the water position after being evaporated is dissolved with acetonitrile, filtering
Afterwards, filtrate is evaporated up to Sievers wormwood active component.
Embodiment 4
A, dry Sievers wormwood herb 100g is taken, is crushed, is placed in 2L round-bottomed flask, flask is placed in heating device, use is small
Polar organic solvent ethyl acetate heating and refluxing extraction 3 times, temperature are 77 DEG C, each 1.5L, and each extraction time is 1h, are merged
Extracting solution, filtering, in being concentrated to dryness in Rotary Evaporators, obtains the low polar fraction of Sievers wormwood；
B, the low polar fraction 4g of the dried Sievers wormwood obtained step a adds the dissolution dispersion of 40mL water, is extracted with n-hexane
3 times, each 40mL, the water position of n-hexane part and raffinate is evaporated respectively, the water position after being evaporated is dissolved with acetonitrile, filtering
Afterwards, filtrate is evaporated up to Sievers wormwood active component.
Embodiment 5
A, dry Sievers wormwood herb 100g is taken, is crushed, 2L tool plug flask is placed in, flask is set into ultrasonic wave extraction instrument, use is small
Polar organic solvent ether room temperature ultrasonic extraction 3 times, each 1.5L, each extraction time is 0.5h, and combined extract filters,
Filtrate decompression is distilled to recover solvent, is collected simultaneously concentrate, obtains the low polar fraction of Sievers wormwood；
B, the low polar fraction 4g of the dried Sievers wormwood obtained step a adds the dissolution dispersion of 40mL water, is extracted with n-hexane
3 times, each 40mL, the water position of n-hexane part and raffinate is evaporated respectively, the water position after being evaporated is dissolved with acetonitrile, filtering
Afterwards, filtrate is evaporated up to Sievers wormwood active component.
Embodiment 6
A, dry Sievers wormwood herb 10Kg is taken, crushes, is placed in 50L extractor, with big 70% ethyl alcohol of polar solvent concentration, temperature
20 DEG C of degree, cold soaking extract 4 times, and each ethanol consumption is 30L, and each extraction time is 7 days, combined extract, in rotary evaporation
It is concentrated to dryness in instrument, aqueous solution 2L dissolution dispersion is added, and with chloroform extraction 3 times, each 2L, combining extraction liquid, Yu Xuan
Turn to be concentrated to dryness in evaporimeter, obtains the low polar fraction of Sievers wormwood；
B, the low polar fraction 4g of the dried Sievers wormwood obtained step a adds the dissolution dispersion of 40mL water, is extracted with n-hexane
3 times, each 40mL, the water position of n-hexane part and raffinate is evaporated respectively, the water position after being evaporated is dissolved with acetonitrile, filtering
Afterwards, filtrate is evaporated up to Sievers wormwood active component.
Embodiment 7
A, dry Sievers wormwood herb 100g is taken, is crushed, 1L tool plug flask is placed in, flask is set in micro-wave oven, with big polarity
95% ethyl alcohol microwave radiation exaraction of solvent strength 3 times, each 600mL, each extraction time are 20min, combined extract, mistake
Filter is dissolved with water after extracting solution is concentrated and is dispersed, and with petroleum ether extraction 3 times, each 1.5L；Combining extraction liquid is in rotary evaporation
It is concentrated to dryness in instrument, obtains the low polar fraction of Sievers wormwood；
B, the low polar fraction 4g of the dried Sievers wormwood obtained step a adds the dissolution dispersion of 40mL water, is extracted with n-hexane
3 times, each 40mL, the water position of n-hexane part and raffinate is evaporated respectively, the water position after being evaporated is dissolved with acetonitrile, filtering
Afterwards, filtrate is evaporated up to Sievers wormwood active component.
