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Immunoassays are efficient for the phytochemical analysis of various matrices. However, producing an appropriate recombinant antibody for small molecules is challenging, resulting in costly analyses. In this study, we aimed to develop recombinant fragment antigen-binding (Fab) antibodies against miroestrol, a potent phytoestrogen marker of Pueraria candollei. Two expression cassettes of Fab were established for the production of active Fab antibodies using SHuffle® T7 Escherichia coli cells. The orientation of variable fragment heavy chain (VH) and variable fragment light chain (VL) in the expression vector constructs influences the reactivity, stability, and binding specificity of the resultant Fab. Stability testing of antibodies demonstrated that Fab is a more stable form of recombinant antibody than a single-chain variable fragment (ScFv) antibody in all conditions. Based on the obtained Fab, the ELISA specifically detected miroestrol in the range of 39.06–625.00 ng/mL. The intra- and inter-assay precisions were 0.74–2.98% and 6.57–9.76%, respectively. The recovery of authentic miroestrol spiked into samples was 106.70–110.14%, and the limit of detection was 11.07 ng/mL. The results for P. candollei roots and products determined using our developed ELISA with Fab antibody and an ELISA with anti-miroestrol monoclonal antibody (mAb) were consistent (R2 = 0.9758). The developed ELISA can be applied for the quality control of miroestrol derived from P. candollei. Therefore, the appropriate expression platform of Fab resulted in the stable binding specificity of the recombinant antibody and was applicable for immunoassays.Key points• ELISAs with Fab has higher sensitivity than that with ScFv.• Fab is more stable than ScFv.• Fab-based ELISA can be used for miroestrol determination of Pueraria candollei.

To address the high demand for Pueraria candollei var. mirifica (PM) used as the active ingredient in health products and its difficulty to cultivate in the field, the growth and production of deoxymiroestrol (DME) and isoflavonoid (ISF) phytoestrogens in PM cell suspensions were studied. In a 125-mL shake flask, the cell suspension produced DME [78.7 ± 8.79–116 ± 18.2 μg/g dry weight (DW)] and ISF (140 ± 6.83–548 ± 18.5 μg/g DW), which are the predominant ISF glycosides. While ISF aglycones accumulated in the PM cell suspension cultured in the airlift bioreactor. The DME content was increased to 976 ± 79.6 μg/g DW when the PM cell suspension was cultured in the 5-L scale bioreactor. The production of DME and ISF was enhanced by elicitors including methyl jasmonate (MJ), yeast extract (YE), and chitosan (CHI). MJ produced the highest induction of DME accumulation, while ISF accumulation was the highest with YE treatment. Analysis of catalase activity implied that the elicitors enhanced ROS production, which resulted in the enhancement of DME and ISF production and accumulation in PM cell suspension cultures. PM cell suspension culture is a promising source of beneficial PM phytoestrogens that exhibit bioactivity that may useful for the treatment of menopausal symptoms.

The expression of recombinant antibody fragments in the cytoplasmic space of Escherichia coli and the refolding process for restoring the structure and activity of such antibodies are not efficient. Herein, fragment antigen-binding (Fab) antibodies against miroestrol and deoxymiroestrol (MD-Fab) and their fusions with a green fluorescent protein (GFP) were expressed. The reactive MD-Fabs were successfully expressed as soluble and active forms in the cytoplasm of the SHuffle® T7 E. coli strain. Regarding the construct of MD-Fab alone, V H –C H 1 could associate V L –C L into Fab in the oxidizing cytoplasm of the E. coli strain, and no additional in vitro refolding was needed. In the case of the fusions with GFP, when the C-terminus of V H –C H 1 was linked with the N-terminus of GFP, the MD-Fab binding reactivity was retained, but the fluorescent activity of GFP interfered. When the C-terminus of GFP was linked to the N-terminus of V L –C L , the binding activity of MD-Fab was not observed. The constructed MD-Fabs had higher specificity toward deoxymiroestrol than the parental monoclonal antibody clone 12G11. In conclusion, MD-Fabs could be expressed using SHuffle® T7 E. coli cells. This process could be considered an economical, productive, and effective method to produce antibody fragments for immunoassay techniques.

The effects of the phytoestrogen-enriched plant Pueraria mirifica (PM) extract on ovari-ectomy (OVX)-induced cognitive impairment and hippocampal oxidative stress in mice were investigated. Daily treatment with PM and 17β-estradiol (E2) significantly elevated cognitive behavior as evaluated by using the Y maze test, the novel object recognition test (NORT), and the Morris water maze test (MWM), attenuated atrophic changes in the uterus and decreased serum 17β-estradiol levels. The treatments significantly ameliorated ovariectomy-induced oxidative stress in the hippocampus and serum by a decrease in malondialdehyde (MDA), an enhancement of superoxide dismutase, and catalase activity, including significantly down-regulated expression of IL-1β, IL-6 and TNF-α proinflammatory cytokines, while up-regulating expression of PI3K. The present results suggest that PM extract suppresses oxidative brain damage and dysfunctions in the hippocampal antioxidant system, including the neuroinflammatory system in OVX animals, thereby preventing OVX-induced cognitive impairment. The present results indicate that PM exerts beneficial effects on cognitive deficits for which menopause/ovariectomy have been implicated as risk factors.

