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.

Estrogenic Activity of Derris scandens Stem Extract and its Major Compounds Using MCF-7 Cell Proliferation Assay and Estrogen-Related Gene Expression.

Derris scandens, which contains isoflavones and prenylated derivatives, has analgesic and anti-inflammatory properties and is an ingredient in traditional Thai medicine for perimenopause and menopause. However, the estrogenic activity of D. scandens has not yet been explored. Therefore, this study aimed to examine the estrogenic activity of the stem extract of D. scandens and its isoflavone derivatives. In this study, we conducted a proliferation assay in MCF-7 cells, and used quantitative reverse transcription polymerase chain reaction to assess gene expression. We found that the relative cell proliferation of the compounds (1 µM) was ranked in the following order as compared to 0.1 nM 17ß-estradiol (100%): genistein (97.84%) > derrisisoflavone A (83.17%) > genistein-7-O-[α-rhamnopyranosyl-(1 → 6)-glucopyranoside] (69.55%) > 6,8-diprenylgenistein (51.91%) > lupalbigenin (18.72%). Furthermore, cotreatment with 1 µM lupalbigenin and 0.1 nM 17ß-estradiol was performed, which decreased cell proliferation to 80.38%. In vitro results suggest that lupalbigenin has an estrogen-antagonistic effect. At a dose of 1 µM, genistein had the strongest efficacy in increasing the expression of human estrogen receptor ß by 4.0-fold compared to the control. Furthermore, genistein-7-O-[α-rhamnopyranosyl-(1 → 6)]-ß-glucopyranoside augmented the gene expression of human estrogen receptor α and human estrogen receptor ß by 1.5- and 3.4-fold, respectively. Prenylated derivatives of genistein (derrisisoflavone A, 6,8-diprenylgenistein, and lupalbigenin) significantly suppressed the gene expression of the human androgen receptor. The administration of the crude extract at 10 µg/mL significantly suppressed human androgen receptor (0.6-fold) and transmembrane protease serine 2 (0.1-fold) expression but did not significantly affect human estrogen receptor α and human estrogen receptor ß gene expression. This herbal medicine may be safe for estrogen-exposed breast cancer patients.

Development and characterisation of highly specific monoclonal antibody-based immunoassays for the detection and quantification of genistein-7-O-[α-rhamnopyranosyl-(1→6)]-ß-glucopyranoside in Derris scandens (Roxb.) Benth.

INTRODUCTION: The stem of the plant species Derris scandens (Roxb.) Benth. (DS) contains genistein-7-O-[α-rhamnopyranosyl-(1→6)]-ß-glucopyranoside (GTG), which is a unique marker. Previous analyses of GTG using antibody-based immunoassays were compromised because of their high cross-reactivity with structurally related compounds of DS, thereby limiting their applicability in DS quality control. OBJECTIVE: Conjugation of GTG with carrier proteins was achieved using the Mannich reaction to produce a highly specific monoclonal antibody (mAb) targeting GTG (anti-GTG mAb). METHODS: The anti-GTG mAb was generated using hybridoma technology and characterised using an indirect competitive enzyme-linked immunosorbent assay (icELISA). Both lateral-flow immunoassay (LFIA) and icELISA were developed to detect and quantify GTG in DS raw materials and associated products. RESULTS: icELISA using the anti-GTG mAb showed 100% specificity for GTG, with only 1.77% cross-reactivity with genistin and less than 0.01% cross-reactivity with other compounds. icELISA demonstrated a linear range for GTG determination between 62.5 and 2000 ng/mL. The limits of detection (LOD) and quantification were 49.68 and 62.50 ng/mL for GTG, respectively. The precision of the analysis ranged from 1.28% to 4.20% for repeatability and from 1.03% to 7.05% for reproducibility. The accuracy of the analysis ranged from 101.97% to 104.01% for GTG recovery. GTG levels determined via icELISA were consistent with those confirmed via high-performance liquid chromatography (HPLC) (R2 = 0.9903). Moreover, the LOD of LFIA for GTG was 500 ng/mL. CONCLUSION: Immunoassays utilising specific anti-GTG mAbs were successfully developed, including LFIA for rapid GTG detection and icELISA for GTG quantification.

