Cancer cell growth inhibition by black bean (Phaseolus vulgaris L) extracts

Abstract

A group of phytochemical compounds extracted from whole germinated or not germinated black beans ( Phaseolus vulgaris L) and/or their dry milled fractions such as seed coats or hulls and cotyledons is described. These phytochemicals are classified as phenolics, such as polyphenols, flavonoids, coumarins, and tannins, triterpenes such as saponins, phytosterols and other antioxidant compounds that proved to be effective to decrease cancer proliferation of hormone dependent mammary (MCF-7), hormone independent hepatic (HepG2) and colon (Caco2) cancer cells. They also effectively protect against chemical damage induced with DMBA and furthermore prevented cancer induced by this chemical in Wistar rats and also reduced tumor size when consumed after tumor detection. Methanol, acetone, ethanol, water and mixtures of these solvents were used to obtain the extracts from raw, cooked, and germinated black beans of different varieties, both from the whole grain and the hulls. The black bean malting process produced extracts rich in aglycone forms of the previous mentioned compounds that were more biologically active against MCF-7 cancer cell proliferation. Both raw extracts, C-18 HPLC refined extracts and fractionated extracts may be used in the treatment, prophylaxis, amelioration or defense against a variety of hormone dependent and independent cancers. The extracts and compounds have additional utilities, e.g., lower cholesterol or lower oxidation of LDL and/or inhibit cholesterol synthesis (or the enzyme therefor) and/or reduce liver fibrosis and/or reduce symptoms of menopause and/or stimulate calcium absorption and/or have estrogenic activity such as feminizing estrogenic activity and/or are antioxidants, for instance, as an active ingredient in a nutritional supplement and/or as a food, cosmetic or pharmaceutical antioxidants or colorants.

1 . An isolated black bean ( Phaseolus vulgaris ) extract prepared by process which comprises:
 (a) extracting a mixture of compounds from the black bean or hulls of the black bean with a polar solvent system consisting essentially of (i) one or more C 1 -C 6  alcohol or (ii) water and a C 1 -C 6 -alcohol;
 (b) subjecting the mixture of compounds from extracting step (a) to a chromatographic separation step which comprises of a first elution step, wherein a first fraction is obtained, and a second elution step, wherein a second fraction, which is the isolated black bean extract, is obtained, wherein: 
 (i) the first fraction has less flavonols, flavones and isoflavones than the second fraction; 
 (ii) the first fraction also contains phenolic acids and other water soluble compounds; and 
 (iii) the second fraction contains flavonols, isoflavones, anthocyanins and a compound with a mass spectral ion peak of 981 Daltons. 
   
     
     
         2 . The isolated black bean extract of  claim 1 , wherein the black bean is selected from the genotypes of making a black bean extract, wherein the black bean is from the genotype Mex 332, Negro Coaxtla 91, Negro 8025, Negro San Luis, Negro Altiplano, Negro 150, NG-Sahuatoba, Negro Tacana, Negro Viscaya, Negro Otomi, Negro Perla or Negro INIFAP. 
     
     
         3 . The isolated black bean extract of  claim 1 , wherein said mass spectral ion peak of 981 Daltons when subjected to double ionization, gives mass spectral ion peaks of 797, 635, 599 and 441 Daltons. 
     
     
         4 . A composition which comprises of the black bean extract of  claim 1  and a pharmaceutically acceptable carrier and/or excipient. 
     
     
         5 . A composition which comprises of the black bean extract of  claim 2  and a pharmaceutically acceptable carrier and/or excipient. 
     
     
         6 . A composition which comprises of the black bean extract of  claim 3  and a pharmaceutically acceptable carrier and/or excipient. 
     
     
         7 . A composition which comprises the black bean extract of  claim 1 . 
     
     
         8 . A composition which comprises the black bean extract of  claim 2 . 
     
     
         9 . A composition which comprises the black bean extract of  claim 3 . 
     
     
         10 . The isolated black bean extract of  claim 1 , wherein the solvent for the first elution is a mixture of water and a C 1 -C 6 -alcohol and the solvent for the second elution is a C 1 -C 6  alcohol. 
     
     
         11 . The isolated black bean extract of  claim 10 , wherein the C 1 -C 6 -alcohol is methanol or ethanol. 
     
     
         12 . The isolated black bean extract of  claim 11 , wherein the C 1 -C 6 -alcohol is methanol. 
     
     
         13 . The isolated black bean extract of  claim 1 , wherein the first fraction contains less than 1% by weight of flavonols, flavones or isoflavones based on the dry weight of the compounds in the first fraction. 
     
     
         14 . The isolated black bean extract of  claim 13 , wherein the amount of anthocyanins ranges from about 15 ppm to about 105 ppm. 
     
     
         15 . The isolated black bean extract of  claim 14 , wherein the amount of anthocyanins ranges from about 15 ppm to about 60 ppm. 
     
