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.

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.

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.

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.

An estimated quantitative lateral flow immunoassay for determination of artesunate using monoclonal antibody.

There have been reports of fake artesunate (ART), which has led to deaths from untreated malaria in South East Asia. To rapidly screen for fake and adulterated ART products in the drug market, a lateral flow immunoassay (LFIA) based on a colloidal gold-monoclonal antibody probe for detection of ART within samples was developed. With this method, the calibration curve for ART was determined by the intensity ratio of the test and control bands at various ART concentrations. The linearity range was 12.5-200 µg/ml of ART. Samples were tested by the developed LFIA and can be calculated for ART contents. The levels of ART in the samples were also confirmed by enzyme-linked immunosorbent assay. The results of the two methods were in good conformance. The proposed LFIA was demonstrated to be a simple and rapid analytical method for detecting ART in the pharmaceutical formulation.

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.

Development of a colloidal gold nanoparticle-based immunochromatographic strip for the one-step detection of miroestrol and puerarin.

Pueraria candollei or White Kwao Krua (Leguminosae) is an indigenous plant in Thailand which has long been used in Thai traditional medicine. The tuberous root of this plant is widely used for rejuvenation, particularly in elder women. Among the bioactive compounds in P. candollei, miroestrol and puerarin exhibit estrogenic activity. This study aims to develop an immunochromatographic strip (ICS) with a colloidal gold-based detection system for the simultaneous detection of miroestrol and puerarin in a one-step analysis. The developed method is sensitive and specific for the detection of miroestrol and puerarin in raw materials and marketed products. The detection limits of miroestrol and puerarin were 0.15 and 4.5 µg, respectively. In addition, the results from the developed ICS were confirmed with an enzyme-linked immunosorbent assay and presented a good correlation between these two methods. This is the first report on the development of an ICS that can detect miroestrol and puerarin in one step. The developed ICS provides a simplified method for the detection of miroestrol and puerarin in P. candollei and Pueraria spp.

A pilot pharmacokinetic study of miroestrol and deoxymiroestrol on rabbit sera using polyclonal antibody-based icELISA analysis.

Miroestrol (ME) and deoxymiroestrol (DME) are the most potent phytoestrogens and bioactive markers in Pueraria candollei var. mirifica tuberous roots. To understand their pharmacokinetic profiles, a pharmacokinetic study of ME and DME, at 0.43 and 0.21 mg per kg body weight, respectively, in three rabbits was performed after orally administering a single dose of P. candollei var. mirifica enriched fraction extract. Two established polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assays were validated to determine ME and DME in rabbit sera. In rabbits, the area under the 0- to 48-hr concentration-time curve of ME and DME were 854.92 and 1,692.84 ng·h/ml, respectively. The maximum concentration of ME was measured 1 hr after administration as 69.62 ± 8.28 ng/ml, and the maximum concentration of DME was measured at 3 hr as 81.8 ± 5.43 ng/ml. These results provide an initial approach for designing and studying the relationship between the ME and DME levels and their therapeutic effects based on their pharmacokinetic profiles.

Development of an Enzyme-Linked Immunosorbent Assay for Determination Of Miroestrol Using an Anti-miroestrol Monoclonal Antibody.

Miroestrol is a chromene with potent estrogenic activity present in Pueraria candollei, commonly known as White Kwao Krua. Although this compound is only present in low amounts in the plant, it plays an important role in the estrogenic action of P. candollei products. As a tool for further studies about the efficacy and safety of P. candollei as a phytoestrogenic supplement, we generated a novel monoclonal antibody against miroestrol. This anti-miroestrol monoclonal antibody was used to develop an immunoassay for the determination of miroestrol content, which can be used for quality control purposes of P. candollei. The developed ELISA against miroestrol has a calibration range of 10-780 ng/mL miroestrol, a limit of detection of 3.5 ng/mL, and a limit of quantitation of 12.2 ng/mL. According to the validation analysis, the established ELISA is precise, accurate, specific, and sensitive for miroestrol detection in plants. Furthermore, the anti-miroestrol monoclonal antibody was used to prepare an immunoaffinity column for the isolation of miroestrol from the tuberous root of P. candollei. The column provides a simple procedure for miroestrol isolation, with a capacity of 3.91 µg of miroestrol per 1 mL of immunogel.

