Monoclonal antibody-based enzyme-linked immunosorbent assay for quantification of majonoside R2 as an authentication marker for Nngoc Linh and Lai Chau ginsengs.

Background: Recent years have witnessed increasing interest in the high amount of ocotillol-type saponin in Panax vietnamensis, particularly in relation to majonoside R2 (MR2). This unique 3%-5% MR2 content impart Ngoc Linh and Lai Chau ginsengs with unique pharmacological activities. However, in the commercial domain, unauthentic species have infiltrated and significantly hindered access to the authentic, efficacious variety. Thus, suitable analytical techniques for distinguishing authentic Vietnamese ginseng species from others is becoming increasingly crucial. Therefore, MR2 is attracting considerable attention as a target requiring effective management measures. Methods: An enzyme-linked immunosorbent assay (ELISA) was developed by producing monoclonal antibodies against MR2 (mAb 16E11). The method was thoroughly validated, and the potential of the immunoassay was confirmed by high-performance liquid chromatography with ultraviolet spectroscopy. Furthermore, ELISA was applied to the assessment of the MR2 concentrations of various Panax spp., including Korean, American, and Japanese ginsengs. Results and conclusions: An icELISA using mAb 16E11 exhibited linearity between 3.91 and 250 ng/mL of MR2, with detection and quantification limits of 1.53 and 2.50 - 46.6 ng/mL, respectively. Based on this study, the developed icELISA using mAb 16E11 could be a valuable tool for analyzing MR2 level to distinguish authentic Ngoc Linh and Lai Chau ginsengs from unauthentic ones. Furthermore, the analysis of the samples demonstrated that Ngoc Linh and Lai Chau ginsengs exhibit a notably higher MR2 value than all other Panax spp. Thus, MR2 might be their ideal marker compound, and various bioactivities of this species should be explored.

Application of Monoclonal Antibodies against Naturally Occurring Bioactive Ingredients.

Monoclonal antibodies (Mabs) are widely used in a variety of fields, including protein identification, life sciences, medicine, and natural product chemistry. This review focuses on Mabs against naturally occurring active compounds. The preparation of Mabs against various active compounds began in the 1980s, and now there are fewer than 50 types. Eastern blotting, which was developed as an antibody staining method for low-molecular-weight compounds, is useful for its ability to visually represent specific components. In this method, a mixture of lower-molecular-weight compounds, particularly glycosides, are separated by thin-layer chromatography (TLC). The compounds are then transferred to a membrane by heating, followed by treatment with potassium periodate (KIO4) to open the sugar moiety of the glycoside on the membrane to form an aldehyde group. Proteins are then added to form Schiff base bonds to enable adsorption on the membrane. A Mab is bound to the glycoside moiety on the membrane and reacts with a secondary antibody to produce color. Double Eastern blotting, which enables the simultaneous coloration of two glycosides, can be used to evaluate quality and estimate pharmacological effects. An example of staining by Eastern blotting and a component search based on the results will also be presented. A Mab-associated affinity column is a method for isolating antigen molecules in a single step. However, the usefulness of the wash fractions that are not bound to the affinity column is unknown. Therefore, we designated the wash fraction the "knockout extract". Comparing the nitric oxide (NO) production of a glycyrrhizin (GL)-knockout extract of licorice with a licorice extract revealed that the licorice extract is stronger. Therefore, the addition of GL to the GL-knockout extract of licorice increased NO production. This indicates that GL has synergic activity with the knockout extract. The GL-knockout extract of licorice inhibited high-glucose-induced epithelial-mesenchymal transition in NRK-52E cells, primarily by suppressing the Notch2 pathway. The real active constituent in licorice may be constituents other than GL, which is the causative agent of pseudohyperaldosteronism. This suggests that a GL-knockout extract of licorice may be useful for the treatment of diabetic nephritis.

An aptamer-based fluorometric method for the rapid berberine detection in Kampo medicines.