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.

Immunochromatographic assay for miroestrol and deoxymiroestrol, its cross-reactivity, and application in Pueraria mirifica (white Kwao Krua) analysis.

INTRODUCTION: Miroestrol (Mi) and deoxymiroestrol (Dmi) are trace, yet potent, phytooestrogens found in white Kwao Krua [Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham, PM]. However, the analysis of these substances is difficult because of complex matrix effects and their various analogues. In addition, alteration in the cross-reactivity of a gold nanoparticle (AuNP)-based immunochromatographic assay (ICA) resulting from the electrostatic adsorption between antibodies and AuNPs has not yet been evaluated. OBJECTIVES: This study aims to develop, characterise, and validate ICA with a monoclonal antibody exhibiting similar reactivity against Mi and Dmi (MD-mAb). MATERIALS AND METHODS: The ICA performance was validated for cross-reactivity and performance in comparison with those of indirect competitive enzyme-linked immunosorbent assays (icELISAs) with MD-mAb and mAb exhibiting specificity against Mi (Mi-mAb). RESULTS: The ICA showed a limit of detection (LOD) at 1 and 16 µg/mL for Mi and Dmi, respectively. The cross-reactivity of the ICA with Dmi was lower (6.25%) than that observed with the icELISA (120%). Cross-reactivity of ICA against other compounds of the PM was also correlated with those of icELISA; no false-positive/negative results were observed. The repeatability and reproducibility of the ICA were confirmed. The results obtained using ICA in samples of PM are correlated with the concentrations determined through icELISAs. CONCLUSION: An ICA with MD-mAb was constructed and validated. However, direct conjugation via the electrostatic adsorption of mAb-AuNPs was expected to alter the cross-reactivity of ICA, especially that of the analyte analogue Dmi.

Immunoaffinity separation of miroestrol and deoxymiroestrol from Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham using fragment antigen-binding antibody produced via Escherichia coli.

INTRODUCTION: Miroestrol and deoxymiroestrol are potent phytoestrogens and are oestrogen markers of Pueraria candollei var. mirifica. However, purifying these compounds is difficult because they only exist in trace amounts. OBJECTIVES: Active fragment antigen-binding (Fab) antibodies were produced via Escherichia coli SHuffle® T7 and used to selectively separate these compounds. MATERIALS AND METHODS: Two immunoaffinity separation approaches were developed, namely the immunoaffinity column (IAC) and a cell-based method. Group-specific Fab antibodies against miroestrol and deoxymiroestrol (anti-MD Fab) were used as biological binding reagents for selective separation. RESULTS: The Fab-based IAC effectively separated miroestrol and deoxymiroestrol (0.65 and 2.24 µg per 2 mL of resin, respectively) from P. mirifica root extract. When P. mirifica extract was added to E. coli cultures during Fab expression via a cell-based method, the target compound accumulated in intracellular compartments and, thus, were separated from E. coli cells after the removal of other compounds. A yield of 1.07 µg of miroestrol per gram of cell pellet weight was obtained. Miroestrol and deoxymiroestrol were successfully purified from P. mirifica extract using anti-MD Fab via the IAC and an intracellular cell-based method. CONCLUSION: The proposed methods can simplify the miroestrol and deoxymiroestrol extraction process and provide a basis for applications utilising recombinant antibodies to separate target compounds.

Harringtonine Ester Derivatives with Enhanced Antiproliferative Activities against HL-60 and HeLa Cells.

