Machine Learning-Based Analysis Reveals Triterpene Saponins and Their Aglycones in Cimicifuga racemosa as Critical Mediators of AMPK Activation.

Cimicifuga racemosa (CR) extracts contain diverse constituents such as saponins. These saponins, which act as a defense against herbivores and pathogens also show promise in treating human conditions such as heart failure, pain, hypercholesterolemia, cancer, and inflammation. Some of these effects are mediated by activating AMP-dependent protein kinase (AMPK). Therefore, comprehensive screening for activating constituents in a CR extract is highly desirable. Employing machine learning (ML) techniques such as Deep Neural Networks (DNN), Logistic Regression Classification (LRC), and Random Forest Classification (RFC) with molecular fingerprint MACCS descriptors, 95 CR constituents were classified. Calibration involved 50 randomly chosen positive and negative controls. LRC achieved the highest overall test accuracy (90.2%), but DNN and RFC surpassed it in precision, sensitivity, specificity, and ROC AUC. All CR constituents were predicted as activators, except for three non-triterpene compounds. The validity of these classifications was supported by good calibration, with misclassifications ranging from 3% to 17% across the various models. High sensitivity (84.5-87.2%) and specificity (84.1-91.4%) suggest suitability for screening. The results demonstrate the potential of triterpene saponins and aglycones in activating AMP-dependent protein kinase (AMPK), providing the rationale for further clinical exploration of CR extracts in metabolic pathway-related conditions.

Polyacetylenic caffeoyl amides from Ammodaucus leucotrichus.

Six undescribed polyacetylenic caffeoyl amides, five known flavones and three known lignans were obtained from the fruits of the North African traditional medicinal plant Ammodaucus leucotrichus Coss. & Durieu (Apiaceae). Isolation was achieved by a combination of chromatographic methods, and structures were established by extensive 1D and 2D NMR spectroscopy, mass spectrometry, electronic circular dichroism, and by GC-MS analysis of sugar derivatives. Polyacetylenic caffeoyl amides are reported for the first time as specialized metabolites.

Clerodane Diterpenes from Casearia corymbosa as Allosteric GABAA Receptor Modulators.

An EtOAc extract of Casearia corymbosa leaves led to an allosteric potentiation of the GABA signal in a fluorometric imaging plate reader (FLIPR) assay on Chinese hamster ovary (CHO) cells stably expressing GABAA receptors with an α1ß2γ2 subunit composition. The activity was tracked by HPLC-based activity profiling, and four known (2, 3, 4, and 8) and five new clerodane-type diterpenoids (1, 5-7, and 9) were isolated. Compounds 1-8 were obtained from the active time window. The absolute configuration of all compounds was established by ECD. Compounds 3, 7, and 8 exhibited EC50 values of 0.5, 4.6, and 1.4 µM, respectively. To explore possible binding sites at the receptor, the most abundant diterpenoid 8 was tested in combination with diazepam, etazolate, and allopregnanolone. An additive potentiation of the GABA signal was observed with these compounds, while the effect of 8 was not inhibited by flumazenil, a negative allosteric modulator at the benzodiazepine binding site. Finally, the activity was validated in voltage clamp studies on Xenopus laevis oocytes transiently expressing GABAA receptors of the α1ß2γ2S and α1ß2 subtypes. Compound 8 potentiated GABA-induced currents with both receptor subunit compositions [EC50 (α1ß2γ2S) = 43.6 µM; Emax = 809% and EC50 (α1ß2) = 57.6 µM; Emax = 534%]. The positive modulation of GABA-induced currents was not inhibited by flumazenil, thereby confirming an allosteric modulation independent of the benzodiazepine binding site.

Ingadosides A-C, acacic acid-type saponins from Inga sapindoides with potent inhibitory activity against downy mildew.

As part of a project aiming at the discovery of environmentally friendly alternatives to copper in organic agriculture, a 96% ethanolic extract from the leaves of Inga sapindoides showed potent inhibitory activity against grapevine downy mildew (Plasmopara viticola) in vitro (MIC100 25 µg/mL). Separation of the n-BuOH soluble fraction by silica gel column chromatography followed by a combination of RP18 and HILIC HPLC resulted in the isolation of a series of bidesmosidic saponins characterized by the presence of a monoterpenoid unit attached to a triterpenoid aglycone, a p-methoxycinnamoyl residue, and rare sugar residues such as N-acetyl-d-glucosamine, d-quinovose, and d-fucose. The isolated compounds inhibited the formation or activity of P. viticola zoospores with MIC100 values of 3 or 6 µg/mL, respectively. I. sapindoides, a tree which is often cultivated for shading coffee plantations in Central America, may represent a sustainable source of fungicidal products to be used in the replacement of copper.

