UPLC-TQD-MS/MS Method Validation for Quality Control of Alkaloid Content in Lepidium meyenii (Maca)-Containing Food and Dietary Supplements.

Lepidium meyenii Walp. (Brassicaceae), also known as Maca or Peruvian ginseng, is a common ingredient in food supplements with many claimed health benefits, such as improved endurance, increased energy level, and enhanced sexual properties. Due to potential toxicity of its chemicals, including alkaloids, some regulatory authorities, e.g., in Belgium, Germany, the United States, expressed concerns about the safe consumption of Maca root. However, due to the lack of commercial standards, no established analytical method currently exists for this purpose. The current project focuses on the quantitative determination of potentially toxic alkaloids from Maca. The current study presents the first analytical method for quality control of alkaloid content in Maca-containing food and dietary supplements, assessing the presence of 11 major compounds belonging to three different classes, i.e., imidazole, ß-carboline, and pyrrole alkaloids. An accurate, rapid, and sensitive UPLC-TQD-MS/MS method is reported, which was fully validated according to the International Council for Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and SANTE/11312/2021 guidelines. To ensure the method's applicability and practicability in the absence of primary standards, validation of secondary standards (SSs) alongside primary standards (PSs) was also conducted for imidazole alkaloids. As a result, in Maca raw powder, total alkaloid content was found to vary from 418 to 554 ppm (mg/kg). Furthermore, all quantified imidazole alkaloids were ascertained to be the major alkaloids with the total content from 323 to 470 ppm in Maca raw powder, followed by the ß-carboline and pyrrole alkaloids. It was also observed that the commercial preparation of finished products affects the total alkaloid content, evidenced by the large variation from 56 to 598 ppm. Ultimately, from a regulatory point of view, it seems advisible not to request the complete absence of the alkaloids but to impose a maximum level based on safety considerations. In addition to the analytical method, a low-cost, simple, and scalable synthetic scheme of macapyrrolins A, C, and G was reported for the first time.

HILIC UPLC/ QTof MS Method Development for the Quantification of AGEs Inhibitors - Trouble Shooting Protocol.

OBJECTIVE: The paper reports an attempt to develop and validate a HILIC UPLC/ QTof MS method for quantifying N-ε-carboxymethyl-L-lysine (CML) in vitro, testing N-ε-carboxy[D2]methyl-L-lysine (d2-CML), and N-ε-carboxy[4,4,5,5-D4]methyl-L-lysine (d4-CML) as internal standards. METHOD: During the method development, several challenging questions occurred that hindered the successful completion of the method. The study emphasizes the impact of issues, generally overlooked in the development of similar analytical protocols. For instance, the use of glassware and plasticware was critical for the accurate quantification of CML. Moreover, the origin of atypical variation in the response of the deuterated internal standards, though widely used in other experimental procedures, was investigated. RESULT: A narrative description of the systematic approach used to address the various drawbacks during the analytical method development and validation is presented. CONCLUSION: Reporting those findings can be considered beneficial while bringing an insightful notion about critical factors and potential interferences. Therefore, some conclusion and ideas can be drawn from these trouble-shooting questions, which might help other researchers to develop more reliable bioanalytical methods, or to raise their awareness of stumbling blocks along the way.

Identification and Quantification of Polymethoxylated Flavonoids in Different Citrus Species Using UPLC-QTOF-MS/MS and HPLC-DAD.

Many species from the genus Citrus are used in traditional medicine and contain polymethoxylated flavonoids. These compounds show anti-inflammatory and chemopreventive activities, among others, and therefore have a big potential to be developed as therapeutic agents or dietary supplements. Citrus species are different in their profile and yield of polymethoxylated flavonoids. Therefore, polymethoxylated flavonoids were identified and quantified in seven different Citrus species, including wild-type and commercially available species. All species were profiled using UPLC-QTOF-MS/MS analysis combined with mass spectral molecular networking. A total of 38 polymethoxylated flavonoids were detected and 8 of them were present in every species. As the yield of polymethoxylated flavonoids was different for each species, a generally applicable HPLC-diode array detection method was developed and validated according to the ICH guidelines to quantify the amount of nobiletin and the total amount of polymethoxylated flavonoids expressed as nobiletin. Analysis of the seven samples showed evidence that wild-type Citrus species (e.g., Citrus depressa) contain higher yields of polymethoxylated flavonoids compared to commercially available species (e.g., Citrus limon). Qualitative analysis revealed the broadest variety of different PMFs in C. depressa, Citrus reticulata, and Citrus reticulata × Citrus sinensis, which makes them interesting sources of polymethoxylated flavonoids for future development as therapeutic agents or dietary supplements.

