Acetylation of Oleanolic Acid Dimers as a Method of Synthesis of Powerful Cytotoxic Agents.

Oleanolic acid, a naturally occurring triterpenoid compound, has garnered significant attention in the scientific community due to its diverse pharmacological properties. Continuing our previous work on the synthesis of oleanolic acid dimers (OADs), a simple, economical, and safe acetylation reaction was performed. The newly obtained derivatives (AcOADs, 3a-3n) were purified using two methods. The structures of all acetylated dimers (3a-3n) were determined based on spectral methods (IR, NMR). For all AcOADs (3a-3n), the relationship between the structure and the expected directions of pharmacological activity was determined using a computational method (QSAR computational analysis). All dimers were also tested for their cytotoxic activity on the SKBR-3, SKOV-3, PC-3, and U-87 cancer cell lines. HDF cell line was applied to evaluate the Selectivity Index of the tested compounds. All cytotoxic tests were performed with the application of the MTT assay. Finally, all dimers of oleanolic acid were subjected to DPPH and CUPRAC tests to evaluate their antioxidant activity. The obtained results indicate a very high level of cytotoxic activity (IC50 for most AcOADs below 5.00 µM) and a fairly high level of antioxidant activity (Trolox equivalent in some cases above 0.04 mg/mL).

Determination of Biologically Active Compounds and Antioxidant Capacity In Vitro in Fruit of Small Cranberries (Vaccinium oxycoccos L.) Growing in Natural Habitats in Lithuania.

The composition of flavonols, proanthocyanidins, anthocyanins, triterpene compounds, and chlorogenic acid in small cranberry fruit samples collected in natural habitats in Lithuania and variation in the antioxidant capacity of cranberry fruit extracts was determined. This study showed that in the flavonol group, hyperoside and myricetin-3-O-galactoside predominated in cranberry fruit samples; in the anthocyanin group, the predominant compounds were cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside, peonidin-3-O-galactoside, and peonidin-3-O-arabinoside, and in the group of triterpene compounds, ursolic acid was predominant. The highest total amounts of flavonols and anthocyanins were found in the samples collected in Cepkeliai State Strict Nature Reserve (2079.44 ± 102.99 µg/g and 6993.79 ± 350.22 µg/g, respectively). Cluster analysis of the chemical composition of small cranberry fruit samples revealed trends in the accumulation of bioactive compounds in cranberry fruit. Cranberry fruit samples collected in central Lithuania had higher levels of triterpene compounds. Statistical correlation analysis showed the strongest correlation between the quantitative composition of cyanidin-3-O-arabinoside and peonidin-3-O-arabinoside and the reducing capacity of the ethanolic extracts of the cranberry fruit samples assessed in vitro by the FRAP assay (r = 0.882, p < 0.01 and r = 0.805, p < 0.01, respectively). Summarizing the results, the geographical factor affects the variation of the quantitative composition of biologically active compounds in cranberry fruit samples.

Chemical constituents of Coccoloba peltata Schott leaves and their cytotoxic activities.

Chromatographic fractionation of CH2Cl2:MeOH (1:1) extract of Coccoloba peltata Schott cultivated in Egypt afforded four compounds, lupeol acetate (1), ent-13-epi-manoyl oxide (2), 5-methoxymethyl-7,8-dimethyltocol (3), and α-tocopherol quinone (4). The planar structures of the isolated compounds were concluded based on HRESIMS and NMR spectroscopy. X-ray crystallography of 2 is reported herein for the first time and its unambiguous absolute configuration was deduced from anomalous dispersion. To the best of our knowledge, this is the first known report on the isolation of compounds 1-4 from this species. Among the isolated compounds, only compound 4 showed a moderate level of cytotoxic activity against six cancer cell lines out of seven cell lines tested.

Lupeol: A dietary and medicinal triterpene with therapeutic potential.

Lupeol, a triterpene derived from various plants, has emerged as a potent dietary supplement with extensive therapeutic potential. This review offers a comprehensive examination of lupeol's applications across diverse health conditions. By meticulously analyzing current scientific literature, we have synthesized findings that underscore lupeol's impact on cancer, diabetes, gastrointestinal disorders, neurological diseases, dermatological conditions, nephrological issues, and cardiovascular health. The review delves into molecular studies that reveal lupeol's ability to modulate disease pathways and alleviate symptoms, positioning it as a promising therapeutic agent. Moreover, we discuss the potential role of lupeol in clinical practice and public health strategies, emphasizing its substantial benefits as a natural compound. This thorough analysis serves as a critical resource for researchers, providing insights into the multifaceted therapeutic properties of lupeol and its potential to significantly enhance health outcomes.

Isolation of a new iso-quinoline alkaloid and cytotoxicity studies of pure compounds and crude extracts of Ravenia spectabilis engl.