Immunosuppressant flavonoids from Scutellaria baicalensis.

Some plants used in Traditional Chinese Medicine serve as treatment for disease states where a suppression of the cellular immune response is desired. However, the compounds responsible for the immunosuppressant effects of these plants are not necessarily known. The immunosuppressant compounds in the roots of Scutellaria baicalensis, one of the most promising plants identified in a previous screening, were tracked by HPLC activity profiling and concomitant on-line spectroscopic analysis. Compounds were then isolated by preparative chromatography, and structures elucidated by spectroscopic methods. Twelve flavonoids (5-16) were identified from the active time windows, and structurally related flavones 2, 4, and 17, and flavanones 1 and 3 were isolated from adjacent fractions. All flavonoids possessed an unusual substitution pattern on the B-ring, with an absence of substituents at C-3 and C-4. Compounds 11, 13, 14, and 16 inhibited T-cell proliferation (IC50 values at 12.1-39 µM) at non-cytotoxic concentrations. The findings may support the use of S. baicalensis in disorders where a modulation of the cellular immune response is desirable.

Identification of an Oxidizing Leachable from a Clinical Syringe Rubber Stopper.

Leaching of toxic or reactive chemicals from polymeric materials can adversely affect the quality and safety of biopharmaceuticals. It was therefore the aim of the present study to analyze leachables from a disposable clinical administration syringe using a polysorbate-containing surrogate solution and to assess their chemical reactivity. Analytical methods did include (headspace) GC-MS, Fourier-transform-infrared spectroscopy, a ferrous oxidation-xylenol orange assay, and nuclear magnetic resonance analysis. In the syringe leachables solution, the carcinogenic 1,1,2,2-tetrachloroethane (TCE) was detected in concentrations above the ICH M7-derived analytical evaluation threshold. TCE was shown to be an oxidation product of dichloromethane used during sample preparation. Since TCE was only isolated from incubations with the contained rubber stopper, we hypothesized that a stopper-derived leachable acted as a reactive oxidant promoting this chemical reaction. Subsequently, the leachable was identified to be the polymerization initiator Luperox® 101. Combining different analytical approaches led to the structural elucidation of a chemical reactive oxidant, which has the potential to interact and alter drug products. We conclude that chemically reactive compounds, such as the newly identified rubber stopper leachable Luperox® 101, may be of concern and therefore should be routinely considered if a prolonged exposure of polymers with drug products can be anticipated.

Phylobioactive hotspots in plant resources used to treat Chagas disease.

Globally, more than six million people are infected with Trypanosoma cruzi, the causative protozoan parasite of the vector-borne Chagas disease (CD). We conducted a cross-sectional ethnopharmacological field study in Bolivia among different ethnic groups where CD is hyperendemic. A total of 775 extracts of botanical drugs used in Bolivia in the context of CD and botanical drugs from unrelated indications from the Mediterranean De Materia Medica compiled by Dioscorides two thousand years ago were profiled in a multidimensional assay uncovering different antichagasic natural product classes. Intriguingly, the phylobioactive anthraquinone hotspot matched the antichagasic activity of Senna chloroclada, the taxon with the strongest ethnomedical consensus for treating CD among the Izoceño-Guaraní. Testing common 9,10-anthracenedione derivatives in T. cruzi cellular infection assays demarcates hydroxyanthraquinone as a potential antichagasic lead scaffold. Our study systematically uncovers in vitro antichagasic phylogenetic hotspots in the plant kingdom as a potential resource for drug discovery based on ethnopharmacological hypotheses.

Identification of a novel di-C-glycosyl dihydrochalcone and the thermal stability of polyphenols in model ready-to-drink beverage solutions with Cyclopia subternata extract as functional ingredient.