An Enzyme-linked Immunosorbent Assay for Genistein 7-O-[α-rhamnopyranosyl-(1→6)]-ß-glucopyranoside Determination in Derris scandens using a Polyclonal Antibody.

INTRODUCTION: Genistein 7-O-[α-rhamnopyranosyl-(1→6)]-ß-glucopyranoside (GTG) is a major bioactive compound in Derris scandens. It is responsible for anti-inflammatory activity by inhibition of cyclooxygenase and lipoxygenase. There are many commercial products of D. scandens available in Thailand. OBJECTIVE: To develop an enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of GTG in plant material and derived products using a polyclonal antibody. METHODS: An immunogen was synthesised by conjugating GTG with a carrier protein. The polyclonal antibody against GTG (GTG-PAb) was produced in New Zealand white rabbits. The ELISA method was validated for specificity, sensitivity, accuracy, precision and correlation with HPLC. RESULTS: The polyclonal antibody was specific to GTG and genistin within the range of compounds tested. The GTG ELISA was applied in the range 0.04-10.00 µg/mL with a limit of detection of 0.03 µg/mL. The recovery of GTG in spiked Derris scandens extracts ranged from 100.7 to 107.0%, with a coefficient of variation less than 7.0%. The intra- and inter-assay variations were less than 5.0%. The ELISA showed a good correlation with HPLC-UV analysis for GTG determination in samples, with a coefficient of determination (r2 ) of 0.9880. CONCLUSION: An ELISA was established for GTG determination in Derris scandens. The GTG-PAb can react with GTG and genistin, but genistin has not been found in the plant. Therefore, the ELISA can be used for high throughput quality control of GTG content in D. scandens and its products. Copyright © 2016 John Wiley & Sons, Ltd.

Development of a Rapid Immunochromatographic Strip Test for the Detection of Mulberroside A.

INTRODUCTION: Mulberroside A (MuA) is the major active anti-tyrosinase compound in the root bark extract of Morus alba L. (Moraceae). Typically, MuA is widely employed as an active ingredient in whitening cosmetics. A rapid and simple assay system utilizing a small quantity of test sample is essential for the detection of MuA in large number of samples. An immunoassay using highly specific MuA polyclonal antibodies may be useful for the determination of small quantities of MuA in test samples. OBJECTIVE: To establish a rapid qualitative MuA test, an immunochromatographic strip test was developed using anti-MuA polyclonal antibodies (anti-MuA PAb). METHODOLOGY: The qualitative assay was based on a competitive immunoassay where the detection reagent consisted of anti-MuA PAb colored with colloidal gold particles. The capture reagent was a MuA-ovalbumin (MuA-OVA) conjugate immobilized on the test strip membrane. RESULTS: A sample containing MuA and the detection reagent were incubated together with immobilized capture reagent on a nitrocellulose membrane. When MuA was present, it competed with the immobilized conjugates on the strip membrane to bind a limited amount of colored antibodies; thus, a positive sample showed no color on the capture spot zone. The detection limit for the strip test was 2 µg/mL. The developed immunochromatographic strip test was utilized to determine MuA in plants, medical preparations and cosmetic samples. CONCLUSION: This immunochromatographic strip test is advantageous as a rapid, simple and sensitive screening method for the detection of MuA in plant extracts, cosmetic samples and pharmaceutical products.

Exploring anti-diabetic potential of compounds from roots of Dendrobium polyanthum Wall. ex Lindl. through inhibition of carbohydrate-digesting enzymes and glycation inhibitory activity.

Eight compounds, including one anthraquinone, two bibenzyls, one phenanthrene, three dihydrophenanthrenes, and one flavonoid, were isolated from the roots of Dendrobium polyanthum Wall. ex Lindl. Among these, six compounds were investigated for inhibitory activities against alpha-glucosidase, alpha-amylase, and advanced glycation end products (AGEs) production. Additionally, molecular docking was conducted to analyze the interactions of the test compounds with alpha-glucosidase. Moscatin, the only isolated phenanthrene, displayed the strongest anti-alpha-glucosidase activity with an IC50 of 32.45 ± 1.04 µM, approximately 10-fold smaller than that of acarbose. Furthermore, moscatilin most strongly inhibited alpha-amylase and AGEs production with IC50 values of 256.94 ± 9.87 and 67.89 ± 9.42 µM, respectively. Molecular docking analysis revealed the effective binding of all substances to alpha-glucosidase with smaller lowest binding energy values than acarbose. Moscatin was selected for kinetics studies, and it was identified as a non-competitive inhibitor with approximately 9-fold greater inhibitory capability than acarbose. This study represents the first report on the phytochemical constituents and antidiabetic potential of compounds derived from the roots of D. polyanthum Wall. ex Lindl.

