Bowman-Birk inhibitors in lentil: Heterologous expression,functional characterisation and anti-proliferative properties in human colon cancer cells

A full-length cDNA, encoding a Bowman-Birk protease inhibitor (BBI), was isolated from lentil immature seeds. The deduced amino acid sequence was longer than that of the BBI extracted from lentil seeds and contained two binding sites; the first inhibitory site inhibits trypsin whereas the second one inhibits chymotrypsin. In order to characterize this lentil BBI, a longer (complete) and its C-terminally processed (mature) form were heterologously expressed in the yeast Pichia pastoris. The recombinant BBI proteins proved to be active against trypsin and chymotrypsin, showing K_i values at nanomolar levels. Mass spectrometry analysis revealed that complete BBI was composed of an array of molecular masses, whereas mature BBI showed the presence of a major peak of the expected size. The effects of mature BBI on the growth of human colon adenocarcinoma HT29 and colonic fibroblast CCD-18Co cells were evaluated. Lentil BBI was able to inhibit the growth of such cells at concentrations higher than 19 μM, in a concentration-dependent manner; by contrast, the CCD18-Co cells were unaffected. These data broaden our knowledge of the beneficial biological activities of naturally-occurring BBI proteins and address the need for systematic evaluation of natural variants in order to design novel strategies in preventive medicine.     

DOUBLE-HEADED TRYPSIN/CHYMOTRYPSIN INHIBITORS FROM HORSE GRAM (DOLICHOS BIFLORUS): PURIFICATION, MOLECULAR AND KINETIC PROPERTIES

Four proteinase inhibitors were purified to homogeneity from horse gram (Dolichos biflorus). These inhibitors are double-headed and inhibit trypsin and chymotrypsin simultaneously and independently. Dissociation constants range between 0.87 and 4.6 × 10^?7 M. Each of the four isoinhibitors possesses a crucial lysine residue at the trypsin reactive site. These inhibitors have molecular masses of 8.5 kDa and isoelectric points of 4.6 to 5.6. They exist mainly as dimers under physiological conditions. Amino acid analysis revealed high levels of half-cystine, serine, aspartate and proline but low levels of methionine and aromatic amino acids. Amino-terminal sequence analysis revealed that each of the four isoinhibitors have a conserved core sequence but are divergent at the N-terminal end. These inhibitors belong to the Bowman-Birk (BBI) family of proteinase inhibitors as reflected by their inhibitory properties, amino acid composition and homology to other BBIs.     

Immunoassays for Bowman-Birk and Kunitz Soybean Trypsin Inhibitors in Infant Formula

ABSTRACT: Because the consumption of soybean inhibitors of digestive enzymes in processed foods may have both beneficial and adverse health-related effects, reliable and rapid analytical methods for these inhibitors are needed. Monoclonal antibody-based sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for the 2 major soybean protease inhibitors, the Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI) of trypsin and chymotrypsin. The ELISAs had limits of quantification of approximately 1 and 3 ng/mL for BBI and KTI, respectively, and were used to measure active inhibitors in soy infant formulas. Results were compared with enzymatic analyses and demonstrated that most of the trypsin- and chymotrypsin-inhibitory activities of infant formula were due to constituents other than KTI and BBI. The sandwich ELISA for BBI was also effective in detecting soybean germplasm with atypically low levels of BBI.     

Inhibitor activities of chickpeas (Cicer arietinum L) against bovine,pocine and human trypsin and chymotrypsin

Twenty-two chickpea samples (7 genotypes, 4 desi and 3 kabuli) representing three different sites of cultivation and three years of harvest were examined for their inhibitory activities against bovine, porcine and human trypsin/chymotrypsin, and compared for their isoinhibitor patterns. No significant difference in inhibitor activity between the desi and kabuli seed types was found for any of the enzymes used, when data from different locations and years were averaged. However, some significant differences were observed between individual cultivars when compared at the same location and in the same year. Inhibitor activities against the three trypsins and three chymotrypsins were statistically different with activities against bovine (5.46 ± 1.21) > porcine (5.24 ± 1.18) > human (4.45 ± 0.93) trypsin and those against bovine (4.89 ± 1.27) < porcine (8.91 ± 2.14) < human (10.54 ± 2.50) chymotrypsin (means ± standard deviation, expressed as mg active enzyme completely inhibited per g seed meal). Levels of activity of both trypsin and chymotrypsin inhibitor were influenced by the location and year of cultivation. Isoelectric focusing yielded identical inhibitor patterns for all samples, indicating seven isoinhibitors acting against trypsin and chymotrypsin. Results are discussed in relation to nutritional significance of these inhibitors and perspectives for breeding genotypes low in protcase inhibitors.     