Heat processing of ready-to-drink beverages is required to ensure a microbiologically safe product, however, this can result in the loss of bioactive compounds responsible for functionality. The objective of this study was to establish the thermal stability of a novel dihydrochalcone, 3',5'-di-ß-d-glucopyranosyl-3-hydroxyphloretin (2), 3',5'-di-ß-d-glucopyranosylphloretin (3) and other Cyclopia subternata phenolic compounds, in model solutions with or without citric acid and ascorbic acid. The solutions were heated at 93, 121 and 135 °C, relevant to pasteurisation, commercial sterilisation and ultra-high temperature (UHT) pasteurisation, respectively. For most compounds, the acids decreased the second order reaction rate constants, up to 27 times. Compound 2 (46.29 ± 0.53 (g/100 g)-1 h-1), and to a lesser extent compound 3 (5.94 ± 0.01 (g/100 g)-1 h-1) were the most thermo-unstable compounds when treated at 135 °C without added acids. Even though differential effects were observed for compounds at different temperatures and formulations, overall, the phenolic compounds were most stable under UHT pasteurisation conditions.

Saponins from Saffron Corms Inhibit the Gene Expression and Secretion of Pro-Inflammatory Cytokines.

Corms are obtained as a byproduct during the cultivation of saffron (Crocus sativus). In a project aimed at the valorization of this waste product, we observed that a 70% EtOH extract of the corms and a sugar-depleted MeOH fraction of the extract inhibited the TNF-α/IFN-γ-induced secretion and gene expression of the chemokines IL-8, MCP-1, and RANTES in human HaCaT cells. The effects were in part stronger than those of the positive control hydrocortisone. For preparative isolation, the 70% EtOH extract was partitioned between n-BuOH and water. Separation of the n-BuOH-soluble fraction by centrifugal partition chromatography, followed by preparative and semipreparative HPLC, afforded a series of bidesmosidic glycosides of echinocystic acid bearing a 3,16-dihydroxy-10-oxo-hexadecanoic acid residue attached to the glycosidic moiety at C-28. They include azafrines 1 and 2, previously reported in saffron, and eight new congeners named azafrines 3-10. Saffron saponins significantly inhibited TNF-α/IFN-γ-induced secretion of RANTES in human HaCaT cells at 1 µM (p < 0.001). Some of them further lowered TNF-α/IFN-γ-induced gene expression.

Unusual derivatives from Hypericum scabrum.

Three new compounds (1-3) with unusual skeletons were isolated from the n-hexane extract of the air-dried aerial parts of Hypericum scabrum. Compound 1 represents the first example of an esterified polycyclic polyprenylated acylphloroglucinol that features a unique tricyclo-[4.3.1.11,4]-undecane skeleton. Compound 2 is a fairly simple MPAP, but with an unexpected cycloheptane ring decorated with prenyl substituents, and compound 3 has an unusual 5,5-spiroketal lactone core. Their structures were determined by extensive spectroscopic and spectrometric techniques (1D and 2D NMR, HRESI-TOFMS). Absolute configurations were established by ECD calculations, and the absolute structure of 2 was confirmed by a single crystal determination. Plausible biogenetic pathways of compounds 1-3 were also proposed. The in vitro antiprotozoal activity of the compounds against Trypanosoma brucei rhodesiense and Plasmodium falciparum and cytotoxicity against rat myoblast (L6) cells were determined. Compound 1 showed a moderate activity against T. brucei and P. falciparum, with IC50 values of 3.07 and 2.25 µM, respectively.

Comprehensive off-line CCC × LC-DAD-MS separation of Cyclopia pubescens Eckl. & Zeyh. phenolic compounds and structural elucidation of isolated compounds.

