CN104688828B - The application of the total glycosides induction antiviral cell factor of purplestem privet leaf - Google Patents
The application of the total glycosides induction antiviral cell factor of purplestem privet leaf Download PDFInfo
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Abstract
The invention discloses the application of the total glycosides induction antiviral cell factor of the new opplication of the total glycosides of purplestem privet leaf, particularly purplestem privet leaf, the total glycosides of the purplestem privet leaf are isolated with chromatographic column after being extracted by ethanol.The invention also discloses a kind of total glycosides formulation of purplestem privet leaf, the preparation contains the total glycosides of purplestem privet leaf, and pharmaceutical carrier, auxiliary material or diluent.The present invention proves that the total glycosides of purplestem privet leaf can induce body in vivo and produce endogenous antiviral cell factor by a series of experiments, also can produce antiviral cell factor by inducing cell in vitro.
Description
Technical field
The present invention relates to the total glycosides of purplestem privet leaf, is specifically the application that the total glycosides of purplestem privet leaf induces antiviral cell factor.
Background technology
Purplestem privet leaf (Ligustrum purpurascensY.C Yang) is Ligustrum, is mainly distributed on
The ground such as Yunnan in Southwestern China area, Guizhou, Sichuan, are the medicinal plants of folk tradition, are used as medicine more with the tender leaf or leaf of plant,
Nature and flavor bitter sweet, has effects that wind and heat dispersing, improving eyesight promote the production of body fluid；Cure mainly headache due to pathogenic wind-heat, dentalgia, hot eyes, Ting ear, aphtha, pyreticosis polydipsia,
The diseases such as dysentery, in China's medicinal history more than 2,000 years existing among the people.
Purplestem privet leaf mainly glycosides compound containing Phenylpropanoid Glycosides, flavone compound, terpenoid etc..Wherein Phenylpropanoid Glycosides class
Compound includes：Purplestem privet leaf glycosides (ligupurpuroside) A, B, M, N, Verbascoside (acteoside), cis purplestem privet leaf
Glycosides (cis-ligupurpuroside) B, acteoside isomer (isoacteoside), echinacoside (echinacoside), Echinacea purpurea
Glycosides (echinacoside) A and bignoniad glycosides (campenoside) II；Flavone compound includes Quercetin (quercentin),
Hyperoside (hyperoside), luteolin-7-glucoside (leuteolin-7-glucoside), cosmosiin
(cosmosiin), Rhoifolin (rhoifolin), Kaempferol (kaempferol), Isorhamnetin (isorhamnetin), cigarette
Flower glycosides (nicotiflorin) and apiolin (apigenin) etc..
Antiviral cell factor is secreted by body cell, has linkage body inherent immunity and specific immunity should
Answer, and then suppress or catch and kill the small molecular protein of internal virus.Antiviral cell factor can be divided to two major classes：One kind can be acted on directly
Body cell produces antiviral protein, i.e. (1) interferon：IFN-α, IFN-β, IFN-γ and IFN- λ (IL-28, IL-29), (2) are swollen
Tumor necrosis factor：TNF-α and TNF-beta；Interferon and tumor necrosis factor can JAK (Janus directly in active cell
Activated kinase)-STAT (Signal transducer and activator of transcription) signal
Path, then causes downstream signal cascade reaction, including MTOR signal paths, PI3K signal paths, MAPK signal paths quilt
Activate, some crucial protein gene are transcribed in final active cell core, translate antiviral protein and then to bacterium, disease
Poison and cancer cell etc. produce defense reaction.Another kind of is energy activating macrophage, natural killer cells and toxic T lymphocyte
Deng cell, and then catch and kill or the cell factor of phagosome inner virus, i.e. interleukin：IL-1β、IL-2、IL-6、IL-8、IL-12、
IL-15, IL-17, IL-18, IL-21, IL-27 etc..
Shown according to current data, not yet retrieve the application in relation to the total glycosides induction antiviral cell factor of purplestem privet leaf
Report.
The content of the invention
It is an object of the invention to propose the new opplication of the total glycosides of purplestem privet leaf, the particularly total glycosides induction of purplestem privet leaf is antiviral
The application of cell factor.
