MXPA02009412A

MXPA02009412A - Method of profiling a plant extract.

Info

Publication number: MXPA02009412A
Application number: MXPA02009412A
Authority: MX
Inventors: Vassilios Papadopoulos
Original assignee: Sod Conseils Rech Applic
Priority date: 2000-03-31
Filing date: 2001-03-27
Publication date: 2005-02-03
Also published as: WO2001075181A3; IL151856A0; PL365418A1; AU4783201A; AU2001247832B2; CA2403303A1; NO20024611L; CZ20023275A3; WO2001075181A2; JP2003529377A; CN1185354C; NO20024611D0; RU2271393C2; KR100479151B1; KR20020089409A; CN1439059A; HUP0301997A3; HUP0301997A2; NZ521728A; HK1053150A1

Abstract

The present invention is directed to a method of establishing an identity of Ginkgo biloba leaves or isolated ginkgolide B (GKB) or another component of the extract of Ginkgo biloba leaves, by obtaining a gene regulation profile. The invention is also directed to a method of verifying the identity of a Ginkgo biloba extract by comparing the gene regulation profile of a Gingko biloba extract of unknown or questionable origin with the gene regulation pofile of a Gingko biloba extract of known origin. The present invention is further directed to a method of establishing a gene expression profile of Ginkgolide A, Ginkgolide B or any other component isolated from a Ginkgo biloba extract, more particularly, from EGB 761reg;.

Description

METHOD TO CHARACTERIZE THE PROFILE OF A VEGETABLE EXTRACT DECLARATION REGARDING SPONSORED RESEARCH FOR THE FEDERAL GOVERNMENT The present invention was sponsored in part by grants ES-07747 NIEHS, from the National Institutes of Health (NIH) and, therefore, the US government. may have certain rights over the present invention.

BACKGROUND OF THE INVENTION The present invention is directed to a method for establishing an "identity" of the leaves of Ginkgo biloba or the isolated Gingkolide B (GKB), a component of the extract of the leaves of Ginkgo biloba, by obtaining a "profile of gene regulation. " The invention also addresses a method for verifying the identity of an extract of Ginkgo biloba by comparing the profile of gene regulation of a Ginkgo biloba extract of unknown or questionable origin, with the profile of gene regulation of an extract of Ginkgo biloba. Ginkgo biloba of known origin. The term "known origin" refers to the commercial source of the extract. A preferred aspect of the present invention is where the Ginkgo biloba extract of known origin is EGB 761®, produced and marketed by IPSEN of Paris, France. More particularly, this invention is directed to a method for determining the authenticity of an extract of unknown origin which claims to be EGB 761®. The present invention, moreover, is directed to a method for establishing a gene expression profile of Gingkolide A, Gingkolide B or any other component isolated from an extract of Ginkgo biloba, more particularly, of EGB 761®. Pharmaceutical manufacturing is based on the control over the composition and the consistency of the profile of the biological activity of a batch manufactured. This normalization and control provides a reproducible material in the predictable and consistent treatment of patients.

This use of standardization and control to protect against the commercialization of counterfeit extracts claiming to be EGB 761® is beneficial for patients since it assures patients that they are obtaining / receiving an extract with a particular profile of biological activity . Ginkgo biloba is one of the oldest trees, and extracts of its leaves have been used in traditional medicine for several hundred years. There are several studies that describe the beneficial effects of Ginkgo biloba extracts in patients with disturbances of concentration, memory and cognitive functions associated with aging and senility, and for those with all types of dementias, mood swings and the ability to deal with the daily sources of stress. A standardized extract of Ginkgo biloba leaves, called EGB 761®, has been used in most of these studies. It is also known that this extract has cardioprotective effects (DeFeudis FV Ginkgo biloba extract (EGB 761®): from chemistry to clinic, Ullstein Medical, Wisbaden, Germany, 400 P. 1998, Tosaki, A., Droy-Lefaix, MT Pali, T., and Das, DK, Free Rad. Biol. Med., 14: 361-370, 1993). These effects have been attributed, at least in part, to the properties of elimination of free radicals by the EGB 761®, probably due to the presence of flavonoid or terpenoid constituents in the extract. Recent studies in vivo and in vi tro demonstrate that the terpene constituents of EGB 761®, Gingkolids and bilobalides, 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 on the EGB 761® have reported the medicinal value of the product for the treatment of a variety of clinical disorders including peripheral and cerebrovascular insufficiencies associated with aging and senility. 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. iesbaden, DeFeudis, F. V. The extract contains 24% ginkgoflavonglucosides, 6% terpene lactones (Gingkolids and bilobalide), approximately 7% proanthocyanidins and several other constituents. See, Boralle, N., et al., In: Ginkgolides, Chemistry, Biology, Pharmacology and Clinical Perspectives, Ed: Braquet, P., J.R. Prous Science Publishers, 1988. More frequently, counterfeit formulations claiming to be EGB 761® are placed on goods in commercial motion. These fakes do not have the same composition or components that make up the authentic EGB 761®. Patients who obtain the false 761® EGB, who believe that the falsification is authentic, are deprived of the benefit of a full range of the biological activity of the EGB 761®. In addition, the good faith associated with EGB 761® is being weakened. Therefore, there is a need for a method to establish the profile of the biological activity of the EGB 761®, which can then be used to compare with the biological activity profile of the false EGB 761® to detect counterfeits in the market.

