Development of monoclonal antibody-based enzyme-linked immunosorbent assay for quantitative quality control of Derris scandens (Roxb.) Benth.

Derris scandens (Roxb.) Benth. is a medicinal plant used for treatment of musculoskeletal pain in Thai traditional medicines. Its stem contains active compound genistein-7-O-[α-rhamnopyranosyl-(1 to 6)-ß-glucopyranoside] (GTG) which is used as a biomarker for standardization of D. scandens extracts. As an alternative for rapid quantitation of GTG, a monoclonal antibody against GTG was prepared and applied for an indirect competitive enzyme-linked immunosorbent assay (ELISA) to determine GTG in plants and herbal products. The established method provided a quantification range of 0.31-10 µg/mL with a limit of detection of 0.29 µg/mL. The assay was validated for precision and accuracy by intra- and interassay variation analyses, recovery test, and comparison analysis between the amounts of GTG determined by ELISA and HPLC. The results exhibited that the developed ELISA is sensitive and effective for determination of GTG in D. scandens plant materials and herbal products.

An Enzyme-linked Immunosorbent Assay for Genistein 7-O-[α-rhamnopyranosyl-(1→6)]-ß-glucopyranoside Determination in Derris scandens using a Polyclonal Antibody.

INTRODUCTION: Genistein 7-O-[α-rhamnopyranosyl-(1→6)]-ß-glucopyranoside (GTG) is a major bioactive compound in Derris scandens. It is responsible for anti-inflammatory activity by inhibition of cyclooxygenase and lipoxygenase. There are many commercial products of D. scandens available in Thailand. OBJECTIVE: To develop an enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of GTG in plant material and derived products using a polyclonal antibody. METHODS: An immunogen was synthesised by conjugating GTG with a carrier protein. The polyclonal antibody against GTG (GTG-PAb) was produced in New Zealand white rabbits. The ELISA method was validated for specificity, sensitivity, accuracy, precision and correlation with HPLC. RESULTS: The polyclonal antibody was specific to GTG and genistin within the range of compounds tested. The GTG ELISA was applied in the range 0.04-10.00 µg/mL with a limit of detection of 0.03 µg/mL. The recovery of GTG in spiked Derris scandens extracts ranged from 100.7 to 107.0%, with a coefficient of variation less than 7.0%. The intra- and inter-assay variations were less than 5.0%. The ELISA showed a good correlation with HPLC-UV analysis for GTG determination in samples, with a coefficient of determination (r2 ) of 0.9880. CONCLUSION: An ELISA was established for GTG determination in Derris scandens. The GTG-PAb can react with GTG and genistin, but genistin has not been found in the plant. Therefore, the ELISA can be used for high throughput quality control of GTG content in D. scandens and its products. Copyright © 2016 John Wiley & Sons, Ltd.

In vivo immunomodulatory effects of plant flavonoids in lipopolysaccharide-challenged broilers.

Plant flavonoids are generally regarded as natural replacers of synthetic growth promoters in poultry production. This study investigated the immunomodulatory effects of plant flavonoids, such as genistein and hesperidin, in lipopolysaccharide (LPS)-challenged broilers. A total of 700 21-day-old commercial Arbor Acres broiler chicks were randomly assigned into six treatment groups, each having six pens of 20 chicks/pen. Chicks were fed a basal diet without any additive (control, CON), 5 mg genistein/kg feed (G5), 20 mg hesperidin/kg (H20), or a basal diet with a combination of genistein and hesperidin (1 : 4) with doses of 5 mg/kg feed (GH5), 10 mg/kg (GH10) and 20 mg/kg (GH20) for 6 weeks. Half of the birds from each treatment were separated, and either challenged with 0·9% sodium chloride solution or Escherichia coli LPS (250 µg/kg BW) on days 16, 18 and 20. The results showed that both genistein and hesperidin improved (P<0.01) the plasma antioxidant status of growing broilers, by increasing total antioxidant capacity (TAOC), superoxide dismutase (SOD) activity and decreasing malondialdehyde production. LPS challenge further increased (P<0.05) TAOC and SOD levels. Regardless of LPS challenge, both genistein and hesperidin improved the humoral and mucosal immunity by increasing the intestinal intraepithelial lymphocyte numbers (P<0.01), as well as anti-Newcastle disease and anti-avian influenza antibody titers (P<0.05). Supplementation of both the plant flavonoids generally increased (P<0.05) the immune organs indices (spleen, thymus and bursa). Thus, supplementation of basal diet of broiler chicks, either with genistein or hesperidin, improved immune and antioxidant status of growing broilers. In addition, combined supplementation of both the flavonoids showed further improvement than individual compounds.