Alkaloids from Lepidium meyenii (Maca), structural revision of macaridine and UPLC-MS/MS feature-based molecular networking.

Lepidium meyenii Walp., known as Peruvian ginseng, is widely used in ethnomedicine. To date, L. meyenii is cultivated worldwide at high-altitude and is commonly used as a food supplement. However, its medicinal value is still controversial and its mechanism of action remains unknown, due to limited knowledge about the phytochemical constituents of this plant species. In this study, a multidisciplinary approach comprising conventional NMR- and HRMS-based structure elucidation, quantum mechanical calculation of NMR chemical shifts and UPLC-MS/MS feature-based molecular networking was applied to analyse the phytochemical profile of L. meyenii. In the current work, three previously undescribed imidazole alkaloids were identified using extensive spectroscopic techniques (HRMS, NMR), for which the names lepidiline E, F and G were adopted. In addition, two amidine alkaloids were reported, representing an undescribed class of alkaloids in L. meyenii, and 1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid, a well-known ß-carboline alkaloid, was also isolated from L. meyenii for the first time. Molecular networks of imidazole, amidine and ß-carboline alkaloids in L. meyenii were constructed by the Global Natural Products Social Molecular Networking (GNPS) web platform, resulting in the tentative identification of three undescribed analogues. In addition, the structure of a previously reported compound named 'macaridine' was revised as macapyrrolin C based on density functional theory (DFT) calculations and comprehensive comparison of NMR data.

The presence of benzophenone in sunscreens and cosmetics containing the organic UV filter octocrylene: A laboratory study.

BACKGROUND: The reason why patients photosensitized to the drug ketoprofen (KP) may develop severe photoallergic skin reactions to octocrylene (OCT), an organic ultraviolet filter in sunscreens and cosmetics, remains largely unknown. OCT can be synthesized by using unsubstituted benzophenone (BP), a possible human carcinogen. OBJECTIVES: To verify if, and to what extent, BP residues are present in OCT-containing consumer products. METHODS: The raw material of OCT and 39 skincare products, of which 28 contain OCT, were chemically analysed for the presence of BP by means of liquid chromatography. RESULTS: In the OCT raw material and in all 28 OCT-containing products the presence of BP could be demonstrated, mostly in concentrations above 10 ppm (0.001%), whereas a majority of OCT-free products (8/11, 73%) did not contain BP. Moreover, BP concentrations significantly increased, in a time- and temperature-dependent manner, likely due to the additional degradation of OCT. CONCLUSIONS: Photoallergic contact dermatitis from OCT in patients photosensitized to KP might rely on residual BP impurities. Toxicological and ecological studies that evaluate the safety of OCT might also need to consider the concomitant presence of BP.

In vitro antigenotoxic activity, in silico ADME prediction and protective effects against aflatoxin B1 induced hepatotoxicity in rats of an Erythrina latissima stem bark extract.

Stem bark of Erythrina latissima E. Mey (Leguminosae) contains a wide range of prenylated flavonoids able to counteract the genotoxic properties of aflatoxin B1 (AFB1). Thus, the hypothesis was raised that E. latissima stem bark extracts (ELBE) may counteract the in vivo hepatotoxic effects of aflatoxins, contaminants in food and feed. An HPLC-DAD method was developed and validated to determine the level of flavonoid aglycones (11.82%) and glycosides (16.17%). ADME, pharmacokinetic and drug-likeness assessment of major flavonoids of ELBE, using the web tool SwissADME, showed good oral bioavailability. The protective effect of ELBE against AFB1 induced genotoxicity in the Vitotox assay after metabolic activation was confirmed (IC50 of 44.32 µg/ml), followed by evaluation of its inhibitory effect on hepatotoxicity in rats induced by the same agent. Male Wistar rats were orally treated with ELBE (20 mg/kg, 50 mg/kg and 100 mg/kg) or curcumin (500 mg/kg) combined with piperine (20 mg/kg) - positive control, for 8 days prior to AFB1 exposure (1 mg/kg). The ELBE group showed a decreased activity of ALP and γ-GT compared to the AFB1 group. Histopathological examination of the liver demonstrated ameliorative effects of ELBE. Thus, ELBE could have a protective effect against hepatotoxins such as AFB1.