BACKGROUND: Berberine (BBR), a key component in Kampo medicine, is a cationic benzylisoquinoline alkaloid whose detection plays a critical role in the quality control of these traditional remedies. Traditional methods for detecting BBR often involve complex procedures, which can be time-consuming and costly. To address this challenge, our study focuses on developing a simpler, faster, and more efficient detection method for BBR in Kampo medicine formulations. RESULTS: We successfully developed a rapid fluorometric detection method for BBR using colloidal gold nanoparticle-based systematic evolution of ligands by exponential enrichment (GOLD-SELEX). Initially, specific single-stranded DNA (ssDNA) sequences were selected for their ability to enhance BBR's fluorescence intensity. The optimal ssDNA sequence, identified as BBR38, was further truncated to produce BBR38S, a stem-loop ssDNA that improved fluorescence upon interaction with BBR. To further enhance the fluorescence, the BBR38S aptamer underwent additional modifications, including stem truncation and nucleotide mutations, resulting in the higher fluorescence variant BBR38S-3 A10C. The final product, TetBBR38S, a tetramer version of BBR38S-3 A10C, exhibited a linear detection range of 0.780-50.0 µg mL-1 and a limit of detection of 0.369 µg mL-1. The assay demonstrated sufficient selectivity and was successfully applied to analyze 128 different Kampo medicine formulations, accurately detecting BBR content with high precision. SIGNIFICANCE: This study represents an advancement in Kampo medicine research, marking the first successful application of an aptamer-based approach for BBR detection in complex matrices. The developed method is not only simple and rapid (with a detection time of 5 min) but also cost-effective, which is crucial for widespread application.

Advanced quality assessment of Sanshishi (Gardenia jasminoides Ellis) and Kampo medicines using a monoclonal antibody against geniposide.

Gardenia jasminoides Ellis, a plant widely used in traditional medicine, is known for its array of biological activities. A key bioactive compound, geniposide (GE), an iridoid glycoside, significantly contributes to the medicinal properties of the plant, with potential side effects. Thus, a reliable and efficient method for GE detection is required to ensure the quality of medicinal-grade G. jasminoides Ellis. This study developed such a method by first synthesizing GE-bovine serum albumin conjugates to function as immunizing agents in mice. This led to the production of a monoclonal antibody (mAb 3A6) against GE from the fusion of splenocytes from immunized mice with myeloma cells (P3U1), resulting in a hybridoma that produces mAb 3A6. Thereafter, we developed a mAb 3A6-based indirect competitive enzyme-linked immunosorbent assay (icELISA). The icELISA exhibited satisfactory sensitivity (0.391-12.5 µg/ml) and repeatability (coefficients of variation <10%). The accuracy of this method was validated through a spike-recovery assay (recovery of 101-112%). Furthermore, the icELISA was employed to determine the GE content in plant and Kampo medicine samples. The GE content positively correlated with those determined by high-performance liquid chromatography-ultraviolet. The proposed icELISA is rapid, cost-effective, and reliable for high-throughput GE detection in G. jasminoides Ellis, thereby contributing to the improved quality control and standardization of this valuable medicinal plant.

Simultaneous rapid detection of glycyrrhizin and sennoside A in Daiokanzoto samples by lateral flow immunoassay.

INTRODUCTION: Glycyrrhizin (GLY) and sennoside A (SA) are characteristic bioactive marker compounds of the Kampo medicine Daiokanzoto. Their accurate detection in blends of Rhei rhizoma and Glycyrrhizae radix of several species (4:1 or 4:2) is essential for quality control and to ensure therapeutic efficacy. A rapid, efficient assay can significantly facilitate their detection. OBJECTIVE: To establish a rapid qualitative assay for GLY and SA detection, a lateral flow immunoassay (LFA) was developed using specific monoclonal antibody (mAb) nanoparticles. METHODOLOGY: This assay harnesses the competitive binding of mAb nanoparticles to the immobilized analytes on test strips and free analytes in the samples. Two conjugates for detecting GLY and SA, GLY-bovine serum albumin and SA-human serum albumin, were separately immobilized on the test zones of LFA strips. The detection mechanism is reliant on the visual detection of color changes in the test zones. RESULTS: When GLY and SA were present in samples, they contended with the immobilized conjugates on the strip to bind with the mAb nanoparticles and produced distinct color patterns in the test zones. The limits of detection of the assay for GLY and SA were both 3.13 µg/mL. The capability of the LFA was substantiated using plant samples and Daiokanzoto, and its alignment with indirect competitive ELISA results was confirmed. CONCLUSION: The introduced LFA is a groundbreaking procedure that offers a rapid, straightforward, and sensitive method for simultaneously detecting GLY and SA in Daiokanzoto samples. It is instrumental in ensuring product quality.

Highly sensitive indirect competitive enzyme-linked immunosorbent assay based on a monoclonal antibody against saikosaponin b2 for quality control of Kampo medicines containing Bupleuri radix.