The biochemical and functional food properties of the bowman-birk inhibitor

The Bowman-Birk inhibitor (BBI) is a small water-soluble protein present in soybean and almost all monocotyledonous and dicotyledonous seeds. The molecular size of BBI ranges from 1,513 Da to about 20,000 Da. BBI is to seeds what alpha(1)-antitrypsin is to humans. Soy-based food products rich in BBI include soybean grits, soymilk, oilcake, soybean isolate, and soybean protein concentrate. BBI is stable within the pH range encountered in most foods, can withstand boiling water temperature for 10 min, resistant to the pH range and proteolytic enzymes of the gastrointestinal tract, bioavailable, and not allergenic. BBI reduces the proteolytic activities of trypsin, chymotrypsin, elastase, cathepsin G, and chymase, serine protease-dependent matrix metalloproteinases, urokinase protein activator, mitogen activated protein kinase, and PI3 kinase, and upregulates connexin 43 (Cx43) expression. Several studies have demonstrated the efficacy of BBI against tumor cells in vitro, animal models, and human phase IIa clinical trials. FDA considers BBI as a drug. FDA also approves labels claiming that consumption of at least 3 to 4 oz of tofu or 8 oz of soymilk or soy protein may reduce the risk of coronary heart disease and breast cancer. This review highlights the biochemical and functional food properties of the Bowman-Birk inhibitor.     

Active Bowman–Birk inhibitors survive gastrointestinal digestion at the terminal ileum of pigs fed chickpea‐based diets

BACKGROUND: Protease inhibitors of the Bowman–Birk family have been demonstrated to be naturally occurring chemopreventive agents in a wide range of in vitro and in vivo models. In vitro and in vivo experiments have reported that Bowman–Birk inhibitors (BBIs) may exert colorectal chemopreventive effects. To exert such effects, these proteins have to survive, at least to some extent, the digestive process within the gastrointestinal tract. RESULTS: In order to determine the survival rates of functional BBI proteins in vivo, five castrated male pigs (100 ± 2 kg body weight) fitted with T‐shaped cannulas at the terminal ileum were fed a chickpea‐based diet. Pigs fed hydrolysed casein as the only protein source were used as an experimental control diet. The survival rates of BBI proteins from chickpea‐based diets at the terminal ileum, expressed in terms of trypsin (TIA) and chymotrypsin inhibitory activity (CIA), were 7.3 and 4.4%, respectively. The presence of BBI proteins in ileal samples from pigs fed chickpea‐based diet was confirmed by SDS‐PAGE and peptide mass fingerprinting. CONCLUSION: It is concluded that significant amounts of active BBI proteins reach the large intestine of the pig. Further pharmacological studies are necessary in order to determine the potential of dietary BBI proteins as colorectal chemopreventive agents in humans. Copyright © 2007 Society of Chemical Industry     

PURIFICATION AND PARTIAL CHARACTERIZATION OF TWO PROTEINACEOUS α-AMYLASE INHIBITORS FROM TRITICALE

A total of six α-amylase inhibitory proteins (isoinhibitors) were extracted from triticale (Triticum X Secale) seeds and two of them were purified to homogeneity. The isoinhibitors were extracted by 70% ethanol and produced, by Sephadex G-100 chromatography, two peaks that exhibited α-amylase inhibitory activity. Further purification of the most active peak by DEAE-cellulose chromatography resulted in six active fractions. Two of them designated as TAI-5 and TAI-6, considered to be homogeneous by both acidic and alkaline electrophoresis, were partially characterized. The isoelectric points were 4.80 and 4.70, and the molecular weights 39, 200 and 29, 200, respectively. Under dissociating conditions the molecular weights were 13, 500 and 13, 000, suggesting that the isoinhibitors are composed of three and two subunits, respectively. Both isoinhibitors were stable at different pHs, relatively stable at 98C, and resistant to proteolysis by trypsin, chymotrypsin and pepsin. The optimum interaction pH for both isoinhibitors with human salivary amylase was 7.9. They exhibited specificity to human salivary and porcine pancreatic α-amylases, but had no inhibitory activity on Bacillus subtillis, Aspergillus oryzae and endogenous triticale α-amylases.     

