CN100352825C - Extractive form active ingredient of bitter bamboo, prepartion method and application - Google Patents

Abstract

The present invention provides an extract of an effective component from bitter bamboo, a preparation method thereof and an application thereof in cosmetics and pharmacy. The main effective component of the extract is Taxiphyllin (2R-beta-D-glucopyranosyl-2-p-Hydroxybenzylcyanide); the extract has favorable anti-oxidative performance and obvious tyrosinase inhibitory activity at the same time; the extract can be used for cosmetics for whitening skin color, or pigment increasing dermatosis drugs.

Description

The purposes of extract of active ingredient in hairy bamboo

Technical field

The present invention relates to subjects such as Natural Medicine Chemistry and biological chemistry, what be specifically related to extract from the herbal medicine bitter bamboo has the application of the inhibiting effective constituent of tyrosinase activity in makeup, medicine.

Background technology

Tyrosine oxidase is the main rate-limiting enzyme in the melanocyte biosynthetic pathway.Most of materials of skin depigmentation that can make all block the generation of skin melanocyte by suppressing this enzymic activity, belong to tyrosine oxidase inhibition type Solu-Eze (Depigmenting agent).In recent years, the tyrosinase inhibitor of separating natural has obtained people's common concern from microorganism and plant amedica.It not only is used for the clinical treatment that pigment increases dermatoses (chloasma, freckle etc.), and can be used for makeup the colour of skin is brightened.

In general, can suppress this enzymic activity with the similar single phenolic compound quinhydrones of the substrate of tyrosine oxidase, many single phenol or polyphenolic substances with similar structure of functional groups also can the restraint of tyrosinase activity.But the external preparation unstable chemcial property of quinhydrones and other phenolic compound, oxidation stain easily also has this class material to cause the report of the permanent hickie of skin in addition, has limited the development and application of this compounds.Vitamins C and arbutin are additives relatively more commonly used in the current skin-lightening cosmetic.But vitamins C equally also is very unstable, can't directly add in the product, adopts ascorbic derivative (as ascorbic phosphoric acid salt etc.) usually.And arbutin finds also that in research in recent years its restraint of tyrosinase activity is not very stable.Therefore, seek the focus that activity is high, safe tyrosinase inhibitor is still present research.

This has the existing many-sided development and use of plant of affluent resources at home and abroad bamboo.Aspect cosmetics production, except raw material as some makeup subsidiary productss, series products as the packing box of the handle of lash curler, eye-lash and blush brush etc. and some makeup, bamboo also is used as effective additive of natural cosmetics and functional cosmetics with the form of bamboo class extract, have preserve moisture, whiten, multiple function such as anti-ageing, anti-inflammatory, cleaning and hair care.For example, gondola Erbasol company extracts from bamboo shoots and obtains alpha hydroxy acid and have the cell of renewal, keep the effect of pH equilibrated, is applied to the production of moisture-keeping shower cream; The extract that Japan NOEVIA company obtains from a kind of bamboo that blooms can effectively suppress the generation of the T3 isomerase of tyrosine oxidase in the B16 melanophore, is applied to the novel whitening cosmetic development.The domestic Folium Bambosae flavone product of then being developed by the Sheng Shi biological products company limited of Anji, Zhejiang can be used as the natural biological antioxidant and is applied in the multiple industries such as medicine, health care and makeup.

Less relatively to the research of bitter bamboo (Pleioblastus amarus Keng (Keng) f. (Gramineae)) at present.Shoot ofBitter Bamboo has heat-clearing in the Chinese medicine record, dehumidifying, tomorrow, treatment order pain, aphtha, functions such as burn.

Summary of the invention

The objective of the invention is to, the above-mentioned purposes of Chinese herbal medicine extract in makeup and pharmaceutical industries as tyrosinase inhibitor is provided.

