CN102389535B - Extraction and separation method and use of active ingredients of red yeast rice - Google Patents
Extraction and separation method and use of active ingredients of red yeast rice Download PDFInfo
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- CN102389535B CN102389535B CN201110297613.2A CN201110297613A CN102389535B CN 102389535 B CN102389535 B CN 102389535B CN 201110297613 A CN201110297613 A CN 201110297613A CN 102389535 B CN102389535 B CN 102389535B
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Abstract
The invention relates to an extraction and separation method and use of active ingredients of red yeast rice. The method comprises the following steps: drying red yeast rice and crushing red yeast rice into powder, extracting ethanol, concentrating, and obtaining extract of red yeast rice powder; and obtaining the active ingredients by silica column chromatography and gradient elution. A red yeast rice protein tyrosine phosphatase 1B (PTP1B) inhibitor is an active ingredient obtained by elution with eluent prepared from petroleum ether and ethyl acetate according to a weight ratio of 10:1. The inhibitor can be used for preparing medicines for preventing and treating PTP1B-targeted diabetes or obesity. Compared with the prior art, a lower-polarity axanthein active part F1 prepared by the method has a high inhibiting effect on PTP1B, as is proved by initial in-vitro PTP1B inhibition tests; and thus, the axanthein active part F1 can be used for preparing medicines for preventing and treating PTP1B-targeted diabetes or obesity.
Description
Technical field
What the present invention relates to is extraction, separation method and the purposes of the active Chinese drug component component in a kind of field of Chinese herbal, specifically a kind of extraction of red colouring agent for food, also used as a Chinese medicine activeconstituents, separation method and purposes.
Background technology
Red kojic rice (red yeast rice), claims again red colouring agent for food, also used as a Chinese medicine or red bent, is medicine-food two-purpose type traditional Chinese medicine material, take polished rice or long-grained nonglutinous rice as raw material gets the science rarity of ，Wei China greatness through monascus ruber fermentation, has and exceedes history in thousand so far.As far back as between year in the Northern Song Dynasty, the clear different record > > of < < that Tao Gu writes has just had the earliest and has recorded as the effect of food antiseptics it.In the < < Compendium of Materia Medica > > that pharmaceutical use ，Ze Ming Dynasty LI Shi-Zhen is write and the preparation technology of Red kojic rice and invigorating the spleen thereof are invigorated blood circulation etc., there is comparatively perfect record.Along with deepened continuously modern age to the research of Red kojic rice, it is subject to Asia other countries, the even great attention of European and American areas researcher gradually in the superior efficacy of food, medicine and other fields performance, and constantly explores, illustrates its elementary and beneficial functional secondary metabolites.Japan rattan doctor Zhang far away was at the < < of 1979
the Journal of Antibioticsthe 852nd page of < < Monacolin K of > >, in A new hypocholesterolemic agent produced by a monascus species > > mono-literary composition, mention, he finds from red monascus (Monascus ruber) success and separation obtains Monacolin K(Fig. 1, and in figure, R group is-CH
3), and the effect that Monacolin K has reduction blood cholesterol levels is proposed first.Verified, Monacolin K is 3-hydroxy-3-methylglutaryl coenzyme A reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase, HMG-CoA reductase) a kind of competitive inhibitor, it has hindered HMG-CoA and under its reductase enzyme effect, has generated mevalonic acid (mevalonate) final synthetic cholesterol and other products.
In recent years, along with the development to biologically active substance research in red colouring agent for food, also used as a Chinese medicine, the scholar of various countries has carried out dark people to it and research widely, find that red colouring agent for food, also used as a Chinese medicine can produce many useful secondary metabolites, as monascorubin, cholesterol inhibitor help Lovastatin, and there is hypotensive, antibacterial, to fall the physiologically actives such as blood ammonia, antitumor, hypoglycemic composition, make traditional red colouring agent for food, also used as a Chinese medicine increase new intension.
