Identification of Major Bioactive Anti-inflammatory Compounds of Derris scandens Stem Using RAW 264.7 Cells and HPLC-UV Analysis.

Derris scandens (DS) is widely recognized for its therapeutic properties, specifically its analgesic effects, which significantly alleviate muscle pain. The chemical constituents of DS stem include various isoflavone derivatives. However, there is currently a lack of specified anti-inflammatory chemical markers and analytical methods for quality control. The present study aimed to evaluate the anti-inflammatory activity of DS and its constituents using the RAW 264.7 cell model. The expression of inflammatory genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and 5-lipoxygenase (5-LOX) was examined using quantitative RT-PCR. An high-performance liquid chromatography with a UV detection method was developed to quantitatively analyze genistein-7-O-[α-rhamnopyranosyl-(1 → 6)]-ß-glucopyranoside, genistein, derrisisoflavone A, lupalbigenin, and 6,8-diprenylgenistein in DS stem. The developed HPLC-UV method demonstrated high sensitivity with limits of detection and quantification ranging from 0.01 to 0.06 µg/mL and 0.03 to 0.18 µg/mL, respectively. The accuracy of the method ranged from 93.3 to 109.6%. Furthermore, the repeatability and reproducibility of the method were suitable, as indicated by the relative standard deviations of ≤ 3.02% and ≤ 6.22%, respectively. The DS extract notably inhibited NO production, exhibiting effects comparable to those of 500 µM diclofenac, and substantially suppressed the expression of iNOS, COX-2, IL-6, and 5-LOX of lipopolysaccharide (LPS)-induced genes. As to the pure isoflavone derivatives, the order of NO production inhibition was found to be genistein > lupalbigenin > derrisisoflavone A > 6,8-diprenylgenistein > genistein-7-O-[α-rhamnopyranosyl-(1 → 6)]-ß-glucopyranoside. Genistein, derrisisoflavone A, and 6,8-diprenylgenistein significantly suppressed the upregulation of all LPS-induced genes. Consequently, these compounds are recommended as anti-inflammatory markers for the quantitative chemical analysis of DS.

Comparative stability and analytical performance of anti-miroestrol recombinant antibody in different cassettes.

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.

Fragment antigen-binding (Fab) antibody-based lateral flow immunoassay for rapid and sensitive detection of potent phytoestrogen, deoxymiroestrol.

Pueraria candollei is an ingredient of Thai herbal medicine, dietary supplements, and cosmetics. The in vitro and in vivo studies of this plant supported anti-osteoporotic activity and used for hormone replacement therapy. Deoxymiroestrol shows the most potent phytoconstituent in tuberous root of P. candollei with estrogenic activity. The quality controls are important for good agricultural practice (GAP) and good manufacturing practice (GMP) of plant-derived raw materials. The rapid detection of lateral flow immunoassay (LFIA) using colloidal gold is simply method, easy visualize detection and produce less waste than conventional chromatographic detection. In this study, LFIA for qualitative detection of deoxymiroestrol using antigen-binding fragment antibody (Fab) was developed. The result showed that the developed LFIA displays specific detection of deoxymiroestrol. Cross reactivity of this method was analyzed with miroestrol, isomiroestrol and methylisomiroestrol which showed 39.97%, 7.71% and 5.72%, respectively. After optimal condition, limit of detection (LOD) for deoxymiroestrol is 250 ng/ml. Plant samples were applied to strip test compare with indirect competitive ELISA using polyclonal antibody to confirm the application of LFIA. The results of LFIA method were comparable with those from ELISA. This developed lateral flow immunoassay can apply to detect deoxymiroestrol for the rapid testing. The developed method can use for quality control in plant samples as deoxymiroestrol is biomarker compound in P. candollei.

Plant-made antibody against miroestrol: a new platform for expression of full-length immunoglobulin G against small-molecule targets in immunoassays.

KEY MESSAGE: Plant 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.

Artificial color light sources and precursor feeding enhance plumbagin production of the carnivorous plants Drosera burmannii and Drosera indica.

Plumbagin is the main pharmacologically active compound of carnivorous plants in the genera Drosera. It possesses various pharmacological activities, including anticancer and antimalarial activities, and is used in traditional medicine. In this study, we reported a sustainable production system of plumbagin by adding sodium acetate and L-alanine as precursors to in vitro cultures of Drosera burmannii Vahl and Drosera indica L. In addition, plumbagin production was reported in the cultures subjected to different color LED lights. The highest plumbagin level (aerial part 14.625 ±â€¯1.007 mg·g-1 DW and root part 1.806 ±â€¯0.258 mg·g-1 DW) was observed in D. indica cultured under blue LED light for 14 days, and further culturing did not increase plumbagin production. In addition, plumbagin enhancement by precursor feeding (9.850 ±â€¯0.250 mg·g-1 DW, 1.2-fold) was observed in the aerial part of D. indica treated with 50 mg·L-1 sodium acetate for 3 days. Comparing both plants, up to 700-fold higher plumbagin was observed in D. indica than in D. burmannii. Moreover, in both plants, the aerial part accumulated higher plumbagin (up to 10-fold) than the roots. This is the first report on the effect of artificial LED lights on the plumbagin level of Dorsera plants. The culturing of D. indica under blue LED light showed enhanced plumbagin levels and suggests a fast and simple system for the in vitro production of plumbagin.