RU2271393C2 - Method for preparing pattern of vegetable extract - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention proposes a method for detection of pattern of genetic regulation with Ginkgo biloba extract, ginkgolide B of Ginkgo biloba extract component. At the first stage cells of the strain MDA-231 are treated with one of indicated components followed by isolation of poly-A + RNA from treated and untreated cells MDA-231 that are used for generation of labeled cDNA probes. Prepared cDNA probes are hybridized with a matrix comprising one or some cDNA, hybridization degree of labeled cDNA probes from treated and untreated cells MFA-231 with indicated matrix is compared and the pattern of genetic regulation is prepared. Also, invention gives the corresponding names of genes and degree of genes expression alteration (as %). Also, invention describes a method for detection of identity of Ginkgo biloba extract of unknown or doubtful origin with the prepared pattern of genetic regulation with Ginkgo biloba extract of the known origin. Using the invention provides detection of patterns of biological activity of Ginkgo biloba extract, ginkgolide B or component from Ginkgo biloba extract, and the commercial preparation EGB 761 ® that can be used for detection of counterfeit preparation in market.

EFFECT: improved preparing method.

5 cl, 1 dwg, 1 tbl

Description

Statement regarding federal research funding

This study was funded in part by grant ES-07747 NIEHS, from the National Institutes of Health, and thus the US government may have certain rights to the present invention.

BACKGROUND OF THE INVENTION

The present invention relates to a method for establishing the “identity” of Ginkgo biloba leaves or isolated Ginkgolide B (GKB), a component of the Ginkgo biloba leaf extract, by obtaining a “gene regulation profile”. The invention also relates to a method for verifying the identity of a Ginkgo biloba extract by comparing the gene regulation profile of a Ginkgo biloba extract of unknown or doubtful origin with a gene regulation profile of a Ginkgo biloba extract of known origin. The term "known origin" refers to a commercial source of extract. In a preferred aspect of the present invention, the Ginkgo biloba extract is EGB 761®, which is manufactured and sold by IPSEN in Paris, France. More specifically, this invention relates to a method for determining the authenticity of an extract of unknown origin, which is believed to be EGB 761®. The present invention also relates to a method for establishing a gene expression profile of ginkgolide A, ginkgolide B or other components isolated from Ginkgo biloba extract, more specifically from EGB 761®.

Pharmaceutical production is based on the control of the composition and conformity of the biological activity profile of the batch produced. This standardization and control provides reproducible material for predictable and appropriate patient care. This application of standardization and control in order to protect against fake extracts issued as EGB 761® is beneficial for patients, as it gives patients confidence that they receive an extract with a specific biological activity profile.

Ginkgo biloba is one of the relic plants, and extracts from its leaves have been used in traditional medicine for several hundred years. There are numerous studies describing the beneficial effects of Ginkgo biloba extracts on patients with impaired attention, memory and cognitive functions associated with aging and decrepitude, and those with all types of dementia, mood changes and the ability to cope with daily stresses. Most of these studies used the standardized Ginkgo biloba EGB 761® leaf extract. It is also known that this extract has a cardioprotective effect (DeFeudis FV Ginkgo biloba extract (EGB 761®): from chemistry to clinic. Ullstein Medical, Wisbaden, Germany. 400 pp. 1998; Tosaki, A., DroyLefaix, M. T., Pali, T., and Das, DK, Free Rad. Biol. Med., 14: 361-370, 1993). These effects are due, at least in part, to the fact that EGB 761® has the ability to capture free radicals, possibly due to the presence of flavonoid and terpenoid components in the extract. Recent in vivo and in vitro studies have demonstrated that the terpene constituents of EGB 761®, ginkgolides and bilobalide, have antioxidant properties (Pietri, S., Maurelli, E., Drieu, K., and Culcasi, M., J. Mol. Cell . Cardiol., 29: 733-742, 1997; Yao, Z., Boujrad, N., Drieu, K., and Papadopoulos, V., Adv. Ginkgo Biloba Res. 7: 129-138, 1998). Other studies of EGB 761® reported the medical value of this product in the treatment of various clinical disorders, including cerebrovascular insufficiency and peripheral vascular insufficiency associated with aging and decrepitude. See, for example, Ginkgo biloba Extract (EGB 761®) Pharmacological Activities and Clinical Applications, DeFeudis, F. V., Eds, Elsevier, 1991; and Ullstein Medical 1998, Ginkgo biloba extract (EGB 761®), Eds. Wiesbaden, DeFeudis, F. V. The extract contains 24% ginkgo-flavone glycosides, 6% terpene lactones (ginkgolides and bilobalide), about 7% proanthocyanidins and some other constituents. See Boralle, N., et al. In: Ginkgolides, Chemistry, Biology, Pharmacology and Clinical perspectives, Ed: Braquet, P., J. R. Prous Science Publishers, 1988.