Further By consulting literatures is found, Hever et al. was at the < < Journal o of 1999
f Clinical Nutritionthe 69th phase of > > 231 pages of < < Cholesterol-lowering effects of a proprietary Chinese red-yeast rice dietary supplement, in American > > mono-literary composition, mention Monacolin K the inhibition activity of HMG-CoA reductase enzyme is better than to lovastatin (Lovastatin) greatly, and its toxic side effect is very micro-, thereby block safer and more effectively, reduce the synthetic of endogenous cholesterol.Man et al. was at the < < of 2002
molecular and Cellular Biochemistryin > > the 233rd phase 153 pages of < < Cholestin inhibits cholesterol synthesis and secretion in hepatic cells (HepG2) > > mono-literary composition, mention, by clinical trial, prove: functional red yeast rice contains potent lipopenicillinase material because of it, not only HMG-CoA reductase enzyme is had to good restraining effect, make liver cell (Hep G2) cholesterol, thereby reduce the synthetic and secretion of the interior nonesterified cholesterol of liver cell (Hep G2) and cholesteryl ester (cholesteryl ester), and then alleviate patch danger, and liver function is not caused to disruptive impact.Hong, et, al., In 2008's "JOURNAL, OF, MEDICINAL, FOOD" Section 11 657 "Chinese, Red, Yeast, Rice, Versus, Lovastatin, Effects, on, Prostate, Cancer, Cells, With, and, Without, Androgen, Receptor, Over, expression "in an article proves Lovastatin on hormone-dependent prostate cancer, (LNCaP) and androgen-independent prostate cancer (LNCaP-AR) cell proliferation inhibition good; however compared and words, the same dose concentration Monacolin, K (5.93μM) inhibition of cell proliferation LnCaP Lovastatin 2 times, and the inhibition of cell proliferation LNCaP-AR type of Lovastatin is four times, and even better drug stability .In addition, about other beneficial functionals of functional red yeast rice, in recent years, also repeatedly reported as hypotensive, treatment osteoporosis etc., and received fervent concern.For example, Yang Meng etc. has just mentioned in the literature and has produced in various degree vitamins D because of the many bacterial strains in monascus
2precursor---ergosterol, after uviolizing, ergosterol is converted into vitamins D
2prevention Infant Rickets and help human body phosphorus are absorbed with to obvious effect.
Data shows, 2010-2030, and due to factors such as aging population and population growths, Chinese cardiovascular disorder generation number also will continue to rise; If do not paid attention to, the year two thousand thirty China cardiovascular patient will increase by 2,130 ten thousand, cardiovascular death number will increase by 7,700,000.This impels people to increase the demand of reducing blood-fat similar drug, healthcare products, makes hypolipidemic, food products market be continued expansion, especially brings the greatest business opportunity of monascus product with natural fat-reducing effect.
Summary of the invention
The object of the invention is to the deficiency existing for prior art, a kind of extraction, separation method and purposes of red colouring agent for food, also used as a Chinese medicine activeconstituents is provided.By preliminary external Protein Tyrosine Phosphatases 1B(PTP1B) inhibition test, confirm effectively arrestin matter tyrosine phosphatase 1B(PTP1B of a class red colouring agent for food, also used as a Chinese medicine class PTP1B inhibitor provided by the present invention) activity, can be for the preparation for the treatment of diabetes and fat similar drug, healthcare products.
The object of the invention is to be achieved through the following technical solutions:
The extraction, the separation method that the present invention relates to a kind of red colouring agent for food, also used as a Chinese medicine activeconstituents, comprise the steps:
(a) Red kojic rice is dried and crushed into after powder, after extraction using alcohol, concentration extraction obtains Hongqu powder (red colouring agent) extract;
(b) the Hongqu powder (red colouring agent) extract in step (a) obtains active ingredient after silica gel column chromatography gradient elution.
Preferably, the described extraction using alcohol in step (a), is specially with 75% ethanol of 8～10 times of amounts of relatively described Red kojic rice xeraphium quality and extracts three times under 40 ℃ of conditions, each two hours.
Preferably, in the described silica gel column chromatography gradient elution in step (b), eluent used is sherwood oil and ethyl acetate, and its weight ratio is respectively 10:1,5:1,3:1,2:1,1:1,1:5.
Preferably, the active ingredient obtaining in described step (b) is 6.
The invention still further relates to a kind of red colouring agent for food, also used as a Chinese medicine class PTP1B inhibitor that preparation obtain according to above-mentioned method, the active ingredient that the eluent wash-out that described red colouring agent for food, also used as a Chinese medicine class PTP1B inhibitor is the employing weight ratio sherwood oil that is 10:1 and ethyl acetate obtains.
The invention still further relates to a kind of purposes of take in the medicine of diabetes that PTP1B is target spot or obesity in preparation prevention and treatment according to above-mentioned red colouring agent for food, also used as a Chinese medicine class PTP1B inhibitor.
Compared with prior art, the beneficial effect that the present invention has is: the less xanthein reactive site F1 of polarity that the present invention's preparation obtains, through external PTP1B activity experiment, confirm, there is stronger PTP1B and suppress active, can be used for preparation prevention and treatment and take PTP1B(PTP 1B) be the medicine of diabetes or the obesity of target spot.
Accompanying drawing explanation
Fig. 1 is the structural formula of Monacolin K;
Fig. 2 is Hongqu powder (red colouring agent) extraction, separation and active testing schema;
Fig. 3 is the active result schematic diagram of each efficient part vitro inhibition of red colouring agent for food, also used as a Chinese medicine PTP1B;
Fig. 4 is each efficient part cross-over experiment of red colouring agent for food, also used as a Chinese medicine and dusting cover result schematic diagram.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art further to understand the present invention, but not limit in any form the present invention.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can also make some distortion and improvement.These all belong to protection scope of the present invention.
the extraction of embodiment 1, red colouring agent for food, also used as a Chinese medicine active ingredient, separation
In the present invention, to be purchased from Japanese prefecture be heath food center to Red kojic rice used.After Red kojic rice is dried to 12 under 60 ℃ of conditions, be ground into meal stand-by.