SUMMARY OF THE INVENTION The present invention is directed to a method for establishing the profile of the gene regulation of a Ginkgo biloba extract or a component of the Ginkgo biloba extract, comprising the steps of: obtaining at least one batch of cells without treatment; treating a first batch of cells with a Ginkgo biloba extract or a component of Ginkgo biloba extract to obtain a treated batch of cells; quantify an effect on the expression of one or more genes of the treated cells to obtain a number of affected genes; and comparing the number of affected genes with a number of genes from cells that have not been treated with Ginkgo biloba or a component of Ginkgo biloba extract to obtain the profile of gene regulation of Ginkgo biloba extract or a component of the extract of Ginkgo biloba. A preferred method of the above method is wherein the quantification step comprises: isolating poly A + RNA from the treated cell batch to obtain the poly A + RNA treated; isolating the poly A + RNA from the untreated cell batch to obtain unaddressed poly A + RNA; generating the labeled cDNA probes from the treated poly A + RNA to obtain the labeled probes of the processed cDNA; generating the labeled cDNA probes from the unreacted poly A + RNA to obtain the labeled probes from the untreated cDNA; hybridizing the probes of the cDNA treated in a sequence having one or more cDNA to obtain a hybridized and treated sequence of the cDNA; hybridizing the untreated cDNA probes to form a sequence having one or more cDNA to obtain a hybridized and untreated cDNA sequence; quantifying each of the cDNAs of the hybridized and treated sequence of the cDNA to obtain amounts of treated cDNA; quantifying each of the cDNAs of the hybridized and untreated cDNA sequence to obtain amounts of untreated cDNA; and comparing the amounts of each of the treated cDNAs with the amounts of untreated cDNA to characterize the profile of gene regulation. A preferred method of the immediately preceding method is wherein the cells are DA-231 cells; Ginkgo biloba extract is EGB 761®; and the sequence is a gene piece that has a multiplicity of genes. A preferred method of the immediately preceding method is wherein the gene regulation profile of the EGB 761® comprises the increased expression of the c-Mic proto-oncogene, and a diminished expression of the following genes: protimosin-a, CDK2, p55CDC, myeloblastin nuclear antigen cell proliferation pl20, NET1, ERK2, Adenosine A2A Receptor, Flt3 ligand, Grb2, Clusterin, X-β, Glutathione S-transferase P, N-Mic, 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 a, B-lymphocyte CD19 antigen, L1CAM, β-catenin, integrin a3 subunit, integrin a4 subunit, integrin a6 subunit, integrin ß5 subunit, integrin aM subunit, APC, PE-1, RhoA, c-Jun, protymosin-a, CDK2, p55CDC and myeloblastin . A preferred method of the immediately preceding method is wherein the profile of the gene regulation of the EGB 761® is approximately c-Mic - + 75%, c-Jun = -78%, RhoA = -93%, APC = -59%, PE-1 = -42%, Protimosin-a = -79%, Myeloblastin = -66%, p55CDC = -63%, Nuclear Antigen of Cell Proliferation pl20 = -68%, CDK2 = -83%, NET1 = -55%, ERK2 = -46%, A2A Receptor of Adenosine = -40%, ligand Flt3 = -58%, Grb2 = -70%, Clusterine = -54%, RXR-ß = -55%, Glutathione S-transferase P = -39%, N-Míc = -74%, d TRADD = -51%, ??? - 1 = -40%, Id-2 = -65%, ATF4 = -42%, ETR103 = -65%, ETR101 = -60%, B-lymphocyte antigen CD19 = - 62%, L1CAM = -72%, β-catenin = -58%, Integrin Subunit = -41%, Integrina Subunit ß5 = -55%, Integrin Subunit 4 = -49%, Integrina Subunit 3 = -77 %, Subunit a6 of Integrin = -53%, Factor-1 Stimulant of Colonies of Macrophages (CSF-1) = - 31%, Growth Factor similar to EGF of Heparin Binding (HB-EGF) = -62%, Protein similar to the Hepatocyte Growth Factor (HGFLP) = -81%, and Inhibin = -69%, where the percentages shown can be ± 20%. A preferred method of any of the above methods is wherein the cells are MDA-231 cells; the component of Ginkgo biloba extract is Gingkolide B; and the sequence is a gene piece that has a multiplicity of genes.