Provided by the invention from bitter bamboo (Pleioblastus amarus Keng (Keng) f. (Gramineae)), particularly in the extract for preparing in the shoot ofBitter Bamboo, main effective constituent is Taxiphyllin, 2R-β-D-glucopyranosyl-2-p-hydroxybenzylcyanide, and molecular formula is C ₁₄H ₁₇NO ₇(molecular weight is 311) can account for more than 98% of extract, and structure is:

Extract of the present invention is separated purification can obtain white needle (chloroform-methanol), water-soluble, methyl alcohol.Point sample launches on prefabricated silica-gel plate, and with the iodine colour developing, spot colors becomes green gradually by yellow, becomes blueness again, and places in air, and color does not disappear; When developping agent is chloroform-methanol (2: 1); During ethyl acetate-methyl alcohol (1: 3) benzene-methyl alcohol (2: 1), R _fValue is respectively 0.56,0.17,0.42.Through electrospray ionization mass spectrometry (ESI-MS), high resolution electron impact mass spectra (HREI-MS), ¹³C NMR and ¹Data such as H NMR show that the molecular formula of this compound is C ₁₄H ₁₇NO ₇Ultimate analysis shows that this compound contains an itrile group. ¹H-NMR (400MHZ, methyl-sulphoxide-d ₆) show, this compound exist the benzene ring structure that a contraposition replaces (δ 7.32 and δ 6.82, J=8.6Hz, 4H) and hydroxyl (δ 9.84, and δ 5.23, and δ 5.05, δ 4.98 and δ 4.55,5H). ¹H-NMR hydrogen spectrum data and the Cyanogenic glucoside taxiphyllin data consistent of having delivered. ¹³C-NMR carbon spectrum data are as shown in table 1.

Table 1 bitter bamboo extract ¹³C-NMR carbon spectrum data

C	The bitter bamboo extract
		CN
1 2 3 4 5 6 7 1’ 2’ 3’ 4’ 5’ 6’	118.5 123.0 128.9 115.2 157.8 115.2 128.9 65.9 99.7 72.9 76.3 69.8 77.1 61.1

The concrete preparation method of Chinese herbal medicine extract of the present invention is:

Method one:

1. shoot ofBitter Bamboo obtains medicinal extract with 50～90% times of volume of ethanol refluxing extraction 2 times;

Shoot ofBitter Bamboo medicinal extract be dissolved in water (about 5～10 times of volumes) centrifugal, filter;

3. filtrate is with propyl carbinol or chloroform, the extraction of ethyl acetate equal solvent;

4. the medicinal extract silicagel column is used the chloroform-methanol wash-out;

5. recrystallization.

Method two:

1. shoot ofBitter Bamboo obtains medicinal extract with 50～90% alcohol reflux of 10 times of volumes 2 times;

2. shoot ofBitter Bamboo medicinal extract be dissolved in water (about 5～10 times of volumes);

3. the aqueous solution adds 95% ethanol sedimentation, and placement is spent the night;

4. filtrate thickening filtration, and with chloroform, sherwood oil or etc. the propyl carbinol solvent extraction;

5. use absorption with macroporous adsorbent resin, with 30～60% ethanol elutions;

6. silica gel column chromatography is used the chloroform-methanol gradient elution;

7. with recrystallizations such as methyl alcohol or acetone, dehydrated alcohols.

Method three:

1. shoot ofBitter Bamboo obtains medicinal extract with 50～90% alcohol reflux of 10 times of volumes 2 times;

Shoot ofBitter Bamboo medicinal extract be dissolved in water (about 8～12 times of volumes) centrifugal, filter;

3. filtrate concentrates back macroporous resin wash-out, alcohol-water gradient elution a little;

4. elutriant separates with silica gel column chromatography, and solvent for use is chloroform-methanol (2: 1～5: 1);

5. recrystallization.

Extract of active ingredient in hairy bamboo of the present invention has good antioxidant activity, has tangible tyrosinase inhibitory activity simultaneously.

