Untargeted metabolomics reveals alterations in metabolites of lipid metabolism and immune pathways in the serum of rats after long-term oral administration of Amalaki rasayana.

Amalaki rasayana, a traditional preparation, is widely used by Ayurvedic physicians for the treatment of inflammatory conditions, cardiovascular diseases, and cancer. Metabolic alterations induced by Amalaki rasayana intervention are unknown. We investigated the modulations in serum metabolomic profiles in Wistar rats following long-term oral administration of Amalaki rasayana. Global metabolic profiling was performed of the serum of rats administered with either Amalaki rasayana (AR) or ghee + honey (GH) for 18 months and control animals which were left untreated. Amalaki rasayana components were confirmed from AR extract using HR-LCMS analysis. Significant reductions in prostaglandin J2, 11-dehydrothromboxane B2, and higher levels of reduced glutathione and glycitein metabolites were observed in the serum of AR administered rats compared to the control groups. Eleven different metabolites classified as phospholipids, glycerophospholipids, glucoside derivatives, organic acids, and glycosphingolipid were exclusively observed in the AR administered rats. Pathway analysis suggests that altered metabolites in AR administered rats are those associated with different biochemical pathways of arachidonic acid metabolism, fatty acid metabolism, leukotriene metabolism, G-protein mediated events, phospholipid metabolism, and the immune system. Targeted metabolomics confirmed the presence of gallic acid, ellagic acid, and arachidonic acid components in the AR extract. The known activities of these components can be correlated with the altered metabolic profile following long-term AR administration. AR also activates IGF1R-Akt-Foxo3 signaling axis in heart tissues of rats administered with AR. Our study identifies AR components that induce alterations in lipid metabolism and immune pathways in animals which consume AR for an extended period.

A Recombinant Fab Antibody Against Kwakhurin as a Tool for Sensitive Indirect Competitive ELISA.

BACKGROUND: Pueraria candollei var. mirifica (P. candollei), known as White Kwao Krua in Thai, has long been used in traditional Thai medicine for the symptoms of menopause due to the potent estrogenic activity exhibited by the isoflavonoids and chromenes it contains. Recently, health hazards caused by P. candollei-derived products have arisen in Japan, and demands for analytical methods to standardize the P. candollei have been increasing. Previously, we have focused on quantifying the unique P. candollei-derived isoflavonoid kwakhurin (Kwa) and developed an indirect competitive enzyme- linked immunosorbent assay (icELISA) using a monoclonal antibody (MAb) against Kwa. However, MAb preparation requires the use of costly culture medium and sophisticated techniques. OBJECTIVE AND METHOD: In this study, we produced a recombinant antigen-binding fragment (Fab) against Kwa, as an alternative to MAb, for use in icELISA for quantitative analysis of Kwa. The VHCH1 and VL-CL proteins were individually expressed in Escherichia coli BL21 (DE3) strain and were then refolded to form active anti-Kwa Fab. RESULTS AND CONCLUSION: Characterization of anti-Kwa Fab revealed that it possessed high specificity (cross-reactivities with other Kwa-related compounds, <0.03%) and high sensitivity (limit of detection, 8.16 ng/mL). Additionally, validation analyses indicated that icELISA using anti-Kwa Fab is highly precise, accurate, and sufficiently reliable for use in quantitative analysis of Kwa. Consequently, an icELISA incorporating anti-Kwa Fab was developed for the analysis of P. candollei-derived products, to assure consumer safety.

Preparation and application of a monoclonal antibody against the isoflavone glycoside daidzin using a mannich reaction-derived hapten conjugate.