Isoflavones, genistein and daidzein, regulate mucosal immune response by suppressing dendritic cell function.

Lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls, has been shown to have a strong adjuvant effect towards inhaled antigens contributing to airway inflammation. Isoflavones are anti-inflammatory molecules present in abundant quantities in soybeans. We investigated the effect of isoflavones on human dendritic cell (DC) activation via LPS stimulation and subsequent DC-mediated effector cell function both in vitro and in a mouse model of upper airway inflammation. Human monocyte-derived DCs (MDDC) were matured with LPS (or TNF-α) +/- isoflavones (genistein or daidzein). The surface expression levels of DC activation markers were analyzed by flow cytometry. Mature DCs +/- isoflavones were washed and cultured with freshly-isolated allogenic naïve CD4⁺ T cells for 5 days or with autologous natural killer (NK) cells for 2 hours. The percentages of proliferating IFN-γ⁺ CD4⁺ T cells and cytokine levels in culture supernatants were assessed. NK cell degranulation and DC cytotoxicity were measured by flow cytometry. Isoflavones significantly suppressed the activation-induced expression of DC maturation markers (CD83, CD80, CD86) and MHC class I but not MHC class II molecules in vitro. Isoflavone treatment inhibited the ability of LPS-DCs to induce IFN-γ in CD4⁺ T cells. NK cell degranulation and the percentage of dead DCs were significantly increased in isoflavone-treated DC-NK co-culture experiments. Dietary isoflavones suppressed the mucosal immune response to intra-nasal sensitization of mice to ovalbumin. Similar results were obtained when isoflavones were co-administered during sensitization. These results demonstrate that soybean isoflavones suppress immune sensitization by suppressing DC-maturation and its subsequent DC-mediated effector cell functions.

Immunomodulatory effects and improved prognosis of experimental autoimmune encephalomyelitis after O-tetradecanoyl-genistein treatment.

Experimental autoimmune encephalomyelitis (EAE) is a murine autoimmune disease used to study multiple sclerosis (MS), a human inflammatory demyelinating disease of the central nervous system. Genistein, an isoflavonoid phytoestrogenic compound found in soy, is known to reverse clinical signs of EAE. Although genistein has some potential in clinical application, it has some disadvantages related to its chemical structure, such as rapid in vivo metabolism and a fast decline in serum after oral administration. The present work investigates the treatment of EAE by using 7-O-tetradecanoyl-genistein (TDG), a more lipophilic analog of genistein obtained by esterification. The clinical course of EAE was investigated in C57Bl/6 mice immunized with myelin oligodendrocyte glycoprotein peptide (MOG)(35-55) in complete Freund's adjuvant supplemented with Mycobacterium tuberculosis H37RA. After 14 days of MOG immunization, mice were treated with TDG for seven days. Numbers of IL-17-producing cells and Foxp3 by CD4(+) T cells and CTLA-4 expression by CD3(+) T cells from brain were determined by flow cytometry. Levels of IL-6, IFN-γ and IL-10 were evaluated by ELISA. Brain sections were stained by hematoxylin and eosin method. The data obtained indicate that TDG treatment ameliorates the clinical signs of EAE, which correlates with a decrease of IL-17-producing cells and an increase in Foxp3(+)CD4(+) cells in the brain. TDG is also shown to enhance IL-10 production and CTLA-4 expression and to reduce IFN-γ and IL-6. Altogether, these findings suggest an immunomodulatory therapeutic role for TDG in EAE and multiple sclerosis.

[Targeted killing of the Nalm-6 cells with 2E8-Genistein immunotoxin and its mechanism].