Most often, fake products issued as EGB 761® fall into the trade flow. Such fakes do not have the same component composition that makes up the authentic EGB 761®. Patients who receive a fake EGB 761®, believing that the fake is authentic, do not receive the benefits of the full spectrum of biological activity of EGB 761®. Moreover, the favorable reputation associated with the EGB 761® comes to naught. Therefore, there is a need for a method for establishing the biological activity profile of EGB 761®, which can then be used to compare with the biological activity profile of a fake EGB 761® to identify such counterfeits in retail outlets.

SUMMARY OF THE INVENTION

The present invention relates to a method for establishing a gene regulation profile with a Ginkgo biloba extract or a component of a Ginkgo biloba extract, which comprises the steps of:

obtaining at least one group of untreated cells;

treating the first group of cells with Ginkgo biloba extract or a component of the Ginkgo biloba extract to obtain a treated group of cells;

quantifying the effect on the expression of one or more genes in the treated cells, to obtain the number of affected genes; and

comparing the number of affected genes with the number of genes in cells not treated with Ginkgo biloba extract or a component of Ginkgo biloba extract, to obtain a gene regulation profile of Ginkgo biloba extract or component of Ginkgo biloba extract.

A preferred method for implementing the above method is one in which the step of quantifying includes

the selection of polyA + RNA from the treated group of cells, to obtain the processed polyA + RNA;

isolation of polyA + RNA from an untreated group of cells to obtain an untreated polyA + RNA;

generating labeled cDNA probes from the treated polyA + RNA, to obtain processed labeled cDNA probes;

generating labeled cDNA probes from untreated polyA + RNA to produce untreated labeled cDNA probes;

hybridization of the treated cDNA probes with a field (field means a ranked row) containing one or more cDNAs to obtain a processed hybridized cDNA field;

hybridization of untreated cDNA probes with a field containing one or more cDNA to obtain an untreated hybridized cDNA field;

quantification of each cDNA in the treated hybridized cDNA field, to obtain quantities of the processed cDNA;

quantification of each cDNA in an untreated hybridized cDNA field to obtain amounts of untreated cDNA; and

comparing the amounts of each of the treated cDNA with the amounts of each of the untreated cDNA to obtain a gene regulation profile.

A preferred method for implementing the immediately preceding method is a method in which the cells are MDA-231 cells; Ginkgo biloba extract is EGB 761®; and the field is a gene chip carrying multiple genes.

A preferred method for implementing the immediately preceding method is a method in which the gene regulation profile of EGB 761® includes increased expression of the c-Myc proto-oncogen and reduced expression of the following genes: protimosin-α, CDK2, p55CDC, myeloblastin, nuclear antigen of proliferating cells p120, NET1, ERK2, A2A adenosine receptor, FIt3 ligand, Grb2, clusterin, RXR-β, glutathione-S-transferase P, N-Myc, TRADD, SGP-2, NIP-1, Id-2, ATF-4, ETR101, ETR -103, macrophage colony stimulating factor-1, heparin-binding EGF-like growth factor, protein, under hepatocyte growth factor, inhibin-α, B-lymphocyte antigen CD19, L1CAM, β-catenin, integrin subunit α3, integrin subunit α4, integrin subunit α6, integrin subunit β5, integrin subunit αM, APC, PE-1, RhoA, c -Jun, protimosin-α, CDK2, p55CDC and myeloblastin.

A preferred method for implementing the immediately preceding method is a method in which the gene regulation profile of EGB 761® is approximately:

s-mus = 75%

c-Jun = -78%,

RhoA = -93%,

APC = -59%,

PE-1 = -42%,

protimosin-α = -79%,

myeloblastin = -66%,

p55CDC = -63%,

nuclear antigen of proliferating cells p120 = -68%,

CDK2 = -83%,

NET1 = -55%,

ERK2 = -46%,

A2A adenosine receptor = -40%,

Flt3 ligand = -58%,

Grb2 = -70%,

clusterin = -54%,

RXR-β = -55%,

glutathione-S-transferase P = -39%,

N-Myc = -74%,

TRADD = -51%,

NIP-1 = -40%,

Id-2 = -65%,

ATF4 = -42%,

ETR103 = -65%,

ETR101 = -60%,

antigen of B-lymphocytes CD19 = -62%,

L1CAM = -72%,

β-catenin = -58%,

integrin subunit αM = -41%,

integrin subunit β5 = -55%,

integrin subunit α4 = -49%,

integrin subunit α3 = -77%,

integrin subunit α6 = -53%,

macrophage colony stimulating factor-1 (CSF-1) = - 31%,

heparin binding EGF-like growth factor (HB-EGF) = - 62%,

protein similar to hepatocyte growth factor (HGFLP) = - 81%, and

inhibin-α = -69%,

where the percentages shown may be ± 20%.