Harringtonine (HT), produced from Cephalotaxus species, is known to exhibit potent antiproliferative activity against myeloid leukemia cells by inhibiting protein synthesis. A previous study using acute promyelocytic leukemia (HL-60) cells raised the possibility that the C-5' methyl group of HT plays an important role in regulating leukemia cell line antiproliferative activity. In order to investigate the effect of hydrocarbon chains at C-5' on the resultant activity, the C-5' methyl group was replaced with various straight- and branched-chain hydrocarbons using the corresponding alcohols, and their antiproliferative activity against HL-60 and HeLa cells was investigated. As a result, 4'-n-heptyl-4'-demethylharringtonine (1f, n-heptyl derivative) showed the most potent cytotoxicity among the HT ester derivatives produced, with IC50 values of 9.4 nM and 0.4 µM for HL-60 and HeLa cells, respectively. Interestingly, the cytotoxicity of derivative 1f against HL-60 and HeLa cells respectively was ∼5 (IC50 = 50.5 nM) and ∼10 times (IC50 = 4.0 µM) those of HT and ∼2 (IC50 = 21.8 nM) and ∼4 times (IC50 = 1.7 µM) more than homoharringtonine (HHT). These results demonstrate the potential of the derivative 1f as a lead compound against leukemia.

Evaluating the in Vitro Efficacy of Quassinoids from Eurycoma longifolia and Eurycoma harmandiana against Common Cold Human Coronavirus OC43 and SARS-CoV-2 Using In-Cell Enzyme-Linked Immunosorbent Assay.

Coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, has become a pandemic and public health crisis. SARS-CoV-2 and the seasonal common cold coronavirus (HCoV-OC43) belong to the beta genus of human coronaviruses (HCoVs). In-cell ELISA assays were performed using HCoV-OC43 and SARS-CoV-2 and evaluated the antiviral activity of herbal plants. Eurycoma longifolia (EL) and Eurycoma harmandiana (EH) roots (antipyretic properties) and their constituent quassinoids, especially chaparrinone and eurycomalactone, showed potent anti-HCoV-OC43 and SARS-CoV-2 activities, and the low IC50 values of the mentioned constituents were observed in the range of 0.32-0.51 µM. Eurycomanone and 13ß,21-dihydroeurycomanone may contribute to the antiviral activity of EL, whereas chaparrinone is the major and active antiviral constituent of EH root. The content of quassinoids, ß-carboline, and canthin-6-one alkaloids and the cytotoxicity profile of EL and EH extracts were varied regarding extraction solvents. The boiled water and 50% EtOH extractions of both plants were less toxic than those with 95% EtOH as the extraction solvent. Our research suggests that quassinoids, which come from EL and EH roots and are anti-coronavirus compounds, are potential treatment candidates for COVID-19 and merit further in vivo investigations.

Improvement in the binding specificity of anti-isomiroestrol antibodies by expression as fragments under oxidizing conditions inside the SHuffle T7 E. coli cytoplasm.

Sensitive and specific analysis of isomiroestrol (Iso) is required for the quality control of Pueraria candollei, a herb used to treat menopausal disorders. The anti-isomiroestrol monoclonal antibody (Iso-mAb) exhibits cross-reactivity with miroestrol and deoxymiroestrol, which impacts the analytical results. Here, the active and soluble forms of the single-chain variable fragment (Iso-scFv) and fragment antigen-binding (Iso-Fab) against Iso were expressed using Escherichia coli SHuffle® T7 to alter the binding specificity. The Iso-scFv format exhibited a higher binding activity than the Iso-Fab format. The reactivity of Iso-scFv towards Iso was comparable with that of the parental Iso-mAb. Remarkably, the binding specificity of the scFv structure was improved and cross-reactivity against analogs was reduced from 13.3-21.0% to ˂ 1%. The structure of recombinant antibodies affects the binding characteristics. Therefore, the immunoassays should improve specificity; these findings can be useful in agricultural processes and for quality monitoring of P. candollei-related materials.

Phosphodiesterase-5 Inhibitory Activity of Canthin-6-One Alkaloids and the Roots of Eurycoma longifolia and Eurycoma harmandiana.