     
         16 . The isolated black bean extract of  claim 15 , wherein the anthocyanins are delphinidin, petunidin and malvidin. 
     
     
         17 . The isolated black bean extract of  claim 16 , wherein the amount of delphinidin ranges from about 5 ppm to about 20 ppm, the amount of petunidin ranges from about 5 ppm to about 20 ppm and malvidin ranges from about 5 ppm to about 20 ppm. 
     
     
         18 . The isolated black bean extract of  claim 14 , wherein the flavonols are one or more compounds selected from the group consisting of myricetin, myricetin 3′-rhamnoside, quercetin, quercetin 3-β-galactoside, quercetin 3-glucopyranoside, quercetin 4′-galactoside, kaempferol, kaempferol 3-O-glucoside, 3′,4′,5′,5,7-pentahydroxyflavonol, 3′,4′,5,7-tetrahydroxyflavonol and 4′,5,7-trihydroxyflavonol. 
     
     
         19 . The isolated black bean extract of  claim 18 , wherein the solvent for the first elution is a mixture of water and a C 1 -C 6 -alcohol and the solvent for the second elution is a C 1 -C 6  alcohol. 
     
     
         20 . A process of making a black bean extract which comprises:
 (a) extracting the black bean or black bean hulls with at least one polar solvent; selected from the group consisting of (i) one or more C 1 -C 6  alcohol or (ii) water and a C 1 -C 6 -alcohol; and optionally,   (b1) is a chromatographic separation step which results in an extract which contains glycosidic forms of delphinidin, petunidin and malvidin; or   (b2) is a chromatographic separation step wherein a first fraction is obtained, which has less flavanols, flavones and isoflavones than a second fraction and wherein the first fraction also contains phenolic acids and other water soluble compounds and a second fraction which contains flavanoids and anthocyanins; and   (c) at least one additional extraction with at least one polar solvent.   
     
     
         21 . The process of  claim 20 , wherein the black bean is selected from the genotypes Mex 332, NG-Coaxtla 91, NG-8025, NG-San Luis, NG Altiplano, NG-150, NG-Sahuatoba; NG Tacana, NG-Viscaya, Negro Otomi, NG Perla and NG-INIFAP. 
     
     
         22 . The process of any one of  claim 20  or  21  wherein (a) is malted, sprouted or germinated black bean. 
     
     
         23 . The process of  claim 20 , wherein the at least one polar solvent in step (c) is selected from the group consisting of C 6 -C 10  heteroaryl, C 1 -C 6 -alcohol, C 1 -C 6 -aldehyde, C 1 -C 6 -amines, C 1 -C 6 -ketones, C 2 -C 6  esters, C 2 -C 6 -ethers, acetone, ethanol, methanol, water and mixtures thereof. 
     
     
         24 . The process of  claim 23 , wherein the at least one polar solvent in step (c) is selected from the group consisting of C 1 -C 6 -alcohol, C 2 -C 6  esters, C 2 -C 6 -ethers, and mixtures thereof. 
     
     
         25 . An isolated black bean extract of produced by the process which comprises:
 (a) extracting the black bean or black bean hulls with at least one polar solvent selected from the group consisting of (i) one or more C 1 -C 6  alcohol or (ii) water and a C 1 -C 6 -alcohol;
 and optionally either (b1) or (b2) wherein, 
   (b1) is a chromatographic separation step which results in an extract which contains glycosidic forms of delphinidin, petunidin and malvidin; or   (b2) is a chromatographic separation step wherein a first fraction is obtained, which has less flavanols, flavones and isoflavones than a second fraction and wherein the first fraction also contains phenolic acids and other water soluble compounds and a second fraction which contains flavanoids and anthocyanins; and optionally, further comprises,   (c) at least one additional extraction with at least one polar solvent.   
     
     
         26 . The isolated black bean extract of  claim 25 , wherein the black bean is selected from the genotypes Mex 332, NG-Coaxtla 91, NG-8025, NG-San Luis, NG Altiplano, NG-150, NG-Sahuatoba; NG Tacana, NG-Viscaya, Negro Otomi, NG Perla and NG-INIFAP. 
     
     
         27 . The isolated black bean extract of any one of  claim 25  or  26  wherein (a) is malted, sprouted or germinated black bean. 
     
     
         28 . The isolated black bean extract of  claim 25 , wherein the at least one polar solvent in step (c) is selected from the group consisting of C 6 -C 10  heteroaryl, C 1 -C 6 -alcohol, C 1 -C 6 -aldehyde, C 1 -C 6 -amines, C 1 -C 6 -ketones, C 2 -C 6  esters, C 2 -C 6 -ethers, acetone, ethanol, methanol, water and mixtures thereof. 
     
     
         29 . The isolated black bean extract of  claim 28 , wherein the at least one polar solvent in step (c) is selected from the group consisting of C 1 -C 6 -alcohol, C 2 -C 6  esters, C 2 -C 6 -ethers, and mixtures thereof.