OBJECTIVE: Leukemia is the most common hematopoietic malignancies in children. Chemotherapy is currently the primary modality of treatment for this fatal disease. Although chemotherapy is very effective in terms of cell killing, severe side effects such as severe infections, intracranial hemorrhage etc. are frequently encountered due to its poor selective damage between normal and malignant cells or tissues. Thus, a new therapy with highly selective killing of malignant cells which leaves the normal cells unaffected is desperately desired. The aim of this study was to investigate the targeting efficacy in vitro with a new clone of anti-human CD19 antibody immunotoxin 2E8-Genistein on B lineage leukemia cell line Nalm-6 cells and its mechanisms in order to provide the evidence of target therapy on B lineage leukemia and lymphoma. METHODS: 2E8-Genistein immunotoxin was generated by conjugating Mab 2E8 with a tyrosine kinase inhibitor, Genistein (Gen) via the Sulfo-SANPAH, an ultra-violet sensitive reagent. Nalm-6, a CD19+ B cell leukemia cell line, was used as target cells, while Molt-3, a CD19-T cell leukemia cell line, was taken as the negative control. The morphology of the cells was observed under the reverted reversed light microscope and the viability was checked with either trypan blue exclusion or MTT methods. Two-color flow cytometry was applied to study the mechanism of cell killing. RESULTS: After 24 hours of culture, 2E8-Genistein showed marked target killing on Nalm-6 cells at nine different concentrations from 20 nmol/L through 100 nmol/L with cell survival rates from (71.8 +/- 7.9)% down to (16.6 +/- 12.9)%, respectively (n = 3), which were all significantly lower than that of control group (100 +/- 13.9)% (P < 0.05). The killing effect was even more significant when the concentration was over 80 nmol/L. The growth inhibition rates of this immunotoxin on Nalm-6 cells were 82%, 84% and 94%, respectively at 24, 48 and 72 hours of culture in a time dependent manner. Significant difference was observed between the cell growth curve of Nalm-6 cultured with 100 nmol/L of 2E8-Gen and those of Nalm-6 cultured with medium (blank), PBS (negative control) or the same concentration of pure 2E8 antibody (negative control) groups (F = 152.15, P = 2.15 x 10(-7)), but there was no significant difference among the three control groups (F = 1.51, P = 0.29). When Molt-3 cells were used as target cells, the cell growth curves of Molt-3 cultured with 2E8-Gen (100 nmol/L) and with negative control of blank did not show any significant difference (F = 0.34, P = 0.59). PI/FITC Annexin V double staining analysis with flow cytometry showed that the positive rate (33.45 +/- 8.77)% of early apoptosis on Nalm-6 cells induced by 100 nmol/L of 2E8-Genistein was significantly higher than that of negative control of blank (10.44% +/- 1.28%, t = -4.39, P = 0.001) at 24 hours of culture. CONCLUSION: 2E8-Genistein immunotoxin can significantly target the Nalm-6 cells in vitro in a time response manner and the apoptosis induction is involved in the course of this killing effect.

Genistein enhances antigen-specific cytokine production in female DO11.10 transgenic mice.

Genistein is a phytoestrogen contained at high levels in soy products and has been shown to regulate immunoresponse. In this study, we evaluated the effects of genistein on the production of cytokines from antigen (Ag)-specific T cells using DO11.10 transgenic mice because the direct effect of genistein on Ag-specific cytokine production has not been elucidated. The oral administration of 20 mg/kg genistein increased IFN-gamma and IL-4 production from DO11.10+ T cells in response to ovalbumin (OVA)323-339 peptide in female DO11.10 mice. Analysis of intracellular cytokine synthesis revealed that the percentages of cytokine-producing cells in the control and genistein-treated groups were not different, indicating that increased cytokine production occurred at the single-cell level. In contrast to the female mice, genistein did not increase cytokine production in male mice, suggesting that the effect of genistein on cytokine production is gender-dependent.

Lipoteichoic acid induces nuclear factor-kappaB activation and nitric oxide synthase expression via phosphatidylinositol 3-kinase, Akt, and p38 MAPK in RAW 264.7 macrophages.

We previously demonstrated that lipoteichoic acid (LTA) might activate phosphatidylcholine-phospholipase C (PC-PLC) and phosphatidylinositol-phospholipase C (PI-PLC) to induce protein kinase C activation, which in turn initiates nuclear factor-kappaB (NF-kappaB) activation and finally induces inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release in RAW 264.7 macrophages. In this study, we further investigated the roles of tyrosine kinase, phosphatidylinositiol 3-kinase (PI3K)/Akt, and p38 mitogen-activated protein kinase (MAPK) in LTA-induced iNOS expression and NO release in RAW 264.7 macrophages. Tyrosine kinase inhibitors (genistein and tyrphostin AG126), PI3K inhibitors (wortmannin and LY 294002), and a p38 MAPK inhibitor (SB 203580) attenuated LTA-induced iNOS expression and NO release in concentration-dependent manners. Treatment of RAW 264.7 macrophages with LTA caused time-dependent activations of Akt and p38 MAPK. The LTA-induced Akt activation was inhibited by wortmannin, LY 294002, genistein, and tyrphostin AG126. The LTA-induced p38 MAPK activation was inhibited by genistein, tyrphostin AG126, wortmannin, LY 294002, and SB 203580. The LTA-induced formation of an NF-kappaB-specific DNA-protein complex in the nucleus was inhibited by wortmannin, LY 294002, genistein, tyrphostin AG126, and SB 203580. Treatment of macrophages with LTA caused an increase in kappaB-luciferase activity, and this effect was inhibited by tyrphostin AG126, wortmannin, LY 294002, the Akt dominant negative mutant (AktDN), and SB 203580. Based on those findings, we suggest that LTA might activate the PI3K/Akt pathway through tyrosine kinase to induce p38 MAPK activation, which in turn initiates NF-kappaB activation, and ultimately induces iNOS expression and NO release in RAW 264.7 macrophages.