Eurycoma longifolia (EL) and Eurycoma harmandiana (EH) are natural medicinal plants belonging to the Simaroubaceae family, and are well-known for their ability to enhance male sexual performance. The present study investigated the phosphodiesterase-5 (PDE-5) inhibitory activity of intact roots of EL and EH. Additionally, canthin-6-one alkaloids, ß-carboline alkaloids, and quassinoids were also screened for PDE-5 inhibitory activity. We developed in vitro root and callus cultures of EL and EH to determine their PDE-5 inhibitory activity. Our results indicated that canthin-6-one alkaloids, which include canthin-6-one-9-O-ß-D-glucopyranoside, 9-methoxycanthin-6-one, canthin-6-one, and 9-hydroxycanthin-6-one, exhibited PDE-5 enzymatic inhibitory activity, with IC50 values of 2.86±0.23, 3.30±1.03, 4.31±0.52, and 4.66±1.13 µM, respectively. The ethanolic extract of the intact roots of EL and EH, and the in vitro root culture of EH had large amounts of canthin-6-one alkaloids (1.50±0.04, 2.12±0.03, and 3.48±0.08 mg/g dry weight, respectively), and showed potent PDE-5 inhibition. Our findings indicate that in vitro root cultures of EH may be used to replace intact plants, and canthin-6-one-9-O-ß-D-glucopyranoside should be further investigated for development as a health supplement.

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.

Competitive immunochromatographic test strips for the rapid semi-quantitative analysis of the biologically active bitter glycoside, amarogentin.

Amarogentin (AG), a biologically active secoiridoid glycoside, is responsible for the efficacy of Gentianaceae based medications. Thus, qualitative and quantitative analyses of AG are of significance for batch to batch quality control purposes. By conjugating colloidal gold nanoparticles with the AG-specific monoclonal antibody, MAb 1E9, we were able to develop a single-step competitive immunochromatographic assay (ICA) for simple quantification of the AG content in plant samples. With a limit of detection of 250 ng/mL, the analytical results were obtained after immersing the ICA test strip in the detection mixture for 15 min. This new ICA is superior to conventional ICAs as it is considerably faster due to the speed with which the test strips can be produced and the omission of the time-consuming preparation phase that was previously required to make the fiber pad. Moreover, our ICA only needs a small amount of analyte (20 µL).The reliability of the reported test strip was confirmed by comparing its semi-quantitative results with those obtained via an indirect competitive enzyme-linked immunosorbent assay (icELISA). The positive correlation between these methods (R2 = 0.984) indicated that this new ICA could be applied for the semi-quantitative analysis of the AG content in plant samples.

Improvement of stilbene production by mulberry Morus alba root culture via precursor feeding and co-elicitation.

Large amounts of Morus alba L. (MA) roots are needed as the source of active stilbenes in the industrial production of traditional medicines and cosmeceuticals. A recent investigation demonstrated resveratrol and its derivatives to be promising anti-COVID-19 agents. However, conventional cultivation of MA does not meet the demand for its stilbenes, and root quality usually varies between crops. This study established the in vitro non-GMO root culture of MA and optimized the root density, precursor feeding, and elicitors for stilbene productivity. A root culture with optimal inoculum density (3 g/flask of 30 mL medium) accumulated mulberroside A, oxyresveratrol, and resveratrol at 18.7 ± 1.00 mg/g, 136 ± 5.05 µg/g, and 41.6 ± 5.84 µg/g dry weight (DW), respectively. The feeding of L-tyrosine shortened the time required to reach the stilbene productive stage. Root cultures co-treated with 200 µM methyl jasmonate and 2 mg/mL yeast extract accumulated the highest contents of mulberroside A (30.3 ± 2.68 mg/g DW), oxyresveratrol (68.6 ± 3.53 µg/g DW), and resveratrol (10.2 ± 0.53 µg/g DW). In summary, root culture is a promising and sustainable source of stilbenes for the development of health products and agents for further investigation as potential anti-COVID-19 agents.

Immunochromatographic assay for the detection of kwakhurin and its application for the identification of Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham.

