CN109922657A - Cell line culture from claw hook grass platymiscium - Google Patents

Abstract

The present invention relates to claw hook grass plant cell cultures, cell line and extract and their preparation methods and purposes.

Description

Cell line culture from claw hook grass platymiscium

Technical field

The present invention relates to careless (Harpagophytum procumbens) plant cell cultures, cell line and the extractions of claw hook Object and their preparation method and purposes.

Background technique

Claw hook grass is the plant of a category in sesame section or sesame family.It is most common to be called devil's pawl or radix ranunculi ternati, It is a kind of weeds, have visually noticeable fruit perennial tuberous plant (Mncwangi et al., 2012； Journal of Ethnopharmacology 143 p775-771)。Harpagophytum procumbens (H.procumbens) (Burch.) DC.Ex Mesin originates in the Kalahari Desert area in South Africa, is considered to have very high Medical value (125 p171-178 of Gyurkovska et al., 2011 Food Chemistry), secondary piece of plant Stem is harvested because of its medicinal characteristic.Up to 36 kinds of compounds (Mncwangi, 2012) are isolated from H.procumbens；So And not reporting chemicals is separated from entire plant or from secondary tuberization.For thousands of years, H.procumbens It is more than 30 kinds of medical conditions that stem tuber, which is utilized as herbtherapy, and has been demonstrated the anti-inflammatory property for having strong (Georgiev et al.,2010；Food Chemistry 121 p967-972).

In Namibia, wild harvest H.procumbens is the livelihood of many rural communities.To this medicinal plant Increase in demand is that primary producer brings more chances, but also makes natural resources nervous.According to Namibia in 1975 " conservation of nature regulations ", H.procumbens was listed in protected species in 1977.For the regulations, need to permit ability Harvest and outlet H.procumbens.Its also protection by Botswana's legislation similar with South Africa.By encyclopedic receipts The field harvest that the person of obtaining carries out tends to protection species, but with the increase of demand and financial motivation, result in it is more (and Knowledge is less) harvester excessive harvest, this produces significant impact to the resources bank of H.procumbens.In nanometer ratio It is sub-, it is stated that per hectare is only left one plant of plant, and has 1000-2000 plants of plants in natural population in the past.Therefore, 1975 are based on Year " conservation of nature regulations " carry out tightened up regulation and protection, and preceding environment and travel service department are in 1977 by H.procumbens (locality is known as gamagu) is classified as protected species.

It is verified to be difficult to cultivate needs of the H.procumbens to replace wild harvest, and cannot be guaranteed anyway Yield from wild and cultivated plant bioactive compound.Yield depends entirely on their vegetation type and is undergone Growth conditions, and even if fluctuation can not be eliminated when environmental factor is optimized for cultivated plant.In addition, although according to Report produces H.procumbens vegetable material (Georgiev, 2010) by cell culture technology, but due to reproducibility and sense The problem of contaminating incidence, method used is not suitable for the industrial production of bioactive compound.Finally, regardless of vegetable material Source how, with viable commercial amount and the form used is suitble to extract bioactive compound from H.probumbens There are further difficulties.

Therefore, it is necessary to prepare the alternative of hook grass vegetable material, and bioactivity chemical combination is extracted from the material The alternative of object.

Summary of the invention

The inventors discovered that can by cell culture technology produce H.procumbens, so as to avoid harvest it is wild or The needs of cultivated plant.Method of the invention provides callus and suspension culture, and particularly suitable for scaling up To commercial production levels.The present inventor is also prepared for stable H.procumbens cell line, and it is especially suitable for these methods.

The present inventor further developed a kind of side that secondary metabolite is produced in H.procumbens culture cell Method, and the method for obtaining from the cell plant extracts, the extract are also provided by the present invention.The extract can be with It is stem cell extract.The extract includes the bioactive compound of the particular category of viable commercial yield, benzyl carbinol Glycoside (especially including verbascoside).This method optionally includes that one or more benzyl carbinol glycosides are separated from extract. Extract or composition comprising the extract can be used for treating and beauty (non-treatment) purpose.

Benzyl carbinol glycoside is a kind of bioactive compound, is naturally present in plant, and benzene is characterized as in structure The derivative of formic acid, containing phenethyl ring, by ester or glycosidic bond be added thereto β-glucopyranose (apiose, galactolipin, Rhamnose or xylose).Benzyl carbinol glycoside can be subdivided into five kinds of main compounds: verbascoside, Isoverbascoside, forsythin B, Burnt glutinous rehmannia benzyl carbinol glucoside D (jionoside D) and people from day grass glucoside B (leucosceptoside B).Other benzyl carbinol glycosides include 2- O- acetyl group acteoside (2-O-AcetyleAcetoside).

The pharmaceutical research of in vitro and in vivo shows that these compounds have an extensive bioactivity, including antibacterium, anti- Tumour, antiviral, anti-inflammatory, neuroprotection, anti-oxidant, liver protection, immunological regulation and tyrosinase inhibitory action.Verbascoside, Referred to as Kusaginin is a kind of caffeic acid derivative, was initially illustrated in 1963 with the title of acteoside.Verbascoside relates to And many treatments and cosmetic applications.Have been described it as a kind of anti-aging components (WO2004069218) and its skin-color Plain calm active (JP2005082522 and WO2001026670).Plant extracts (and composition comprising it) of the invention is suitable For any of these applications.

The present invention provides:

A method of the callus of culture H.procumbens, comprising:

A. to generate rice shoot outside the sterilizing seed body of H.procumbens；

B. from being extracted in the rice shoot between at least one stipes；

C. it is formed by cultivating the internode evoked callus on solid medium, the culture medium and condition of culture It is suitable for callus generation；

D. the callus generated by step c is cultivated on solid medium, the culture medium and condition of culture are suitble to be cured Injured tissue growth；

And optionally:

E. it i.e. by the way that periodically the health part of callus is transferred in fresh solid medium, and keeps being suitble to more The condition of injured tissue growth, come the callus for maintaining incubation step d to generate；Or

F. resulting callus is stored by the sample of the healthy part of the freezing callus.

The callus of the H.procumbens generated according to the method for the present invention.

A method of suspend culture H.procumbens cell, comprising:

It a. will be in the fluid nutrient medium of H.probumbens cell inoculation to suitable suspension cell growth；

B. the cell obtained by culture under conditions of being suitable for suspension cell growth；

And optionally:

C. by periodically dividing any close agglomerate, the cell is resuspended in fresh culture or is added is new Fresh culture medium, and remain adapted to the condition of suspension cell continued propagation, with the culture gained cell that maintains to suspend；Or

D. obtained cell is saved as into cell line, alternately through the sample for freezing the cell.

A kind of H.procumbens cell line, is generated by following steps:

It a. will be in the fluid nutrient medium of H.probumbens cell inoculation to suitable suspension cell growth；

B. the cell obtained by culture under conditions of being suitable for suspension cell growth；

And optionally:

C. by periodically dividing any close agglomerate, the cell is resuspended in fresh culture or is added is new Fresh culture medium, and remain adapted to the condition of suspension cell continued propagation, with the culture gained cell that maintains to suspend；Or

D. gained cell is saved as into cell line, alternately through freezing, the preferably sample of cell described in freezen protective.

The cell line of H.probumbens produced above, wherein cell used in step a, which is used as, comes from callus The sample of material provide, the callus generates by following steps:

I) to generate rice shoot outside the sterilizing seed body of H.procumbens；

Ii) from being extracted in the rice shoot between at least one stipes；

Iii it) is formed by cultivating the internode evoked callus on solid medium, the culture medium and culture item Part is suitable for callus generation；

Iv the callus generated by step c) is cultivated on solid medium, the culture medium and condition of culture are suitable for Callus growth；

And optionally:

V) it by the way that periodically the health part of callus is transferred in fresh solid medium, and maintains to be suitble to callus The condition of tissue growth, come the callus for maintaining incubation step d to generate；Or

Vi resulting callus) is stored by the sample of the healthy part of the freezing callus,

Preferably, Callus material and culture medium ratio are between 1:2 and 1:10w/w.

H.procumbens cell line produced above, wherein the culture medium of step a and/or c is to be supplemented at least one The basic element of cell division, at least one are not the auxin (2,4-D) of 2,4- dichlorophenoxyacetic acid, and MS the or GB5 culture medium of sugar, Wherein the basic element of cell division is preferably the fast cry of certain animals (BAP) of 6- benzyl amino, and auxin is preferably α-naphthylacetic acid (NAA), and sugar is preferably sucrose.

H.procumbens cell line produced above, the wherein basic element of cell division in the culture medium of step a and/or c: growth The ratio of element is each independently 2:1 to 20:1.

H.procumbens cell line produced above, wherein the culture medium in step a is supplemented with 5mg/ml BAP, 1mg/ Culture medium in ml NAA and 30g/l sucrose and/or step c is supplemented with 2mg/ml BAP, 0.1mg/ml NAA and 30g/l Sucrose.

H.procumbens cell line produced above, wherein the condition in step b and c includes black at about 22 to 28 DEG C In the dark, it cultivates, and/or wherein carries out within step C about every 2-3 weeks primary periodic with orbital oscillation or on wave reactor It is resuspended or culture medium adds.

H.procumbens cell line produced above, wherein culture medium has magnanimity shown in following Table A or B and micro- Secondary element and vitamin combination.

A kind of H.procumbens cell line, is deposited in NCIMB preservation mechanism, and deposit number is NCIMB 42467.

A method of generate secondary metabolite in H.procumbens cell, this method be included in be suitable for it is described Cell growth under conditions of, in the medium first suspend culture H.procumbens cell, then change condition of culture and/or Culture medium makes them suitable for generating secondary metabolite by the cell.

The method that one or more benzyl carbinol glycosides are extracted from H.procumbens cell, this method include in the medium The sample for obtaining the cell generates the plant extracts comprising one or more benzyl carbinol glycosides from the cell, and optionally Separate one or more benzyl carbinol glycosides from the plant extracts, preferably verbascoside.

According to extracted from H.procumbens cell one or more benzyl carbinol glycosides method produce plant extracts, This method includes the sample for obtaining the cell in the medium, is generated from the cell comprising one or more benzyl carbinol glycosides Plant extracts, and one or more benzyl carbinol glycosides, preferably verbascoside optionally are separated from the plant extracts, it is optional Wherein the yield of benzyl carbinol glycosides is at least 5%w/w in extract on ground, and the yield of verbascoside is at least 3%w/w, and 2-O- second The yield of acyl group acteoside is at least 1.5%w/w of extract.

As above the plant extracts generated, wherein the cell sample is to generate secondary from H.procumbens cell It is obtained in the obtained culture medium of the method for metabolite, this method includes the culture that suspends first in the medium H.procumbens cell (under conditions of being suitable for cell growth), then changes condition of culture and/or culture medium, so that They are suitable for generating secondary metabolite by the cell.For example, can by close at the end of growth cycle and before harvest extremely Generation that is 4 days few, adding the methyl jasmonate that concentration is 100 μM to realize secondary metabolite.

Plant extracts produced above, wherein the culture that suspends first is carried out by following steps:

It a. will be in the fluid nutrient medium of H.probumbens cell inoculation to suitable suspension cell growth；

B. the cell obtained by culture under conditions of being suitable for suspension cell growth；

And optionally:

C. by periodically dividing any close agglomerate, the cell is resuspended in fresh culture or is added is new Fresh culture medium, and remain suitable for the condition of suspension cell continued propagation, come the culture gained cell that maintains to suspend；Or

D. it is stored obtained cell as cell line, alternately through the sample for freezing the cell,

And the change carries out after step b or step c, and/or wherein cultivates first for described in described Cell is the sample from the cell line invented as described herein.