Inhibitors of human and bovine trypsin and chymotrypsin in fenugreek (Trigonella foenum-graecum L.) seeds. Reaction with the human and bovine proteinases.

The reaction between the three Bowman-Birk proteinase inhibitors isolated from fenugreek seeds (TFI-B2, TFI-N2 and TFI-A8) and the human and bovine proteinases was investigated by studying the complexes formed and their properties. TFI-B2, the Lys-Leu trypsin chymotrypsin inhibitor, can bind 1.9 mol human trypsin (HT), 1.3 mol bovine trypsin (BT) and/or 0.4 mol human (HCT) or bovine (BCT) chymotrypsin per mole of inhibitor. HT was bound at the two reactive sites and BT mainly at the lysine-containing trypsin-reactive site, whereas HCT and BCT were only bound at the leucine-containing chymotrypsin-reactive site. TFI-N2, the Arg-Leu trypsin chymotrypsin inhibitor, could bind 1 mol BT and BCT, but 1.3 mol HT and 1.2 mol HCT per mole of inhibitor. In addition to the usual binding, the human enzymes could also be bound at the respective "wrong" reactive site. TFI-A8, the Arg-Arg trypsin inhibitor, binds 2 mol HT or BT per mole of inhibitor at the two trypsin-reactive sites, whereas HCT and BCT (about 0.2 mol/mol) are bound to one of the two "wrong" reactive sites.     

Antiproliferative effects of prenylflavonoids from hops on human colon cancer cell lines

In recent years, interest in hop‐derived constituents, especially for prenylflavonoids has grown, as they have a wide range of biological properties including antioxidant, anticarcinogenic and antimicrobial activities. Two main hop prenylflavonoids, xanthohumol and isoxanthohumol, and hop extract enriched in prenylflavonoids, were tested for their antiproliferative activities on colon cancer cell lines, HT‐29 and SW620, and a noncancerous cell line, IEC‐6. It was confirmed that both xanthohumol and isoxanthohumol inhibited cell proliferation, even at micromolar concentrations. For cell line HT‐29, the IC₅₀ was 1.2 ± 0.9 and 16.9 ± 0.9 µmol dm^?3 for xanthohumol and isoxanthohumol, respectively. Similar values were obtained for SW620 cells (2.5 ± 0.2 and 37.3 ± 3.2 µmol dm^–3). None of the pure prenylflavonoids that were tested affected the proliferation of the noncancerous cell line, IEC‐6. The effect of the hop extract containing xanthohumol was also tested for antiproliferative activities on the cancer cell lines, HT‐29 (IC₅₀ = 3.1 ± 0.2 µmol dm^–3) and SW620 (IC₅₀ = 1 ± 0.2 µmol dm^?3), and on the cell line, IEC‐6 (IC₅₀ = 65.5 ± 11.3 µmol dm^?3). The results showed a similar trend to that for pure compounds, suggesting a possible future application of hop extracts in the pharmaceutical industry. Copyright © 2014 The Institute of Brewing & Distilling     

Response of intestinal proteinase activities to the feeding of isolated winged bean (Psophocarpus tetragonolobus) and soya bean (Glycine max) trypsin inhibitors in rats