In another aspect, the present invention provides a method for verifying the identity of the Ginkgo biloba extract comprising the steps of: obtaining a profile of the gene regulation of the Ginkgo biloba extract to obtain a profile of the gene regulation; obtain a profile of the gene regulation of EGB 761® to produce a profile of the gene regulation of EGB 761®; to compare the profile of gene regulation of Ginkgo biloba extract with the profile of gene regulation of EGB 761 ®; determine if the values of the gene regulation profile of the Ginkgo biloba extract is within ± 10% of the profile values of the GBS 761® gene regulation to obtain verification of the identity of the Ginkgo biloba extract. A preferred method of the immediately preceding method is wherein the method for obtaining a profile of the gene regulation of the Ginkgo biloba extract and the profile of the gene regulation of EGB 761® comprises the steps of: isolating poly A + RNA from the batch treated cells to obtain the poly A + RNA; isolating the poly A + RNA from a batch of untreated cells to obtain unaddressed poly A + RNA; generating the labeled cDNA probes from the treated poly A + RNA to obtain the labeled probes of the treated cDNA, - generating the labeled cDNA probes from unreacted poly A + RNA to obtain the labeled probes of untreated cDNA; hybridizing the treated cDNA probes to obtain a sequence having one or more cDNAs to obtain a hybridized and treated cDNA sequence; hybridizing the untreated cDNA probes to obtain a sequence having one or more cDNA to obtain a hybridized and untreated cDNA sequence; quantifying each of the cDNAs of the hybridized and treated cDNA sequence to obtain amounts of treated cDNA; quantifying each of the cDNAs of the hybridized and untreated sequence of the cDNA to obtain amounts of untreated cDNA; and comparing the amounts of each of the cDNAs treated with the amounts of the untreated cDNA to obtain the profile of the gene regulation.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. The transcriptional response for EGB 761® suggests an effect on the genes involved in cell proliferation. The results shown represent a quantitative analysis of the sequence for the expression of the human Atlas cDNA containing 588 cDNA fragments amplified by PCR (Clontech Inc.). The mRNAs were obtained from control or treated MDA-231 cells, for 48 hours, with EGB 761® (20 μg / ml). To normalize the abundance of mRNA, the densitometric values obtained from the image analysis were normalized using maintenance genes provided within the sequence. Only consistent and significant changes were considered above 30%.