Key messagePlant expression platform is the new source of immunoglobulin G (IgG) toward small low-molecular-weight targets. The plant-made monoclonal antibody-based immunoassay exhibits comparable analytical performance with hybridoma antibody.Immunoassays for small molecules are efficiently applied for monitoring of serum therapeutic drug concentration, food toxins, environmental contamination, etc. Immunoglobulin G (IgG) is usually produced using hybridoma cells, which requires complicated procedures and expensive equipment. Plants can act as alternative and economic hosts for IgG production. However, the production of free hapten (low-molecular-weight target)-recognizing IgG from plants has not been successfully developed yet. The current study aimed at creating a plant platform as an affordable source of IgG for use in immunoassays and diagnostic tools. The functional IgG was expressed in Nicotiana benthamiana leaves infiltrated with Agrobacterium tumefaciens strain GV3101 with recombinant geminiviral vectors (pBY3R) occupying chimeric anti-miroestrol IgG genes. The appropriate assembly between heavy and light chains was achieved, and the yield of expression was 0.57 µg/g fresh N. benthamiana leaves. The binding characteristics of the IgG to miroestrol and binding specificity to related compounds, such as isomiroestrol and deoxymiroestrol, were similar to those of hybridoma-produced IgG (monoclonal antibody, mAb). The plant-based mAbs exhibited high sensitivity for miroestrol (IC50, 23.2 ± 2.1 ng/mL), precision (relative standard deviation ≤ 5.01%), and accuracy (97.8–103% recovery), as determined using quantitative enzyme-linked immunosorbent assay. The validated enzyme-linked immunosorbent assay was applicable to determine miroestrol in plant samples. Overall, the plant-produced functional IgG conserved the binding activity and specificity of the parent IgG derived from mammalian cells. Therefore, the plant expression system may be an efficient and affordable platform for the production of antibodies against low-molecular-weight targets in immunoassays.

The effect of abiotic and biotic elicitors (methyl jasmonate, chitosan, salicylic acid, Agrobacterium, and yeast extract) at various concentrations on total isoflavonoid accumulation was studied in the hairy root cultures of Pueraria candollei. All elicitors stimulated isoflavonoid production. Yeast extract (0.5 mg/ml) was the most efficient giving total isoflavonoids at 60 ± 1 mg/g dry wt, which was 4.5-fold higher than control hairy roots on day 3 of elicitation.

The consumer and cosmetic industries have recently placed a greater emphasis on ecofriendly solvents for botanical extraction, including natural deep eutectic solvents (NADES). In this study, NADES were prepared for Morus alba callus extraction. The efficiency of extraction from the NADES and methanol was investigated by comparison of the stilbenoids yield and anti-melanogenesis activity. Prior to testing the irritability of a suitable NADES on the reconstructed human epidermis (RhE), the effect of the selected NADES on stilbenoids stability was determined. The results showed that the highest yields of stilbenoids were obtained from choline chloride-glycerol mixtures (Ch1G2) and methanol extracts, with no significant difference in yields (5.06 ± 0.05 and 6.32 ± 0.40 mg/g callus dry weight, respectively). The NADES extracts of M. alba callus showed comparable anti-melanogenesis activity compared to methanol. In term of stability, stilbenoids in Ch1G2 remained stable after six months of storage at 4 °C except resveratrol. Furthermore, Ch1G2 had no irritation effect on RhE. Thus, based on the findings of this study, Ch1G2 is an intriguing green solvent alternative for the extraction of M. alba callus and may be advantageous for the preparation of skin-lightening cosmetics.

Artemisinin production by hairy roots of Artemisia annua L. was increased 6-fold to 1.8 microg mg(-1) dry wt over 6 days by adding 150 mg chitosan l(-1). The increase was dose-dependent. Similar treatment of hairy roots with methyl jasmonate (0.2 mM) or yeast extract (2 mg ml(-1)) increased artemisinin production to 1.5 and 0.9 microg mg(-1) dry wt, respectively.

The phytochemical investigation of Huberantha jenkinsii resulted in the isolation of two new and five known compounds. The new compounds were characterized as undescribed 8-oxoprotoberberine alkaloids and named huberanthines A and B, whereas the known compounds were identified as allantoin, oxylopinine, N-trans-feruloyl tyramine, N-trans-p-coumaroyl tyramine, and mangiferin. The structure determination was accomplished by spectroscopic methods. To evaluate therapeutic potential in diabetes and Parkinson’s disease, the isolates were subjected to assays for their α-glucosidase inhibitory activity, cellular glucose uptake stimulatory activity, and protective activity against neurotoxicity induced by 6-hydroxydopamine (6-OHDA). The results suggested that mangiferin was the most promising lead compound, demonstrating significant activity in all the test systems.

Morus alba L. (Moraceae) has been used in traditional medicine for the treatment of several illnesses. Recent research also revealed several pharmacological activities from many groups of secondary metabolites, including the stilbenoids mulberroside A, oxyresveratrol, and resveratrol, which are promising compounds for cosmetic and herbal supplement products. In our previous study, cell cultures of M. alba showed high productivity of these compounds. In this study, we attempted to develop immobilized cell cultures of M. alba and to test the effect of elicitors and precursors on the production of stilbenoids. The immobilization of the M. alba cells significantly promoted the secretion of mulberroside A into the extracellular matrix and culture media to 60%, while enhancing the level of oxyresveratrol and resveratrol by 12- and 27-fold, respectively. The elicitation of immobilized cells with a combination of 50 µM methyl jasmonate and 0.5 mg/mL yeast extract for 24 h promoted a twofold increase in the production of all three stilbenoids. Furthermore, the addition of 0.05 mM l-phenylalanine, 0.03 mM l-tyrosine, or a combination resulted in the enhancement of mulberroside A production for up to twofold. The addition of l-tyrosine significantly enhanced the production of oxyresveratrol and resveratrol. This is the first report of stilbenoid production using immobilized cell cultures of M. alba. The cultures have benefits over normal cell suspension cultures by promoting the secretion of mulberroside A and enhancing the levels of oxyresveratrol and resveratrol. Thus, it could be a candidate method for the production of these stilbenoids.