A preferred method for implementing any of the preceding methods is a method in which the cells are MDA-231 cells; the component of the Ginkgo biloba extract is ginkgolid B; and the field is a gene chip carrying multiple genes.

In another aspect, the present invention relates to a method for verifying the identity of a Ginkgo biloba extract, comprising the steps of

obtaining a gene regulation profile with Ginkgo biloba extract in order to obtain a gene regulation profile;

EGB 761® gene regulation profile with the EGB 761® gene regulation profile;

comparing the gene regulation profile of Ginkgo biloba extract with the EGB 761® gene regulation profile;

determining whether the regulation profile of the Ginkgo biloba extract is within the range of ± 10% of the profile of the gene regulation of EGB 761®, to obtain a check of the identity of the Ginkgo biloba extract.

A preferred method for implementing the immediately preceding method is one in which the method for obtaining a gene regulation profile with Ginkgo biloba extract and EGB 761® comprises the steps of

the selection of polyA + RNA from the treated group of cells, to obtain the processed polyA + RNA;

isolation of polyA + RNA from an untreated group of cells to obtain an untreated polyA + RNA;

generating labeled cDNA probes from the treated polyA + RNA, to obtain processed labeled cDNA probes;

generating labeled cDNA probes from untreated polyA + RNA to produce untreated labeled cDNA probes;

hybridization of the treated cDNA probes with a field (ranked adjacent) containing one or more cDNAs to obtain a processed hybridized cDNA field;

hybridizing untreated cDNA probes with a field containing one or more cDNAs to obtain an untreated hybridized cDNA field;

quantifying each cDNA in the treated hybridized cDNA field to obtain amounts of the treated cDNA;

quantifying each cDNA in an untreated hybridized cDNA field to obtain amounts of untreated cDNA; and

comparing the amounts of each of the treated cDNA with the amounts of each of the untreated cDNA to obtain a gene regulation profile.

Brief Description of the Drawing

Drawing. The transcriptional response to EGB 761® means the effect on genes involved in cell proliferation.

The results shown are a quantitative analysis of the Atlas expression field of human cDNA containing 588 cDNA fragments amplified by PCR (Clontech Inc.). mRNA was obtained from control or treated with EGB 761® within 48 hours of MDA-231 cells. To normalize the abundance of mRNA, densitometric values obtained by image analysis were normalized using the control and dispatch genes available on the field. Only stable significant changes of more than 30% were considered.

Detailed description

The term "ginkgo terpenoid" includes all naturally occurring terpenes that are derived from the gymnosperm tree Ginkgo biloba, as well as synthetically produced ginkgo terpenoids and their pharmaceutically active derivatives and salts, and mixtures thereof. Examples of ginkgo terpenoids include ginkgolides. Ginkgolides, Chemistry, Biology, Pharmacology, and Clinical Perspectives, J. R. Provs. Science Publishers, Edited by P. Braguet (1988); F. V. DeFeudis, Ginkgo Biloba Extract (EGB 761®); Pharmacological Activities and Clinical Applications, Elsevier, Chapter II (1991).

As used herein, the term “ginkgolid” includes various ginkgolides described in the books cited above, as well as non-toxic pharmaceutically active derivatives thereof. Examples of ginkgolide derivatives include tetrahydro derivatives, acetyl derivatives and alkyl esters such as monoacetate derivatives and triacetate derivatives described by Okabe, et al., J. Chem. Soc. (c), pp. 2201-2206 (1967). Ginkgolide B has the following structure and when used in this description refers to an isolated ginkgolide B:

Used in this description, the term "Ginkgo biloba extract" includes a set of natural molecules, including terpenoids isolated from leaves of the Ginkgo biloba tree. Preferably, the extract is a specific Ginkgo biloba extract preparation known as EGB 761®.