In vitro gastrointestinal biotransformation and characterization of a Desmodium adscendens decoction: the first step in unravelling its behaviour in the human body.

OBJECTIVES: The isolation and identification of the flavonoids present in a decoction of Desmodium adscendens was performed. In view of the oral use of the decoction, this work focused on the stability in gastrointestinal conditions and biotransformation by intestinal microflora in the colon of D-pinitol, vitexin and the flavonoid fraction of the decoction, as a first step in unravelling its behaviour in the human body. METHODS: The freeze-dried decoction was first subjected to column chromatography. Subsequently an enriched flavonoid fraction, was separated by repeated semi-preparative high-performance liquid chromatography (HPLC) or by HPLC-SPE. The isolated compounds were elucidated by NMR. Biotransformation experiments were carried in an in vitro gastrointestinal dialysis model. KEY FINDINGS: The major flavonoids of a decoction of D. adscendens were characterized as vicenin-2, isoschaftoside, schaftoside, 2″-O-xylosylvitexin, 2″-O-pentosyl-C-hexosyl apigenin and a O-hexosyl-C-hexosyl apigenin, tentatively identified as 2″-O-glucosyl-vitexin. During their passage in the gastrointestinal dialysis model, vitexin and C-glycosides thereof were found to be stable. Only the O-glycosidic bonds of O-glycosides of vitexin or isovitexin were hydrolysed during the colonic phase. CONCLUSIONS: A D. adscendens decoction was found to be rich in vitexin and isovitexin glycosides from which vitexin and the C-glycosides thereof were found to be stable in the simulated gastrointestinal tract.

Advantages of a validated UPLC-MS/MS standard addition method for the quantification of A-type dimeric and trimeric proanthocyanidins in cranberry extracts in comparison with well-known quantification methods.

The berries of Vaccinium macrocarpon, cranberry, are widely used for the prevention of urinary tract infections. This species contains A-type proanthocyanidins (PACs), which intervene in the initial phase of the development of urinary tract infections by preventing the adherence of Escherichia coli by their P-type fimbriae to uroepithelial cells. Unfortunately, the existing clinical studies used different cranberry preparations, which were poorly standardized. Because of this, the results were hard to compare, which led sometimes to conflicting results. Currently, PACs are quantified using the rather non-specific spectrophotometric 4-dimethylaminocinnamaldehyde (DMAC) method. In addition, a normal phase HPTLC-densitometric method, a HPLC-UV method and three LC-MS/MS methods for quantification of procyanidin A2 were recently published. All these methods contain some shortcomings and errors. Hence, the development and validation of a fast and sensitive standard addition LC-MS/MS method for the simultaneous quantification of A-type dimers and trimers in a cranberry dry extract was carried out. A linear calibration model could be adopted for dimers and, after logaritmic transformation, for trimers. The maximal interday and interconcentration precision was found to be 4.86% and 4.28% for procyanidin A2, and 5.61% and 7.65% for trimeric PACs, which are all acceptable values for an analytical method using LC-MS/MS. In addition, twelve different cranberry extracts were analyzed by means of the newly validated method and other widely used methods. There appeared to be an enormous variation in dimeric and trimeric PAC content. Comparison of these results with LC-MS/MS analysis without standard addition showed the presence of matrix effects for some of the extracts and proved the necessity of standard addition. A comparison of the well-known and widely used DMAC method, the butanol-HCl assay and this newly developed LC-MS/MS method clearly indicated the need for a reliable method able to quantify A-type PACs, which are considered to be the pharmacologically active constituents of cranberry, since neither the DMAC or butanol-HCl assays are capable of distinguishing between A and B-type PACs and therefore cannot detect adulterations with, for example, extracts with a high B-type PAC content. Hence, the combination of the DMAC method or butanol-HCl assay with this more specific LC-MS/MS assay could overcome these shortcomings.

Development and validation of a robust high-performance liquid chromatographic method for the analysis of monacolins in red yeast rice.

A robust analytical method, using reversed phase high-performance liquid chromatography with diode array detection, was developed and validated for the quantification of monacolins in red yeast rice bulk products. Tests on the composition of the extraction solvent, extraction time and the number of repetitions of extraction were evaluated with the aim of complete extraction of the monacolins and minimal transitions between the monacolins during analysis. Monacolin K (acid form), monacolin K (lactone form) and minor monacolin peaks were separated on a C18 column (250×4.6mm, 5µm) using acetonitrile/0.1% trifluoroacetic acid as the mobile phase. For the calibration curve of monacolin K (lactone form), a linear correlation in the range 6-119µg/mL was found. The precision of the method for time and concentration gave a relative standard deviation of less than 5%, which was deemed acceptable. The recovery of the method was 98.75%.