Further, the total glycosides of the purplestem privet leaf is extracted and obtained in accordance with the following methods：Take Ligustrum purple stem
The cured leaf of glossy privet, adds ethanol, and heating and refluxing extraction, gained ethanol extract is dissolved with water, hydrotrope Diaion or big
Macroporous adsorbent resin column chromatography for separation, is eluted with water-ethanol or water-methanol mixture, and concentrate eluant obtains the total glycosides of purplestem privet leaf.
Further, the amount for adding ethanol is 8~10 times of purplestem privet leaf cured leaf weight.
Further, the number of the heating and refluxing extraction is three times.
Further, the temperature of the heating and refluxing extraction is 60~100 DEG C.
Further, the volume ratio of the water-ethanol or water-methanol mixture is 0.5~1.5：1.
Further, the volume ratio of the water-ethanol or water-methanol mixture is 1：1.
Further, the total glycosides of the purplestem privet leaf is pale yellow powder, savory, soluble easily in water, its aqueous solution is yellowish
Color or brown color.
Further, the total glycosides of the purplestem privet leaf has seven spots on the silica gel plate of thin-layer chromatography, and Rf value Rf is successively
For 0.035,0.129,0.259,0.483,0.603,0.638,0.922.
Further, the antiviral cell factor is IFN-α, IFN-β and IFN-γ.
Further, the antiviral cell factor is lymphocyte and/or macrophages secrete.
A kind of total glycosides formulation of purplestem privet leaf, the preparation contain the total glycosides of purplestem privet leaf of any of the above-described claim, with
And pharmaceutical carrier, auxiliary material or diluent, those skilled in the art can know these pharmaceutically by this area professional book
Acceptable pharmaceutical carrier, auxiliary material or diluent.
The total glycosides formulation of purplestem privet leaf of the present invention can be capsule, tablet, pill, granule, injection, dripping pill, powder pin
One kind in agent and oral solutions.
The present invention is experimentally confirmed the total glycosides of purplestem privet leaf can inducing mouse lymphocyte and macrophages secrete in vitro
Cell factor：Isolated mouse lymphocyte and macrophage, add the total glycosides of purplestem privet leaf of various concentrations, pass through ELISA
The level of antiviral cell factor (IFN-α, IFN-β and IFN-γ) in experiment detection cell culture supernatant, it was demonstrated that purple stem female
Loyal total glycosides has facilitation to lymphocyte and macrophages secrete IFN-α, IFN-β and IFN-γ.When the total glycosides of purplestem privet leaf is dense
When degree is 50 μ g/ml, the amount of the total glycosides induction of lymphocyte secretion IFN-α of purplestem privet leaf and IFN-β reaches highest, works as purplestem privet leaf
When total glycosides concentration is 25 μ g/ml, the total glycosides induction of lymphocyte secretion of gamma-IFN of purplestem privet leaf reaches highest, when the total glycosides of purplestem privet leaf
When concentration is 50~100 μ g/ml, the amount of the total glycosides inducing macrophage secretion IFN-α of purplestem privet leaf and IFN-β reaches highest, when
When the total glycosides concentration of purplestem privet leaf is 50 μ g/ml, the total glycosides inducing macrophage secretion of gamma-IFN of purplestem privet leaf reaches highest.
The present invention, which is experimentally confirmed the total glycosides of purplestem privet leaf, can induce hypoimmunity mice secrete cytokines in vivo：Will
Mice group, with endoxan modeling, gavage gives the total glycosides of purplestem privet leaf of various dose, passes through ELISA measurings periphery
IFN-β in blood, IFN-γ content, it was demonstrated that the total glycosides of purplestem privet leaf is low to IFN-β in serum caused by endoxan and IFN-γ
With certain restitution, wherein, the effect of IFN-β and IFN-γ level is most obvious in middle dose group up-regulation serum.
The beneficial effects of the invention are as follows：
1. the present invention extracts total glycosides from purplestem privet leaf, and it is characterized using means such as HPLC, TLC.
2. the method that extraction of the present invention prepares the total glycosides of purplestem privet leaf is fast and effective, securely and reliably, and raw material sources are extensive,
It is adapted to large-scale production.