DETAILED DESCRIPTION OF THE INVENTION The term "ginkgoterpenoid" includes all naturally occurring terpenes derived from the gymnosperms of the Ginkgo biloba tree as well as synthetically produced ginkgoterpenoids and pharmaceutically active derivatives and salts thereof and mixtures thereof. of these. Examples of ginkgoterpenoides include Gingkolids. Examples of ginkgoterpenoides are described in 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). The term "Gingkolide" as used herein includes the various Gingkolides described in the aforementioned books as well as the non-toxic and pharmacologically active derivatives thereof. Examples of Gingkolide derivatives include tetrahydro derivatives, acetyl derivatives and alkyl esters such as the monoacetate derivatives and triacetate described in Okabe, et al., J. Chem. Soc. (c), pgs. 2201-2206 (1967). He Gingkolide B has the following structure and, as used herein, refers to Gingkolide B in its isolated form: The term "Ginkgo biloba extract" as used herein includes a collection of natural molecules, including terpenoids, derived from the leaves of the Ginkgo biloba tree. Preferably, the extract is the specific formulation of the Ginkgo biloba extract known as EGB 761®. A profile of the gene expression of an extract of Ginkgo biloba or a component thereof can be obtained by methods known in the art. Traditionally, this profile was obtained by a Northern blot analysis of ?? or a ribonuclease protection assessment for each individual gene product. However, these assessments required a lot of time and took approximately 2-3 days to analyze each gene. Currently, a gene expression profile can be established by using technology for nucleic acid sequencing, such as sequencing I for the expression of the human Atlas cDNA from Clontech (Palo Alto, CA); GeneFilters Microarrays by Research Genetics (Huntsville, AL); and Gene Expression Microarrays by Genome Systems, Inc. (St. Louis, MO). The Gene Filters Microarray are high density DNA sequences that are produced on membranes of 5 cm X 7 cm. Currently, there are four membranes available for human genes and one for rat genes. Each membrane contains approximately 5,000 sequences. Some of these sequences are known genes, while the majority of the sequences represent EST of unknown function. Research Genetics will soon make gene sequences available on the Affymetrix Gene pill platform, where the genes are immobilized on a silicon wafer. In the case of silicon wafers, the hybridization results (with the mRNA of choice) are detected by fluorescence and analyzed by pattern recognition in comparison to either fluorescence or radioactivity that can be used for detection of the results of hybridization in the sequences on membrane. The Genome System method uses GEM technology where a collection of complementary DNA molecules (cDNAs) containing the genetic information of the biological systems of interest are deposited and linked onto a glass surface in a sequence format. Then, the larger portions of a half of the double strand of DNA are removed, and in this way the individual elements of the sequence are activated, they are prepared to react with their uniquely equivalent DNA counterparts in the cells being analyzed. GEM technology can accommodate 10,000 unique genes in a single sequence. The GEM technology also uses a color coding technique to examine the difference in expression between the two mRNA samples. A cDNA sequence contains a diversity of animal cDNA fragments, as a rat or human, preferably human, amplified by PCR and immobilized on a positively charged nylon membrane or glass slides or silicon wafer or any other surface to be able to be revealed, where a DNA / matrix interaction is allowed. A cell type of interest is subjected to treatment with and without Ginkgo biloba extract or a component thereof, for about 48 hours. The poly A + RNA is isolated from control cells and cells subjected to treatment with the extract. 