Plant extracts produced above, wherein the plant extracts is generated by following steps:

A. cellular material is filtered out from the sample, is rinsed with water, it is then dry between about 0 DEG C to about 30 DEG C；

B. obtained dry matter is suspended in water-alcohol solution；

C. at room temperature, gained suspension is exposed to ultrasonic wave about 1 hour to about 24 hours at room temperature, then in room temperature It is lower by the suspension impregnation about 1 hour to about 48 hours；And

D. filtering gained suspension is to remove solid；And optionally

E. extract obtained to be concentrated by evaporating residual solvent between about 0 DEG C to about 30 DEG C.

Plant extracts produced above, wherein in the plant extracts that step d or e are obtained:

The yield of benzyl carbinol glycosides be extract at least 1.5%, at least 2%, at least 3%, at least 4% or preferably at least 5%w/w；And/or

The yield of verbascoside is at least 1%, at least 2% or preferably at least 3%w/w of extract；And/or

The yield of 2-O- acetyl group acteoside is at least 0.5%, at least 1% or preferably at least 1.5%w/ of extract w。

A kind of composition, comprising plant extracts of the present invention and suitable for cosmetics, therapeutic agent, nutriment, food and/or The carrier of animal feed applications.

A kind for the treatment of is reduced or at least one sign of prevention individual's skin aging or at least one ageing-related skin The method that skin damages sign, this method includes giving the plant extracts of the invention of the individual effective dose or mentioning comprising described Take the composition of object.

For treating the plant extracts or composition of the invention of the method for disease or illness.

The purposes of plant extracts or composition of the invention in medicine preparation.

The purposes of plant extracts or composition of the invention in treatment, mitigation or prevention disease or illness.

The purposes of plant extracts or composition of the invention, the plant extracts or composition treatment, reduction or pre- At least one of anti-individual skin aging relevant to one or more of or the sign of skin injury: pigmentation, collagen Protein expression, inflammation, saccharification, barrier function destruction or any combination thereof.

Plant extracts or composition of the invention is such as scorching in the skin injury for the treatment of UV (ultraviolet light) and/or pollution induction Disease, pigmentation, collagen expression, barrier function destroy in purposes.

The purposes of plant extracts or composition of the invention in medicine preparation.

Preferably, the purposes of plant extracts of the invention or composition is cosmetic use.

A kind of at least one sign treated, reduce or prevent individual's skin aging or at least one related to skin aging Skin injury sign method, this method includes giving the plant extracts or composition of the present invention of the individual effective dose.

A kind of at least one sign treated, reduce or prevent individual's skin aging or at least one related to skin aging Skin injury sign method, this method includes giving the plant extracts or composition of the present invention of the individual effective dose. Wherein the skin injury is that abiotic oxidation stress is exposed to due to skin, is optionally drawn by being exposed to UV radiation or pollution It rises.

A kind of at least one sign treated, reduce or prevent individual's skin aging or at least one related to skin aging Skin injury sign beauty method, this method includes giving plant extracts of the present invention or the combination of the individual effective dose Object.

A kind of at least one sign treated, reduce or prevent individual's skin aging or at least one related to skin aging Skin injury sign beauty method, this method includes giving plant extracts of the present invention or the combination of the individual effective dose Object.Wherein the skin injury is that abiotic oxidation stress is exposed to due to skin, optionally by being exposed to UV radiation or dirt Dye causes.

Detailed description of the invention

Fig. 1: illustrating the structure of verbascoside, and verbascoside is an example of benzyl carbinol glycosides.

Fig. 2: Harpagophytum procumbes (H.procumbens) cell line extract (water/ethyl alcohol 25%w/w) Representative RP-HPLC chromatogram.UV wavelength: 330nm.In Zorbax Eclipse C18 column (4.6 × 250mm × 5mm； Agilent Technology) on, it is separated in 35 DEG C with the water of gradient concentration and acetonitrile.

Fig. 3: screening is measured to the DPPH of several extracts.Two batches H.procumbens, ALD53 are tested in the measurement And ALD64, similar radicals scavenging result is shown in all test concentrations.

Fig. 4: the gene of HMOX1 in the fibroblast stimulated by H.procumbens (ALD97) or verbascoside (VB) Expression.TGFb is the positive control of measurement.

Fig. 5: to UV (50mJ/cm²UVB) primary fibroblast induced (uses active material in 1 hour before ultraviolet radiation Pretreatment) in H₂O₂The normalization % of release inhibits.

Fig. 6: after display is handled with H.probumbens extract (0.2mg/ml) or positive control (1mM), to collagen egg The data that the % of white enzyme inhibits.

Fig. 7 A: after display H.probumbens extract (0.2mg/ml) pretreatment, the marker of inflammation of UV induction % inhibit data.MMP1, MMP3, IL-6 and GM-CSF protein level are measured by multiple analysis.It is summarised in different donations The data of n=4 experiment are carried out on person.

Fig. 7 B: after display is pre-processed with H.probumbens extract (0.2mg/ml) or (30 μM) of verbascoside, to UV The data that the % that the MMP1 of induction is generated inhibits.MMP1 protein level is measured by ELISA.It is summarised on different contributors and carries out The data of n=5 experiment.

Fig. 7 C: after display is pre-processed with H.probumbens extract (0.2mg/ml) or (50 μM) of verbascoside, to dirt The data that the % that the MMP1 of dye induction (DPM2 μ g/ml) is generated inhibits.MMP1 protein level is measured by ELISA.Summarize n=3 The data of a difference contributor.

Fig. 8: in the fibroblast with H.probumbens extract (0.2mg/ml) pretreated UV- paracrine stimulation Collagen I, collagen III, collagen IV and fibrillin gene expression analysis.Data are different contributors N=3 summary.Conspicuousness compared with UVB processing: p < 0.05 *, p < 0.01 * *, p*** < 001.Untreated=unused work Property agent pretreatment, the KC culture medium not stimulated is then added.UVB=is not pre-processed with activating agent, and UV stimulation is then added KC culture medium.UVB+H procumbens=H procumbens extract pre-processes 1 hour, and the KC of UV stimulation is then added Culture medium.

Fig. 9 A: by being pre-processed 48 hours with H.probumbens extract (0.2mg/ml) or (16 μM) of verbascoside The generation of fibroblastic intracellular collagen protein I albumen of UV- paracrine stimulation.It is analyzed by immunofluorescence, institute Registration evidence is that the contributor different to n=2 summarizes.The only conspicuousness compared with UVB: p < 0.01**, p < 0.001***.

Fig. 9 B and C: fluorescent image show collagen I dyeing fluorescent image, represent only with or without One contributor of H.probumbens processing, and in the presence of UV handles culture medium.

Figure 10: H.probumbens extract (0.2mg/ml) or verbascoside (16 μM) are used in non-UV processing culture medium After stimulation 24 hours, the gene of collagen I, collagen III, collagen IV and fibrillin in fibroblast Expression analysis.Data are the summary to the n=3 of different contributors (age represents 54,56,74 years old).It is aobvious compared with not treating Work property p < 0.05*, p < 0.01**.

Figure 11 A: in non-UV processing culture medium, with H.probumbens extract (0.2mg/ml) or verbascoside (16 μ M the collagen I protein that 48 hours fibroblasts generate in the cell) is handled.It is analyzed, is shown by immunofluorescence Data be summary to different contributor n=2.Conspicuousness p < 0.05* compared with untreated.

Figure 11 B and C: fluorescent image shows collagen I dyeing, represents only with or without the one of H.procumbens processing A contributor (in the presence of non-UV handles culture medium).

Figure 12 A: data show that the % being saccharified in cell free system inhibits.By PROTEIN B SA, sugared D-ribose (0.5M) and survey Surely control rutin (1mM) mixing, or mixed with the extract of H.procumbens (0.2mg/ml), AGE is quantified with spectrophotometric Product (Advanced Glycation final product).Summarize the data of n=4.Conspicuousness compared with untreated samples (only BSA and sugar).p< 0.01**、p<0.001**。

Figure 12 B: data show that the saccharification % to human fibroblasts inhibits.With sugared 0.5mM glyoxal processing cell to lure Lead 100% saccharification.Then with H.probumbens extract (0.2mg/ml) or measurement control (1mM melbine) processing saccharification Then cell passes through glycated protein carboxymethyl lysin (CML) existing for immunofluorescence dyeing.It summarizes and comes from different contributor n=4 Data.Conspicuousness compared with glyoxal treatment: p < 0.01**, p < 0.001**.

Figure 13: with H.probumbens extract (0.2mg/ml) or the cutin of the UV of (16 μM) of verbascoside processing stimulation Form the gene expression analysis of cell.Data are the summaries to different contributor n=2.Conspicuousness compared with UVB: p < 0.05*、p<0.01**、p<0.001**。

Figure 14: with H.probumbens extract (0.2mg/ml) or the keratinocyte of (16 μM) of verbascoside processing Gene expression analysis.Data are the summaries to different contributor n=2.P < 0.05 conspicuousness * compared with not treating

Figure 15 A: the quantitative melanin from people's pigmentation epidermis extracted with soluble reagents, people's pigmentation It epidermis H.probumbens, verbascoside and treated with kojic acid 4 days to 10 days, is challenged without UV.Histogram is indicated from every kind The value that 3 tissues (n=3) of condition obtain.P < 0.01 conspicuousness * compared with solvent control.

Figure 15 B: the quantitative melanin from people's pigmentation epidermis extracted with soluble reagents, people's pigmentation Epidermis H.probumbens, verbascoside and treated with kojic acid 4 days to 10 days, and challenged with UV.Histogram is indicated from every kind The value that 3 tissues (n=3) of condition obtain.P < 0.01 conspicuousness * compared with solvent control.

Specific embodiment

No matter hereinbefore or hereinafter herein cited all publications, patents and patent applications are integrally incorporated by reference Herein.

It should be understood that the different application of disclosed product and method can be customized according to the specific needs of this field.Also It should be understood that terms used herein are only used for the purpose of description the particular embodiment of the present invention, rather than it is restrictive.

In addition, as used in the specification and the appended claims, singular " one (a) ", " one (an) " and "the" includes plural referents, except non-content is expressly stated otherwise.Thus, for example, referring to that " cell " includes " cell " etc.. " plant extracts " being mentioned above refers to the preparation obtained from vegetable material (usually dry vegetable material), including culture Plant cell or callus, said preparation can be liquid, semisolid or solid form.

There is provided herein a kind of methods for cultivating H.procumbens callus.Callus is that amorphous thin-walled is thin The amorphous agglomerate of born of the same parents.When plant injury, callus is formed at cutting surfaces, and it is considered as seals damaged The protective response of tissue.In vitro, by the way that plant tissue segment (explant) is aseptically placed in solid medium On generate callus.Callus is induced and is formed by the proliferative cell of explantation tissue's cutting surfaces.The present invention also mentions The callus for generating and maintaining by means of the present invention is supplied.The callus is stable, it is meant that plant cell Keep undifferentiated.

In the method for the invention, plant tissue ready for use carrys out the sterilizing seed of free H.procumbens in vitro Between the rice shoot stipes of generation.Which ensure that there is no virus or bacterium infections for crude plant material, with many cell culture technologies Used in leaf or other plant cutting (including Georgiev et al, described in 2010) compare, substantially seed is more It is easy sterilizing.

It is the tissue between the node of stem between stipes, node generally remains leaf, bud or inflorescence.The sterilizing of seed and from going out The generation of the rice shoot of strain can be realized by any suitable method known in the art.Suitable method, which is disclosed in, " plants In object cell culture ", i.e., Evans et al. delivered in Taylor&Francis 2003 ISBN 185996320X (referring particularly to Agreement 6.1 part A and B).