The antinutritional activities of trypsin inhibitors (TIs) were compared between winged beans (Psophocarpus tetragonolobus) and soya beans (Glycine max). The inhibitors of the two beans were isolated by trypsin‐bound Sepharose 4B, and 50 mg of lyophilised powders were intubated intragastrically into 24 h fasted rats. The activities of trypsin and chymotrypsin were compared after 30, 60 and 180 min in the washings of the upper, middle and lower parts of the small intestine. The elution profiles of TI and non‐TI compounds in the affinity chromatography were similar in the two beans, and the antitryptic activities were concentrated 5.5 and 6.2 times (based on specific activity) for winged beans and soya beans respectively. Regardless of the TI fed to rats, trypsin activity in the upper intestine was suppressed to almost undetectable levels at 30 and 60 min after intubation. The activities in the middle and lower intestines were also substantially lowered when rats were fed winged bean TI, and significant differences were detected at 30 and 60 min after intubation when compared with rats fed soya bean TI. However, at 180 min after feeding, no differences were found in the trypsin activity in any gut segments. Similar inhibitory properties of isolated TIs were observed in chymotrypsin activities in the small intestine. The results suggest that winged bean TI may have greater inhibitory activity on the intestinal proteinase compared with soya bean TI. © 2001 Society of Chemical Industry     

REACTION OF LENTIL PROTEINASE INHIBITORS WITH HUMAN AND BOVINE TRYPSIN AND CHYMOTRYPSIN1

The two main trypsin-chymotrypsin isoinhibitors previously purified from lentils (Lens culinaris Medik.), LCI-1 and LCI-4, inhibited one mol of human trypsin (1.05 and 1.00), more than one mol of bovine trypsin (1.53 and 1.38) and human chymotrypsin (1.70 and 1.43) as well as less than one mol of bovine chymotrypsin (0.62 and 0.54, respectively) per mol of inhibitor. Complex formation, together with chemical and enzymatic modification studies, showed that they were Bowman-Birk inhibitors with two independent reactive sites. One of these sites, mainly reacting with trypsin, contained arginine and bound tightly to bovine trypsin, less tightly to human trypsin and loosely to human chymotrypsin. The other reactive site, preferring chymotrypsin, contained tyrosine and bound tightly to human chymotrypsin, less tightly to bovine chymotrypsin and loosely to bovine trypsin. The amounts of bound enzyme exceeding one mol per mol of inhibitor reacted with the “wrong” sites: bovine trypsin with the chymotrypsin-reactive and human chymotrypsin with the trypsin-reactive one. The much higher inhibition of human chymotrypsin compared to that of bovine chymotrypsin resulted from a combination of two effects: the additional binding of human chymotrypsin at the “wrong” reactive site and the weak binding of the bovine chymotrypsin.     

Antinutritional and/or toxic factors in soybean (Glycine max (L) Merril) seeds: comparison of different cultivars adapted to the southern region of Brazil

Soybean (Glycine max (L) Merril) seeds are known to contain different proteins displaying antinutritional and/or toxic effects, such as soybean agglutinin (an N‐acetylgalactosamine‐specific lectin), proteinase inhibitors (Kunitz‐ and Bowman–Birk‐type trypsin and chymotrypsin inhibitors) and urease (seed and tissue isoforms). Two other toxic proteins were previously isolated from soybeans, soyatoxin (21 kDa) and soybean toxin (18.4 kDa), which are immunologically related to canatoxin, a toxic protein from Canavalia ensiformis (jackbean) seeds. In this work we have screened crude extracts from seeds of six different soybean cultivars, which together represent most of the crop harvested in the southern region of Brazil, for the presence of urease, trypsin inhibitory and haemagglutination activities, intraperitoneal toxicity in mice and immunoreactivity against anti‐canatoxin antibodies. Significant differences were found in the contents of proteinase inhibitors, lectin, urease activity and lethality in mice. The relevance of these findings to the agronomic qualities and to the choice of soybean cultivars to be used as food or feed is discussed. Copyright © 2004 Society of Chemical Industry     

Inhibitors of the epidermal growth factor receptor in apple juice extract

The polyphenol-rich extract of a consumer-relevant apple juice blend was found to potently inhibit the growth of the human colon cancer cell line HT29 in vitro. The epidermal growth factor receptor (EGFR) and its subsequent signaling cascade play an important role in the regulation of cell proliferation in HT29 cells. The protein tyrosine kinase activity of an EGFR preparation was effectively inhibited by the polyphenol-rich apple juice extract. Treatment of intact cells with this extract resulted in the suppression of the subsequent mitogen-activated protein kinase cascade. Amongst the so far identified apple juice constituents, the proanthocyanidins B1 and B2 as well as quercetin-3-glc (isoquercitrin) and quercetin-3-gal (hyperoside) were found to possess substantial EGFR-inhibitory properties. However, as to be expected from the final concentration of these potential EGFR inhibitors in the original polyphenol-rich extract, a synthetic mixture of the apple juice constituents identified and available so far, including both proanthocyanidins and the quercetin glycosides, showed only marginal inhibitory effects on the EGFR. These results permit the assumption that yet unknown constituents contribute substantially to the potent EGFR-inhibitory properties of polyphenol-rich apple juice extract. In summary, the polyphenol composition of apple juice possesses promising growth-inhibitory properties, affecting proliferation-associated signaling cascades in colon tumor cells.     