32 P-labeled, fluorescent, chemiluminescent or colorimetric cDNA probes, preferably fluorescently or colorimetrically labeled when silicon or glass chip sequences are used, are generated from each poly A + RNA and hybridized to the sequence as the manufacturer's recommendations. An autoradiography is performed by exposing the transfers on a film at a temperature of about -70 ° C for a period of time of 4-96 hours. The quantification of hybridization is carried out using an imaging system, which can detect fluorescence or chemiluminescence, then capture the image and analyze the data, such as the SigmaGel software. Several exposures are used in order to detect the. genes expressed at low levels. The three internal controls, ubiquitin, G3PDH and β-actin are used to compare the relative expression levels of the gene products detected in the control and in the cells treated with the extract. Experimental variations are corrected using the proportions of gene expression against internal controls. The effect of the treatment with extract on each gene product is expressed as the% of control cells (untreated). An example of the above type of gene expression profile is as follows. The sequence for the expression of human cDNA Atlas I of Clontech (Palo Alto, CA) contains 588 fragments of human cDNA amplified by PCR of 200-500 bp long and immobilized on a positively charged nylon membrane. MDA-231 cells are treated with and without 20 g / ml of EGB 761® for 48 hours. The poly A + AR was isolated from the control and from the cells treated with EGB 761®. 32 P-labeled cDNA probes are generated from each poly A + AR and hybridized to the Atlas sequence according to the manufacturer's recommendations. An autoradiography is performed by exposing the transfers to X-OMAT AR film (Kodak, Ochester, NY) at -70 ° C for 4-96 hours. The quantification of the hybridization observed is carried out using the SigmaGel software (Jandel Scientific, San Rafael, CA). Several exposures are used to detect genes expressed at low levels. The three internal controls, ubiquitin, G3PDH and β-actin are used to compare the relative expression levels of the gene products detected in the control and in the cells treated with EGB 761®. Experimental variations are corrected using the proportions of gene expression against internal controls. The effect of EGB 761® treatment on each gene product is expressed as the% of control cells (untreated). The results of this experiment, presented in Table 1, show the affected genes consistently, at a level above 30% control, due to the treatment with EGB 761®. In summary, the Table. 1 shows that the treatment increased the expression of the c-Mic proto-oncogene and decreased the expression of 35 gene products, including the oncogenes (AP-1, PE-1, RhoA, n-Mic), the cell cycle regulators (CDK2, p55CDC , PCNA pl20), modulators of signal transduction (NET1, ERK2), receptors (A2A, RXR-beta, Grb2) of products (SGP-2, NIP1) related to apoptosis, transcription factors (Id -2, ATF-4, ETR101, ETR-103), growth factors (HB-EGF, similar-HGF), and cell adhesion molecules (CD19, L1CAM, integrins a3, 0t4, a6, ß5; Mac-1, catenin-ß) that are directly involved in the various pathways that regulate cell proliferation. Gene expression profiles can be established for extracts of Ginkgo biloba of known origin and can then be compared with the profile of gene expression of Ginkgo biloba extracts of unknown origin or extracts that claim to be a certain commercial extract. The comparison of the profiles can thus be used as a means of detection to authenticate the origin of an extract.