3. the present invention is experiments prove that the total glycosides of purplestem privet leaf can induce body in vivo, to produce endogenous antiviral thin
Intracellular cytokine, also can produce antiviral cell factor by inducing cell in vitro, can develop into treatment and antiviral cell factor phase
The preparation of the disease of pass, its preparation be it is any through intestines and stomach or non-through gastrointestinal administration formulation, including capsule, tablet, pill,
Granule, injection, dripping pill, powder-injection and oral solutions etc..
Brief description of the drawings
Fig. 1 is the HPLC chromatogram of the total glycosides of 1 purplestem privet leaf of embodiment；
Fig. 2 is the thin-layer chromatogram of the total glycosides of 1 purplestem privet leaf of embodiment；
Fig. 3 is that the total glycosides induction of lymphocyte of purplestem privet leaf of 2 various concentrations of embodiment produces the figure of IFN-α；
Fig. 4 is that the total glycosides induction of lymphocyte of purplestem privet leaf of 2 various concentrations of embodiment produces the figure of IFN-β；
Fig. 5 is the figure that the total glycosides of purplestem privet leaf of 2 various concentrations of embodiment and ConA induction of lymphocyte produce IFN-γ；
Fig. 6 is that the total glycosides inducing macrophage of purplestem privet leaf of 2 various concentrations of embodiment produces the figure of IFN-α；
Fig. 7 is that the total glycosides inducing macrophage of purplestem privet leaf of 2 various concentrations of embodiment produces the figure of IFN-β；
Fig. 8 is the figure that the total glycosides of purplestem privet leaf of 2 various concentrations of embodiment and ConA inducing macrophages produce IFN-γ；
Fig. 9 is that the total glycosides Immune inducing in vivo hypoimmunity mice of 3 purplestem privet leaf of embodiment secretes IFN-β, the figure of IFN-γ.
Embodiment
Reagent, the material used in the present invention is that well known to a person skilled in the art can be bought by commercial undertaking and obtained
.Method used in the present invention, such as HPLC, ELISA are method well known in the art, can pass through textbook or related text
The description offered carries out, and repeats no more, is described in description of the invention, is carried out with reference to method described in the present invention.
Embodiment 1
The extraction of the total glycosides of purplestem privet leaf
1.92kg purplestem privet leaf cured leafs are taken, 19.2kg ethanol is added, 60 DEG C of refluxing extractions is heated to three times, by gained ethanol
Extract is dissolved with water, hydrotrope Diaion column chromatography for separation, using volume ratio as 1：1 water-ethanol mixed liquor elution, concentration
Eluent obtains the total glycosides of 230.4g purplestem privet leafs.
The total glycosides of gained purplestem privet leaf is pale yellow powder, savory, and soluble easily in water, aqueous solution is light brown yellow.
The total glycosides of gained purplestem privet leaf is detected with HPLC, chromatographic condition is：Mobile phase is the Shui Hejia containing 0.1% formic acid
Alcohol, water：Methanol=90：10~10：90 gradient elutions, time 60min, flow velocity 1ml/min, 10 μ l of sampling volume, detect ripple
Long 227nm, in 7min, 20min, 31min, 38min, 44min as shown in Figure 1, there is main peak in chromatogram.
The total glycosides of gained purplestem privet leaf is detected with thin-layered chromatography, solvent is CMW (chloroforms：Methanol：Water=7：3：1) system
System upper strata, is developed the color, the results are shown in Figure 2, there is seven spots on silica gel plate, and Rf value Rf is followed successively by with iodine colour developing and sulfuric acid respectively
0.035,0.129,0.259,0.483,0.603,0.638,0.922.