Saikosaponins are naturally occurring oleanane-type triterpenoids that are found in Bupleuri radix (root of Bupleurum falcatum) and exhibit a broad biological activity spectrum. Saikosaponin b2 (SSb2) is the main saikosaponin in Kampo medicine extracts and is a designated quality control marker for the same in the Japanese Pharmacopeia. Although some monoclonal antibodies (mAbs) against saikosaponins have been produced to evaluate the quality of Bupleuri radix and related products, anti-SSb2 mAbs have not been used to quantify SSb2 in Kampo medicines. To address this knowledge gap, we herein established a new hybridoma cell line secreting a highly specific anti-SSb2 mAb and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this mAb for the detection of SSb2 in Bupleuri radix-containing Kampo medicines. The generated SSb2-recognized mAb exhibited high specificity to SSb2 in icELISA. The developed assay featured high sensitivity (linearity range = 1.95-125 ng/ml), accuracy, precision and reproducibility (coefficient of variation < 5%), and the thus determined SSb2 contents were strongly correlated with those obtained using liquid chromatograph-mass spectrometer. These results suggest that the anti-SSb2 mAb-based icELISA method can be used for the quality control and standardization of Kampo medicines containing Bupleuri radix.

Quality assessment method for Chinpi, dried Citrus spp. peel and its derived Kampo medicines using specific monoclonal antibody against hesperidin.

INTRODUCTION: Hesperidin (hesperetin 7-rutinoside, HP), a flavonoid glycoside found in Citrus unshiu Marcowicz or Citrus reticulata Blanco (Rutaceae), has been reported to exert a variety of pharmacological effects. As the efficacies and qualities of their dried peel, Chinpi and its derived Kampo medicines can be evaluated by their HP contents, a method for HP detection must be developed. OBJECTIVES: To produce a specific monoclonal antibody against HP (mAb 5D12) to detect the HP contents in Japanese traditional medicines via indirect competitive enzyme-linked immunosorbent assay (icELISA). METHOD: BALB/c mice were immunised with many haptens of HP-bovine serum albumin (BSA) conjugates that were prepared using sodium periodate (NaIO4 ) to cause an immune response. In addition, conventional hybridoma techniques were utilised to generate mAb 5D12. RESULTS: The detection range of HP by the mAb 5D12-based icELISA was 1.56-25.0 ng/mL, with a detection limit of 1.12 ng/mL. The maximum coefficient of variation, as evaluated from the intra- and inter-assays, was <10.0%, and the percentages of recovery, as determined by the spike-recovery tests, were 105%-115%. Moreover, the HP content, which was obtained from the developed icELISA, correlated well with that obtained via high-performance liquid chromatography-ultraviolet (HPLC-UV). CONCLUSION: These validation analyses revealed that the established icELISA technique exhibited high precision and accuracy. Notably, this is the first report on the development of icELISA for the HP content-based quality control of Chinpi and its derived Kampo medicines.

Correction: Analysis of canthin-6-one alkaloids derived from Eurycoma spp. by micellar liquid chromatography and conventional high-performance liquid chromatography: a comparative evaluation.

[This corrects the article DOI: 10.1039/D2RA07034K.].

Construction of a monoclonal antibody against glabridin (2G4) and development of an enzyme-linked immunosorbent assay.

INTRODUCTION: Glabridin is a unique isoflavonoid found only in Glycyrrhiza glabra L. The pharmacological effects of glabridin are well established, especially for beauty- and wellness-related uses, such as antioxidant, anti-inflammatory, ultraviolet (UV) protection, and skin-lightening effects. Therefore, glabridin is often found in commercial products such as creams, lotions, and dietary supplements. OBJECTIVE: This study aimed to develop an enzyme-linked immunosorbent assay (ELISA) using a glabridin-specific antibody. METHOD: Immunogen conjugation of glabridin-bovine serum albumin was performed via the Mannich reaction, and the resulting conjugates were injected into BALB/c mice. Subsequently, hybridomas were produced. An ELISA method for glabridin determination was developed and validated. RESULT: A highly specific antibody against glabridin was produced using clone 2G4. The assay range for the determination of glabridin was 0.28-7.02 µg/ml, with a detection limit of 0.16 µg/ml. The validation parameters in terms of accuracy and precision met the acceptable criteria. Standard curves of glabridin in various matrices were compared to evaluate the matrix effect on human serum using ELISA. Standard curves of the human serum and water matrix were obtained in the same manner, and the measurement range was 0.41-10.57 µg/ml. CONCLUSION: The developed ELISA method was used to quantify glabridin in plant materials and products with high sensitivity and specificity, and has potential applications in quantifying compounds in plant-derived products and human serum samples.