INTRODUCTION: The minor phenolic constituents of Cyclopia pubescens Eckl. & Zeyh. are unknown and one dimensional (1D) liquid chromatography (LC) is unable to provide sufficient separation. METHODOLOGY: A two-dimensional (2D) LC method incorporating normal-phasehigh performance countercurrent chromatography (NP-HPCCC) in the first dimension (1 D) and reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) as the second dimension (2 D) was developed. The analytical HPCCC method was subsequently scaled up to semi-preparative mode and fractions pooled based on phenolic sub-groups. The phenolic compounds in selected fractions were subsequently isolated using RP-HPLC on a C18 column. Isolated compounds were identified by nuclear magnetic resonance (NMR) spectroscopy. The absolute configurations of compounds were determined by optical rotation and electronic circular dichroism spectra. Sugars were identified by gas chromatography-mass spectrometry (GC-MS) analysis. RESULTS: The comprehensive off-line 2D CCC × LC method gave a good spread of the phenolic compounds. Orthogonality calculated using both the convex hull and conditional entropy methods were 81%. High-resolution mass spectrometric fragmentation spectra obtained from a quadrupole-time-of-flight instrument and ultraviolet-visible (UV-vis) spectral data were used to (tentatively) identify 32 phenolic compounds from the analytical CCC fractions. Of the seven isolated compounds, (2S)-5-O-[α-l-rhamnopyranosyl-(1 → 2)-ß-d-glucopyranosyl]eriodictyol (3) and (2S)-5-O-[α-l-rhamnopyranosyl-(1 → 2)-ß-d-glucopyranosyl]-5,7,3',4'-tetrahydroxyflavan (4) were newly identified in all plants. The other isolated compounds were identified as (2S)-5-O-[α-l-rhamnopyranosyl-(1 → 2)-ß-d-glucopyranosyl]naringenin (1), R-neo-eriocitrin (2), 3-O-α-l-arabinopyranosyl-3,4-dihydroxybenzoic acid (5), 4-O-ß-d-glucopyranosyl-Z-4-hydroxycinnamic acid (6) and 4-(4'-O-ß-d-glucopyranosyl-4'-hydroxy-3'-methoxyphenyl)-2-butanone (7). CONCLUSIONS: Among the 32 compounds (tentatively) identified, only six were previously identified in Cyclopia pubescens using 1D LC. Most of the isolated compounds were also identified for the first time in Cyclopia spp., improving the knowledge of the minor phenolic compounds of this genus.

Compounds from Toddalia asiatica: Immunosuppressant Activity and Absolute Configurations.

In a screening of an extract library from plants used in Traditional Chinese Medicine the MeOH extract of Toddalia asiatica inhibited proliferation of human primary T cells with an IC50 of 25.8 µg/mL. Activity in the extract was tracked by HPLC activity profiling, and a total of 15 compounds were characterized. Three compounds, toddalic acid (6) and both enantiomers (7a and 7b) of toddanolic acid (7), were new natural products, and two recently published compounds, (2'R)-toddalolactone 3'-O-ß-d-glucopyranoside (10) and (2'S)-toddalolactone 2'-O-ß-d-glucopyranoside (11), were described in detail for the first time. The absolute configurations of compounds 8, 9, 10, 12, 13, and 15 were determined by comparison of experimental and calculated ECD spectra. For glucosides 9 and 10, ECD data and chiral-phase HPLC of the aglycones after enzymatic hydrolysis confirmed the results. Nitidine chloride (4) inhibited proliferation of primary human T cells with an IC50 of 0.4 µM.

HPLC-Based Activity Profiling for Antiprotozoal Compounds in Croton gratissimus and Cuscuta hyalina.

In a screening of Sudanese medicinal plants for antiprotozoal activity, the chloroform fractions obtained by liquid-liquid partitioning from ethanolic extracts of fruits of Croton gratissimus var. gratissimus and stems of Cuscuta hyalina Roth ex Schult. exhibited in vitro activity against axenically grown Leishmania donovani amastigotes. This antileishmanial activity was localized by HPLC-based activity profiling. Targeted preparative isolation afforded flavonoids 1-6, 3-methoxy-4-hydroxybenzoic acid (7), and benzyltetrahydroisoquinoline alkaloids laudanine (8) and laudanosine (9) from C. gratissimus, and pinoresinol (10), isorhamnetin (11), (-)-pseudosemiglabrin (12), and kaempferol (13) from C. hyalina. The antiprotozoal activity of 1-13 against L. donovani (axenic and intracellular amastigotes), Trypanosoma brucei rhodesiense (bloodstream forms), and Plasmodium falciparum (erythrocytic stages), and the cytotoxicity in L6 murine myoblast cells were determined in vitro. Quercetin-3,7-dimethylether (6) showed the highest activity against axenic L. donovani (IC50, 4.5 µM; selectivity index [SI], 12.3), P. falciparum (IC50, 7.3 µM; SI, 7.6), and T. b. rhodesiense (IC50, 2.4 µM; SI, 23.2). The congener ayanin (2) exhibited moderate antileishmanial (IC50, 8.2 µM; SI, 12.2), antiplasmodial (IC50, 7.8 µM; SI, 12.9), and antitrypanosomal activity (IC50, 11.2 µM; SI, 8.9). None of the compounds showed notable activity against the intramacrophage form of L. donovani.