Anti-inflammatory activity of verbascoside- and isoverbascoside-rich Lamiales medicinal plants.

Verbascoside and isoverbascoside are two active phenylethanoid glycosides mainly found in plants of the order Lamiales. This study analyzes the verbascoside and isoverbascoside levels and the total phenolic contents in the water and ethanolic extracts of 20 medicinal plants of the order Lamiales commonly used in Thailand. The related bioactivities, including the antioxidant activity via the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reduction activity potential assays and anti-tyrosinase and -inflammatory activities via the cyclooxygenase and nitric oxide assays are also investigated. The extracts of several plant species, including Barleria prionitis, B. lupulina, Rhinacanthus nasutus, Orthosiphon aristatus, and Nicoteba betonica, exhibit high verbascoside and isoverbascoside content levels. The correlation analysis between the bioactive activities and the active compounds demonstrates a significant association between the verbascoside level in the water extracts and both the DPPH antioxidant activity and the nitric oxide level in the anti-inflammatory assays. This study provides the first report on the verbascoside and isoverbascoside quantification of several plant samples. The findings provide valuable insights for future research on lesser-studied plants possessing high verbascoside and isoverbascoside levels, which exhibit promising anti-inflammatory activities.

Establishment of Afgekia mahidolae B.L. Burtt & Chermsir in vitro culture and effect of elicitation on its bioactive compounds.

Afgekia mahidolae is a rare plant species that possesses antioxidant, antimicrobial, and wound healing properties. This study aimed to establish the in vitro culture of A. mahidolae and investigate the effects of elicitors on their phenolic and flavonoid production, including the antioxidant activities. The established callus was prepared in the form of cell suspension cultures to determine the effect of elicitors. After elicitation for 3 days, A. mahidolae cell suspension cultures treated by 5 µM salicylic acid or 100 mg/L yeast extract exhibited significantly higher levels of total phenolic and total flavonoid content than untreated cultures, which correlated to the antioxidant activities. In addition, the profiles of phenolic and flavonoid compounds in the callus and intact leaves of A. mahidolae were determined by LC-MS, which showed different phytochemicals. The findings of this study may encourage more sustainable development of A. mahidolae cultivation.

Effect of andrographolide and deep eutectic solvent extracts of Andrographis paniculata on human coronavirus organ culture 43 (HCoV-OC43).

BACKGROUND: Andrographis paniculata (Burm. f.) Nees has demonstrated potential for treating infections caused by coronaviruses. However, no antiviral activity of andrographolide or A. paniculata extracts against human coronavirus organ culture 43 (HCoV-OC43) has been reported. PURPOSE: This study aimed to evaluate the anti-HCoV-OC43 effect of andrographolide and A. paniculata as well as the correlation between andrographolide concentration and the anti-HCoV-OC43 activity of A. paniculata extracts. METHODS: This study evaluated and compared the in vitro anti-HCoV-OC43 activities of various A. paniculata extracts and andrographolide. To obtain A. paniculata extracts with different concentrations of andrographolide and its components, methanol and deep eutectic solvents (DES) were used to extract the aerial parts of A. paniculata. Andrographolide content was determined using UV-HPLC, and antiviral activity was assessed in HCT-8 colon cells. RESULTS: The methanol and five acidic DES (containing malic acid or citric acid) extracts of A. paniculata exerted anti-HCoV-OC43 activity. Antiviral activity had a moderately strong positive linear relationship (r = 0.7938) with andrographolide content. Although the methanol extract contained the highest andrographolide content (2.34 mg/ml), its anti-HCoV-OC43 activity was lower than that of the DES extracts containing lower andrographolide concentrations (0.92-1.46 mg/ml). CONCLUSION: Methanol and the five acidic DES extracts of A. paniculata exhibited anti-HCoV-OC43 activity. However, the in vitro antiviral activity of A. paniculata extracts did not have a very strong positive linear relationship (r < 0.8) with andrographolide concentration in the extract. As a result, when comparing A. paniculata extracts, the anti-HCoV-OC43 test could provide a different result from the andrographolide concentration determination.