Recovery at the terminal ileum of some legume non‐nutritional factors in cannulated pigs

In order to exert an effect, either local or systemic, putative beneficial dietary substances have to survive at least to some extent the digestive process within the gastrointestinal tract. For that purpose, five castrated male pigs (100 ± 2 kg mean live b.w.) fitted with T‐shaped ileal cannulas were used to determine the recovery up to the terminal ileum of various non‐nutritional factors (NNF) from legumes (defatted soybean, raw lupin, raw chickpea and raw or autoclaved kidney beans). Kidney bean lectin (PHA) recovery within the small intestine ranged between 2.5 and 4.8 mg from 5.96 mg ingested. Two different methods were used to determine ileal digestibility of protease inhibitors (PI) in pigs. According to the enzyme‐linked immunosorbent assay (ELISA) method, apparent digestibility of Kunitz trypsin inhibitor and Bowman–Birk inhibitor for pigs fed the defatted soybean‐based diet were 0 and 58%, respectively. On the other hand, the apparent digestibility of PI along the small intestine, expressed in terms of trypsin inhibitory activity, was 96, 98 and 95% of activity in the diet, while data expressed in terms of chymotrypsin inhibitory activity were 95, 99 and 79% of activity in the diet for defatted soybean, raw chickpea and autoclaved kidney bean diets respectively. Differences found in ileal digestibility values of PI using both methodologies, ELISA and enzymatic inhibition, might be explained by the detection of inactive forms by specific antibodies. Intestinal apparent digestibility of phytate was 0% for defatted soybean and autoclaved kidney bean diets, whereas values for raw lupin and chickpea diets were 4.1 and 24.5%, respectively. In conclusion, significant amounts of several NNF survived the passage through the stomach and small intestine of cannulated pigs, which might have biological relevance. Copyright © 2006 Society of Chemical Industry     

Purification of trypsin/chymotrypsin inhibitor from jack fruit seeds

A trypsin/chymotrypsin inhibitor (JSTI) was isolated from jack fruit seeds (Artocarpus integrifolia Hook f) by ammonium sulphate fractionation and chromatography on DEAE–cellulose and Sephadex G-100. During all stages of purification, the ratio of trypsin and chymotrypsin inhibitory activities remained constant. The purified preparation was found to be homogeneous by gel filtration, polyacrylamide gel electrophoresis (PAGE) and ultra-centrifugation. From the sedimentation coefficient, S _20w value of 3·5 ± 0·15 S. the molecular weight of JSTI was calculated to be 30·00 ± 2·50 kamu. The inhibitor showed a molecular weight of 24·55 kamu on a Sephadex G-75 column when eluted with 6 M guanidine hydrochloride, Under non-denaturing conditions, JSTI exhibited anomalous behaviour on a Sephadex G-200 column. On SDS–PAGE, the inhibitor showed two major bands with molecular weights of 26·30 and 15·00 kamu and two minor bands with molecular weights of 19·50 and 12·00 kamu. The carboxyamidomethylated JSTI showed three trypsin inhibitory activity bands on PAGE, suggesting the presence of isoinhibitors.     