Embodiment 2
The experiment of the external evoked mouse lymphocyte of the total glycosides of purplestem privet leaf (LPGs) and Factor of Macrophage
1. the preparation and purification of lymphocyte
(1) take off neck and put to death mouse, then mouse is put into 75% alcohol and soaks 5min, make its skin as much as possible
Alcohol exposure；
(2) mouse disinfected is put into superclean bench, cuts off left belly, take out spleen, be put into the serum-free of precooling
In RPMI-1640 nutrient solutions (3~5ml), fat, connective tissue are rejected, then washed with the culture medium of precooling serum-free；
(3) clean mouse spleen is shredded, is ground with glass syringe bolt, 200 mesh nylon wires are crossed, with precooling without blood
Clear culture medium rinses nylon wire；
(4) upper strata lymphocyte suspension is drawn in test tube, 4~5ml mouse lymphocyte separating liquids is added, with 800rcf
(relative centrifugal force) centrifuges 30min；
(5) test tube is taken out, reject supernatant, adds 1640 culture mediums, overturns for several times, and 10min is centrifuged with 250rcf；
(6) the suitable RPMI-1640 nutrient solutions for containing 10% hyclone of cell precipitation are resuspended, it is thin obtains single lymph
Born of the same parents' suspension；
(7) cell count：The 100 μ l of lymphocyte suspension in test tube are drawn, platform expects blue dyeing counting, ensures viable count
More than 95%, and cell concentration is adjusted as 1.5 × 106A/ml.
2. the separation of macrophage
4~5ml RPMI-1640 culture mediums are injected to mouse peritoneal, soft mouse web portion 1min stands 5min, and cervical vertebra takes off
Mortar is put to death, and is soaked about 5min in 75% alcohol, is collected peritoneal fluid, is centrifuged 5min with 1500r/min, supernatant is abandoned, with 10% tire
1640 culture mediums of cow's serum adjust cell concentration to 1 × 106A/ml, is seeded to 24 well culture plates, in 5%CO2, 37 DEG C of cultures
4h is incubated in case, abandons supernatant, then non-adherent cell is washed away with 1640 culture mediums, up to macrophage.
3. cell culture
(1) reagent group is handled：The total glycosides of purplestem privet leaf is configured to 1650 μ g/ml, 1100 μ g/ml, 550 μ g/ml, 275 μ g/
Ml, 110 μ g/ml, 55 μ g/ml, 11 μ g/ml series concentration solution, are divided into two groups of A, B, every group of 21 holes are accurate to inhale by culture plate
The total 100 μ l of glycosides solution of purplestem privet leaf of various concentrations are taken to add in the hole of culture plate, each concentration repeats to add three holes, A groups
1ml lymphocytes suspension (1.5 × 10 is added per hole6A/ml), B groups add 1ml macrophages (1 × 10 per hole6A/ml), make
For the medicine group of various concentrations, medicine final concentration be respectively 150 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 10 μ g/ml,
5 μ g/ml, 1 μ g/ml, are 1100 μ l per hole cumulative volume；
(2) positive controls are handled：By canavalin (ConA) be configured to 1650 μ g/ml, 1100 μ g/ml, 550 μ g/ml,
275 μ g/ml, 110 μ g/ml, 55 μ g/ml, 11 μ g/ml series concentration solution, are divided into two groups of C, D by culture plate, every group of 21 holes,
The accurate 100 μ l of ConA solution for drawing various concentrations are added in the hole of culture plate, and each concentration repeats to add three holes, and C groups are every
Hole adds 1ml lymphocytes suspension (1.5 × 106A/ml), D groups add 1ml macrophages (1 × 10 per hole6A/ml), as
Positive controls, ConA final concentrations are respectively 150 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 10 μ g/ml, 5 μ g/ml, 1
μ g/ml, are 1100 μ l per hole cumulative volume；
(3) blank control group is handled：Add the sky of 100 μ l serum free mediums and 1ml lymphocytes suspension as A, C group
White control, adds the blank control of 100 μ l serum free mediums and 1ml macrophages as B, D group.
4. antiviral cell factor (IFN-α, IFN-β and IFN- in detection cell culture supernatant are tested by ELISA
Level γ), the result is shown in Fig. 3~8：
Fig. 3 is that the total glycosides induction of lymphocyte of purplestem privet leaf of various concentrations produces the figure of IFN-α；Fig. 4 is various concentrations
The total glycosides induction of lymphocyte of purplestem privet leaf produces the figure of IFN-β；Fig. 5 is the total glycosides of purplestem privet leaf and the ConA induction leaching of various concentrations
Bar cell produces the figure of IFN-γ；Fig. 6 is that the total glycosides inducing macrophage of purplestem privet leaf of various concentrations produces the figure of IFN-α；Fig. 7
It is that the total glycosides inducing macrophage of purplestem privet leaf of various concentrations produces the figure of IFN-β；Fig. 8 is the total glycosides of purplestem privet leaf of various concentrations
The figure of IFN-γ is produced with ConA inducing macrophages.