The method of the present invention includes between extracting at least one stipes in rice shoot, and it is layered on solid medium.It can make With any suitable solid dielectric.Suitable culture medium include but is not limited to Murashige and Skoog's culture medium (MS), Gamborg B5 medium (GB5), McCowan's Woody Plant Medium (McC), Andersons Rhododendrum culture medium (AR), Shenk and Hildebrandt culture medium (SH) and Litvay ' s culture medium, every kind It can be solidified with any suitable curing agent.Preferred culture medium for the method for the present invention includes cured with plant agar Murashige and Skoog's culture medium (MS) or Gamborg B5 (GB5) culture medium.Culture medium for the method for the present invention It is preferred that respectively including the magnanimity listed in Table A and B and microelement and vitamin combination.

By maintaining culture medium suitable under conditions of callus induction, and/or by being lured with suitable callus The specific components supplementing culture medium led carrys out evoked callus and is formed." supplement " refers to that specific components can be before the start of the method Comprising in the medium, or can be added in culture medium in the appropriate stage of method.Those skilled in the art can be easily Determine callus induction and growth/maintenance felicity condition.Representative condition is about 22 to about 28 DEG C under low intensity fluorescence lamp Temperature, dark/light circulates in about 8 hours: about 16 hours.The preferred method of the present invention uses about 23 DEG C to about 25 DEG C in the dark Temperature, preferably from about 24 DEG C.

The supplemental components of culture medium generally include:

At least one basic element of cell division is generally selected from zeatin (Z), ribosylzeatin (ZR), isopentenyl gland purine (IP), 6-benzyl aminopurine (BAP), 6-nonylaminopurine (kinetin), N⁶(m- hydroxybenzyl) adenine (topolin), Thidiazuron (TDZ), chloropyuril (CPPU or 4PU-30)；

At least one is not the auxin of 2,4- dichlorophenoxyacetic acid (2,4-D), is generally selected from indole-3-acetic acid (IAA), indole -3-butyric acid (IBA), 1- methyl α-naphthyl acetate (NAA), phenylacetic acid (PAA), 2,4,5 T 2,4,5 trichlorophenoxy acetic acid (2,4,5- T), picloram, dicamba, p-chlorophenoxyacetic acid (CPA)；With

It is generally selected from the sugar of sucrose, glucose, fructose and maltose.

In needing all methods of the invention comprising auxin, 2,4-D are not used.Although this auxin is usually used In culture plant cell, but present inventor have determined that it is the component for not being suitable for the method for the present invention, the method for the present invention purport In the stabilization callus or suspended cell culture of the plant cell for generating H.procumbens.2,4-D be it is inappropriate, because It may cause the variation of mitotic activity and chromosome and chromatin Structure and the variation in the cell cycle for it, lead Cause the exception of plant cell.

Those skilled in the art can readily determine that the debita spissitudo of every kind of component.However, culture medium when culture starts In the basic element of cell division: auxin ratio is preferably from about 2:1 to about 20:1.The ratio can be, for example, about 2:1, about 4:1, about 5:1, about 6:1, about 10:1 or about 20:1, but preferably the ratio is about 2:1 or about 5:1.In the method for the invention, cell division Element is preferably 6-benzyl aminopurine (BAP), and auxin is preferably α-naphthylacetic acid (NAA), and sugar is preferably sucrose.The amount of BAP is preferred It is about 5mg/l.The amount of NAA is preferably from about 1mg/l.The amount of sucrose is preferably from about 20g/l to about 50g/l, preferably from about 30g/l.

Once induction, by continue on solid medium cultivate callus come cultivate/maintain callus.This passes through Under conditions of culture medium is maintained suitable callus growth, and/or by changing culture media composition, make it suitable for being cured Injured tissue growth.Those skilled in the art can readily determine that the felicity condition of callus growth, and can be used for The condition of callus induction is identical.In a preferred method of the invention, callus growth condition generally includes in the dark, It is cultivated at about 22 DEG C to about 28 DEG C, preferably from about 23 DEG C to about 25 DEG C, most preferably from about 24 DEG C.Changing culture medium composition may include The supplemental components of different level are added into existing culture medium, or healthy Callus material is transferred in fresh culture, The wherein suitable callus growth of level of various components.

Appropriate change to be adapted to grow is carried out to culture media composition, it may include change relative to for cultivating starting When the basic element of cell division: auxin ratio.The ratio is usually maintained in about 2:1 between about 20:1, but preferably with respect to being used for The ratio of callus induction increases.The ratio can be, for example, about 2:1, about 4:1, about 5:1, about 6:1, about 10:1 or about 20: 1, condition is its ratio identical as ratio used in callus induction or preferably higher than used.The most preferred basic element of cell division: raw Long element ratio is about 20:1.The amount of sugar is usually maintained in the similar level with level needed for callus induction.In the present invention Method in, the amount of BAP is preferably decreased to about 2mg/l.The amount of NAA is preferably decreased to about 0.1mg/l.The amount of sucrose is preferably from about 20g/l is to about 50g/l, preferably from about 30g/l.

It is suitable for the fresh solid training of callus growth by being periodically transferred to the Callus material of health It supports in base, and remains suitable for the condition of callus growth, optionally to maintain culture callus.Technical staff can be easy Ground determines the appropriate intervals periodically shifted.However, in the method for the invention, the transfer carries out one in generally about every 2-3 weeks It is secondary.

Callus is stored alternately through the sample for the healthy part for freezing the callus, such as from suspension The cell of the callus in the medium.Can be carried out by any appropriate method known in the art callus or its The freezing of his vegetable material.

The method for the culture H.procumbens cell that suspends is also provided herein.The culture that suspends refers to cell in liquid It is grown in culture medium.Any suitable fluid nutrient medium can be used.Suitable culture medium includes but is not limited to Murashige And Skoog ' s culture medium (MS), Gamborg B5 medium (GB5), McCowan's Woody Plant Medium (McC), Andersons Rhododendrum culture medium (AR), Shenk and Hildebrandt culture medium (SH) and Litvay ' s culture medium.Preferred culture medium for the method for the present invention include Murashige and Skoog ' s (MS) or Gamborg B5(GB5).Culture medium for the method for the present invention preferably respectively includes the magnanimity listed in Table A and B and micro member Element and vitamin combination.

The present invention also provides the cells of suspension culture method production and/or maintenance through the invention.The cell is Stable, it is meant that they keep undifferentiated, and can be used as cell line preservation." stable cell lines " are defined as having at any time There is the cell culture system of high and constant proliferation rate, identical phenotypic characteristic (cellular colours, group are kept in various subcultures Poly- brittleness, size etc.), and have reproducible secondary metabolites horizontal during various subculture steps.By this hair The exemplary cells system of bright offer has been preserved in following preservation mechanism:

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AB21 9YA

Scotland.

Preservation date: on September 18th, 2015；Preservation accession number: NCIMB 42467.

Preservation side: Oriflame Cosmetics AG, c/o Global Management AG, Bleicheplatz 3, CH-8200 Schaffhausen, Switzerland.

Suspension culture method of the invention includes by H.probumbens cell inoculation to the liquid for being suitable for suspension cell growth In body culture medium, and the cell obtained by culture under conditions of being suitable for suspension cell growth.The cell of inoculation preferably includes to pass through The Callus material sample that callus culture method according to the present invention generates, in this case, Callus material 1:2 is preferably from about to about 1:10 with the ratio of culture medium.Alternatively, the cell of inoculation can come from any suitable source, such as It can be the stable H.probumbens cell line previously according to method culture of the invention.One example of this cell line It is the cell line NCIMB 42467 of preservation.

The felicity condition of suspension cell growth can be readily determined by technical staff, and can be used for callus The condition of growth is identical.Representative condition is under low intensity fluorescence lamp, and temperature is about 22 DEG C to about 28 DEG C, and dark/light circulation is about 8 Hour: about 16 hours.The preferred method of the present invention in the dark, uses at a temperature of about 23 DEG C to about 25 DEG C, preferably from about 24 DEG C. It is usually gently stirred for suspended cell culture in the whole process, such as with track concussion or on wave reactor.

If fluid nutrient medium is supplemented with the component for being suitable for the growth, fluid nutrient medium is suitable for the life of suspension cell It is long." supplement " refers to that specific components can be before the start of the method comprising in the medium, or can be in the appropriate rank of method Section is added in culture medium.The supplemental components of culture medium generally include:

At least one basic element of cell division is generally selected from zeatin (Z), ribosylzeatin (ZR), isopentenyl gland purine (IP), 6-benzyl aminopurine (BAP), 6-nonylaminopurine (kinetin), N6- (m- hydroxybenzyl) adenine (topolin), Thidiazuron (TDZ), chloropyuril (CPPU or 4PU-30)；

At least one is not the auxin of 2,4- dichlorophenoxyacetic acid (2,4-D), is generally selected from indole-3-acetic acid (IAA), indole -3-butyric acid (IBA), 1- methyl α-naphthyl acetate (NAA), phenylacetic acid (PAA), 2,4,5 T 2,4,5 trichlorophenoxy acetic acid (2,4,5- T), picloram, dicamba, p-chlorophenoxyacetic acid (CPA)；With

It is generally selected from the sugar of sucrose, glucose, fructose and maltose.

Those skilled in the art can readily determine that the debita spissitudo of every kind of component.However, culture medium when culture starts In the basic element of cell division: auxin ratio is preferably from about 2:1 to about 20:1.The ratio can be for example, about 2:1, about 4:1, about 5: 1, about 6:1, about 10:1 or about 20:1, but preferably the ratio is about 2:1 or about 5:1.In the method for the invention, the basic element of cell division Preferably 6-benzyl aminopurine (BAP), auxin are preferably α-naphthylacetic acid (NAA), and sugar is preferably sucrose.The amount of BAP is preferably About 5mg/l.The amount of NAA is preferably from about 1mg/l.The amount of sucrose is preferably from about 20g/l to about 50g/l, preferably from about 30g/l.

By periodically dividing any close agglomerate, the cell is resuspended in fresh culture or is added Fresh culture, and remain suitable for the condition of suspension cell continued propagation, optionally to maintain suspension cell culture.The process can To be known as " secondary culture ".Technical staff can be readily determined the suitable of these cyclic activities based on the growth rate of cell Work as interval.However, in the method for the invention, interval typically about 1-3 weeks.

The maintenance of cell, which may also include, changes culture medium composition.Changing culture media composition may include to existing culture medium The supplemental components of middle addition different level, or cell is transferred in the fresh culture for having changed various composition levels.

Be adapted to maintain the cell in suspension culture may include appropriate change of culture media composition, relative to In cultivating when starting, change the basic element of cell division: auxin ratio.The ratio generally remains in about 2:1 between about 20:1, but Increase preferably with respect to ratio when starting for cultivating.The ratio can be for example, about 2:1, about 4:1, about 5:1, about 6:1, about 10:1 or about 20:1, condition are its ratios identical as ratio used when starting or preferably higher than used.Most preferred cell division Element: auxin ratio is about 20:1.The amount of sugar is usually maintained in the similar level with level needed for callus induction.At this In the method for invention, the amount of BAP is preferably decreased to about 2mg/l.The amount of NAA is preferably decreased to about 0.1mg/l.The amount of sucrose is preferred It is about 20g/l to about 50g/l, preferably from about 30g/l.

Alternately through according to any appropriate method known in the art freeze healthy cell sample, or by generally acknowledging Carry out preservation as cell line in preservation mechanism.