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.

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.

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.

Ground Reaction Force and Moment Estimation through EMG Sensing Using Long Short-Term Memory Network during Posture Coordination.

Motion prediction based on kinematic information such as body segment displacement and joint angle has been widely studied. Because motions originate from forces, it is beneficial to estimate dynamic information, such as the ground reaction force (GRF), in addition to kinematic information for advanced motion prediction. In this study, we proposed a method to estimate GRF and ground reaction moment (GRM) from electromyography (EMG) in combination with and without an inertial measurement unit (IMU) sensor using a machine learning technique. A long short-term memory network, which is suitable for processing long time-span data, was constructed with EMG and IMU as input data to estimate GRF during posture control and stepping motion. The results demonstrate that the proposed method can provide the GRF estimation with a root mean square error (RMSE) of 8.22 ± 0.97% (mean ± SE) for the posture control motion and 11.17 ± 2.16% (mean ± SE) for the stepping motion. We could confirm that EMG input is essential especially when we need to predict both GRF and GRM with limited numbers of sensors attached under knees. In addition, we developed a GRF visualization system integrated with ongoing motion in a Unity environment. This system enabled the visualization of the GRF vector in 3-dimensional space and provides predictive motion direction based on the estimated GRF, which can be useful for human motion prediction with portable sensors.

Analysis of canthin-6-one alkaloids derived from Eurycoma spp. by micellar liquid chromatography and conventional high-performance liquid chromatography: a comparative evaluation.

Extracts of Eurycoma longifolia Jack (EL) and Eurycoma harmandiana Pierre (EH) contain numerous bioactive compounds and varying matrices that are challenging to separate using chromatographic techniques. Herein, micellar liquid chromatography (MLC) was used to analyze canthin-6-one alkaloids contained in these extracts, and the achieved performance was compared with that of a conventional high-performance liquid chromatography (HPLC) method. The optimal mobile phase of MLC corresponded to 15 : 85 (v/v) acetonitrile : water (pH 3) containing 110 mM sodium dodecyl sulfate and 10 mM NaH2PO4. The retention times of canthin-6-one-9-O-ß-d-glucopyranoside, 9-hydroxycanthin-6-one, canthin-6-one, and 9-methoxycanthin-6-one were 4.78/15.42, 17.64/24.11, 32.84/38.27, and 39.04/39.86 min, respectively, in the cases of isocratic MLC and conventional HPLC. In both cases, the analyte resolution exceeded 1.5. The MLC elution behavior of the examined analytes was largely determined by their hydrophobicity and ionization. The sensitivity, precision, accuracy, and per-run acetonitrile consumption of the MLC method were comparable to those of the conventional HPLC method. However, the latter method exhibited higher performance for application to EL and EH samples, particularly those with low analyte concentrations and varying sample matrices. Overall, the analysis of canthin-6-one alkaloids using MLC was limited to trace analytes due to interference by the matrix.

From Nobeyama Radio Observatory to the international project ALMA -Evolution of millimeter and submillimeter wave astronomy in Japan.

The establishment of the Nobeyama Radio Observatory (NRO) in 1982 was an important event that greatly influenced the subsequent development of Japanese astronomy. The 45 m radio telescope and the Nobeyama Millimeter Array (NMA) pushed Japanese radio astronomy to the forefront of the world. As a plan beyond the Nobeyama telescopes, the Japanese radio astronomy community considered a large array to achieve unprecedented resolution at millimeter and submillimeter wavelengths under the project name of the Large Millimeter and Submillimeter Array (LMSA). After long and patient discussions and negotiations with the United States and Europe, the LMSA plan eventually led to the ALMA (Atacama Large Millimeter/submillimeter Array) as an international joint project, and the ALMA was inaugurated in 2013. This paper reviews the process from the establishment of the NRO to the realization of the ALMA, including planning of the LMSA, international negotiations, site survey, instrumental developments, and initial science results.

Bioimprinting as a Receptor for Detection of Kwakhurin.