Embodiment 8
A, dry Sievers wormwood herb 100g is taken, is crushed, is placed in 1L round-bottomed flask, flask is set in heating device, with big pole
Property solvent absolute ethyl alcohol, 78 DEG C of temperature heating and refluxing extraction 3 times, each 1.5L, each extraction time be 1h, combined extract,
Filtering, concentration remove alcohol, are dissolved and are dispersed with water after being evaporated, and with petroleum ether extraction 3 times, each 1.5L；Combining extraction liquid, Yu Xuanzhuan
It is concentrated to dryness in evaporimeter, obtains the low polar fraction of Sievers wormwood；
B, the low polar fraction 4g of the dried Sievers wormwood obtained step a adds the dissolution dispersion of 40mL water, is extracted with n-hexane
3 times, each 40mL, the water position of n-hexane part and raffinate is evaporated respectively, the water position after being evaporated is dissolved with acetonitrile, filtering
Afterwards, filtrate is evaporated up to Sievers wormwood active component.
Embodiment 9
A, dry Sievers wormwood herb 100g is taken, is crushed, is placed in 1L round-bottomed flask, flask is set in heating device, with big pole
Property 50% ethanol water of solvent strength, 80 DEG C of temperature heating and refluxing extraction 3 times, each 1.5L, each extraction time be 1h, close
And extracting solution, filtering, concentration remove alcohol, are dissolved and are dispersed with water after being evaporated, and with methylene chloride extraction 3 times, each 1.5L；Merge extraction
It takes liquid in being concentrated to dryness in Rotary Evaporators, obtains the low polar fraction of Sievers wormwood；
B, the low polar fraction 4g of the dried Sievers wormwood obtained step a adds the dissolution dispersion of 40mL water, is extracted with n-hexane
3 times, each 40mL, the water position of n-hexane part and raffinate is evaporated respectively, the water position after being evaporated is dissolved with acetonitrile, filtering
Afterwards, filtrate is evaporated up to Sievers wormwood active component.
Embodiment 10
A, dry Sievers wormwood herb 100g is taken, is crushed, is placed in 1L round-bottomed flask, flask is set in heating device, with big pole
Property solvent anhydrous methanol, 65 DEG C of temperature heating and refluxing extraction 3 times, each 1.5L, each extraction time be 1h, combined extract,
Filtering, concentration remove alcohol, are dissolved and are dispersed with water after being evaporated, and with chloroform extraction 3 times, each 1.5L；Combining extraction liquid is steamed in rotation
It is concentrated to dryness in hair instrument, obtains the low polar fraction of Sievers wormwood；
B, the low polar fraction 4g of the dried Sievers wormwood obtained step a adds the dissolution dispersion of 40mL water, is extracted with n-hexane
3 times, each 40mL, the water position of n-hexane part and raffinate is evaporated respectively, the water position after being evaporated is dissolved with acetonitrile, filtering
Afterwards, filtrate is evaporated up to Sievers wormwood active component.
Embodiment 11
Dry Sievers wormwood herb 100g is taken, is crushed, 1L tool plug flask is placed in, flask is set in micro-wave oven, it is molten with big polarity
50% methanol of agent concentration, microwave radiation exaraction 3 times, each 600mL, each extraction time is 20min, and combined extract filters,
It is dissolved and is dispersed with water after extracting solution is concentrated, and with petroleum ether extraction 3 times, each 1.5L；Combining extraction liquid is in Rotary Evaporators
It is inside concentrated to dryness, obtains the low polar fraction of Sievers wormwood；
B, the low polar fraction 4g of the dried Sievers wormwood obtained step a adds the dissolution dispersion of 40mL water, is extracted with n-hexane
3 times, each 40mL, the water position of n-hexane part and raffinate is evaporated respectively, the water position after being evaporated is dissolved with acetonitrile, filtering
Afterwards, filtrate is evaporated up to Sievers wormwood active component.