The method that the present invention also provides a kind of to generate secondary metabolite in H.procumbens cell, this method packet It includes under conditions of being suitable for cell growth, suspend culture H.procumbens cell first in the medium, then changes Become condition of culture and/or culture medium, makes them suitable for generating secondary metabolite by the cell.The training that suspends first Supporting can carry out according to above-mentioned the method for the present invention.The cell used can come from being generated according to method present invention as described above Cell line, such as preservation cell line NCIMB 42467 cell.The change is usually in the life of the culture that suspends first Occur at the end of wanting for a long time, can be readily determined by technical staff.In the method for the invention, the change is usually suspending Growth occurs after 1-3 weeks, but can occur sooner or later, this depends on the growth rate of suspension cell.

The change may include any one or more of following:

Increase precursor compound concentration in culture medium, such as sucrose is added；

The concentration of secondary metabolites in culture medium is reduced, such as adsorbs metabolin by situ；

Biological exciton is added in the medium, such as: oligosaccharide, chitosan, glucan, glycoprotein, at high steam The pathogen mycelium of reason, fermentoid, plant signal conducting compound

Abiotic exciton, such as heavy metallic salt, pH adjusting agent are added in the medium

Adjust temperature and/or illumination condition

By the fixed suspension culture of any suitable method, for example, being captured in inert base.Suitable fixative packet Include calcium alginate, polyphenylene oxide, fibrous polypropylene, alginate+nylon, reticulated polyurethane, agarose, agar, carragheen, bright Glue, polyacrylamide, alginate+gelatin.

When the change includes the additive into culture medium, technical staff can be readily determined suitable concentration. In the case where described change including adjusting condition, technical staff can be readily determined suitable adjusting.Technical staff also knows Road is used to adsorb the appropriate method of secondary metabolites and fixed suspension culture.

In a preferred method of the invention, the change includes that biological exciton is added into culture medium.The biology swashs Hair is preferably plant signal conducting compound, most preferably methyl jasmonate.Biological exciton is preferably with about 0.1 μM to about 500 μM, most preferably from about 100 μM of concentration addition.

After the usual change, in the dark, about 22 DEG C to about 28 DEG C, preferably from about 23 DEG C to about 25 DEG C, most preferably It about 24 DEG C, is cultivated by orbital oscillation or on wave reactor at least 4 days.

This method can be semicontinuous or continuous process, optionally include the periodicity of fresh Callus material or cell Culture medium exchange and/or periodically addition, and/or a part of cell in fresh culture is periodically resuspended, and/or periodically add Add biological exciton.

The present invention also provides extract one from H.procumbens cell or other plant cell comprising benzyl carbinol glycosides The method of kind or a variety of benzyl carbinol glycosides, this method includes the sample for obtaining the cell in the medium, from comprising a kind of or more The cell of kind benzyl carbinol glycosides generates plant extracts, wherein the benzyl carbinol glycosides are preferably verbascoside.Extract is optimal Choosing includes verbascoside and 2-O- acetyl group acteoside.Alternatively, this method can be described as the cell from H.procumbens Generate plant extracts, the plant extracts includes one or more benzyl carbinol glycosides, preferred verbascoside, most preferably wherein institute Stating extract includes verbascoside and 2-O- acetyl group acteoside.This method optionally includes from the plant extracts Separate one or more benzyl carbinol glycosides.

Cell sample used in this method can be obtained from any suitable source, such as can be previously according to this hair The stable H.procumbens cell line of bright method culture.One example of this cell line is the cell line of preservation NCIMB 42467.Alternatively, the sample of the cell as obtained by carrying out suspension cell culture method of the invention and take out, comes Obtain cell.

Extracting method of the invention is the cold extraction method of optimization, uses mild extraction conditions and sustainable molten Agent." sustainable " refer to solvent hypotoxicity, low price, extensive availability and good reproducibility (such as be easy to by Biomass material production) aspect be advantageous.The selection of temperate condition and sustainable solvent, it is ensured that high yield and optimal time The product of grade metabolite profile, generation does not contain potential noxious pollutant.

This method includes harvesting cell first.Harvest can be carried out by any suitable method.It describes in instances Illustrative methods.The preferred method for harvesting cell includes filtering.For example, cell can be harvested by Buchner funnel flask suction strainer, make Suitable filter material (such as the Miracloth for being about 22-25 μm with aperture^TM).Then it is usually rinsed with water (preferably distilled water) The cell material of filtering, it is then dry under the conditions of mild temperature, usually between about 0 DEG C to about 38 DEG C, preferably about 20 DEG C between about 30 DEG C.

Then it is suitable as in the water-alcohol solution of solvent obtained dry matter to be suspended in, such as water/ethyl alcohol, water/ Propyl alcohol, water/isopropanol, water/n-butanol, preferably water/ethyl alcohol.Water-alcohol solution is usually 10-95%v/v, preferably from about 25%v/v. By gained suspension in about 45-65Hz, preferably from about 45Hz, it is ultrasonically treated (being exposed to ultrasonic wave) at room temperature about 1 hour to about 24 Hour, preferably from about 1 to 2 hour.After ultrasonic treatment, by suspension impregnation about 0.5 hour to about 48 hours, preferably from about 0.5 to 1 small When, it is then filtered to remove solid again.Any appropriate method known in the art can be used to be filtered.These methods include The quantitative paper filter for the use of aperture being about 11 μm.Illustrative methods describe in instances.

After filtering, plant extracts then is concentrated alternately through evaporation residual solvent at low temperature, usually at about 0 DEG C Rotary evaporation is used between to about 30 DEG C, until reaching azeotropic point.This is usually required about 5 to about 20 minutes.It can be by light brown The presence of residue is as the acquired indicator of plant extracts needed for indicating.

It may then pass through any suitable method and the extract of concentration be freeze-dried or be spray-dried.Preferred freezing Drying proposal includes that the plant extracts of concentration is freezed to about 1 hour at about -20 DEG C to about -150 DEG C, preferably from about -80 DEG C extremely About 3 hours, preferably from about 1 hour.Then the solution of freezing is placed in freeze-dryer and obtains dry crude extract.The mistake The canonical parameter of journey is as follows:

Condenser temperatures: -50 to -120 DEG C, preferably from about -110 DEG C

Pressure limit: 10 millibars to 1 microbar, preferably from about 7 microbars

Time range: 1-72 hours, preferably from about 48 hours

Plant extracts can be used as in a part incorporation product of freeze-drying or spray drying process, i.e., by with it is suitable It is freeze-dried or is spray-dried together together in the carrier of cosmetics, therapeutic agent, nutriment, food and/or animal feed applications.? Suitable carrier includes maltodextrin, starch, cellulose and cyclodextrin in context.Alternatively, plant extracts can be by outstanding It floats in the carrier suitable for cosmetics, therapeutic agent, nutriment, food and/or animal feed applications and mixes in product.

Suitable carrier includes glycerol (glycerol), glycerol (glycerine), butanediol, propylene glycol, D-sorbite list Only or any of above combination, denatured alcohol, PEG-40, rilanit special and isopropyl myristate.

In the plant extracts produced by means of the present invention, usually:

The yield of benzyl carbinol glycosides is at least 1.5%, at least 2%, at least 3%, at least the 4% or excellent of the biomass of extract Select at least 5%w/w；And/or

The yield of verbascoside is at least 1%, at least 2% or preferably at least 3%w/w of the biomass of extract；And/or

The yield of 2-O- acetyl group acteoside be extract biomass at least 0.5%, at least 1% or preferably at least 1.5%w/w.

The present invention also provides the plant extracts and include its composition.The extract and the composition can Suitable for any cosmetics, therapeutic agent, nutriment, food and/or animal feed applications.The product is optionally incorporated into suitable In the excipient or carrier of the purposes.

The present invention also provides treatment, at least one sign of reduction or prevention individual's skin aging or at least one and always Change the method for relevant skin injury sign, this method includes giving the plant extracts or packet of the present invention of the individual effective dose Composition containing the extract.Individual is usually people.Skin can at any position of body, but preferably face or head Skin.Extract or giving for composition are typically directly applied to skin, i.e., locally, and correspondingly prepare extract or composition.

Human skin is made of two primary layers, is played an important role to maintenance overall health of skin.Top exterior layer, i.e. table Skin plays the role of protective barrier, is prevent mechanical damage, external microorganism and other external aggression objects such as UV and pollution One of defence line.Corium is located at below epidermis, constitutes about 90% skin thickness.Corium contains intensive protein network, assigns skin Skin intensity and elasticity.Fibroblast is the major cell types found in the dermis, and is responsible for generating being used as in corium and send out The collagen of existing most one of abundant protein.

Skin aging is bioprocess, by internal (natural biological aging) and external (leading to skin texture variation) because The combined influence of element generates, and usually passes through the various protein bio marks in the epidermis and skin corium of degradation skin Object.External factor includes being exposed to UV (photoinduction or photoaging), being exposed to pollution or other stimulants and pressure.These Each in factor results in skin and is exposed to abiotic oxidation stress, this again results in skin aging and ageing-related Skin injury.Therefore, method of the invention is alternatively described as treatment, reduces or prevent caused by being coerced as abiotic oxidation The method of damage to skin.

It is believed that the damaged portion is generated due to the formation of free radical in skin.It is led for example, being repeated exposure to UV Cause forms peroxy radical, is decomposed to form malonaldehyde, subsequently cross-linked and polymerize collagen.It is reported that free radical can be with Activator metal protease, such as Collagenase, they can decompose skin collagen and elastin laminin.Radical damage also can Cause skin corium thickness to reduce, again results in cutis laxa.Free radical results in the of aging sign to the comprehensive function of skin One of one and most apparent sign-skin elasticity are harmful to be lost, and prematurity wrinkle is then formed.It is anti-oxidant in skin histology The presence of agent is that system is effectively protected in one kind, can resist the destruction of these reactive free radicals.However, with year The growth in age, the anti-oxidative defense ability in skin reduces, to enhance the destruction of free radical.Other mechanism also result in Skin aging and ageing-related skin injury, and these mechanism may include makes corium and epidermis, synthesis and/or are repaired The ability of protein is impaired.

As proved in example, plant extracts of the invention plays the work of antioxidant (reducing radical effect) With, as the active reinforcing agent of endogenous anti-oxidative, and the stimulant as generation skin and epidermal protein matter, therefore this hair Bright method is directly based upon many factors relevant to skin aging and ageing-related skin injury and acts on, to control It treats, reduce or prevention skin aging or the sign of ageing-related skin injury.

Method of the invention can treat, reduce or prevent skin aging or ageing-related at least the one of skin injury Kind sign: wrinkle, skin lines, dry skin, skin lack flexibility, lack the colour of skin, thinning of skin, skin collagen fiber Degradation, cutis laxa, skin sagging, skin internal degradation, inflammation, rubescent, spot, eyes edema or black eye, capillary Expansion, solar lentigines elastic fiber hyperplasia, appearance keratin and/or cutaneous pigmentation disease.

This method can also be used in following at least one: improve skin wound healing；By reducing rubefaction, spot or inflammation Disease promotes the colour of skin uniform；Desalinate the color of (whitening) skin and/or reduces the appearance of cutaneous pigmentation；Promote skin again It is raw, with generate more evenly, it is more compact, more have the colour of skin and more flexible skin；Promote Skin Cell long-lived；Promote skin bright Degree；Promote dermatoglyph and the colour of skin uniform；It treats or prevents because of ulcer caused by being exposed to UV or contact stimulus object or pollution Region or skin stress or damage field；And treat acne or other skin blemishes.