INTRODUCTION: Daidzin and its aglycone daidzein are major pharmacologically active compounds of soybean (Glycine max), kudzu (Pueraria lobata), and kwao kruea khao (P. mirifica). Pharmacological activities of daidzin are mediated by its more potent metabolites daidzein and equol; however, daidzin is the predominant compound found in these medicinal plants, and the efficacy and safety of equol depend on the amount of daidzin consumed. OBJECTIVE: To develop a specific monoclonal antibody (MAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) for standardisation of daidzin content in herbal medicines or nutraceuticals. METHODOLOGY: The Mannich reaction was used for the synthesis of a highly immunogenic conjugate between daidzin and a cationised carrier protein. Splenocytes of hyperimmunised mice were fused with myeloma cells to generate a hybridoma secreting antibody against daidzin. RESULTS: The icELISA showed high selectivity and acceptable sensitivity for daidzin determination (1.56-100 ng/mL) with high reproducibility (coefficients of variation were < 5%). The icELISA was a reliable analytical method for daidzin in Glycine max, Pueraria lobata and P. mirifica, for which daidzin recoveries from spiked samples were 98.99-104.94%. Daidzin content of these plant-derived products determined using the icELISA were in close agreement with those determined by a HPLC-UV method. CONCLUSION: The icELISA is useful for specific daidzin determination because of its reliability, low cost, speed and high throughput.

Enzyme-linked immunosorbent assay for total isoflavonoids in Pueraria candollei using anti-puerarin and anti-daidzin polyclonal antibodies.

Pueraria candollei (White Kwao Khuer) is a medicinal plant containing puerarin, daidzin, genistin, daidzein, and genistein as major isoflavonoids used for its rejuvenating and estrogenic effects. In order to analyze these compounds, a single enzyme-linked immunosorbent assay (ELISA) for total isoflavonoids was developed using anti-puerarin and anti-daidzin polyclonal antibodies (PAbs). The range for calibration of isoflavonoids by ELISA was 0.05-6.25 microg/mL. Total isoflavonoid concentrations in P. candollei samples determined by the newly developed assay system showed good agreement with those analyzed by HPLC. Based on validation analysis, this analytical method by ELISA is a precise, accurate, and sensitive method for the determination of total isoflavonoids in P. candollei.

Isolation and immunomodulatory effect of homoisoflavones and flavones from Agave sisalana Perrine ex Engelm.

Three known flavones and seven known homoisoflavonoids were isolated from the methanolic extract of the leaves of Agave sisalanaPerrine ex Engelm. Their structures were elucidated on the basis of spectroscopic analysis. The isolated compounds were also evaluated for immunopharmacological activity. PBMC were used as target cells, and cell proliferation was determined by (3)H-thymidine uptake. (+/-)-3,9-Dihydroeucomin (4), dihydrobonducellin (5), and 5,7-dihydroxy-3-(4'-hydroxybenzyl)-4-chromanone (7) showed inhibitory effects on PBMC proliferation activated by PHA with IC(50) values 19.4, 73.8, and 58.8 microM, respectively. All three compounds significantly inhibited the production of IL-2 and IFN-gamma in activated PBMC in a concentration-dependent manner.

Immunomodulatory activity of isoflavones isolated from Iris germanica (Iridaceae) on T-lymphocytes and cytokines.

The immunomodulatory activities of two isoflavones, 5,7-dihydroxy-6,4'-dimethoxyisoflavone (irisolidone) (1) and 5,4'-dihydroxy-6,7-methylenedioxyisoflavone (irilone) (2) isolated from Iris germanica (Iridaceae) is reported. Their influence on production of T-lymphocytes (CD4+ and CD8+ cells) and T-cell cytokines, namely Th1: IL-2, IFN-gamma and Th2: IL-4 and IL-5 in a dose-dependent manner was studied by flow cytometric method in Balb/c mice. Oral administration of drugs at doses of 0.1-0.8 mg/kg per oral dose showed 1 to possess stimulatory activity on T-cells and Th1 cytokine production, while as 2 acted as an immunosuppressant for both cells and cytokines. The methylated products of 1 and 2 showed a similar trend to that of their parent compounds but their activity was drastically decreased revealing the importance of free phenolic groups for their immunomodulating activities.

Lead compounds for anti-inflammatory drugs isolated from the plants of the traditional oriental medicine in Korea.