The gene expression profile of an extract of Ginkgo biloba or its components can be obtained by methods known in the art. Typically, such a profile was obtained by Northern blotting of RNA or by analysis of the protection against ribonuclease used for each individual gene product. However, these analysis methods are time consuming and require approximately 2–3 days to analyze each gene. Currently, the gene expression profile can be determined by applying nucleic acid field technology such as the Atlas I expression field of human cDNA from Clontech (Palo Alto, CA); GeneFilters microfields from Research Genetics (Huntsville, AL); and microfields of gene expression from Genome Systems, Inc. (St. Louis, MO). Microfields Gene Filters are high-density DNA fields that are produced on 5 cm x 7 cm membranes. Four membranes for human genes and one for rat genes are currently available. Each membrane contains approximately 5000 sequences. Some of these sequences are known genes, while most of the sequences are ESTs with unknown function. Research Genetics will soon have access to gene fields on the Affymetrix Gene chip platform, where the genes are immobilized on a silicone chip. In the case of silicone chips, hybridization results (with mRNA of choice) are detected by fluorescence and analyzed by pattern recognition by comparing either fluorescence or radioactivity, which can be used to detect hybridization results on membrane fields.

The Genome System method uses GEM technology, in which a set of complementary DNA molecules (cDNAs) that contain genetic information from biological systems of interest is deposited and attached to a glass surface in a field format. Then, most of the half of the double DNA strand is removed, thus activating the individual elements of the field, preparing them for reaction with their uniquely suitable additions from the tested cells. Using GEM technology, 10,000 unique genes can be placed on one field. GEM technology also employs a color coding method to evaluate differences in expression between two DNA samples.

A cDNA field contains multiple PCR amplified cDNA fragments of an animal, such as a rat or human, preferably a human, immobilized on a positively charged nylon membrane or slide, silicone chip, or any other surface to be developed where DNA / template interaction is possible. The cell type of interest is treated or not treated with Ginkgo biloba extract or its component for 48 hours. PolyA + RNA is isolated from control or extract-treated cells. ³² P-labeled, fluorescent, chemiluminescent or colorimetric cDNA probes are generated from each polyA + RNA using glass or silicone chip based fields, preferably chemiluminescent or colorimetric, and hybridize them on the field according to the manufacturer's recommendations. Autoradiography is performed by exposing the spots to the film at about -70 ° C for 4-96 hours. Hybridization assays are performed using image processing systems that can detect fluorescence or chemiluminescence during image capture and analyze data such as SigmaGel software. For the detection of genes expressed at a low level, multiple exposures are used. Three internal controls, ubiquitin, G3PDH and β-actin, are used to compare the relative expression levels of the detected gene products in the control and the cells treated with the extract. Variations associated with the experiment are corrected using the relationships of gene expression to internal controls. The effect of processing the extract for each gene product is expressed in% of the value obtained for control (untreated) cells.

The following example of the preceding type of gene expression profile is provided. Clontech's human Atlas I cDNA expression field (Palo Alto, CA) contains 588 PCR-amplified 200-500 bp cDNA fragments immobilized on a nylon membrane. MDA-231 cells were treated or not treated with 20 mg / ml EGB 761® for 48 hours. PolyA + RNA was isolated from control or EGB 761® treated cells. ³² P-labeled probes were generated from each polyA + RNA and hybridized on an Atlas field according to the manufacturer's recommendations. Autoradiography was performed by exposing the spots to X-OMAT AR film (Kodak, Rochester, NY) at -70 ° C for 4-96 hours. Quantitative analysis of observed hybridization was performed using SigmaGel software (Jandel Scientific, San Rafael, CA). For the detection of genes expressed at a low level, multiple exposures were used. Three internal controls, ubiquitin, G3PDH and β-actin, were used to compare the relative expression levels of the detected gene products in the control and EGB 761® treated cells. Variations associated with the experiment were corrected using the ratios of gene expression to internal controls. The effect of processing EGB 761® for each gene product was expressed as% of the value obtained for control (untreated) cells. The results of this experiment, which are presented in Table 1, show genes stably exposed to levels above 30% of control by treatment with EGB 761®. In fact, the table shows that treatment led to an increase in the expression of rotoncogene c-Myc and a decrease in the expression of 35 gene products, including oncogenes (AP-1, PE-1, RhoA, n-Myc), cell cycle regulators (CDK2, p55CDC, PCNA p120), signal transduction modulators (NET1, ERK2), apoptotic products (SGP-2, NIP1), receptors (A2A, RXR-beta, Grb2), transcription factors (Id-2, ATF-4, ETR101, ETR -103), growth factors (HB-EGF, HGF-like) and cell adhesion molecules (CD19, L1CAM, integrins α3, α4, α6, β5, Mac-1, β-catenin), which are directly involved in various ways of regulating cellular about LIFERATIONS.

Gene expression profiles can be established for Ginkgo biloba extracts of known origin, and then they can be compared with the gene expression profile of Ginkgo biloba extracts of known origin or extracts that are presented as a specific commercial extract. Comparison of profiles can thus be used as a screening tool to confirm the origin of the extract.