It should be appreciated that above-mentioned ageing-related skin aging and the sign of skin injury may not be by potential disease Caused by pathological state, thus treat, the method that reduces or prevent these signs be considered it is single cosmetic, i.e., Non-therapeutic.Therefore, this method can be described as beauty method, or, in other words, this method is treated by therapy Method.Similarly, the present invention can be described as the cosmetic use of plant extracts or composition of the invention, for treating, It reduces or at least one sign of prevention individual's skin aging or at least one ageing-related skin injury sign.

It should again be understood, however, that certain diseases and illness may generate and the same or similar effect of aging in skin. Therefore, in certain embodiments, the method for treating, mitigating or preventing these signs is considered treatment, mitigation or prevention The method of the symptom of these potential diseases or illness.Therefore, the present invention also provides plant extracts of the invention or composition, In the method for the symptom that it is used to treat, mitigates or prevents this disease or illness.The present invention also provides plants of the invention The purposes of extract or composition in the drug for preparing the symptom for treating, mitigating or preventing this disease or illness.Institute It states disease or illness can be the one or more signs for causing skin aging, ageing-related skin injury sign or by upper State any disease or illness of skin injury caused by abiotic oxidation is coerced.These diseases and illness include day hot spot, yellowish-brown Spot, leucoderma and seborrheic keratosis.

Plant extracts and composition of the invention applies also for treating or preventing inflammation and relevant disease and disease Disease, such as rheumatoid arthritis.Therefore, the present invention provides the method for the treatment of inflammation, and this method includes giving the individual to have The plant extracts of the invention of effect amount or composition comprising the extract.The present invention provides plant extracts of the invention Object or composition comprising the extract, the method for being used to treat inflammation.The present invention provides plant extracts of the invention Object or composition are preparing the purposes in the drug for treating inflammation.The inflammation can be due to disease or illness, including Brandy nose, acne, atopic dermatitis and psoriasis.

Table A

The preferred magnanimity and microelement and vitamin combination of MS culture medium for the method for the present invention.

Table B.

The preferred magnanimity and microelement and vitamin combination of GB5 culture medium for the method for the present invention.

It is further illustrated by the examples that follow the present invention, these embodiments should not be construed as further limiting.

Example

All cell culture as described herein aseptically carry out in laminar air flow cabinet.

Example 1: the generation of static culture (callus)

Callus is generated by that will organize to be taped against on culture dish from plant tissue, the culture dish includes:

A) 4.4g/l Murashige&Skoog as defined in Table A (MS) culture medium, it is solid with the plant agar of 6.5g/l Change, is supplemented with the sucrose of the 6-benzyl aminopurine (BAP) of 5mg/l and the α-naphthylacetic acid (NAA) of 1mg/l, 30g/l.

Or

B) 4.4g/l Gamborg B5 medium (GB5) culture medium as defined in table B, with the plant agar of 6.5g/l Solidification, is supplemented with the sucrose of the 6-benzyl aminopurine (BAP) of 5mg/l and the α-naphthylacetic acid (NAA) of 1mg/l, 30g/l.

Culture dish is sealed and is incubated in the dark in 24 DEG C.

Plant tissue for generating each callus is to be originated from the seed that sterilizes from external from according to standard scheme Between the stipes that sterile H.procumbens rice shoot is cut.It is published see, e.g. Evans et al. in Taylor&Francis 2003 " culture plant cell " ISBN 185996320X (especially 6.1 part A and B of agreement).

Example 2: the continued propagation of static culture (callus)

Culture medium in the same manner as in Example 1 is used for the initial growth period of callus.Then callus is made to exist It is grown on culture dish containing the culture medium with the reduced basic element of cell division and auxin level.It is special:

A) 4.4g/l Murashige&Skoog as defined in Table A (MS) culture medium, it is solid with the plant agar of 6.5g/l Change, is supplemented with the sucrose of the 6-benzyl aminopurine (BAP) of 2mg/l and the α-naphthylacetic acid (NAA) of 0.1mg/l, 30g/l.

Or

B) 4.4g/l Gamborg B5 medium (GB5) culture medium as defined in table B, it is solid with the plant agar of 6.5g/l Change, supplements the sucrose of the 6-benzyl aminopurine (BAP) of 2mg/l and the α-naphthylacetic acid (NAA) of 0.1mg/l, 30g/l.

Culture dish is sealed and is incubated in the dark in 24 DEG C.

Usually every three weeks to callus carry out secondary culture (for the faster culture of growth, it may be necessary to culture Time is shorter).Secondary culture is related to for Callus material being transferred to fresh culture, and removes and started any different of differentiation Normal cell or cell.If callus is frangible, material sample is simply layered on to the fresh cultured ware containing new culture medium On.If callus be it is compact, be divided into the block of diameter about 5mm first and be transferred to the fresh of new culture medium In culture dish.The process is repeated until obtaining frangible callus.When seeking to generate suspension culture, frangible callus group It is desirable for knitting, because the callus is easy to break when stirring in liquid medium.

Embodiment 3: suspension culture is generated from callus on static culture base

Fluid nutrient medium, callus (g) and culture medium are inoculated with the Callus material obtained according to Examples 1 and 2 It the ratio between (ml) is 1:2 to 1:10.The liquid medium composition used is as follows:

A) 4.4g/l Murashige&Skoog as defined in Table A (MS) culture medium is supplemented with the 6- benzyl ammonia of 5mg/l The sucrose of the α-naphthylacetic acid (NAA) of base purine (BAP) and 1mg/l, 30g/l.

Or

B) 4.4g/l Gamborg B5 medium (GB5) culture medium as defined in table B is supplemented with the 6- benzyl ammonia of 5mg/l The sucrose of the α-naphthylacetic acid (NAA) of base purine (BAP) and 1mg/l, 30g/l.

Callus material is obtained by scraping then weighing from culture dish, is then transferred to sterile 250-1000ml Conical flask.Culture medium is added to reach suitable ratio.Then flask is sealed with the sealing of gas-permeable, and black at 24 DEG C In the dark, it is incubated for the rotation of the orbital oscillation of 70-90rpm (laboratory scale) or on wave reactor (extensive).

Example 4: the continued propagation of suspension culture

By periodically separating close agglomerate, and suitably adds fresh MS or GB5 culture medium and (have modified, have The tonic spectrum of 30g/l sucrose, 2mg/l BAP and 0.1mg/l NAA), continue to cultivate the suspension culture prepared according to example 3. Usually every 2-3 weeks completes primary (when growing into deadtime) for this, while cell is maintained sterile 250-1000ml and is bored In shape bottle, in the dark in 24 DEG C, (advised greatly with 70-90rpm orbital oscillation rotation (laboratory scale) or on wave reactor Mould).

Example 5: the generation of inducing secondary metabolite

By at the end of growth cycle takes (i.e. deadtime) and 100 μM of methyl jasmonates of preceding addition at least 4 days are harvested, Inducing secondary metabolite generates in the suspension culture prepared according to embodiment 3 and 4.Cell is maintained in 24 DEG C of dark In 250-100ml flask, with 70-90rpm orbital oscillation rotation (laboratory scale) or on wave reactor (extensive).

Example 6: the extraction of product

The cell material that embodiment 5 obtains is passed through and is hadThe cloth of filter (exemplary aperture: 22-25 μm) Family name's funnel flask filters, rinsing and the drying at about 30 DEG C in distilled water, until weight is stablized.

Then dry cell material is suspended in water-alcohol solution (EtOH/H₂O；10 to 95%v/v) in, and will mixing Object is at room temperature with 45Hz ultrasonic treatment 1 hour to 24 hours.After ultrasonic treatment, it is small that suspension is impregnated to 1 to 48 at room temperature When, carry out further filtration step then to remove solid.

As under low temperature (0 to 30 DEG C) rotary evaporation of solvent come plant extracts obtained by being concentrated, until reach it is minimum altogether Boiling point, usually after 5 to 20 minutes.The presence of gained plant extracts is indicated with light brown residue.It then usually will be dense The extract of contracting freezes about 1 hour at about -80 DEG C.Then the solution of freezing is placed in freeze-dryer to obtain drying Crude extract.The canonical parameter of the process is as follows:

Condenser temperatures: -50 to -120 DEG C, preferably from about -110 DEG C

Pressure limit: 10 millibars to 1 microgram, preferably from about 7 microbars

Time range: 1-72 hours, preferably from about 48 hours

With post analysis and illustrate dried extract secondary metabolites spectrum

Embodiment 7: secondary metabolites spectrum analysis

On 1200 Series HPLC System of Agilent equipped with 6520 Q-TOF mass detector of Agilent, use Reversed-phase HPLC-ESI-Q-TOF illustrates the secondary metabolites spectrum of the plant extracts obtained according to embodiment 6.Use reversed-phase column (Agilent, Zorbax C-18,5 μ m 4.6mm × 250mm).

HPLC condition is as follows: flow velocity 0.218ml/min；Oven temperature, 35 DEG C；Solvent A, 0.1% aqueous formic acid；Solvent B, acetonitrile；Gradient: 0.00min, 5% (B), 60.00min, 40% (B), 70.00min, 5% (B), 75.00min, 5% (B)； Injection volume, 10 μ l (10mg/ml).Mass spectrometric data obtains within the scope of m/z 100-1000, acquisition rate 1.35spectra/ Sec, average 10.000 transitions.Parameter adjustment in source is as follows: dry gas temperature, and 250 DEG C；Dry flow velocity 5L/min, atomizer Pressure 45psi, destroyer voltage 150V.Data acquisition and processing (DAP) is completed by Agilent Mass Hunter.

Exemplary yield is as shown in the table:

The weight of " %w/w "=shown compound accounts for the percentage of the vegetable material of extraction.

" g/Kg " is the weight grams of compound shown in the vegetable material of every kilogram of extraction.

The representative RP-HPLC chromatogram of extract is shown in Fig. 2.

Example 8: the oxidation resistance of extract-external biological chemical assay

The H.procumbens extract sample that two different batches (ALD53 and ALD64) prepared as described above obtain Antioxidant activity with standard DPPH measuring method measure (see, e.g. Brand-Williams W et al Lebenson Wiss Technol 1995；28:25-30).After the measurement is by being added substances, the purple free radical state of DPPH is measured (absorbing at 516nm) becomes restoring the color change of yellow product DPPHH, to determine the antioxidation water of substances It is flat.Vitamin C and green-tea extract are used as positive control.For comparison purposes it is also tested for individual verbascoside, and is used In omparison purpose green-tea extract.The measurement includes that the test substances of DPPH solution (Sigma) and various various concentrations are mixed It closes, is then incubated for 30 minutes, subsequently measures the absorbance at 516nm.As a result as shown in Figure 3.Two kinds of H.procumbens are mentioned Object (ALD53 and ALD64) is taken to reach similar under all test concentrations, the antioxidant activity of good level (is reached in 0.2nM To 50-60%).It was found that individual verbascoside reaches and the comparable result of positive control.

The ability of example 9:H.procumbens extract change people's cell gene expression

The purpose of these experiments is handled by quantitative polyase chain reaction (qPCR) measurement test substances and reference material Cell gene expression, the amount of specific RNA in qPCR measurement sample.This method include from all samples preparation RNA and The method of cDNA.By people's primary cell culture (including fibroblast or keratinocyte) in 48 porocyte culture plates It cultivates to about 80% and converges.Then with test substances (H.procumbens extract or verbascoside) or reference material (carrier or TGFb it) handles cell 12-48 hours, then carries out RNA and extract and cDNA synthesis and qPCR.Primer and other materials it is detailed Information is shown in annex 1.