Lignans, Amides, and Saponins from Haplophyllum tuberculatum and Their Antiprotozoal Activity.

A screening of Sudanese medicinal plants for antiprotozoal activities revealed that the chloroform and water fractions of the ethanolic root extract of Haplophyllum tuberculatum exhibited appreciable bioactivity against Leishmania donovani. The antileishmanial activity was tracked by HPLC-based activity profiling, and eight compounds were isolated from the chloroform fraction. These included lignans tetrahydrofuroguaiacin B (1), nectandrin B (2), furoguaiaoxidin (7), and 3,3'-dimethoxy-4,4'-dihydroxylignan-9-ol (10), and four cinnamoylphenethyl amides, namely dihydro-feruloyltyramine (5), (E)-N-feruloyltyramine (6), N,N'-diferuloylputrescine (8), and 7'-ethoxy-feruloyltyramine (9). The water fraction yielded steroid saponins 11-13. Compounds 1, 2, and 5-13 are reported for the first time from Haplophyllum species and the family Rutaceae. The antiprotozoal activity of the compounds plus two stereoisomeric tetrahydrofuran lignans-fragransin B2 (3) and fragransin B1 (4)-was determined against Leishmania donovani amastigotes, Plasmodium falciparum, and Trypanosoma brucei rhodesiense bloodstream forms, along with their cytotoxicity to rat myoblast L6 cells. Nectandrin B (2) exhibited the highest activity against L. donovani (IC50 4.5 µM) and the highest selectivity index (25.5).

Anti-Inflammatory and Cytotoxic Potential of New Phenanthrenoids from Luzula Sylvatica.

Phenanthrenoids have been widely described, in the Juncaceae family, for theirbiological properties such as antitumor, anxiolytic, anti-microbial, spasmolytic, and antiinflammatoryactivities. The Juncaceae family is known to contain a large variety ofphenanthrenoids possessing especially anti-inflammatory and cytotoxic properties. Luzulasylvatica, a Juncaceae species, is widely present in the Auvergne region of France, but has neverbeen studied neither for its phytochemical profile nor for its biological properties. We investigatedthe phytochemical profile and evaluated the potential anti-inflammatory activities of L. sylvaticaaerial parts extracts. A bioassay-guided fractionation was carried out to identify the most activefractions. Nine compounds were isolated, one coumarin 1 and eight phenanthrene derivatives (2-9), including four new compounds (4, 5, 8 and 9), from n-hexane and CH2Cl2, fractions. Theirstructures were established by HRESIMS, 1D and 2D NMR experiments. The biological properties,especially the anti-inflammatory/antioxidant activities (ROS production) and antiproliferativeactivity on THP-1, a monocytic leukemia cell line, of each compound, were evaluated. Threephenanthrene derivatives 4, 6, and 7 showed very promising antiproliferative activities.Phenanthrene derivatives.

Antibacterial and ATP Synthesis Modulating Compounds from Salvia tingitana.

A surface extract of the aerial parts of Salvia tingitana afforded a nor-sesterterpenoid (1) and eight new sesterterpenoids (2-̵9), along with five known sesterterpenoids, five labdane and one abietane diterpenoid, one sesquiterpenoid, and four flavonoids. The structures of the new compounds were established by 1D and 2D NMR spectroscopy, HRESIMS, and VCD data and Mosher's esters analysis. The antimicrobial activity of compounds was evaluated against 30 human pathogens including 27 clinical strains and three isolates of marine origin for their possible implications on human health. The methyl ester of salvileucolide (10), salvileucolide-6,23-lactone (11), sclareol (15), and manool (17) were the most active against Gram-positive bacteria. The compounds were also tested for the inhibition of ATP production in purified mammalian rod outer segments. Terpenoids 10, 11, 15, and 17 inhibited ATP production, while only 17 inhibited also ATP hydrolysis. Molecular modeling studies confirmed the capacity of 17 to interact with mammalian ATP synthase. A significant reduction of ATP production in the presence of 17 was observed in Enterococcus faecalis and E. faecium isolates.

Sesquiterpene Lactones from Artemisia argyi: Absolute Configuration and Immunosuppressant Activity.