INTRODUCTION: The plant Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham (PM), known by its common Thai name as white Kwao Krua, is sometimes misidentified because it presents similar botanical characteristics to those of Butea superba (red Kwao Krua). The phytochemicals in PM are phytoestrogens in the class of isoflavonoids, but Butea superba contains flavonoids that exhibit androgenic and antiestrogen effects. OBJECTIVES: This research aims to develop a simple analytical method for identification and to differentiate PM from red Kwao Krua and other Pueraria species. METHODS: A gold nanoparticle-based immunochromatographic assay (ICA) was developed for the detection of kwakhurin (Kwa), a unique compound found in PM. The parameters, including sensitivity, accuracy, precision, and specificity, were validated. All samples were analyzed using ICA and high-performance liquid chromatography with UV detector (HPLC-UV). The results of the two methods were compared for consistency checking. RESULTS: The cutoff limit of Kwa detection was 160 ng/mL, which was lower than in the HPLC-UV method. The repeatability and reproducibility of the ICA preparation and assembly showed high precision. The cross-reactivity to related isoflavonoids was less than 0.32%, which implied high specificity of the ICA for Kwa. Moreover, false-positive and false-negative results from other plant extracts were not observed. CONCLUSION: The developed ICA is applicable for distinguishing PM from red Kwao Krua and other Pueraria species. This simple analytical method can be applied for the identification of raw PM materials in the industrial and agricultural sectors.

Molecular cloning and characterization of type III polyketide synthase from Plumbago zeylanica.

Two cDNAs encoding type Ш polyketide synthase (PKS1) and chalcone synthase (CHS, PKS2), were cloned from fresh leaves of Plumbago zeylanica L. (P. zeylanica). Their heterologous expression revealed that PKS1 catalyzed the formation of five α-pyrones from three to six acetate units by accepting acetyl-CoA and malonyl-CoA. In contrast, PKS2 catalyzed the formation of naringenin and bisnoryangonin by accepting p-coumaroyl-CoA and malonyl-CoA. Naringenin is thought to be involved in the biosynthesis of various bioactive flavonoids. PKS2 can be used to molecular breeding to enhance the production of these useful secondary metabolites via its overexpression.[Formula: see text].

Effects of Thai Local Ingredient Odorants, Litsea cubeba and Garlic Essential Oils, on Brainwaves and Moods.

The functional food market is growing with a compound annual growth rate of 7.9%. Thai food recipes use several kinds of herbs. Lemongrass, garlic, and turmeric are ingredients used in Thai curry paste. Essential oils released in the preparation step create the flavor and fragrance of the famous tom yum and massaman dishes. While the biological activities of these ingredients have been investigated, including the antioxidant, anti-inflammatory, and antimicrobial activities, there is still a lack of understanding regarding the responses to the essential oils of these plants. To investigate the effects of essential oil inhalation on the brain and mood responses, electroencephalography was carried out during the non-task resting state, and self-assessment of the mood state was performed. The essential oils were prepared in several dilutions in the range of the supra-threshold level. The results show that Litsea cubeba oil inhalation showed a sedative effect, observed from alpha and beta wave power reductions. The frontal and temporal regions of the brain were involved in the wave alterations. Garlic oil increased the alpha wave power at lower concentrations; however, a sedative effect was also observed at higher concentrations. Lower dilution oil induced changes in the fast alpha activity in the frontal region. The alpha and beta wave powers were decreased with higher dilution oils, particularly in the temporal, parietal, and occipital regions. Both Litsea cubeba and turmeric oils resulted in better positive moods than garlic oil. Garlic oil caused more negative moods than the others. The psychophysiological activities and the related brain functions require further investigation. The knowledge obtained from this study may be used to design functional food products.

Expression of actively soluble antigen-binding fragment (Fab) antibody and GFP fused Fab in the cytoplasm of the engineered Escherichia coli.

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, VH-CH1 could associate VL-CL 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 VH-CH1 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 VL-CL, 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.

Kwakhurin-magnetic particles conjugates enable fast enzyme immunoassay for the detection of kwakhurin in Pueraria candollei.