The expression of HMOX1, the HMOX1 coding protein HO-1 or Heme oxygenase are measured to the experimental specificity, It belongs to Vitagenes family, and the Vitagenes further comprises heat shock protein and thioredoxin system.These bases Because raising in stress response, protecting and maintaining to play a crucial role in Cell Homeostasis.HO-1 albumen and ferroheme It is related to biliverdin that (having pro-oxidant properties) is degraded to carbon monoxide, ferrous iron.Most latter two ingredient is referred to as bilirubin and iron The precursor of the anti-oxidant compounds of albumen.HMOX1 well-known (Vile, Basu- because of its protective effect to Oxidative Stress Modak, Waltner , &Tyrrell, 1994).

RNA is extracted

After carrying out cell processing with H.procumbens extract or verbascoside, by the way that RTL lysis buffer is added (RNeasy Mini kit, Qiagen) directly cracks cell on tissue culture plate.Then according to the scheme of manufacturer at Cell lysate is managed to extract for RNA.Contained by the Absorbance Ratios for reading 260nm/280nm with Nanodrop to measure RNA The ultimate density and purity of amount.

CDNA synthesis

According to the scheme of manufacturer, cDNA is synthesized from RNA using iScript Advanced (BioRad).By RNA 42 It is incubated for 30 minutes at DEG C, then inactivates enzyme 5 minutes at 85 DEG C in thermal cycler (BioRad).According to the rich of coded sequence of target transcript It spends, 5-15ml RNA sample is used in the reactant of each 20 μ l.Then by the way that 80ml is added without RNase water by 20ml cDNA Sample is directly diluted in PCR plate with 1:5, and is stored in -80 DEG C before qPCR analysis.

qPCR

Before qPCR, cDNA sample 1:5 is diluted with water.With 5 μ l cDNA, 10 μ l SsoAdvanced SYBR + 4 μ l of green+1 μ l specific primer prepares qPCR reactant without RNase water.Use GAPDH as internal reference gene.PCR is followed Ring parameter；95 DEG C thermal starting 3 minutes, 40 circulations (95 DEG C are for 10 seconds, and 60 DEG C continue 30 seconds).According to being normalized to reference base The Δ Δ Ct method of cause analyzes result.

As a result-HMOX1

H.procumbens extract and pure verbascoside dramatically increase the expression of HMOX1 in human fibroblasts.Two The result of independent experiment is summarised in following table and Fig. 4 A.

H.procumbens extract and verbascoside raise HMOX1, show both through up-regulation endogenous anti-oxidative Response and with free radical resisting protective nature.

Example 10-H.procumbens extract adjusts the ability that reactive oxygen species generate in the human body cell of UV induction

These experiments test H.procumbens extract or verbascoside inhibits UV in human skin fibroblasts to lure The ability that the hydrogen peroxide led generates.According to standard culture protocol, by fibroblast in DMEM/10%FBS culture medium Middle culture.By experimental setup in 48 orifice plates, cell confluency degree is 80-90%.With H.procumbens extract, verbascoside Or reference material (vitamin E (vit E) and resveratrol) pretreatment cell 1 hour, then UVB handles (50mJ/cm²), 37 DEG C be incubated for 2 hours.Then the ROS-Glo kit from Promega is used, analysis H is illustrated according to manufacturer₂O₂It is horizontal.Knot Fruit is shown in Fig. 5.It was found that individual H.procumbens extract and verbascoside have than be used as positive control other Well-known antioxidant (vitamin E (vit E) and resveratrol), what the higher hydrogen peroxide for inhibiting UV induction generated Ability.

The ability of example 11-H.procumbens extract adjusting collagenase activity

Collagen amount in skin is maintained by the balance between collagen synthesis and collagen decomposition, described Decomposition is that the enzyme by Collagenase, i.e., in matrix metalloproteinase (MMP) family mediates.The purpose of the research is determining H.procumbens extract or verbascoside inhibit the representative collagen from clostridium histolyticum in cell free system The ability of enzyme NB4.

In brief, Collagenase (0.3U/ml) and active material (1mM) is incubated for 15 minutes at 25 DEG C.3mM is added The synthetic peptide (structure of simulation collagen) of sequence FALGPA is used as substrate, and is measured at 345nm by spectrophotometry Obtained quantity.As a result as shown in Figure 6.H.procumbens extract inhibits Collagenase 20%.

Material

4 SERVA17454.02 500mg of Collagenase NB from clostridium histolyticum

2- furans acryloyl group-Leu-Gly-Pro-Ala (FALGPA) Sigma, Cat.F513 5-25mg

The ability of example 12- extract adjusting people's cell Inflammatory Mediators

The purpose of these measurements is determining H.procumbens extract or verbascoside, in human skin fibroblasts The inhibitory effect that the marker of inflammation that ultraviolet light or pollution mediate generates.

The level of the measuring method measurement IL-6, GM-CSF, MMP1 and MMP3.IL-6 and GM-CSF is the standard sign of inflammation Object.MMP1 and MMP3 is also the marker of inflammation, but they are especially relevant with the present invention, because they also directly participate in adjusting Skin injury.Every kind is all matrix metalloproteinase (MMP), it is a kind of responsible enzyme for decomposing collagen in skin.MMP-1 (Collagenase) and MMP-3 (stromelysin) play a major role in the degradation of dermis, and known by such as UV With the stimulation of the environment-stress of pollution.Pollutant used in these researchs includes diesel particulate object (DPM).

Keratinocyte condition (KC) culture medium

According to standard culture protocol, keratinocyte is cultivated in EpiLife/HKGS culture medium.When reaching 80- 90% when converging, and stimulates cell with UVB (50mJ/cm 2) or DPM (2 μ g/ml), or do not stimulate cell, is further cultured for 24 hours.So Cell culture medium is collected afterwards and for then stimulating fibroblast.It is believed that UV or the keratinocyte of pollution processing pass through side Secretion signal conduction causes fibroblastic response, this provides the model of stress damage outside skin.

Fibroblast stimulates and is treated with active material

According to standard culture protocol, fibroblast is cultivated in DMEM/10%FBS culture medium.Experiment is set It sets in 48 orifice plates, cell confluency degree is 80-90%.It is small with H.procumbens extract or verbascoside pretreatment cell 1 When, the KC conditioned medium of the keratinocyte from UV or pollution processing is then added.

IL-1 β (100ng/ml) is used to handle cell as positive control.Processing fibroblast 24 hours, then according to system The explanation for making quotient determines Markers of inflammation (MMP1, MMP3, IL-6 in fibroblast culture medium by ELISA or Luminex And GM-SCF) level.

As a result

H.procumbens extract inhibit fibroblast in every kind of UV stimulation Markers of inflammation MMP-1, MMP-3, The generation of IL-6 and GM-CSF (referring to Fig. 7 A).H.procumbens extract completely inhibits the generation of MMP-1 under UV stress (referring to Fig. 7 B), and the MMP-1 for inhibiting pollution to induce generates 75% (referring to Fig. 7 C).

Material

EpiLife keratinocyte culture medium

Example 13-H.probumbens extract adjusts the ability of people's cell gene expression after UV stimulation

The purpose of these measurements is determining H.probumbens extract or verbascoside to the skin for being exposed to UV stress The influence of gene expression in model.Fibroblast is pre-processed with H.probumbens extract, then such as that in embodiment 11 Sample is exposed to (or not stimulating) cell culture medium of UV stimulation.As carried out in embodiment 9, RNA is extracted and cDNA is synthesized, then QPCR is carried out to determine gene expression dose.Measure collagen I, collagen III, collagen IV and fibrillin Expression.

Collagen is the constitutive protein of extracellular matrix, is made of 29 kinds of different types, and collagen I is existed in corium Most important protein, about 85-90%.Collagen III accounts for about the 10-15% of extracellular matrix.Both protein exist It assigns skin texture integrality aspect to play a significant role, in light aging/UV damage skin, collagen I and III are horizontal All significantly reduce (Talwar HS 1995).Collagen IV is present at dermal epidermal junction in (DEJ), facilitates shape At the cohesive force between corium and epidermis.According to description, the reduction of this protein will lead to deeper wrinkle, and lead to skin bullet The forfeiture of property.The skin of light aging is characterized in that the solution of tropoelastin and its relevant microfibril component fibrillin Body.In addition, observing the fibrillin consumption at dermal epidermal junction in the non-photoaging skin of light aging vs.Lead to glue The variation that former protein I, collagen III, collagen IV and fibrillin decompose is the master of the final sign of aging skin Want reason.Therefore, new generate of these protein repairs since skin injury caused by lasting external stress is help Important.

As shown in figure 8, pre-processing fibroblast with H.procumbens extract, it is subsequently exposed to UV- culture medium item Part can dramatically increase the gene expression of collagen I, III, IV and fibrillin relative to control.H.procumbens Up-regulation of the extract to these genes shows that it will be helpful to repair skin from that may send out in the presence of lasting environment-stress Raw damage.

Example 14-H.procumbens extract adjusts the ability that protein generates in people's cell after UV stimulation

Further explore the discovery of H.procumbens extract gene expression of stimulation collagen I after UV stress (referring to Fig. 8), to determine whether the expression of collagen I gene is converted into the relevant protein product of biology.Such as example 13, use Active material pre-processes human fibroblasts, and is stimulated with UV.Then it (is seen below using anti-collagen protein antibodies with immunofluorescence Be described in detail) observation cell in collagen I protein presence.As shown in figure 9, UV stress under, H.procumbens extract and Verbascoside dramatically increases the generation of collagen I.

Immunofluorescence dyeing

After handling cell, they are then fixed 5 minutes with 100% methanol before immunostaining.By in PBS 10% lowlenthal serum closes 1 hour to inhibit unspecific staining.The measurement of collagen I be by with primary antibody, i.e. rat is anti- People's procollagen I (1:100 dilution) is incubated for 1 hour in PBS and 1% lowlenthal serum, followed by and secondary antibody, i.e. goat resists big Mouse Alex 488 (1:100 dilution) is incubated for 1 hour in PBS and 1% lowlenthal serum.It is redyed using the DAPI of 10ng/ml So that nucleus visualizes.It is carried out by using the cell imaging in 3 software of Cytation (Biotek)/image statistics tool Image analysis obtains mark intensity/cell ratio.

Material

The ability of embodiment 15-H.procumbens extract adjusting the elderly's cellular gene expression

Purposes of these measurements be determining H.procumbens extract or verbascoside to from old contributor at Gene expression in fibrocyte influence (three contributors of test: 54,56 and 74 years old).With H.procumbens extract (or untreated) handles fibroblast, and the culture in normal fibroblast culture medium (DMEM+10%FBS).Therefore, This is the capability model of H.procumbens extract, influence cell due to time aging rather than external stress such as UV and Caused variation.If embodiment 9 carries out RNA extraction and cDNA synthesis, qPCR is carried out then to determine gene expression dose.Measurement The expression of collagen I, collagen III, collagen IV and fibrillin.

As shown in Figure 10, it is expressed more with the pretreated cell of H.probumbens extract than the cell that verbascoside is handled High-caliber all four genes.The cell of H.profumbens processing also expresses higher levels of collagen than untreated control Protein I, collagen III, collagen IV, although only collagen I and collagen IV are dramatically increased.Due to these biologies Marker is reduced in time ageing process, these discoveries on the cell obtained from the elderly show that H.profumbens is mentioned Take object that can play the role of helping to rebuild/reinforce dermal layer, this facilitates to mitigate visible aging sign immediately.