From the experimental result of Fig. 3~8, in the range of 1~150 μ g/ml, the total glycosides of purplestem privet leaf is to lymphocyte and huge
Phagocyte secretes IFN-α, and IFN-β and IFN-γ have facilitation.When the total glycosides concentration of purplestem privet leaf is 50 μ g/ml, purple stem female
The amount of loyal total glycosides induction of lymphocyte secretion IFN-α and IFN-β reaches highest, and IFN-α secretory volume is about 2 times of blank control group,
IFN-β secretory volume is about 3 times of blank control group, when the total glycosides concentration of purplestem privet leaf is 25 μ g/ml, the total glycosides induction leaching of purplestem privet leaf
Bar cell secretion of gamma-IFN reaches highest, and IFN-γ secretion amount is suitable with ConA.When the total glycosides concentration of purplestem privet leaf is 50~100 μ
During g/ml, the amount of the total glycosides inducing macrophage secretion IFN-α of purplestem privet leaf and IFN-β reaches highest, and IFN-α secretory volume is blank
About 2 times of control group, IFN-β secretory volume are about 3 times of blank control groups, when the total glycosides concentration of purplestem privet leaf is 50 μ g/ml, purple stem female
Loyal total glycosides inducing macrophage secretion of gamma-IFN reaches highest, and IFN-γ secretion amount is suitable with ConA.It can be seen that from this experiment
The total glycosides of purplestem privet leaf can induce macrophage and lymphocytic emiocytosis antiviral cell factor IFN-α, IFN-β and IFN-γ.
Embodiment 3
The total glycosides of purplestem privet leaf (LPGs) Immune inducing in vivo hypoimmunity mice secrete cytokines are tested
1. kunming mice is grouped at random, every group 12, it is respectively：Blank control group：Physiological saline 10ml/kg；Model
Group：Physiological saline 10ml/kg；Levamisole hydrochloride group：50mg/kg；The total glycosides high dose group of purplestem privet leaf：800mg/kg；Purple stem
The total glycosides middle dose group of glossy privet：400mg/kg；The total glycosides low dose group of purplestem privet leaf：200mg/kg.To each group mouse continuous gavage 10
My god, it is injected intraperitoneally sterile saline 10ml/kg the next day administration starts on the 1st day, blank control group, abdominal cavity is noted the next day remaining each group
Endoxan 80mg/kg is penetrated, totally 5 times.
2. by IFN-β in ELISA measuring peripheral bloods, IFN-γ content, the results are shown in Figure 9.
As shown in Figure 5, endoxan can trigger IFN-β and IFN-γ in serum to lower, and the total glycosides of purplestem privet leaf is to ring phosphinylidyne
IFN-β and IFN-γ lowly have certain restitution in serum caused by amine, wherein, IFN- in middle dose group up-regulation serum
The effect of β and IFN-γ level is most obvious, it was demonstrated that in animal model, the total glycosides of purplestem privet leaf can induce hypoimmunity mice point
Secrete antiviral cell factor IFN-β and IFN-γ.
Embodiment 4
The preparation of capsule
The total glycosides of 20g purplestem privet leafs, 40g edible celluloses and appropriate capsule auxiliary material are taken, is uniformly mixed, fills capsule, glue
Capsule is 0# capsules, and filling specification is 0.45g/.
Embodiment 5
The preparation of tablet
The total glycosides of 30g purplestem privet leafs, 50g microcrystalline celluloses, 10g lactose, 1g magnesium stearates and appropriate additive of tablet are taken, is mixed
Close uniformly, tablet is made by known tablet manufacturing technology and equipment, product specification is 0.5g/ pieces.
Embodiment 6
The preparation of pill
The total glycosides of purplestem privet leaf and microcrystalline cellulose are crossed into 100 mesh sieves respectively, and it is fine to weigh the total glycosides 30g of purplestem privet leaf, crystallite
Element 50g and appropriate pill auxiliary material are tieed up, is uniformly mixed, a certain proportion of refining honey is added into the material mixed, by public affairs
Pill is made in the pill manufacturing technology and equipment known, product specification is 0.25g/.