A library of extracts from plants used in Chinese Traditional Medicine was screened for inhibition of T lymphocyte proliferation. An ethyl acetate extract from aerial parts of Artemisia argyi showed promising activity and was submitted to HPLC-based activity profiling to track the active compounds. From the most active time window, three guaianolides (1, 2, and 5) and two seco-tanapartholides (3 and 4) were identified and, in a less active time window, five new sesquiterpene lactones (8-11, 17), along with six known sesquiterpene lactones and two known flavonoids. The absolute configurations of compounds 1, 2, 5-10, 13-15, 17, and 18 were established by comparison of experimental with calculated electronic circular dichroism (ECD) spectra. For seco-tanapartholides B (3) and A (4), ECD yielded ambiguous results, and their absolute configurations were determined by comparing experimental and calculated vibrational circular dichroism (VCD) spectra. Compounds 1-5 showed significant, noncytotoxic inhibition of T lymphocyte proliferation, with IC50 values between 1.0 and 3.7 µM.

HPLC-Based Activity Profiling for Antiprotozoal Compounds in the Endemic Iranian Medicinal Plant Helichrysum oocephalum.

In a screening of Iranian plants for antiprotozoal activity a dichlomethane extract from the aerial parts of Helichrysum oocephalum showed in vitro antiprotozoal activity against Plasmodium falciparum and Leishmania donovani, with IC50 values of 4.01 ± 0.50 and 5.08 ± 0.07 µg/mL, respectively. The activity in the extract was tracked by HPLC-based activity profiling, and subsequent targeted preparative isolation afforded 24 compounds, including pyrones 22-24, phloroglucinol derivatives 12-19, and compounds containing both structural motifs (1-11, 20, and 21). Of these, 15 compounds were new natural products. The in vitro antiprotozoal activity of isolates was determined. Compound 3 showed good potency and selectivity in vitro against L. donovani (IC50 1.79 ± 0.17 µM; SI 53).

Antiprotozoal Isoprenoids from Salvia hydrangea.

Fractionation of the n-hexane extract of Salvia hydrangea afforded seven isoprenoids including six new compounds (1-6) and salvadione A (7). Their structures were established by comprehensive spectroscopic and spectrometric data analysis (1D and 2D NMR, HRMS). The absolute configuration of salvadione A (7) was established by single-crystal X-ray diffraction analysis with Cu/Kα radiation. In addition, the absolute configuration of all compounds was determined by electronic circular dichroism spectroscopy. A biosynthetic pathway for the formation of the scaffold of 1 is proposed. The antiprotozoal activity of the compounds against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum was determined, and cytotoxicity was assessed in rat myoblast L6 cells. Perovskone C (2) exhibited good activity against P. falciparum (IC50 0.6 µM) and a selectivity index of 62.2.

Bitter Taste Impact and Thermal Conversion of a Naringenin Glycoside from Cyclopia genistoides.

A naringenin derivative, isolated from Cyclopia genistoides, a bitter tasting herbal tea, especially when in green (unoxidized) form, was identified as (2 S)-5-[α-l-rhamnopyranosyl-(1→2)-ß-d-glucopyranosyloxy]naringenin (1). The compound partially epimerizes to (2 R)-5-[α-l-rhamnopyranosyl-(1→2)-ß-d-glucopyranosyloxy]naringenin (2) when heated at different temperatures (80, 90, 100, 110, and 120 °C) for a prolonged period in a phosphate buffer at pH 5. The fractional conversion model predicted the decrease in the concentration of compound 1 the best. The activation energy of the conversion reaction was calculated as 99.16 kJ mol-1. Prolonged heating resulted not only in formation of compound 2 but eventually a decrease in its concentration and the formation of another conversion product, ( E)-2'-[α-l-rhamnopyranosyl-(1→2)-ß-d-glucopyranosyloxy]-4',6',4-trihydroxychalcone (3). In contrast, naringin, glycosylated at C-7, remained stable when heated under the same conditions (100 °C for 6 h at pH 5). The bitter intensity of compound 1 was substantially less than that of naringin, both tested at 0.04 mM, a concentration typical of compound 1 in an herbal tea infusion of green C. genistoides. This comparison indicates that the position of the sugar moiety plays an important role in determining both bitter intensity and heat stability of naringenin glycosides.