Embodiment 16-H.profumbens extract adjusts the ability that protein generates in old people's cell

Further exploring H.profumbens extract stimulates the discovery of the gene expression of collagen I in old cell (referring to Figure 10), to determine that collagen I gene expresses the biology related protein product whether being converted into such cell. And as embodiment 15, the fibroblast of two old contributors (49 and 54 years old) is come from active matter pretreatment, and sudden and violent It is exposed to the KC culture medium not stimulated (referring to embodiment 11).It is as horizontal in passed through immunofluorescence assay collagen I in embodiment 14. As a result as shown in figure 11.It clearly demonstrates, only H.procumbens extract significantly increases the production of collagen I protein It is raw.Which demonstrate embodiments 15 as a result, i.e. H.procumbens extract can stimulate skin fibroblasts in aging The relevant collagen I protein of biology is generated in Skin Cell.

The ability of embodiment 17-H.procumbens extract prevention saccharification

Saccharification is damage of the glycan molecule to protein.It is a kind of process to play an important role in skin aging, wherein The cell combination of glycan molecule and bound fat and protein.With that is, protein is destroyed and changes the shape of cell, eventually lead to The destruction of cell metabolism.Collagen is a kind of protein for being particularly susceptible to saccharification, shows as wrinkling if impaired Line and face line.It is tested to test H.procumbens extract in cell free system and human fibroblasts and inhibit saccharification Ability.The result shows that H.procumbens extract is able to suppress saccharification 56% (Figure 12 A) and cell in cell free system In saccharification 35% (Figure 12 B).This shows that H.procumbens extract can be used for preventing or reversing ageing-related skin Damage.

Cell-less measurement

It is being related to the non-enzymatic reaction of protein (such as collagen) and sugar, is i.e. during Maillard reacts, is forming evening Phase glycation end product (AGE).Anti- sugar can be monitored by measuring the fluorescence of the AGE formed by the saccharification of model protein and sugar Change activity.The measuring method measures saccharification of the sugared D-ribose to bovine serum albumin(BSA) (BSA).It is slow in the sodium phosphate of pH7.4 BSA (10mg/mL) and D-ribose (0.5M) in fliud flushing with H.probumbens extract or rutin (positive control) processing or (negative control) is not handled, is then incubated for 24 hours at 37 DEG C.Then with the micro- disk reader of spectrophotometer in λ exc 370nm Fluorescence AGE is measured with λ em 440nm.

Material

Raji cell assay Raji (immunofluorescence)

According to standard culture protocol, fibroblast is cultivated in DMEM/10%FBS culture medium.Culture is arrived The fibroblast that 50-70% converges 0.5mM glyoxal handles 48 hours to induce saccharification.At the unused glyoxal of control cell Reason.After being exposed to glyoxal, culture medium is replaced with the fresh culture containing H.procumbens extract or verbascoside And it is further cultured for 72 hours.It is handled cell 72 hours with melbine (1mM), the positive control as the measurement.

After handling cell, they are then fixed 5 minutes with 100% methanol before immunostaining.By in PBS 10% lowlenthal serum closes 1 hour to inhibit unspecific staining.Measure carboxymethyl lysin (CML), be due to saccharification and The adduct formed on protein, measurement be by with primary antibody i.e. mouse anti human CML (3 μ g/ml) in PBS and 1% lowlenthal serum It is middle to be incubated for 1 hour, then 1 incubated in PBS and 1% lowlenthal serum with secondary antibody, that is, goat anti-mouse Alex 488 (1:100 dilution) Educate hour.It is redyed using the DAPI of 10ng/ml so that nucleus visualizes.By using 3 software of Cytation (Biotek) cell imaging/image statistics tool in carries out image analysis, obtains mark intensity/cell ratio.

Material

Embodiment 18-H.procumbens extract adjusts the ability of people's cell epiderm gene expression after UV stimulation

The purpose of these measurements is determining H.procumbens extract or verbascoside to the cutin for being subsequently exposed to UV Form the influence of the gene expression in cell.According to standard culture protocol, angle is cultivated in EpiLife/HKGS culture medium Matter forms cell, and handles (or not handling) with H.probumbens extract or verbasocoside.When reaching 80-90% When converging, with UVB (50mJ/cm²) stimulate cell and be further cultured for 24 hours.If embodiment 9 carries out RNA extraction and cDNA synthesis, Then qPCR is carried out to determine gene expression dose.Determine laminin V, aquaporin 3, involurin and keratin Expression.

The elderly's cutaneous manifestations go out the morphological differences of epidermis and corium.The some variations occurred in epidermis include: epidermis thickness Degree reduces, and dermal epidermal junction flattens to be reduced with keratinocyte proliferation.Laminin is a kind of glycoprotein, is being maintained Indispensable role is played in skin texture.They largely exist in the basilar memebrane of skin, and for maintaining corium-table Skin connection is extremely important.The bioactivity of laminin helps to influence various cell functions, such as cell differentiation, migration, glues It echos skin regeneration-maintenance and cures mechanism necessary to skin.During this investigation it turned out, we attempt to measure H.procumbens Or influence of the verbascoside to laminin V subunit γ (gene encodes LAMC2).In the presence of UV, H.procumbens It significantly have stimulated the generation of laminin, this shows that it may play potential invigoration effect to skin-epidermal junction, increases The compact degree and smoothness of strong skin.

Aquaporin is the protein for promoting water transdermal delivery.Aquaporin 3 (AQP3) aquaporin is old by the time Change the strong influence with chronic UV irradiation.The reduction of AQP3 level is observed in the elderly, this can be explained as the age increases Long and generation dry skin.Therefore, AQP is the key protein matter target for improving the drying of UV induction.In our study, It is observed that H.procumbens stimulates the generation of AQP3 in the epidermal keratinocytes of ultraviolet light stimulation.

Keratin is a kind of protein, constitutes epidermal cornified coating.It has knot similar with procollagen Structure tissue-is the important component of cuticula.The expression reduction of keratin is considered as leading in atopic dermatitis patients epidermis The reason of causing the epidermal barrier defect occurred in these individuals (Wu Z et al.2009).A nearest research is also shown that In patient with chronic hand eczema, keratin is significantly lowered, and it is important that this has affirmed that keratin rises in skin barrier function The hypothesis (Molin S et al.2015) of effect.In our study, it is observed that at H.procumbens extract The keratinocyte of reason significantly stimulates keratin gene to express.This discovery shows that H.procumbens extract may have Help maintain skin barrier function under UV stress conditions.

Involurin is Keratinocyte differentiation and mature marker.The report phase that involurin is expressed after UV irradiation Mutual contradiction, involurin expression increases (Bertrand-Vallery 2010) after some authors report UV irradiation, other authors are then Think that involurin expression reduces (Mammone et al.2000).The difference of these results seems to depend on the source UVB and life Object model.Nevertheless, in our study, it is observed that compared with the cell individually stimulated with ultraviolet light, The generation for the involurin in keratinocyte that H.procumbens extract stimulates ultraviolet light to irradiate.The discovery with it is above-mentioned The effect seen on other epidermis biomarkers together, further supports argument-H.prromumbens extract has to adjust The great ability of important epidermis marker can help improve situation, aquation and function that UV coerces lower skin barrier.

As shown in figure 13, relative to untreated cell, lead to laminin with H.probumbens extract-treated V, the gene expression of each in aquaporin 3, involurin and keratin significantly increases.This shows that the extract is available In prevention or ageing-related skin injury is reversed, such as damage caused by being coerced by outside.

Example 19-H.probumbens extract adjusts the expression of people's cell epiderm gene in the case where no UV is stimulated Ability

The purpose of these measurements is gene in determining H.probumbens extract or verbascoside Human Keratinocytes The influence of expression, the keratinocyte are not exposed to UV or other external stress.Therefore, this is the ability mould of extract Type, influences as time aging rather than cellular change caused by (such as UV) is coerced in outside.According to standard cell culture journey Sequence cultivates keratinocyte in EpiLife/HKGS culture medium, and with H.probumbens extract or Verbasocoside handles (or not handling).If embodiment 9 carries out RNA extraction and cDNA synthesis, qPCR is carried out then with determination Gene expression dose.Determine the expression of aquaporin 3.As shown in figure 14, relative to untreated cell, H.procumbens extract and verbascoside processing cause AQP3 expression to dramatically increase.

This shows that the extract can be used for preventing or reversing ageing-related skin injury.

The skin lightening characteristic of embodiment 20-H.procumbens extract

The reconstruction epidermis of the melanocyte containing NHEM- melanin is used to determine H.procumbens under study for action Whether extract or verbascoside are able to suppress melanin production.Genetic analysis is shown, in the case where no UV irradiation, H.procumbens extract and verbascoside reduce the table of WNT16 (Wingless type MMTV integration site family, member 16) Up to (referring to the result in following table).

Gene Name	H.procumbens	Verbascoside
			WNT16	- 6.49 (p=0.002)	- 3.72 (p=0.019)

MITF participates in the key transcription factor of differentiation, growth and the survival of melanocyte, and it is heavy to adjust more than 25 pigments Gene, including B16 cell key enzyme (i.e. tyrosinase, tyrosine-related proteins -1, dopachrome tautomerase), Melanosome structure component, and it is mature and along the relevant protein of melanocyte dendron transport to it.

Control of the MITF by main melanin production approach, α-melanin including stimulating -1 receptor of hormone/melanocortin Cell, stem cell factor/c-Kit, Endothelin/protein kinase C and Wnt/ beta-catenin approach.Itself by transcriptional level and Specific phosphorylation adjusting (Levy, Khaled, &Fisher, 2006；Shibahara et al.,2001).

In Wnt/ β catenin approach, key control is the level of intracellular beta-catenin.Believe Wnt is not present In the case where number, β catenin is successively by glycogen synthase kinase-3beta (GSK-3 β) phosphorylation, the then β catenin of phosphorylation It is identified by ubiquitin ligase complex, is degraded by ubiquitin dependent mechanism.On the contrary, the activation negative regulator GSK-3 of Wnt approach β leads to the accumulation of cytoplasm β catenin, the β catenin indexing to nucleus, and with the T cell factor (TCF) and lymph Cell enhancement factor -1 (LEF) forms compound, raises the expression of MITF.Therefore, the approach of the Wnt/ β catenin is sharp It lives and passes through the active up-regulation of MITF, stimulation melanin generation (Bellei, Pitisci, Catrical à, Larue , &Picardo, 2011；Cadigan,2008；Yaar&Park,2012).

With the significant decrease of the WNT ligand expression in the cell of H.procumbens extract-treated, it may indicate that and pass through Whitening effect may be had by slowing down Wnt/ β catenin melanin production approach.

Experimental method is as follows:

Normal human epidermal's melanocyte-melanin (NHEMs-DP will be contained；Photosensitive type V-VI) recombinant human epidermal In the liquid-vapor interface of serum free medium, in 37 DEG C of humid atmosphere, CO2 5% cultivates 14 days (differentiation completely).With Before H.probumbens extract-treated, epidermis is transferred in 12 orifice plates, triplicate culture (n=3).

For the experiment of UV irradiation, H.prompumbens extract is applied 18 hours in the culture medium of differentiation epidermis. Then tissue is placed in PBS, uses 4J/cm²UVA and 200mJ/cm²UVB (± 15 minutes) irradiate, with photophore Biosun (Vilbert Lourmat, FR) irradiation.After irradiation, epidermis is incubated for 6 hours again together with extract.

When processing terminate, extracts total serum IgE and pass through spectrophotometry and capillary electrophoresis analysis sample integrity.Then CDNA is synthesized from mRNA by reverse transcription.Changes in gene expression is obtained by qPCR.