TABLE 1 Effect of EGB 761® on gene expression of MDA-231 examined using the sequence for the expression of the human Atlas cDNA as described under Nucleic Acid Sequences. Nom e% Function References Oncooenes change v Tumoral oresores c-Mic + 75% - basic factor of the (37) helix zipper transcription -l zo-helix-leucine - Mic / Max heterodimers induce cycle progression Name % Referent Function Cell turnover, apoptosis and malignant transformation c-Jun -78% -part of the (38) transcription factor AP-1 that regulates the genes involved in cell proliferation RhoA -93% -GTP binding protein that ( 39) is an important regulator of cell proliferation-inactivation of RhoA inhibits the (40) proliferation of HL60 APC -59% cells-APC mutations are (41) associated with colorectal cancers both hereditary and sporadic (42) -an post-translational negative regulator of ß-catenin PE-1 -42% -transcription factor (43) Proteins for the Control of the Cell Cycle Protimos- -79% -acidic acid protein that (44) is up-regulated in the proly thymocytes ferantes, lymphocytes of patients with leukemia and cancer lesions Name% of Function ReferenceChange of malignant breast myeloblas- -66% -a serine protease (45) tub involved in the cellular differentiation of leukemia p55CDC -63% -similar to the regulators of (46) mitosis CDC4 and CDC20-expression that correlates positively with the cell proliferation state Antigen -68% -nuclearized protein expressed (47) Nuclear in proliferating cells pl20-a prognostic indicator (48) Prolifera for cancer patients of breast and adenocarcinoma of prostate cell CDK2 -83% -tyrosine kinase dependent on (49) cyclin involved in the progression through the cell cycle (50) -cycline E / Cdk2 inactivates the retinoblastoma tumor suppressor to allow the cells progress to phase S Name% of function Ref renCambio cias -inhibition of Vitamin D proliferation in LNCaP cells coincides with a reduction in the activity of Cdk2 Intracellular Transducers NET1 -55% - exchange factor of (51) guanine specific for RhoA - transforming protein of NIH3T3 ERK2 - 46% - member of the family of the (52) protein kinases related to the extracellular signal - activated at the time of the stimulation of cells Proteins Related to the Auootosis Receiver -40% -receptor coupled to the (53) A2A of G protein involved in the Adenosine cAMP signaling pathway Ligand -58% -linking for tyrosine (54) Flt3 Cytokine receptor Flt3 kinase -induces the proliferation of myeloid cells in leukemia Grb2 -70% -an adapter protein that (55) links tyrosine kinases Name% of Referent FunctionChange of early retinoblastoma TRADD -51% -protein of the death domain (65) associated with TNFR -involved in cell growth and differentiation induced by TNFR NIP-1 -40% -originally described as (66) a transpo protein Nuclear art of yeast (67) -part of the factor 3 of the start of translation (elF3) of the nucleus complex Transcription factors / DNA binding Id-2 -65% - a member of the family Id of (68) the helix-loop-helix of transcription inhibitors - involved in the proliferation of pancreatic cancer cells in humans ATF4 - 42% - a member of the family (69) ATF / CREB of transcription factors - regulates the transformation induced by Ras of cells Name% of Function ReferenChange Cias NIH3T3 ETR103 -65% -an immediate early gene (70) associated with the macrophage ETR101 -60% -an immediate early gene (71) associated with the lymphocyte Adhesion Molecules? Antigens of the Anomalous Cell Surface -62% -integral protein of the lymphocyte membrane (72) -B lymphocyte-expression is regulated to B CD19 down during retinoid inhibition of lymphoblastoid B cell proliferation L1CA -72% -neurial cell adhesion molecule (73) -increased expression of L1CAM is associated with high-grade migration of β-catenin glioma cells -58% -involved in the (74) cell-cell interactions mediated by cadherin -interact with the TCF / LEF transcription factors in the pathway Name% of Function ReferenChange of signaling Wnt Subunit-des of Integrin aM -41% -regulates cell adhesion (75) of the human neutrophils with LFA-? ß -subunit to the β5 elastase receptor -55% -subunit β of the receptor (76) vitronectin (VR) -involved in the determination (77) of the proliferation of oligodendrocytes (78) -involved in murine retinal angiogenesis 4 -49% -the cross-linking of (79) a4 integrins inhibits the proliferation of lymphoma cells LB (80) -also involved in the metastasis of c Melanoma and lymphoma cells a3 -77% -an integrin antibody a3 (81) functionally disturbing Name% of Function References Change inhibits the proliferation of human epithelial cells -53% -over-expression of integrin a6 (82) collaborates with ErbB2 to induce a more malignant phenotype in NIH3.T3 cells Extracellular Protein Signaling / Communication Factor-1 -31% -regulates proliferation, (83) Stimulating differentiation and survival (84) of monocytes, macrophages and their precursor colonies - initiates a mitogenic signal macrophages that is required through the (CSF-1) phase Gl - CSF-1 cells of the granulosa ovarian cells stably transfected exhibit enhanced cell proliferation -62% factor-overexpressed in various (85) glioma cell growths in human-like and in most of the glioblastomas-binding EGF-stimulates proliferation of Name% of Function ReferenCambio cias Heparin human glioma cells (HB-EGF) Protein -81% -a transmembrane protein of (86) similar to tyrosine kinase was discovered (87) factor to be overexpressed in the hepatocyte growth and in the primary hepatocyte liver carcinoma - human cough - induces proliferation and (HGFLP) migration of murine keratinocytes Inhibin ce -69% - a member of the family of (88) inhibin of the heterodimeric (89) growth factors - inhibin oc is a marker of the trophoblastic neoplasia and it is expressed in virilizing adenomas