Saponins and Flavonoids from an Infusion of Herniaria hirsuta.

Stone diseases present a major health problem in the Western society, since both urinary and biliary stones occur with a relatively high prevalence of 10-12 % and 10-20 %, respectively, and demonstrate a high recurrence rate. At the moment treatment is mainly based on interventional procedures, or prophylactic and dissolution therapy. However, many of the current drugs cause severe side effects, and therefore, there is an increasing interest in natural medicines. At the moment no registered herbal medicinal products are available for treatment of gallstones. Since an infusion of Herniaria hirsuta L. has a proven efficacy against urolithiasis and cholelithiasis, its phytochemical composition has been investigated. Two previously undescribed triterpene saponins, 28-O-{[ß-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)]-[ß-D-glucopyranosyl-(1-6)]-ß-D-glucopyranosyl}-medicagenic acid and 3-O-[α-L-rhamnopyranosyl-(1 → 3)-ß-D-glucuronopyranosyl]-28-O-{[ß-D-glucopyranosyl-(1 → 3)-ß-D-xylopyranosyl-(1 → 4)]-[ß-D-apiofuranosyl-(1 → 3)]-α-L-rhamnopyranosyl-(1 → 2)-ß-D-fucopyranosyl}-medicagenic acid and three known flavonoids, quercetin-3-O-(2″-O-α-L-rhamnopyranosyl)-ß-D-glucuronopyranoside, rutin, and narcissin (isorhamnetin-3-O-rutinoside), were isolated using flash chromatography and successive semi-preparative HPLC and were well characterized by MS and 1D and 2D NMR spectroscopic techniques. These findings could contribute to the development of a standardized extract that can be used in prophylaxis and treatment of gall and kidney stones.

Cholesterol lowering effect in the gall bladder of dogs by a standardized infusion of Herniaria hirsuta L.

ETNOPHARMACOLOGICAL RELEVANCE: Infusions of Herniaria hirsuta L., Herniaria glabra L. and Herniaria fontanesii J.Gay are well known in Moroccon folk medicine for the treatment of biliary dyskinesia, (uro)lithiasis or as a diuretic. Herniariae Herba which can contain H. glabra and H. hirsuta is known in Europe as an urological drug. AIM OF THE STUDY: To investigate the efficacy of a standardized infusion of Herniaria hirsuta against choleltihiasis, and evaluation of its genotoxicity. METHODS AND MATERIALS: An analytical HPLC-UV method to quantify flavonoids and saponins present in the extract of H. hirsuta was developed and validated. An in vivo experiment to evaluate the cholesterol lowering effect of a infusion of H. hirsuta in the gall bladder of dogs was carried out. Dogs were divided into 3 groups i.e. control dogs (CG), dogs treated with ursodeoxycholic acid (UDCA) (2×7.35mg/kg body weight/day) and dogs treated with the standardized infusion (HG) (2×48.5mg/kg body weight/day). Dogs were fed a fatty diet during 120 days after which a diet without additional fat was introduced till day 180. Treatment started 30 days after introduction of the fatty diet and lasted till the end of the experiment. A bile and blood sample of each dog was collected every 30 days, after which the concentration of cholesterol was determined. An Ames test was performed according to the OECD-guidelines. RESULTS: The validated HPLC-UV method showed a linear calibration model and an acceptable precision for the total flavonoid content (total content 4.51%) as well as the total saponin content (12.74%). The in vivo experiments already showed a minor difference for bile cholesterol between CG and HG after 30 days of treatment with the infusion, and the difference was more pronounced after 90 days of treatment. Even 30 days after discontinuation of the cholesterol-rich diet a significant difference remained between CG and HG. There was no statistically significant difference in blood cholesterol. The Ames test showed that the infusion of H. hirsuta could be considered as being free from genotoxic risks. CONCLUSION: A method for the standardization of a infusion of Herniaria hirsuta was developed and validated. Prolonged use of this standardized H. hirsuta extract resulted in a cholesterol-lowering effect in the bile of dogs. Since this pharmacological effect prevents the formation of gallstones and can contribute to solving existing gallstones, a standardized infusion of H. hirsuta may have a positive effect in the treatment of gallstones in human patients.