Extract of the present invention and vitamins C and arbutin are made tyrosinase activity suppress contrast under different concns, the results are shown in Figure 1, when reactant concn was higher, extract of active ingredient in hairy bamboo (YF4332) and vitamins C (Vc) activity level were approaching substantially, are higher than arbutin as can be seen; In low strength range, vitamins C and arbutin are active to descend rapidly, and extract of active ingredient in hairy bamboo (YF4332) descends slowly.Therefore, the sphere of action of extract of active ingredient in hairy bamboo (YF4332) is more wide in range than vitamins C and arbutin, helps the reduction of formulation cost.

From foregoing as can be known, extract of active ingredient in hairy bamboo of the present invention extremely is suitable for the makeup that the colour of skin brightens, or pigment increases the dermatoses medicine.

Description of drawings

The accompanying drawing that comprises among the application is a component part of specification sheets, and accompanying drawing and specification sheets and claims one are used from explanation flesh and blood of the present invention, are used for understanding better the present invention.In the accompanying drawing:

Fig. 1 is extract of active ingredient in hairy bamboo of the present invention (YF4332) and vitamins C and the tyrosinase activity inhibition contrast synoptic diagram of arbutin under different concns.

Fig. 2 be in melanophore arbutin and extract of active ingredient in hairy bamboo of the present invention (YF4332) tyrosinase inhibitory activity and concentration concern synoptic diagram.

Fig. 3 is that arbutin and extract of active ingredient in hairy bamboo of the present invention (YF4332) melanocyte generate the synoptic diagram that concerns that suppresses with concentration in melanophore.

Embodiment

The present invention is further elaborated below in conjunction with embodiment, but these embodiment do not constitute any restriction to the present invention.

Embodiment 1

Be prepared according to separation method one: the ethanol extract 882g of shoot ofBitter Bamboo, with 5～10 times of water gaging dissolution precipitations, filtrate filtered is with extractions such as the solvent of 2～5 times of amounts such as chloroform, ethyl acetate, propyl carbinols, extraction liquid is through silica gel column chromatography, with chloroform-methanol (10: 1～1: 1) wash-out, get 1812.7mg bitter bamboo extract behind the recrystallization.

Embodiment 2

Be prepared according to separation method two: the ethanol extract 700g of shoot ofBitter Bamboo, with 5～10 times of water dissolution, the 95% ethanol placement that adds 2～4 times is spent the night.Filter, use propyl carbinol or chloroform, the extraction of sherwood oil equal solvent behind the filtrate thickening filtration.Extraction liquid is through the dissolve with methanol after-filtration, and macroporous adsorbent resin is extremely colourless with 30～60% ethanol elution on the filtrate, gets fraction 26g.Behind dissolve with methanol, go up silica gel column chromatography, use the chloroform-methanol gradient elution, behind recrystallizations such as methyl alcohol or acetone, dehydrated alcohol, get white solid, i.e. extract of active ingredient in hairy bamboo 0.68g.

In the method, activeconstituents extract of active ingredient in hairy bamboo (YF4332) yield is 1 ‰; Its purity can reach 99.8%.

Embodiment 3

Make each according to separation method three: the ethanol extract 500g of shoot ofBitter Bamboo, the water dissolution, the precipitation that add 8～12 times of volumes, filtrate after the centrifuging is concentrated slightly, last macroporous adsorbent resin alcohol-water gradient elution gets fraction 14.8g (can at 231.8nm detect extract of active ingredient in hairy bamboo (YF4332) content with the high-efficient liquid phase chromatogram HPLC analysis this moment is 73.4%).Go up silica gel column chromatography then,, separate out needle-like crystal behind the recrystallization, i.e. extract of active ingredient in hairy bamboo (YF4332) 157.6mg with chloroform-methanol (2: 1～5: 1) wash-out.

Embodiment 4

Anti-oxidant activity is analyzed

Adopt DPPH free radical scavenging method to measure anti-oxidant activity.