Bioimprinting was performed against ovalbumin (OVA) to confer its binding cavities for kwakhurin (Kwa), an isoflavonoid, produced solely by Pueraria candollei var. mirifica (P. candollei). The characterization of bioimprinted-OVA (biOVA), evaluated by an enzyme-linked immunosorbent assay (ELISA), revealed that it functioned as a specific receptor for Kwa. Using biOVA, two systems, i.e., an indirect competitive ELISA (icELISA) and the even simpler and more rapid competitive enzyme-linked bioimprinted-protein assay (cELBIA), were developed as novel techniques for the quantitative analysis of Kwa in P. candollei and its related products. The two analysis methods were found to have limits of detection (LOD) of 4.0 and 2.5 µg/mL, respectively. The high reliability of the developed icELISA and cELBIA using biOVA was also demonstrated by various validation analyses. Subsequently, bioimprinting was performed using eight other proteins to investigate them as candidate scaffolds for the generation of binding cavities for Kwa. Interestingly, two bioimprinted-IgG monoclonal antibodies (biMAbs) recognized Kwa, but their original binding affinity to hapten was lost. That is, the MAbs obtained a new binding ability to Kwa in exchange for their original binding affinity, raising the possibility that biMAb could be alternatively used as a probe for the quantitative analysis of Kwa as well as biOVA. This is the first report of small molecules recognition by MAbs used as proteins for bioimprinting.

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.

Extraction of curcuminoids and ar-turmerone from turmeric (Curcuma longa L.) using hydrophobic deep eutectic solvents (HDESs) and application as HDES-based microemulsions.

The extraction of curcuminoids and aromatic (ar)-turmerone from Curcuma longa L. using organic solvents produces chemical waste, and is therefore incompatible with food applications. To address this issue, this study presents the design of hydrophobic deep eutectic solvents (HDESs) and HDES-based microemulsions. Using the response surface methodology (RSM), the optimal extraction conditions were identified as follows: HDES = OA:menthol (1:3.6 M ratio), solid-to-liquid ratio = 10:1 (mg/mL), and extraction duration = 90 min (prediction accuracy ≥ 85 %). Under these conditions, the HDES extraction yields of bisdemethoxycurcumin, demethoxycurcumin, curcumin, and ar-turmerone were 2.49 ± 0.25, 5.61 ± 0.45, 9.40 ± 0.86, and 3.83 ± 0.19 % (w/w, dry basis), respectively, while those obtained using the HDES-based microemulsion were 2.10 ± 0.18, 6.31 ± 0.48, 12.6 ± 1.20, and 2.58 ± 0.19 % (w/w, dry basis), respectively. The HDES and its microemulsions are more effective and environmentally friendly than conventional organic solvents for the extraction of curcuminoids and ar-turmerone, and these solvents are also compatible with food and pharmaceutical formulations.

Activated Carbon-Based Immunochromatographic Strip Test for the Rapid Qualitative Analysis of Swertiamarin and Sweroside.

BACKGROUND: Swertia japonica (S. japonica) is a medicinal plant that belongs to the Gentianaceae family. Several reports confirm the biological effects of the S. japonica extract. This plant is used mainly as a digestive stimulant, appetite stimulant, and gastrointestinal disease remedy in Japan. Secoiridoid glycosides are a group of compounds related to the beneficial effects of this plant. OBJECTIVE: We developed an immunochromatographic strip test for major secoiridoid glycosides, such as swertiamarin (SM) and sweroside (SS) detection. METHODS: We fabricated an immunoprobe using activated carbon as a reporter molecule and a monoclonal antibody against SM and SS (MAb D2) as a detection molecule. The test and control zones of the strip test contained SM-cBSA and Goat pAb anti-mouse IgM HRP conjugate, respectively. The immunoprobe reacted competitively with free SM and/or SS and immobilized SM-cBSA. The results were read and interpreted by the black spot intensity in the test zone. RESULTS: We succeeded in developing a strip test system with a detection limit (LOD) of 12.5 µg/mL. The selectivity and reliability evaluation revealed that the strip test is suitable for detecting SM and SS in S. japonica. The result was ready to be read in 30 min. CONCLUSIONS: This method can be a useful tool for the screening of biologically active S. japonica samples for further preparation of traditional medicine. HIGHLIGHTS: To the best of our knowledge, this is the first immunochromatographic strip test developed for the detection of SM and SS in S. japonica samples.

Lateral flow immunoassay for small-molecules detection in phytoproducts: a review.

Phytoproducts are involved in various fields of industry. Small-molecule (Mw < 900 Da) organic compounds can be used to indicate the quality of plant samples in the perspective of efficacy by measuring the necessary secondary metabolites and in the perspective of safety by measuring the adulterant level of toxic compounds. The development of reliable detection methods for these compounds in such a complicated matrix is challenging. The lateral flow immunoassay (LFA) is one of the immunoassays well-known for its simplicity, portability, and rapidity. In this review, the general principle, components, format, and application of the LFA for phytoproducts are discussed.