Effectiveness of neutral electrolyzed water in inactivating HCoV-OC43 and SARS-CoV-2 on the surfaces of plastic and the medicinal plant Centella asiatica (L.) urban.

Concerns have been raised about viral contamination, including in crops due to the recent coronavirus disease 2019 pandemic. Limited evidence is available to support the use of sanitizing agents for human coronavirus-contaminated medicinal plants. Thus, we aimed to investigate the persistence of infectious human coronavirus OC43 (HCoV-OC43) as a SARS-CoV-2 surrogate in storage conditions and the capability of neutral electrolyzed water (NEW) to inactivate coronavirus, including in fresh plants such as C. asiatica. The levels of infectious HCoV-OC43 and the triterpenoid content of C. asiatica were quantified using a plaque assay and high-performance liquid chromatography, respectively. The results showed that the persistence of HCoV-OC43 on C. asiatica leaves is identical to that on inert polystyrene. When covered and kept at room temperature with high humidity (>90% RH), HCoV-OC43 can be stable on C. asiatica leaves for at least 24 h. NEW with 197 ppm of available chlorine concentration (ACC) was effective in inactivating both infectious HCoV-OC43 and SARS-CoV-2 in suspension (≥3.68 and ≥4.34 log reduction, respectively), and inactivated dried HCoV-OC43 on the surfaces of C. asiatica leaves (≥2.31 log reduction). Soaking C. asiatica leaves for 5 min in NEW with 205 ppm of ACC or water resulted in significantly higher asiaticoside levels (37.82 ± 0.29 and 35.32 ± 0.74 mg/g dry weight, respectively), compared to the unsoaked group (29.96 ± 0.78 mg/g dry weight). These findings suggest that although coronavirus-contaminated C. asiatica leaves can pose a risk of transmission, NEW could be an option for inactivation.

Quantification of methylisomiroestrol, a phytoestrogen of Pueraria candollei, by enzyme-linked immunosorbent assay in comparison with high-performance liquid chromatography.

Pueraria candollei is a phytoestrogen-rich herb used to treat estrogen deficiency disorders; however, quality control of P. candollei-related health products is required for consistency of clinical outcomes. Estrogenically active (+)-7-O-methylisomiroestrol could be a potential chemical marker that facilitates the prediction of the overall estrogenic activity of P. candollei. The analytical performance of ELISA using newly produced monoclonal antibodies against methylisomiroestrol was compared with HPLC analysis. The developed indirect competitive ELISA (icELISA) was highly sensitive to methylisomiroestrol for detection, with an LOQ of 2.9 ng/mL, whereas the LOQ was 1.15 µg/mL by HPLC. The results from method validation indicated acceptable precision (1.71-6.37 % and 0.13-2.40 %) and accuracy (99.23-102.54 % and 96.84-101.88 %) of the methylisomiroestrol analysis using icELISA and HPLC. These methods were effectively applied for the determination of the methylisomiroestrol content in P. candollei samples. Apart from the plant tubers, the stem was observed as a source of methylisomiroestrol. The developed ELISA was more effective than HPLC in detecting a small quantity of methylisomiroestrol in the plant samples [0.23 × 10-3% (w/w) to 0.628 × 10-3% (w/w) dry weight]. Therefore, the ELISA could be a useful tool for the standardization of P. candollei, which is the crucial step to improve the quality of plant-derived products.

The Deoxymiroestrol and Isoflavonoid Production and Their Elicitation of Cell Suspension Cultures of Pueraria candollei var. mirifica: from Shake Flask to Bioreactor.

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.

Production of carbazole alkaloids through callus and suspension cultures in Clausena harmandiana.

Carbazole alkaloids are major constituents in Clausena spp. and exhibit a wide range of biological activities. The roots of Clausena harmandiana are a rich source of active carbazole alkaloids. However, its roots take several years to grow to be able to harvest. To obtain an alternative source of carbazole alkaloids, in vitro callus cultures of C. harmandiana were induced, and the formation of two active carbazole alkaloids was investigated. The effects of precursor, concentrations of sucrose, elicitors and light were studied to improve carbazole alkaloids formation. In this study, light had a strong effect on the formation of both carbazole alkaloids. The highest yields of clausine K and 7-methoxymukonal were 4.74 ± 0.26 and 0.92 ± 0.04 mg/g DW, respectively, which have more than 10-fold found in intact roots. According to the results of this study, C. harmandiana callus cultures can be used as an alternative source of carbazole alkaloids for additional biological studies.