Claims

CLAIMS: 1. A method for establishing a profile of the gene regulation of Ginkgo biloba extract or a component of Ginkgo biloba extract, comprising the steps of: obtaining at least one batch of untreated cells; treat a first batch of cells and an extract of Ginkgo biloba or a component of Ginkgo biloba extract to obtain a treated batch of cells, - quantify an effect on the expression of one or more genes of the treated cells to obtain a quantity of genes affected; and comparing the amount of genes affected against a number of cell genes that were not treated with Ginkgo biloba or a component of Ginkgo biloba extract to obtain the profile of gene regulation of Ginkgo biloba extract or a component of Ginkgo extract biloba
2. A method according to claim 1, wherein the quantization comprises: isolating poly A + RNA from the treated cell batch to obtain the poly A + RNA treated; isolating the poly A + RNA from a batch of untreated cells to obtain unaddressed poly A + RNA; generating the labeled cDNA probes from the treated poly A + RNA to obtain the labeled probes of treated cDNA; generate labeled cDNA probes from the unreacted poly A + RNA to obtain the labeled probes of untreated cDNA; hybridizing the cDNA probes treated in a sequence having one or more cDNA to obtain a hybridized and treated cDNA sequence; hybridizing untreated cDNA probes to form a sequence having one or more cDNAs to obtain the hybridized and untreated cDNA sequence; quantifying each of the cDNAs of the hybridized and treated sequence of the cDNA to obtain amounts of treated cDNA; quantifying each of the cDNAs of the hybridized and untreated cDNA sequence to obtain amounts of untreated cDNA; and comparing the amounts of each of the treated cDNAs with the amounts of untreated cDNA to obtain the gene regulation profile.
3. A method according to claim 2, wherein the cells are MDA-231 cells; Ginkgo biloba extract is EGB 761®; and the sequence is a gene piece that has several genes.
4. A method according to claim 3, wherein the profile of the gene regulation of the EGB 761® comprises the increased expression of the c-Mic proto-oncogene, and the diminished expression of the following genes: protimosin-ct, CDK2, p55CDC, antigen nuclear myeloblastin cell proliferation pl20, NETl, E K2, Adenosine A2A receptor, ligand Flt3, Grb2, Clusterin, RXR-β, Glutathione S-transferase P, N-Mic, 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-a, lymphocyte-antigen B CD19, L1CAM, β-catenin, oc-3 integrin subunit, oc-4 integrin subunit, integrin α-6 subunit, integrin β-5 subunit, integrin αM subunit, APC, PE-1, RhoA, c-Jun, protimosin-cc, CDK2, p55CDC and myeloblastin. 5. A method according to claim 4, wherein the profile of the gene regulation of the EGB 761® is approximately c-ic = + 75%, c-Jun = -78%, RhoA = -93%, APC = -59 %, PE-1 = -42%, Protimosine- = -79%, Myeloblastine = -66%, p55CDC = -63%, Nuclear Antigen of Cell Proliferation pl20 = -68%, CD 2 = -83%, NETl = - 55%, ERK2 = -46%, A2A Adenosine Receptor = -40%, ligand Flt3 = -58%, Grb2 = -70%, Clusterine = -54%, RXR-β = -55%, Glutathione S-transferase P = -39%, N-ic = -74%, TRADD = -51%, NIP-1 = -40%, Id-2 = -65%, ATF4 = -42%, ETR103 = -65%, ETR101 = - 60%, B-lymphocyte antigen CD19 = -62%, L1CAM = -72%, ß-catenin = -58%, Subunit aM of Integrina = -41%, subunit ß5 of Integrina = -55%, Subunit a4 of Integrin = -49%, Subunit a3 of Integrina = -77%, Subunit oc6 of Integrina = -53%, Factor-1 stimulating of Colonies of Macrophages (CSF-1) = -31%, Growth Factor similar to EGF of Unión a Heparin (HB-EGF) = -62%, Protein similar to Cre Factor Hepatocyte foundation (HGFLP) = -81% and Inhibin a = -69%, where the percentages shown can be ± 20%.
5. A method according to claim 2, wherein the cells are MDA-231 cells; the component of Ginkgo biloba extract is Gingkolide B; and the sequence is a gene piece that has a multiplicity of genes.
6. A method for verifying the identity of a Ginkgo biloba extract comprising the steps of: obtaining a profile of the gene regulation of the Ginkgo biloba extract to obtain a profile of the gene regulation; obtain a profile of the gene regulation of EGB 761® to produce a profile of the gene regulation of EGB 761®; compare the regulation profile. Gene of Ginkgo biloba extract with the gene regulation profile of EGB 761®; determine if the values of the profile of the gene regulation of the Ginkgo biloba extract are within ± 10% of the values of the profile of the gene regulation of EGB 761® to obtain the verification of the identity of the Ginkgo biloba extract.
7. A method according to claim 6, wherein the method for obtaining a profile of the gene regulation of the Ginkgo biloba extract and the gene regulation profile of EGB 761® comprises the steps of: isolating the poly A + RNA from the batch treated cells to obtain the poly A + RNA treated by isolating the poly A + RNA from a batch of untreated cells to obtain the poly A + untreated RNA; generating the labeled cDNA probes from the treated poly A + RNA to obtain the labeled probes of treated cDNA; generating the labeled cDNA probes from the unreacted poly A + RNA to obtain the labeled probes from the untreated cDNA; hybridizing the treated cDNA probes to obtain a sequence having one or more cDNAs to obtain a treated and hybridized sequence of the cDNA; hybridizing the untreated cDNA probes to obtain a sequence having one or more cDNAs to obtain a hybridized and untreated cDNA sequence; quantifying each of the cDNAs of the hybridized and treated sequence of the cDNA to obtain the amounts of treated cDNA; quantifying each of the cDNAs of the hybridized and untreated sequence of the cDNA to obtain amounts of untreated cDNA; and comparing the amounts of each of the cDNAs treated with the amounts of untreated cDNA to characterize the profile of gene regulation.