单糖链和双糖链大豆皂甙对结肠癌细胞增殖和凋亡的影响

反相柱层析法从大豆胚轴提取单糖链大豆皂甙和双糖链大豆皂甙,研究其抗结肠癌细胞增殖的作用。MTT比色法观察大豆皂甙对结肠癌HT-29细胞增殖的影响,TUNEL法检测其对HT-29细胞凋亡的影响。结果表明,不同糖链结构的大豆皂甙均可时间和浓度依赖性地抑制结肠癌细胞增殖和诱导细胞凋亡,其中单糖链大豆皂甙的诱导细胞凋亡作用更明显。大豆皂甙可通过诱导结肠癌细胞凋亡发挥抗结肠癌作用,其抗肿瘤作用机制以及抗肿瘤作用与糖链结构之间关系需进一步的探讨     

Changes in inhibitory activity and secondary conformation of soybean trypsin inhibitors induced by tea polyphenol complexation

BACKGROUND: Tea polyphenol (TP) is a new food additive for antioxidant application, while soybean is an important resource for food and feed processing. It is therefore of rational and practical significance to investigate the influence of TP on soybean trypsin inhibitors (TIs). The aim of this study was to determine the effects of TP on the inhibitory activity of Kunitz (KTI) and Bowman–Birk (BBTI) TIs and to reveal the relationship between the inhibitory activity and conformation of KTI and BBTI by measurement of circular dichroism (CD) spectra. RESULTS: KTI and BBTI were found to be partially deactivated by TP. BBTI exhibited stronger resistance than KTI to TP deactivation. The unchanged K_M value of trypsin for benzoyl‐DL ‐arginine‐p‐nitroanilide hydrolysis indicated that KTI and BBTI inhibited trypsin in a non‐competitive pattern when complexed with TP. As the TP/TI ratio was increased and the inhibitory activity of KTI and BBTI decreased, the conformation of KTI and BBTI showed relevant changes and the major CD negative bands shifted progressively towards the near‐UV region. CONCLUSION: These results show the deactivation effects of TP on KTI and BBTI and reveal preliminarily the relationship between the inhibitory activity and secondary structure of KTI and BBTI. Copyright © 2009 Society of Chemical Industry     

Inhibitors of human and bovine trypsin and chymotrypsin in fenugreek (Trigonella foenum-graecum L.) seeds Isolation and characterization

Summary Three fenugreek inhibitors (TFI-A8, TFI-N2, and TFI-B2) were isolated from an inhibitor preparation by anion exchange chromatography and subsequent preparative isoelectric focusing using immobilized pH gradients and the canal technique. The purified inhibitors inhibited the enzymes tested differently: TFI-A8 exhibited a high inhibition of trypsin (8.2 mg human trypsin/mg and 8.1 mg bovine trypsin/mg) and a very low inhibition of chymotrypsin (0.8 mg human chymotrypsin/mg and 1.0 mg bovine chymotrypsin/mg). TFI-N2 inhibited the four enzymes to about the same extent (5.0 mg/mg human and 4.1 mg/mg bovine trypsin; 4.9 mg/mg human and 3.7 mg/mg bovine chymotrypsin). TFI-B2 displayed a high inhibition of trypsin (7.5 mg/mg human and 5.1 mg/mg bovine) and a low inhibition of chymotrypsin (1.8 mg/mg human and 1.9 mg/mg bovine). On average, the human enzymes were inhibited better than the bovine ones by the purified inhibitors. The inhibitors contained high amounts of cystine (five or six disulfide bridges per molecule), aspartic acid, threonine, serine and proline, no valine and methionine and two of them also no tryptophan. Their molecular masses were about 6 kDa. Their inclusion into the Bowman-Birk soybean proteinase inhibitor family is discussed.

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Isolierung und Charakterisierung dreier Trypsin-Chymotrypsin-Inhibitoren aus Bockshornkleesamen (Trigonella foenum-graecum L.)

Zusammenfassung Drei Bockshornklee-Inhibitoren (TFI-A8, TFI-N2 und TFI-B2) wurden aus einem Inhibitorgemisch durch Anionenaustauscherchromatographie und anschließende präparative isoelektrische Focussierung in immobilisierten pH-Gradienten nach der Kanal-Technik isoliert. Die isolierten Inhibitoren hemmen die getesteten Enzyme unterschiedlich: TFI-A8 hemmt Trypsin stark [8,2 mg Humantrypsin (HT) bzw. 8,1 mg Rindertrypsin (RT) pro mg] und Chymotrypsin sehr schwach [0,8 mg Humanchymotrypsin (HCT) bzw. 1,0 mg Rinderchymotrypsin (RCT) pro mg]. TFI-N2 hemmt Trypsin und Chymotrypsin etwa gleich stark (5,0 mg HT, 4,1 mg RT, 4,9 mg HCT und 3,7 mg RCT pro mg). TFI-B2 hemmt Trypsin stark (7,5 mg HT bzw. 5,1 mg RT pro mg) und Chymotrypsin schwächer (1,8 mg HCT bzw. 1,9 mg RCT pro mg). Im Mittel werden die beiden Humanenzyme durch die isolierten Inhibitoren stärker gehemmt als die beiden Rinderenzyme. Die Inhibitoren enthalten viel Cystin (5–6 Disulfidbrücken pro Molekül), Asparaginsäure, Threonin, Serin und Prolin, kein Valin oder Methionin und zwei auch kein Tryptophan. Ihre Molekulargewichte liegen bei 6000. Ihre Zugehörigkeit zur Familie der Bowman-Birk Sojabohnen-Proteinase-Inhibitoren wird diskutiert.