Measuring method: 0.1ml contains the abundant mixing of DPPH ethanolic soln that different concns adds beautiful drown sample and 3.9ml of heap of stone, and room temperature is placed and measured 517 nanometer absorbances (OD517nm) after 30 minutes.

Table 1: anti-oxidant activity

Reactant	IC 50(％)
		Vitamins C Root of Kirilow Rhodiola oiliness extract Root of Kirilow Rhodiola aqueous extract Flos Chrysanthemi extract *Green tea extract *Green tea aqueous extract extract of active ingredient in hairy bamboo (YF4332)	0.0003 0.0004 0.005 1.51 0.0007 0.05 0.1

Embodiment 5

External tyrosinase activity suppresses

Measuring principle: under the effect of tyrosine oxidase, levodopa (L-DOPA) is oxidized to tan dopachrome (DOPAchrome).

Measuring method: 0.5ml contains the sample of different concns additive and 10u/ml tyrosine oxidase solution and the abundant mixing of 1.5ml damping fluid of 0.5ml, puts 30 ℃ of water-baths and hatches 20 minutes.Then in each test tube, add the 0.2%L-DOPA of 1.0ml, 30 ℃ of water-bath accurate responses 5 minutes, at once with the absorption peak of spectrophotometer in 475nm place mensuration dopachrome,

Table 2: tyrosinase inhibitory activity

Reactant	IC 50(μM)
		Azelaic acid vitamin C sodium ascorbyl phosphate vitamin C ursin kojic acid extract of active ingredient in hairy bamboo (YF4332)	19521 7540 159 327 35 32

Embodiment 6

Tyrosinase activity suppresses in the melanophore

Measuring method: the B16 melanophore is cultivated after 24 hours and is added extract of active ingredient in hairy bamboo (YF4332), cultivates the back through 48 hours and with physiological saline substratum is rinsed well.Add Triton X-100 (Triton X-100) and hatch 30min, pipette 0.2ml, in remaining cell, add Dopa, when hatching 30min for 37 ℃, measure OD475 with the quantitative cell protein of triketohydrindene hydrate at 37 ℃.

Tested the inhibition in melanophore of bitter bamboo extract to tyrosinase activity.The result as shown in Figure 2.Data show that extract of active ingredient in hairy bamboo (YF4332) has the effect of restraint of tyrosinase activity and melanocyte really in melanophore.

Embodiment 7

The melanophore melanocyte generates and suppresses

Measuring method: add extract of active ingredient in hairy bamboo (YF4332) in the melanophore after cultivating 24 hours, cultivated the back through 48 hours and substratum is rinsed well with phosphate buffered saline buffer (PBS).Add trypsin digestion cell.Remove supernatant after centrifugal, pipette 0.2ml equally with the quantitative cell protein of triketohydrindene hydrate, add NaOH and boil 20min in remaining cell, 400 nanometer absorbances (OD400) are measured in the cooling back.

Tested the bitter bamboo extract in melanophore to melanogenic inhibition.The result as shown in Figure 3.Data show that extract of active ingredient in hairy bamboo (YF4332) has the melanogenic effect of inhibition really in melanophore.

Claims (7)

1. Application of bitter bamboo active ingredient extract in the preparation of cosmetics. 2. The use as claimed in claim 1, wherein the active ingredient extract of bitter bamboo has antioxidant activity. 3. The use as claimed in claim 1, wherein the active ingredient extract of bitter bamboo inhibits tyrosinase activity. 4. The use according to claim 3, wherein the tyrosinase activity is an in vitro activity. 5. The use according to claim 3, wherein the tyrosinase activity is tyrosinase activity in melanocytes. 6. The application as claimed in claim 1, wherein the active ingredient extract of bitter bamboo can be used in the preparation of skin whitening cosmetics. 7. The application according to claim 1, wherein the active ingredient extract of bitter bamboo inhibits the formation of melanin.

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