Evaluation of the anti-angiogenic activity of saponins from Maesa lanceolata by different assays.

Angiogenesis, in which a vascular network is established from pre-existing vessels, is a complex multistep process. Mechanisms underlying angiogenesis can be investigated using a variety of in vitro, ex vivo and in vivo approaches. Evaluation of several promising plants and plant metabolites, including terpenoids, revealed promising anti-angiogenic activity. Since the maesasaponins displayed anti-angiogenic activity in the chick chorioallantoic membrane (CAM) assay, their activity was further investigated in several test systems. The rat aorta ring assay was compared with the placental vein assay and then selected for the ex vivo investigation of the saponins. Besides their effect on the viability of HUVEC, the anti-angiogenic capacity of the compounds was also investigated in an in vivo zebrafish assay. The activity of the saponins in the viability assay was more pronounced than in the rat aorta ring assay and similar to the effect observed in the CAM assay. The use of different test systems, however, implies different results in the case of saponins.

Impaired fibrillin-1 function promotes features of plaque instability in apolipoprotein E-deficient mice.

BACKGROUND: Arterial stiffness has been associated with an increased cardiovascular risk. The aim of this study was to investigate the interaction between arterial stiffness and atherosclerosis. METHODS AND RESULTS: Mice with a mutation C1039G+/-) in the fibrillin-1 gene leading to fragmentation of the elastic fibers were crossbred with apolipoprotein E-deficient (ApoE-/-) mice. Subsequently, ApoE-/- and ApoE-/-C1039G+/- mice were fed a Western-type diet for 10 or 20 weeks. Our results show that the interaction between arterial stiffness and atherosclerosis is bidirectional. On the one hand, arterial stiffness in ApoE-/-C1039G+/- mice increased more rapidly in the presence of atherosclerotic plaques. On the other hand, arterial stiffness promoted the development of larger and more unstable plaques in ApoE-/-C1039G+/- mice. The plaque area at the aortic root was increased 1.5- and 2.1-fold in ApoE-/-C1039G+/- mice after 10 and 20 weeks of Western-type diet, respectively. After 10 weeks of Western-type diet, plaques of ApoE-/-C1039G+/- mice showed increased apoptosis of smooth muscle cells, which was associated with a decrease in collagen content, an enlargement of the necrotic core, and an increase in macrophages. After 20 weeks of Western-type diet, the number of buried fibrous caps was increased in atherosclerotic lesions of ApoE-/-C1039G+/- mice, not only at the level of the aortic valves but also in the brachiocephalic artery and in the upper, middle, and lower thoracic aorta. Furthermore, acute plaque rupture was observed. CONCLUSIONS: These results indicate that fragmentation of the elastic fibers leads to increased vascular stiffness, which promotes features of multifocal plaque instability.

LC-MS analysis of 13,28-epoxy-oleanane saponins in Maesa spp. extracts with antileishmanial activity.

INTRODUCTION: Saponins are natural products that are well known for a wide range of biological activities. For saponins of Maesa balansae, selective antileishmanial activity has been described. OBJECTIVE: In view of their pharmacological interest, several Maesa species from the National Botanical Garden of Meise (Belgium) and wild-grown plants from Vietnam were screened for their antileishmanial potential and saponin content. METHODOLOGY: Different parts of the plants (mainly leaves and twigs) were collected, dried and extracted. Plant extracts were evaluated by liquid chromatography/mass spectrometry (LC-MS) using electrospray ionisation in the negative ion mode and their saponin content was compared with those of Maesa balansae (maesabalides) and Maesa lanceolata (maesasaponins). RESULTS: Several Maesa species (M. ambigua, M. argentea, M. brevipaniculata, M. japonica and M. perlarius) showed potent antileishmanial activity (<0.1 microg/mL) and indeed contained known maesasaponins and maesabalides. However the leaves of M. argentea also revealed two new compounds. Two saponins with [M - H]- ions at m/z 1465 and 1477 were characterised. Their mass spectrometric fragmentation pattern revealed a structure that was the same or closely related to maesasaponin V.3 and VI.2, respectively, but had a glycan part with one additional hexose residue. CONCLUSION: Several known as well as new saponins from Maesa species active against leishmaniasis were characterised using LC-MS.

Determination of saponins in Maesa lanceolata by LC-UV: development and validation.