Immunoassay for biochanin A.

Two variants of immunoassay for the determination of biochanin A (5,7-dihydroxy 4'-methoxy isoflavone), i.e., a radioimmunoassay (RIA) and an indirect ELISA, have been developed and evaluated. Both methods employ the same rabbit antiserum to a 7-O-carboxymethyl-5-hydroxy-4'-methoxyisoflavone-bovine serum albumin (BSA) conjugate. A 125I-labeled hapten-tyrosine methyl ester (TME) conjugate was used as a radioligand for the RIA. The indirect ELISA uses immunogen-coated microtitration plates and a peroxidase-labeled antirabbit Ig antibody. Both methods are specific for biochanin A with a comparable sensitivity (3.1 pg/tube for RIA; 5.3 pg/well for ELISA); however, their sensitivity to individual cross-reactants differs. The main cross-reactants are sissotrin (the cross-reactivity 15.7% for RIA; 120% for ELISA), 5-hydroxy, 4',7-dimethoxy isoflavone (51.5% for RIA; 46.5% for ELISA), prunetin (4.5% for RIA; 5.0% for ELISA), genistein (0.8% for RIA; 2.8% for ELISA) and formononetin (0.4% for RIA; 0.3% for ELISA). These methods were used for the analysis of biochanin A in alfalfa and in several nonleguminous plants.

Time-resolved fluoroimmunoassay of plasma daidzein and genistein.

We present a method for the determination of the phytoestrogens daidzein and genistein in plasma (serum). These weakly estrogenic isoflavones occur in soybeans and in smaller amounts in some other beans and plants. It has been suggested that they may afford protection against prostate and breast cancer. The method is based on time-resolved fluoroimmunoassay (TR-FIA) using a europium chelate as a label. After synthesis of 4'-O-carboxymethyl-daidzein and 4'-O-carboxymethyl-genistein the compounds are coupled to bovine serum albumin (BSA), then used as antigens to immunize rabbits. The tracers with the europium chelate are synthesized using the same 4'-O-derivative of the isoflavones. After enzymatic hydrolysis and ether extraction the immunoassay is carried out using the VICTOR 1420 multilabel counter (Wallac Oy, Turku, Finland). The antisera cross-reacted to some extent with some isoflavonoids but not with flavonoids. The cross-reactivity seems not to influence the results, which were highly specific for both compounds. The correlation coefficients between the TR-FIA methods and the reference method based on isotope dilution gas chromatography-mass spectrometry were high; r-values were about 0.95-0.99 depending on concentration. The intra-assay coefficients of variation (CV%) for daidzein and genistein at three different concentrations vary 3.2-4.5 and 3.2-4.1, respectively. The inter-assay CVs vary 5.0-6.3 and 4.5-5.3, respectively. The working ranges of the daidzein and genistein assays are 1.0-216 and 1.7-370 nmol/l, respectively. The plasma values (n = 80) of daidzein and genistein are very low in Finnish subjects (mean for daidzein, 3.8+/-6.8 and for genistein, 3.2+/-7.6 nmol/l; median value for daidzein 1.5 and for genistein 1.4 nmol/l).

Human peripheral blood mononuclear cells stimulated by Schistosoma mansoni antigens: association between protein tyrosine kinases, mitogen-activated protein kinases and cytokine production.

Concerning schistosomiasis, little is known about the intracellular signaling response of human peripheral blood mononuclear cells (PBMC) to Schistosoma mansoni antigens. To understand the critical role of protein tyrosine kinases (PTKs) in PBMC activation by S. mansoni antigens, we investigated how inhibition of PTKs by genistein, a tyrosine kinase inhibitor, affects proliferation, cytokine production and activation of mitogen-activated protein kinases (MAPKs). Our studies showed that PTKs have an important role in proliferation of PBMC from chronic schistosomiasis patients as cells stimulated with S. mansoni soluble antigens in the presence of genistein had an impaired proliferation. In contrast, PTK inhibition failed to cause any effect on MAPKs activity. We also evaluated the cytokine production for interleukin (IL)-2, interferon gamma (IFN-gamma), and IL-10 in culture supernatants of PBMC treated with or without PTKs inhibitor. Our results show that PBMC from chronic patients produced a high amount of IL-10 when stimulated with soluble egg antigen preparation (SEA), however, the amount produced of IL-2 and IFN-gamma was not significant. In the presence of PTKs inhibitor only the production of IL-10 was decreased. The findings suggest that PTKs are involved on signal transduction pathway for PBMC activation, but may not be an absolute requirement for all signaling responses to S. mansoni antigens.