INTRODUCTION: Kwakhurin (Kwa) is one of the unique isoflavonoids produced in Pueraria candollei var. mirifica (P. candollei), which has long been used as folk medicine for rejuvenation in Thailand. Recently, the use of P. candollei-derived products has widely spread among Japanese women for cosmetic purposes. Correspondingly, there has been an increase in the number of reports regarding possible health hazards caused by estrogenic activity inherent to the plant; thus, the need for a detailed evaluation of the phytoestrogen content of P. candollei-derived products has gained a sense of urgency in recent years. OBJECTIVE: This study aims to develop a rapid enzyme immunoassay that can be applied to the quantitative analysis of Kwa in P. candollei and its derived products. MATERIAL AND METHOD: A rapid and sensitive immunoassay was developed with a combination of Kwa-specific monoclonal antibody (MAb 11F) and Kwa-magnetic particles (MPs) conjugates, which increased the surface area of the solid phase, resulting in a decrease in the immunoreaction time. RESULT: This novel MPs-based enzyme immunoassay (MPs-EIA) was used to determine Kwa concentration in the range from 2.44 to 78.1 ng/mL with a limit of detection of 1.90 ng/mL. Validation analyses revealed that the proposed MPs-EIA protocol was sufficiently precise and accurate for effective quantitative analysis of Kwa in P. candollei and its derived products.

Preincubation format for a sensitive immunochromatographic assay for monocrotaline, a toxic pyrrolizidine alkaloid.

INTRODUCTION: Monocrotaline (MCT), which is classified as a 1,2-dehydropyrrolizidine alkaloid (DHPA), is a toxic compound that is mainly produced by Crotalaria spp. MCT contamination in cereals and herbs leads to hepatitis, gastroenteritis, pulmonary vasculitis and hypertension, and different types of cancer. The current analytical methods for MCT are complicated and expensive using liquid chromatography equipped with mass spectrometry detection. OBJECTIVE: The aim of this study was to develop a simple and sensitive preincubation format for an immunochromatographic assay (PI-ICA) for MCT detection. METHODOLOGY: We conducted the PI-ICA via incubation of an MCT-containing sample with an anti-MCT monoclonal antibody conjugated with colloidal gold before strip dipping. We compared the PI-ICA detection sensitivity with that of the conventional ICA (Conv-ICA) format. RESULTS: The PI-ICA was sensitive with a limit of detection (LOD) of 0.61 ng/mL, which is a 16-fold improvement over the Conv-ICA format. These results indicated that the PI-ICA method exhibits high binding specificity for MCT and low cross-reactivity towards retronecine, retrorsine, senecionine and heliotrine. Sample solutions from plants containing MCT and related DHPAs produced positive results via PI-ICA analysis. CONCLUSIONS: The proposed PI-ICA system provides a highly sensitive method compared to Conv-ICA. In addition, the developed PI-ICA method is simple and highly effective for detecting MCT contamination.

Preparation of a highly specific single chain variable fragment antibody targeting miroestrol and its application in quality control of Pueraria candollei by enzyme-linked immunosorbent assay.

INTRODUCTION: Miroestrol is the potent phytoestrogen isolated from White Kwao Krua (Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham, a Thai traditional medicinal plant. Nowadays, various health supplementary products featuring White Kwao Krua are available worldwide. A sensitive and rapid analytical method for quantification of miroestrol is necessary for quality control of these products. OBJECTIVES: To prepare a single-chain variable fragment (scFv) antibody specific to miroestrol and develop a scFv-based enzyme-linked immunosorbent assay (ELISA) for quantitative analysis of miroestrol in plant materials and health supplementary products. METHODS: A gene encoding anti-miroestrol scFv antibody was constructed and expressed in Escherichia coli SHuffle T7 strain. Anti-miroestrol scFv antibody was characterised and applied to ELISA. The developed scFv-based ELISA method was validated for its sensitivity, specificity, accuracy and precision. RESULTS: Anti-miroestrol scFv antibody was highly specific to miroestrol. The scFv-based ELISA was applied to determine miroestrol in the range 0.06-7.81 µg/mL, with the limit of quantification of 0.06 µg/mL miroestrol. The accuracy of the assay was validated by its 95.08-103.99% recovery from the spiked miroestrol recovery experiment and in good correlation with the results from the monoclonal antibody-based ELISA. The relative standard deviation of the intra- and inter-assay were less than 6.0%. CONCLUSION: The developed scFv-based ELISA was sensitive, specific, accurate, and precise for determination of miroestrol and useful for quality control of P. candollei plant raw materials and supplementary products.