Total RNAs extraction and cDNA synthesis

Total serum IgE is extracted using Qiagen RNeasy kit.After processing, cell is rinsed with PBS and is split in buffer Solution, while epidermis being directly immersed in lysis buffer and (carrying out three repetitions for every kind of condition to cultivate).According to supplier's It is recommended that extracting RNA from cell and tissue.The RNA of collection is stored in -80 DEG C.According to the explanation of manufacturer, high capacity is used RNA-to-cDNA kit (Applied Biosystems) carries out reverse transcription from 2 μ g total serum IgEs.Then cDNA is stored in -20 ℃。

Quantitative PCR

Different probes is by Applied Biosystems (MITF-Hs01117294_m1, WNT16-Hs00365138_ M1 it) manufactures.Using the quick real-time system of 7900HT (Applied Biosystems), real-time qPCR quantifies the table of particular target It reaches.In short, by 4 μ l ADNc (4ng) and 10 μ l TaqManFast Universal Master Mix (Applied Biosystems), 1 μ l TaqManGene Expression Assay and 5 μ l is mixed without RNAse water.In order to make result normalizing Change, gene expression is normalized to house-keeping gene β2-microglobulin B2M.Thermocycling program is with an incubation step (at 50 DEG C Lower 2 minutes), then carry out first time denaturing step (at 95 DEG C 10 minutes).Then amplification scheme carries out 40 circulations (95 again At DEG C 1 minute at 15 seconds and 60 DEG C).

Obtain the threshold cycle number (Ct) of each gene.Use SDS RQ Manager software (v1.2.1, Applied Biosystems destination file) is exported from real-time qPCR equipment, and uses the relative quantification for being designed to carry out gene expression DataAssist software (v3.0, Applied Biosystems) analyzes data, and Ct (Ct) method is compared in the software use (Pfaffl, 2001&Livak and Schmittgen, 2001) and combine statistical analysis.For epidermis, by data and as pipe The reference condition (EtOH 1%) of the β2-microglobulin (B2M) of family's gene is compared.As detection threshold value or Ct cutoff value Maximum allowable Ct value is fixed as 36 circulations.

The further skin lightening characteristic of embodiment 21-H.procumbens extract

In the case where existing and challenging there is no UV, the people's epidermis and photosensitive type III-IV melanocyte of reconstruction are used It is studied, to test the whitening properties of H.procumbens extract and verbascoside.Kojic acid as positive control reduces Melanin content (exist and UV is not present all) in epidermis, demonstrates test and analysis method.With EtOH negative control phase Than, there is no UV challenge (Figure 15 A) and there are UV challenge (Figure 15 B) the two in, with H.probumbens extract or Mao Rui The epidermis of flower glycosides processing all observes the significant decrease of melanin deposition.The conclusion of example 19 is supported in the discovery, and is shown H.procumbens extract and verbascoside have the property that can directly result in skin lightening effects.

Experimental method is as follows:

0th day to the 4th day, from the liquid-vapor interface reconstruct of the growth medium containing BPE 100% (ox pituitary extract) People's epidermis.Incubation is then proceeded to, a day growth medium contains 50%BPE from the 4th day to the 7th, day training from the 7th day to the 14th It supports base and is free of BPE.

From the 4th day to the 14th day (n=3), H.procumbens extract is applied 10 days in total in the medium, 7 days to the 11st day update culture mediums, and there is α-MSH (1 μM) and UVA/B challenge (UVA 1J/cm as follows², UVB 50mJ/cm²).(3- (4,5- dimethylthiazole -2- base) -5- (3- carboxyl-methoxyphenyl) -2- (4- sulfo group is measured by MTS Phenyl) -2H- tetrazole) check that there is no cytotoxicities.

It is to brighten reference compound to verify analysis by the effect parallel analysis of kojic acid (250 μM).As preparing culture medium In the solvent of H.procumbens extract be ethyl alcohol that respective concentration is 0.1%, parallel testing.At the 14th day, by tissue From being taken out in its insert and immerse in soluble extraction solution (Perkin Elmer) and heated 1 hour at 80 DEG C.? The optical density of supernatant is measured at 490nm, and passes through the standard curve ratio with synthesis of melanin (Sigma Aldrich M8631) Relatively determine melanin content.

Annex 1

Specific primer and other materials (example 9,13,15,18,19) for qPCR

Claims (22)

1.H.procumbens cell line, is generated by following steps:

It a. will be in the fluid nutrient medium of H.probumbens cell inoculation to suitable suspension cell growth；

B. the cell obtained by culture under conditions of being suitable for suspension cell growth；

And optionally:

C. by periodically dividing any close agglomerate, the cell is resuspended in fresh culture or is added fresh training Base is supported, and remains suitable for the condition of suspension cell continued propagation, come the culture gained cell that maintains to suspend；Or

D. gained cell is saved as into cell line, the optional sample by cell described in freezen protective.

2. cell line as described in claim 1, wherein cell used in step a is as the material sample from callus It provides, the callus is generated by following steps:

I) to generate rice shoot outside the sterilizing seed body of H.procumbens；

Ii) from being extracted in the rice shoot between at least one stipes；

Iii it) is formed by cultivating the internode evoked callus on solid medium, the culture medium and condition of culture are suitable It is generated together in callus；

Iv the callus obtained by step c) is cultivated on solid medium, the culture medium and condition of culture are suitable for callus Tissue growth；

And optionally:

V) it by the way that periodically the health part of callus is transferred in fresh solid medium, and maintains to be suitble to callus The condition of growth, come the callus for maintaining incubation step d to obtain；Or

Vi resulting callus) is stored by the sample of the healthy part of the freezing callus,

Preferably, wherein Callus material and culture medium ratio between 1:2 and 1:10w/w.

3. described in any item cell lines as in claims 1 and 2, wherein the culture medium of step a and/or c is to be supplemented at least A kind of basic element of cell division, at least one auxin, and MS the or GB5 culture medium of sugar, the auxin is not 2,4- Dichlorophenoxy Guanidine-acetic acid (2,4-D), wherein the basic element of cell division is preferably 6-benzyl aminopurine (BAP), and auxin is preferably α-naphthylacetic acid (NAA), sugar is preferably sucrose.

4. cell line as claimed in claim 3, the wherein basic element of cell division in the culture medium of step a and/or c: the ratio of auxin Example is each independently 2:1 to 20:1.

5. the cell line as described in any one of claim 3 and 4, wherein the culture medium in step a is supplemented with 5mg/ml BAP, Culture medium in 1mg/ml NAA and 30g/l sucrose and/or step c is supplemented with 2mg/ml BAP, 0.1mg/ml NAA and 30g/l sucrose.

6. cell line as described in any one of the preceding claims, wherein the condition in step b and c includes in the dark, about It 22 to 28 DEG C, is cultivated with orbital oscillation or on wave reactor, and/or wherein in step c every about the primary week of progress in 2-3 weeks Phase property is resuspended or addition culture medium.

7. the cell line as described in any one of aforementioned claim, wherein the culture medium has magnanimity as shown in the table and micro- Secondary element and vitamin combination:

Table A:

Or table B:

8. a kind of H.procumbens cell line, is preserved in NCIMB preservation mechanism, accession number is NCIMB 42467.

9. a Plant Extracts, according to the method for extracting one or more benzyl carbinol glycosides from the cell of H.procumbens Preparation, this method includes the sample for obtaining the cell in the medium, and generating from the cell includes one or more benzene second The plant extracts of alcohol glycosides, and optionally one or more benzyl carbinol glycosides, preferably feltwort are separated from the plant extracts Glycosides, optionally wherein the yield of benzyl carbinol glycoside is at least 5%w/w in extract, and the yield of verbascoside is at least 3%w/w, And the yield of 2-O- acetyl group acteoside is at least 1.5%w/w of extract.

10. plant extracts as claimed in claim 9, wherein the cell sample is produced from H.procumbens cell It is obtained in the culture medium that the method for raw secondary metabolite obtains, this method includes in the condition for being suitable for the cell growth Under, suspend culture H.procumbens cell first in the medium, then change condition of culture and/or culture medium, so that it Be suitable for by the cell generate secondary metabolite.

11. plant extracts as claimed in claim 10, wherein the culture that suspends first carries out in the following manner:

It a. will be in the fluid nutrient medium of H.probumbens cell inoculation to suitable suspension cell growth；

B. the cell obtained by culture under conditions of being suitable for suspension cell growth；

And optionally:

C. by periodically dividing any close agglomerate, the cell is resuspended in fresh culture or is added fresh training Base is supported, and remains adapted to the condition of suspension cell continued propagation, come the culture gained cell that maintains to suspend；Or

D. it is stored obtained cell as cell line, alternately through the sample for freezing the cell,

And the change carries out after step b or step c, and/or wherein the cell of the culture that suspends first comes From the sample of the cell line of any one of claims 1 to 8.

12. plant extracts described in any one of claim 9-11, wherein the plant extracts is raw by following steps It produces:

A. cellular material is filtered out from the sample, is rinsed with water, it is then dry between about 0 DEG C to about 30 DEG C；

B. obtained dry matter is suspended in water-alcohol solution；

C. gained suspension is exposed to ultrasonic wave about 1 hour to about 24 hours at room temperature, it then at room temperature will be described outstanding Supernatant liquid impregnates about 1 hour to about 48 hours；With

D. filtering gained suspension is to remove solid；And optionally

E. extract obtained to be concentrated by evaporating residual solvent between about 0 DEG C to about 30 DEG C.

13. plant extracts described in any one of claim 9-12, in the plant extracts that wherein step d or e are obtained:

The yield of benzyl carbinol glycoside is at least 1.5%, at least 2%, at least 3%, at least 4% or preferably at least the 5% of extract w/w；And/or

The yield of verbascoside is at least 1%, at least 2% or preferably at least 3%w/w of extract；And/or

The yield of 2-O- acetyl group acteoside is at least 0.5%, at least 1% or preferably at least 1.5%w/w of extract.

14. a kind of composition, it includes the plant extracts described in any one of claim 9-13, and being optionally adapted for Cosmetic, drug, nutriment, food and/or animal feed applications carrier.

15. a kind for the treatment of is reduced or at least one sign of prevention individual's skin aging or at least one ageing-related skin The method that skin damages sign, this method include giving the described in any item plants of claim 9-13 of the individual effective dose to mention Take composition described in object or claim 14.

16. composition described in plant extracts described in any one of claim 9-13 or claim 14 for treat, Mitigate or prevent the purposes of disease or illness.

17. composition described in plant extracts described in any one of claim 9-13 or claim 14 is being treated, is being subtracted Less or the purposes of prevention at least one of individual skin aging or the sign of skin injury, the skin aging or skin injury Sign is related to one or more of: pigmentation, collagen expression, inflammation, saccharification, barrier function destroys or its is any Combination.

18. composition described in plant extracts described in any one of claim 9-13 or claim 14 is in treatment UV And/or pollution induction skin injury such as inflammation, pigmentation, collagen expression, barrier function destroy in purposes.

19. composition described in plant extracts described in any one of claim 9 to 13 or claim 14 is preparing medicine Purposes in object.

20. purposes described in method of claim 15 or claim 16 or 17, wherein the skin injury be due to Skin is exposed to abiotic oxidation stress, is optionally caused by being exposed to UV radiation or pollution.

21. a kind for the treatment of reduces or prevents at least one signs of skin aging of individual or at least one related with skin aging The beauty method of skin injury sign, this method include giving described in any one of claim 9-13 of the individual effective dose Plant extracts or claim 14 described in composition.

22. a kind for the treatment of reduces or prevents at least one signs of skin aging of individual or at least one related with skin aging The beauty method of skin injury sign, this method include any one of the claim 9 to 13 for giving individual effective dose institute Composition described in the plant extracts or claim 14 stated, wherein the skin injury be exposed to due to skin it is abiotic Oxidative stress is optionally caused by being exposed to UV radiation or pollution.