Embodiment 7
The preparation of granule
The total glycosides 40g of purplestem privet leaf, microcrystalline cellulose 70g and appropriate granule auxiliary material is taken to be uniformly mixed, it is dry using spraying
Drying process is pelletized.
Embodiment 8
The preparation of injection
Take the total glycosides of 50g stem glossy privets and optimum amount Injection preparation auxiliary material to add water for injection that 1000ml solution is made, filter to clear and bright, filling
Envelope, high-temperature short-time sterilization to obtain the final product.The solution of injection is prepared, the workshop of filtering, embedding all more than 100,000 grades is completed.
Embodiment 9
The preparation of oral liquid formulations
The total glycosides 10g of purplestem privet leaf, honey 8g and appropriate oral solutions auxiliary material are weighed, is configured to 100ml's with purified water
Solution, filters, is filling, high-temperature short-time sterilization to obtain the final product.
Embodiment 10
The preparation of dripping pill
Add atoleine that by syringe on syringe needle, water is added in water bath in condenser pipe and in dripping pill receiving bottle
To suitable height, heating, maintains temperature 70 C~80 DEG C, allows syringe needle alignment liquid paraffin pipe, and PEG is weighed by prescription
6000 in evaporating dish, and fusing is heated in water-bath, adds the total glycosides 1g of purplestem privet leaf, is stirred to dissolve, is fully transferred to rapidly
In syringe, syringe piston is slowly promoted, or is instilled by gravity in liquid paraffin condensate liquid, after liquid drips off, is placed
1.5~2 it is small when, cooler is removed, atoleine is poured out and remains to recycle, takes out dripping pill, drip is net, and liquid on ball is put on the skin with filter paper
Paraffin, places and spontaneously dries, counting of weighing, and calculates recovery rate, average ball weight and pill weight variation.
Embodiment 11
The preparation of powder-injection
Take the total glycosides of 50g stem glossy privets and appropriate powder-injection auxiliary material to add water for injection that 1000ml solution is made, be sterile filtered,
Packing, lyophilized, Feng Pin, finally carry out leak test to obtain the final product.
Embodiment 12
The extraction of the total glycosides of purplestem privet leaf
Purplestem privet leaf cured leaf is taken, the ethanol of 8 times of weight of cured leaf is added, 100 DEG C of refluxing extractions is heated to three times, by gained second
Alcohol extracting thing is dissolved with water, and the hydrotrope is separated with macroporous adsorbent resin column chromatography, using volume ratio as 0.5：1 water-methanol mixing
Liquid elutes, and concentrate eluant obtains the total glycosides of purplestem privet leaf.
The above description is merely a specific embodiment, but protection scope of the present invention is not limited thereto, any
Belong to those skilled in the art the invention discloses technical scope in, the change or replacement that can readily occur in, all should
It is included within the scope of the present invention.Therefore, protection scope of the present invention should be subject to scope of the claims.
Claims (5)
1. the total glycosides of purplestem privet leaf is preparing the application in inducing antiviral cell factor medicine, wherein the total glycosides of the purplestem privet leaf is pressed
Extract and obtain according to following methods：The cured leaf of Ligustrum purplestem privet leaf is taken, adds ethanol, heating and refluxing extraction will
Gained ethanol extract is dissolved with water, and the hydrotrope is with Diaion or macroporous adsorbent resin column chromatography from water-ethanol or water-first
Alcohol mixed liquor elutes, and concentrate eluant obtains the total glycosides of purplestem privet leaf, and the amount for adding ethanol is the 8 of purplestem privet leaf cured leaf weight
~10 times, the volume ratio of the water-ethanol or water-methanol mixture is 0.5~1.5：1.
2. application according to claim 1, it is characterised in that the number of the heating and refluxing extraction is three times.
3. application according to claim 1, it is characterised in that the total glycosides of purplestem privet leaf is pale yellow powder, savory,
Soluble easily in water, its aqueous solution is faint yellow or brown color.
4. application according to claim 1, it is characterised in that the antiviral cell factor for IFN-α, IFN-β and
IFN-γ。
5. application according to claim 1, it is characterised in that the antiviral cell factor is lymphocyte and/or huge
Phagocyte secretion.
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