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Parts of this study have been presented at EURO FOOD CHEM V, Sept. 27–29, 1989, Versailles/France [1]     

Dicaffeoylquinic acids in Yerba mate (Ilex paraguariensis St. Hilaire) inhibit NF-κB nucleus translocation in macrophages and induce apoptosis by activating caspases-8 and -3 in human colon cancer cells

Scope : The biological functions of caffeoylquinic acid (CQA) derivatives from various plant sources have been partially elucidated. The objectives were to isolate and purify diCQAs from Yerba mate tea leaves and assess their anti‐inflammatory and anti‐cancer capabilities in vitro and explore their mechanism of action. Methods and results : Methanol extracts of dried mate leaves were resolved by flash chromatography and further purified resulting in two fractions one containing 3,4‐ and 3,5‐diCQAs and the other 4,5‐diCQA with NMR‐confirmed structures. Both fractions inhibited LPS‐induced RAW 264.7 macrophage inflammation by suppressing nitric oxide/inducible nitric oxide and prostaglandin E₂/cyclooxygenase‐2 pathways through inhibiting nucleus translocation of Nuclear factor κB subunits, p50 and p65. The diCQA fractions inhibited Human colon cancer cells CRL‐2577 (RKO) and HT‐29 cell proliferation by inducing apoptosis in a time‐ and concentration‐dependent manner, but did not affect the protein levels of p21, p27, p53, and Bax:Bcl‐2 ratio in RKO cells. In HT‐29 cells, however, the diCQA fractions increased Bax:Bcl‐2 ratio. The diCQA fractions increased the activation of caspase‐8 leading to cleavage of caspase‐3 in both RKO and HT‐29 colon cancer cells. Conclusion : The results suggest that diCQAs in Yerba mate could be potential anti‐cancer agents and could mitigate other diseases also associated with inflammation.     

Vitamin B6 suppresses serine protease inhibitor 3 expression in the colon of rats and in TNF-α-stimulated HT-29 cells

Scope: Previous reports in the areas of animal studies and, recently epidemiology, have linked anti‐tumorigenic and anti‐inflammatory effects to dietary vitamin B6. This study investigated the molecular mechanism of these effects of vitamin B6. Methods and results: DNA microarray analysis was used to obtain information on changes in colon gene expression from vitamin B6 (pyridoxine) repletion in vitamin B6‐deficient rats. Pyridoxine supplementation down‐regulated the inflammatory molecule, serine protease inhibitor clade A member 3 (SPI‐3) mRNA expression in the colon. This study also showed that tumor necrosis factor α (TNF‐α) induced SPI‐3 mRNA expression in HT‐29 human colon cancer cells, and vitamin B6 (pyridoxal hydrochloride) pretreatment of HT‐29 cells inhibited TNF ‐induced mRNA expression of SPI‐3. Vitamin B6 inhibited TNF‐α‐induced NF‐κB activation via suppression of IκBα degradation in HT‐29 cells. HT‐29 cells stably expressing epitope‐tagged ubiquitin were generated and vitamin B6 pretreatment was shown to inhibit ubiquitination of the IkB protein in response to TNF‐α‐i. Conclusion: Vitamin B6 suppressed SPI‐3 expression in the colon of rats and in TNF‐α‐stimulated HT‐29 cells. Further, this study showed a possible role of vitamin B6 in the regulation of protein ubiquitination.