Triterpene saponins are a class of plant natural products with a wide range of bioactivities, which makes them an interesting research subject. The small tree Maesa lanceolata, growing in African countries, is used in traditional medicine against various diseases. In previous work a triterpenoid saponin mixture was isolated from the leaves of M. lanceolata and the compounds were identified as closely related oleanane type triterpenes [Apers, S., Foriers, A., Sindambiwe, J.B., Vlietinck, A., Pieters, L., 1998. Separation of a triterpenoid saponin mixture from Maesa lanceolata: semi preparative reversed-phase wide pore high performance liquid chromatography with temperature control. J. Pharm. Biomed. Anal. 18, 737; Apers, S., De Bruyne, T.E., Claeys, M., Vlietinck, A.J., Pieters, L.A.C., 1999. New acylated triterpenoid saponins from Maesa lanceolata. Phytochemistry 52, 1121]. The compounds showed virucidal, haemolytic, molluscicidal and antiangiogenic activity [Apers, S., Baronikova, S., Sindambiwe, J.B., Witvrouw, M., De Clercq, E., Vanden Berghe, D., Van Marck, E., Vlietinck, A., Pieters, L., 2001. Antiviral, haemolytic and molluscicidal activities of triterpenoid saponins from Maesa lanceolata: establishment of structure-activity relationships. Planta Med. 67, 528; Apers, S., Bürgermeister, J., Baronikova, S., Vermeulen, P., Paper, D., Van Marck, E., Vlietinck, A.J., Pieters, L.A.C., 2002. Antiangiogenic activity of natural products: in vivo and in vitro test models. J. Pharm. Belg. 57 (Hors-série 1), 47]. Here we report the development of an extraction and quantification method to analyse saponin compounds in roots and leaves of M. lanceolata. After a purification step using C(18) solid phase extraction (SPE) cartridges, the samples were analysed on a LC-UV/MS system. The identification of the peaks from the different saponins was confirmed based on the retention time and mass spectrum. The quantification was performed using the UV signals. The standard oleanolic acid curve was linear over a concentration range of 2.8-140.0mug/mL. The recovery from the leaves was 94.5%. The precision of the method with respect to time and concentration was acceptable, with relative standard deviation (RSD%) values of 4.9 and 4.3, respectively.

A novel isoflavonoid from Millettia puguensis.

From the roots of Millettia puguensis (Leguminosae), a novel isoflavonoid (5), 2'-methoxy-4',5'-methylenedioxy-7,8-[2-(1-methylethenyl)furo]isoflavone, and four known compounds, i. e., lupeol (1), (-)-maackiain (2), 6,7-dimethoxy-3',4'-methylenedioxyisoflavone (3) and 7,2'-dimethoxy-4',5'-methylenedioxyisoflavone (4) were isolated and identified by 1H-, 13C-NMR and mass spectroscopy. All compounds were evaluated for their antiprotozoal and cytotoxic activities, but only a moderate antileishmanial activity was observed for compound 3 (IC50 = 32 microM against Leishmania infantum), and a moderate cytotoxicity for compound 2 (IC50 = 43 microM on MRC-5 cells).

Flavonoids, NodD1, NodD2, and nod-box NB15 modulate expression of the y4wEFG locus that is required for indole-3-acetic acid synthesis in Rhizobium sp. strain NGR234.

Flavonoids secreted by host plants activate, in conjunction with the transcriptional activator NodD, nod gene expression of rhizobia resulting in the synthesis of Nod factors, which trigger nodule organogenesis. Interestingly, addition of inducing flavonoids also stimulates the production of the phytohormone indole-3-acetic acid (IAA) in several rhizobia. Here, the molecular basis of IAA synthesis in Rhizobium sp. NGR234 was investigated. Mass spectrometric analysis of culture supernatants indicated that NGR234 is capable of synthesizing IAA via three different pathways. The production of IAA is increased strongly by exposure of NGR234 to daidzein in a NodD1-, NodD2-, and SyrM2-dependent manner. This suggests that the y4wEFG locus that is downstream of nod-box NB15 encodes proteins involved in IAA synthesis. Knockout mutations in y4wE and y4wF abolished flavonoid-inducible IAA synthesis and a functional y4wF was required for constitutive IAA production. The promoter activity of NB15 and IAA production both were enhanced by introduction of a multicopy plasmid carrying nodD2 into NGR234. Surprisingly, the y4wE mutant still nodulated Vigna unguiculata and Tephrosia vogelii, although the nodules contained less IAA and IAA conjugates than those formed by the wild-type bacterium.