As shown in Figure 2, dry Hongqu powder (red colouring agent) is extracted three times under 40 ℃ of conditions to each two hours with 8～10 times of amount 75% ethanol.After extraction, cross and filter to remove residue, merging filtrate, the dry red colouring agent for food, also used as a Chinese medicine medicinal extract (being Hongqu powder (red colouring agent) extract) that obtains after concentrating under reduced pressure.
Red colouring agent for food, also used as a Chinese medicine medicinal extract is through silica gel column chromatography, with sherwood oil: ethyl acetate system gradient elution obtains six reactive sites; Eluent proportioning used is respectively petrol ether/ethyl acetate=10:1,5:1,3:1,2:1,1:1,1:5, six ascending F1 of being followed successively by of reactive site polarity that obtain, F2, F3, F4, F5, F6.Wherein, F1 is the xanthein composition that polarity is less; F5 is the brown pigment composition that polarity is larger, and through HPLC, LC-MS data analysis is accredited as Monacolin K.
embodiment 2, PTP1B suppress active testing
Each reactive site of embodiment 1 gained is carried out to PTP1B and suppress active testing, as shown in Figure 2.Adopt molecular biology method, (this project bacterium is applied > > openly at < < PTP1B inhibitor and in synthesizing and preparing treatment diabetes B medicine to the hGST-PTP1B-BL21 E.Coli mankind PTP1B engineering bacteria of structure gene recombination, publication number: CN 102018688 A, open day 2011.4.20), with GST affinity chromatography column purification hGST-PTP1B protein, the polypeptide pNPP that utilization contains phosphoric acid is by Protein Tyrosine Phosphatases 1B(PTP1B) take off a product pNP after phosphoric acid and at wavelength 405nm place, have the principle of absorption peak, to generate the scale of pNP after PTP1B effect, show that PTP1B enzymic activity changes and the inhibition situation of compound to enzymic activity, computerized compound is to PTP1B enzyme inhibition rate.
It is recombinant expressed testing PTP1B albumen used and purifying obtains.The determination of activity system of PTP1B: at test solvent (10 mM Tris, 25 mM NaCl, 1 mM EDTA, pH=7.5) in, add 7.5 μ g/mL PTP1B, 6 mM pNPP and dissolving or be diluted in component to be measured in DMSO, each test concentrations all arranges 3 multiple holes.Adopt the logarithmic value of concentration of component to make regression equation to inhibiting rate, calculate IC50.The compounds of this invention suppresses active result as shown in Figure 3 to PTP1B, test the positive control sample of using be lovastatin and Simvastatin, as shown in table 1 to Data induction in figure:
Table 1
From Fig. 3 and table 1: it is active that reactive site F1 has stronger inhibition to PTP1B, red colouring agent for food, also used as a Chinese medicine medicinal extract, it is active that reactive site F3 and F4 have certain inhibition to PTP1B, reactive site F2 and F5 have faint PTP1B and suppress active, and reactive site F6 and positive control do not demonstrate and suppress active PTP1B.
Each efficient part cross-over experiment of red colouring agent for food, also used as a Chinese medicine and dusting cover result schematic diagram are as shown in Figure 4, as shown in Figure 4: Hongqu powder (red colouring agent) suppresses active be mainly derived from the pigment reactive site F1 that polarity is less, the Monacolin K(reactive site F5 with hypoglycemic effect that traditional institute is thought for PTP1B) PTP1B being suppressed to activity does not play a major role.
Claims (2)
1. a red colouring agent for food, also used as a Chinese medicine class PTP1B inhibitor, is characterized in that, prepare by the following method and obtain,
(a) Red kojic rice is dried and crushed into after powder, after extraction using alcohol, concentration extraction obtains Hongqu powder (red colouring agent) extract;
(b) the Hongqu powder (red colouring agent) extract in step (a) is through silica gel column chromatography, adopts the active ingredient obtaining after the eluent wash-out of sherwood oil that weight ratio is 10:1 and ethyl acetate, i.e. red colouring agent for food, also used as a Chinese medicine class PTP1B inhibitor.
2. the purposes that a red colouring agent for food, also used as a Chinese medicine class PTP1B inhibitor according to claim 1 be take in the medicine of diabetes that PTP1B is target spot or obesity in preparation prevention and treatment.
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| US20140193455A1 (en) * | 2013-01-09 | 2014-07-10 | Byoung Cheon KIM | Pharmaceutical composition for prevention or treatment of steroid-induced diabetes and health functional food |
| CN105566268B (en) * | 2016-03-09 | 2017-12-08 | 福州大学 | A kind of method for preparing purified of red yeast rice Lovastatin |
| CN111388609A (en) * | 2020-03-25 | 2020-07-10 | 杭州天曲生物技术有限公司 | Method for extracting functional red yeast rice with high Moraxelin K content |
| CN115181624A (en) * | 2022-07-11 | 2022-10-14 | 四川省食品发酵工业研究设计院有限公司 | Functional red yeast rice wine rich in lovastatin and brewing process thereof |
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