Effects of compounds isolated from medicinal plants in Korea on prostaglandin E2 (PGE2) production in rat peritoneal macrophages were examined, and mechanism of action of the active constituents was analyzed. The active constituents were as follows; tectorigenin and tectoridin isolated from the rhizomes of Belamcanda chinensis, platycodin D isolated from the roots of Platycodon grandiflorum, imperatorin isolated from the roots of Angelica dahurica, and hyperin isolated from the roots of Acanthopanax chiisanensis. These compounds inhibit the induction of cyclooxygenase-2 (COX-2), thus inhibiting PGE2 production. The chemically synthesized chalcone derivative, 2'-hydroxy-4'-methoxychalcone, also inhibits PGE2 production by suppressing COX-2 induction. In contrast, taiwanin C isolated from the roots of Acanthopanax chiisanensis inhibited PGE2 production by direct inhibition of COX-1 and COX-2.

Synthesis of haptens and conjugates for ELISA of glycitein: development and validation of an immunological test.

Two carboxylic acid haptens of glycitein were synthesized, with a spacer arm at the C2 position. They differed in the length of the spacer arm, with the length of the spacer arms being three or four carbon atoms, and were named Delta3-glycitein and Delta4-glycitein haptens, respectively. The different haptens were coupled to bovine serum albumin (BSA), and the coupling efficiency was assessed by MALDI mass spectrometry. Polyclonal antibodies were generated against the BSA conjugates. An additional conjugate of Delta4-glycitein hapten was generated with swine thyroglobulin (Thyr). Enzyme-linked immunosorbent assays (ELISAs) based on the competition between free glycitein and Delta4-glycitein-Thyr conjugates for specific antibodies were developed. The IC50 of the standard curves was 15.6 ng mL(-1) with anti-Delta3-glycitein and 62.5 ng mL(-1) with anti-Delta4-glycitein, that is, 10.9 and 44 pmol/well, respectively. With the Delta3-glycitein antibody, interassay and intra-assay variations were 12.2 and 11.5%, respectively. Specificity tests did not show any significant cross-reaction with any other soy isoflavone. This specificity is not influenced by the length of the spacer arm. The assay was validated by measurements performed on plasma samples as well as on soy-based foodstuffs and on soy-based food supplements.

[Preclinical study of maxar safety]. / Doklinicheskoe izuchenie bezopasnosti maksara.

The results of preclinical safety evaluation of the new hepatoprotector maxar showed that this drug can be classified as a low-toxicity substance with respect to acute toxicity. No significant functional and structural changes in the systems and organs of experimental animals were observed after a 6-month administration in rats (in a dose of 300, 600, and 1200 mg/kg) and in dogs (500 mg/kg). Maxar exhibited no mutagen and allergen properties, produced no immunotoxicant action, and did not adversely affect the reproduction function.

A flavonoid sulfate antigen activates human alphabeta CD8+ Th2 lymphocytes in pollen allergy.

Cellular immune responses are initiated when T lymphocytes expressing alphabeta TCR recognize peptide antigens bound to MHC molecules or, less frequently, double-stranded glycolipid antigens bound to CD1 molecules. In the allergy to Parietaria judaica, human alphabeta CD8+ Th2 lymphocytes react to a non-peptide pollinic antigen presented by B cells. The environmental allergen was purified and identified as a new flavonoid pigment: 2'-O-sulfate, 6-O-betaD-glucuronopyranosyl, 2',5,6-trihydroxy-isoflavone. Its specific recognition by alphabeta CD8+ Th2 T cells (1) depends upon an MHC- and CD1-independent presentation mediated by B cells, (2) is determined by the flavonoid carbohydrate and sulfate groups and (3) leads to positive skin prick test in allergic patients. Hence, an unusual mode of aromatic sulfated antigen recognition by alphabeta CD8+ Th2 T lymphocytes might underlie the cellular mediation of human allergy to plant allergens.