Embodiment 12
Any one Sievers wormwood active component that embodiment 1-11 is obtained is as the bioactivity of the purposes of anti-inflammatory drug
Screening:
MTT colorimetric method for determining cell viability: logarithmic growth phase RAW264.7 cell inoculation is in 96 well culture plates, cell
Density is 2 × 105A/L, every 100 μ L of hole, at 37 DEG C of temperature, concentration 5%CO2Under the conditions of after overnight incubation, positive control is added
The Sievers wormwood active component solution of andrographis paniculata injection 25 μ g/mL and various concentration 6.25-100 μ g/mL, bacteria lipopolysaccharide
LPS (1 μ g/mL) induces inflammatory reaction to co-culture 16h, investigates the influence after drug and LPS are added to cell, and cell culture is overnight
Afterwards, it inhales and abandons liquid in hole, every hole addition 100 μ L of 0.5mg/mL MTT, 37 DEG C of temperature, concentration 5%CO2Continue to incubate in incubator
After educating 4h, culture is terminated, inhales and abandons liquid in hole, 150 μ L dimethyl sulfoxides (DMSO) are added in every hole, vibrate 10min, make intracellular
Crystallization is sufficiently dissolved, and each hole light absorption value of measurement is shown in Fig. 1 at microplate reader 490nm wavelength.
Detection indicate that: Sievers wormwood active component maximum concentration is right when 25 μ g/mL and LPS act on cell altogether
RAW264.7 macrophage proliferation unrestraint effect, it is safe and non-toxic.
Embodiment 13
Any one Sievers wormwood active component that embodiment 1-11 is obtained is as the bioactivity of the purposes of anti-inflammatory drug
Screening:
This (Griess) method of Gree measures nitric oxide (NO) content: investigating Sievers wormwood active component to lipopolysaccharide-induced
The inhibiting effect of the inflammatory reaction of mouse macrophage RAW264.7；Containing 10% fetal calf serum after mouse macrophage passage
(FBS) in high glucose medium DMEM (Dulbecco ' s minimum essential medium, DMEM, Hyclone company)
Culture, adds the Sievers wormwood active component of positive control and various concentration 6.25-25 μ g/mL, bacteria lipopolysaccharide LPS (1 μ g/mL)
After inducing inflammatory reaction to co-culture 16h, supernatant is collected, Griess method measures nitric oxide production content in cell supernatant, root
According to the influence for RAW264.7 cell release nitric oxide (NO) that active component various concentration induces bacteria lipopolysaccharide, to anti-
It reflects nitric oxide (NO) level and sees Fig. 2.
Detection indicate that: the mouse macrophage RAW264.7 that Sievers wormwood active component can significantly inhibit LPS induction is generated
Excessive inflammatory mediator NO, and its effect is in concentration dependent.
Embodiment 14
Any one Sievers wormwood active component that embodiment 1-11 is obtained is as the bioactivity of the purposes of anti-inflammatory drug
Screening:
ELISA method measures interleukin-6 (Interleukin-6, IL-6): by the RAW264.7 macrophage of logarithmic growth phase
For cell inoculation in 96 well culture plates, cell density is 2 × 105A/mL, every 100 μ l of hole, 37 DEG C of temperature, concentration 5%CO2Condition
Lower culture adds the Sievers wormwood active component of positive control and various concentration 6.25-25 μ g/mL, bacteria lipopolysaccharide LPS (1 μ g/
ML) induction inflammatory reaction is incubated for 16h altogether, and every group of processing repeats 3 holes, and ELISA method measures Sievers wormwood active component, and treated
The content of the IL-6 cell factor of RAW264.7 cell secretion is shown in Fig. 3.
Embodiment 15
Any one Sievers wormwood active component that embodiment 1-11 is obtained is as the bioactivity of the purposes of anti-inflammatory drug
Screening:
ELISA method measures interleukin-1 beta (Interleukin-1 β, IL-1 β): by the RAW264.7 of logarithmic growth phase
Macrophage is inoculated in 96 well culture plates, and cell density is 2 × 105A/mL, every 100 μ L of hole, 37 DEG C of temperature, concentration 5%CO2
Under the conditions of cultivate, add the Sievers wormwood active component of positive control and various concentration 6.25-25 μ g/mL, bacteria lipopolysaccharide LPS (1
μ g/mL) induce inflammatory reaction to be incubated for 16h altogether, every group of processing repeats 3 holes, and ELISA method measures Sievers wormwood active component, and treated
The content of the IL-1 β cell factor of RAW264.7 cell secretion is shown in Fig. 4.