Claims (5)

1. A method for establishing a gene regulation profile with Ginkgo biloba extract, Ginkgolid B, or a component of Ginkgo biloba extract, comprising the steps of: obtaining at least one group of untreated MDA-231 cells; treating the first group of MDA-231 cells with Ginkgo biloba extract, Ginkgolid B, or a component of the Ginkgo biloba extract, to obtain a treated group of MDA-231 cells; quantifying the effect on the expression of one or more genes in the treated MDA-231 cells to obtain the number of exposed genes, where the quantification involves the isolation of polyA + RNA from the treated group of cells to obtain the processed polyA + RNA; isolation of polyA + RNA from an untreated group of cells to obtain an untreated polyA + RNA; generating labeled cDNA probes from the treated polyA + RNA to produce processed labeled cDNA probes; generating labeled cDNA probes from untreated polyA + RNA to produce untreated labeled cDNA probes; hybridization of the processed cDNA probes with a matrix containing one or more cDNA to obtain a processed hybridized cDNA field; hybridization of untreated cDNA probes with a matrix containing one or more cDNA to obtain an untreated hybridized cDNA template; quantification of each cDNA on the processed hybridized cDNA template to obtain amounts of the processed cDNA; quantifying each cDNA on an untreated hybridized cDNA template to obtain amounts of untreated cDNA; comparing the amounts of each of the treated cDNA with the amounts of each of the untreated cDNA to obtain a gene regulation profile; and comparing the number of exposed genes with the number of genes in MDA-231 cells not treated with Ginkgo biloba extract, Ginkgolide B or a component of Ginkgo biloba extract, to obtain a gene regulation profile for Ginkgo biloba extract, Ginkgolide B or a component of Ginkgo biloba extract. 2. The method of claim 1, wherein the Ginkgo biloba extract is EGB 761®, and the matrix is a gene chip carrying multiple genes. 3. The method according to p. 2, where the gene regulation profile of EGB 761® includes increased expression of the c-Myc protooncogen and reduced expression of the following genes: protimosin-α, CDK2, p55CDC, nuclear antigen of proliferating cells p120 myeloblastin, NET1, ERK2, adenosine A2A receptor, Plt3 ligand, Grb2, clusterin, RXR-β, glutathione S-transferase P, N-Myc, TRADD, SGP-2, NIP-1, Id-2, ATF-4, ETR101, ETR-103, macrophage colony stimulating factor-1, heparin-binding EGF-like growth factor, protein similar to hepatocyte growth factor, inhibin-α, B-lymphocyte antigen CD19, LI CAM, β-k atenine, α3 integrin subunit, α4 integrin subunit, α6 integrin subunit, β5 integrin subunit, αM integrin subunit, APC, PE-1, RhoA, c-Jun, protimosin-α, CDK2, p55CDC and myeloblastin. 4. The method of claim 3, wherein the gene regulation profile of EGB 761® is as follows: s-mus = 75% c-Jun = -78%, RhoA = -93%, APC = -59%, PE-1 = -42%, protimosin-α = -79%, myeloblastin = -66%, p55CDC = -63%, nuclear antigen of proliferating cells p120 = -68%, CDK2 = -83%, NET1 = -55%, ERK2 = -46%, A2A adenosine receptor = -40%, Flt3 ligand = -58%, Grb2 = -70%, clusterin = -54%, RXR-β = -55%, glutathione-8-transferase P = -39%, N-Myc = -74%, TRADD = -51%, NIP-1 = -40%, Id-2 = -65%, ATF4 = -42%, ETR103 = -65%, ETR101 = -60%, B-lymphocyte antigen CD 19 = -62%, L1CAM = -72%, β-catenin = -58%, integrin subunit αM = -41%, integrin subunit β5 = -55%, integrin subunit α4 = -49%, integrin subunit α3 = -77%, integrin subunit α6 = -53%, macrophage colony stimulating factor-1 (CSF-1) = - 31%, heparin binding EGF-like growth factor (HB-EGF) = - 62%, protein similar to hepatocyte growth factor (HGFLP) = - 81%, and inhibin-α = -69%, where the percentage values may be ± 20%. 5. The method of establishing the authenticity of the Ginkgo biloba extract, which involves the following steps: establishing a gene regulation profile of the Ginkgo biloba EGB 761® extract according to the method of claim 1, comparing the gene regulation profile of the Ginkgo biloba extract with the gene regulation profile of EGB 761®; confirmation of the authenticity of the Ginkgo biloba extract if the obtained values of the gene regulation profile under the action of the Ginkgo biloba extract are within ± 10% of the values of the gene regulation profile of EGB 761®.