Embodiment 16
Any one Sievers wormwood active component that embodiment 1-11 is obtained is as the bioactivity of the purposes of anti-inflammatory drug
Screening:
ELISA method measures tumor necrosis factor α (Tumor necrosis factor α, TNF-α): by logarithmic growth phase
RAW264.7 macrophage is inoculated in 96 well culture plates, and cell density is 2 × 105A/mL, every 100 μ L of hole, 37 DEG C of temperature,
Concentration 5%CO2Under the conditions of cultivate, add the Sievers wormwood active component of positive control and various concentration 6.25-25 μ g/mL, bacterium
Lipopolysaccharides LPS (1 μ g/mL) induction inflammatory reaction is incubated for 16h altogether, and every group of processing repeats 3 holes, and it is effective that ELISA method measures Sievers wormwood
The content of the TNF-α cell factor for RAW264.7 cell secretion that treated at position is shown in Fig. 5.
Embodiment 17
Any one Sievers wormwood active component that embodiment 1-11 is obtained is as the bioactivity of the purposes of anti-inflammatory drug
Screening:
ELISA method measures the monocyte chemoattractant factor 1 (Monocyte chemoattractant protein-1, MCP-1): will
The RAW264.7 macrophage of logarithmic growth phase is inoculated in 96 well culture plates, and cell density is 2 × 105A/mL, every 100 μ of hole
L, 37 DEG C of temperature, 5%CO2Under the conditions of cultivate, add the effective portion of Sievers wormwood of positive control and various concentration 6.25-25 μ g/mL
Position, bacteria lipopolysaccharide LPS (1 μ g/mL) induction inflammatory reaction are incubated for 16h altogether, and every group of processing repeats 3 holes.ELISA method measures big seed
The content of the MCP-1 cell factor of wormwood artemisia active component treated RAW264.7 cell secretion is shown in Fig. 6；
The testing result of embodiment 14-17 shows: the Sievers wormwood active component that the method obtains through the invention can be shown
Write four kinds of inflammatory cytokines for inhibiting the mouse macrophage RAW264.7 of LPS induction to be overexpressed: tumor necrosis factor-alpha
(TNF-α), interleukin-6 (IL-6), Interleukin -1β (IL-1 β) and the monocyte chemoattractant factor 1 (MCP-1).
As a result:
The Sievers wormwood active component that the method obtains through the invention can significantly inhibit the mouse macrophage of LPS induction
RAW264.7 generates excessive inflammatory mediator NO, while the inflammation of the overexpression for the RAW264.7 cell secretion for inhibiting LPS to induce
Cell factor: tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), Interleukin -1β (IL-1 β) and the monocyte chemoattractant factor 1
(MCP-1).Result above proves that Sievers wormwood active component has good anti-inflammatory activity, can be used as raw material or adjuvant and is preparing
Prevent in anti-inflammatory drug or treats by inflammation caused by NO, TNF-α, IL-6, IL-1 β and MCP-1 excess.
Claims (2)
1. a kind of preparation method of Sievers wormwood active component, it is characterised in that follow these steps to carry out:
A, by Sievers wormwood small polar organic solvent petroleum ether, methylene chloride, chloroform, acetone, ethyl acetate or ether, using straight
It connects immersion, be heated to reflux solvent extraction or the extraction of ultrasonic wave secondary solvent, then vacuum distillation removes solvent, and it is low to obtain Sievers wormwood
Polar fraction；
Or Sievers wormwood is used into big polar solvent concentration 50%- anhydrous methanol or concentration 50%- dehydrated alcohol, 20-82 DEG C of item of temperature
It extracted under part using cold soaking, be heated to reflux solvent extraction or Microwave-assisted solvent extraction, extract is obtained, after extract is concentrated
It is dissolved and is dispersed with water, then extracted with petroleum ether, methylene chloride or chloroform, obtain the low polar fraction of Sievers wormwood；
B, the low polar fraction of Sievers wormwood that step a is obtained is dissolved in water, then with n-hexane extraction, water position extracted is evaporated
Afterwards, it is dissolved in acetonitrile, filters, be evaporated filtrate to get Sievers wormwood active component.
2. the Sievers wormwood active component obtained according to the method for claim 1 is preparing the purposes in anti-inflammatory drug.
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