A new 2-quinolone alkaloid, iso-oligophyline (1), and two very unusual C34 terpenoids, proposed names ravespanol (2) and ravespanone (3), along with two known compounds, ß-sitosterol (4), and methyl linoleate (5), were isolated from the leaf extract of Ravenia spectabilis engl. Methyl linoleate constitutes the first report of isolation from this species. We have already reported the isolation of atanine (6), oligophyline (7), ravenoline (8), and arborinine (9) from the plant. Based on nuclear magnetic resonance (NMR) spectroscopy and mass spectrometric analysis, the structure of the isolated chemicals was determined. The crude fractions and four compounds (6,7,8 and 9) were evaluated for a cytotoxicity study on a panel of six human stomach cancer cell lines (SCL, SCL-6, SCL-37'6, SCL-9, K-3, N21) by MTT assay. Among the plant extracts and isolated compounds, petroleum ether fraction and compound 7 exhibited the highest cytotoxic activity against SCL and SCL-6 cells, where the IC50 values were 17.9 and 16.56 µM, respectively.

[Metabolite identification and metabolic pathway analysis of Platycodonis Radix extracts from Tongcheng city in rats].

In this study, we delved into the prototypical components and metabolites of Platycodonis Radix extracts(PRE) from Tongcheng city in plasma, urine and feces of rats, and revealed its metabolic pathways and metabolic rules in vivo. The prototypical components and metabolites of PRE in rats were characterized and identified by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) and mass defect filter(MDF). The biological samples were analyzed by ACQUITY UPLC BEH C_(18)(2.1 mm×100 mm, 1.7 µm), with 0.1% formic acid water(A)-0.1% formic acid acetonitrile(B) as mobile phase, and the biological samples were analyzed in negative ion mode by electrospray ionization mass spectrometry(ESI-MS). Twelve prototypical saponins and twenty-seven metabolites were detected in plasma, urine and feces of rats treated with PRE by oral administration. Eleven prototypical components and nine metabolites were detected in plasma, eleven prototypical components and eight metabo-lites were detected in urine, and ten prototypical components and twenty metabolites were detected in feces. Further studies showed that the metabolic pathways of PRE in rats mainly include oxidation, reduction, acetylation, stepwise hydrolytic deglycosylation, glucuronidation and so on. This study provides a scientific basis for clarifying the pharmacological basis and mechanism of PRE from Tongcheng city.

Exploring the Potential of Oleanolic Acid Dimers-Cytostatic and Antioxidant Activities, Molecular Docking, and ADMETox Profile.

The presented work aimed to explore the potential of oleanolic acid dimers (OADs): their cytostatic and antioxidant activities, molecular docking, pharmacokinetics, and ADMETox profile. The cytostatic properties of oleanolic acid (1) and its 14 synthesised dimers (2a-2n) were evaluated against 10 tumour types and expressed as IC50 values. Molecular docking was performed with the CB-Dock2 server. Antioxidant properties were evaluated with the CUPRAC method. ADMETox properties were evaluated with the ADMETlab Manual (2.0) database. The results indicate that the obtained OADs can be effective cytostatic agents, for which the IC50 not exceeded 10.00 for many tested cancer cell lines. All OADs were much more active against all cell lines than the mother compound (1). All dimers can inhibit the interaction between the 1MP8 protein and cellular proteins with the best results for compounds 2f and 2g with unsaturated bonds within the linker. An additional advantage of the tested OADs was a high level of antioxidant activity, with Trolox equivalent for OADs 2c, 2d, 2g-2j, 2l, and 2m of approximately 0.04 mg/mL, and beneficial pharmacokinetics and ADMETox properties. The differences in the DPPH and CUPRAC assay results obtained for OADs may indicate that these compounds may be effective antioxidants against different radicals.

Biological Properties of Oleanolic Acid Derivatives Bearing Functionalized Side Chains at C-3.

Triterpene acids are a class of pentacyclic natural carboxylic compounds endowed with a variety of biological activities including antitumor, antimicrobial, and hepatoprotective effects. In this work, several oleanolic acid derivatives were synthesized by structurally modifying them on the C-3 position. All synthesized derivatives were evaluated for possible antibacterial and antiviral activity, and among all the epimers, 6 and 7 demonstrated the best biological activities. Zone-of-inhibition analyses were conducted against two strains, E. coli as a Gram-negative and S. aureus as a Gram-positive model. Subsequently, experiments were performed using the microdilution method to determine the minimum inhibitory concentration (MIC). The results showed that only the derivative with reduced hydrogen bonding ability on ring A possesses remarkable activity toward E. coli. The conversion from acid to methyl ester implies a loss of activity, probably due to a reduced affinity with the bacterial membrane. Before the antiviral activity, the cytotoxicity of triterpenes was evaluated through a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Samples 6 and 7 showed less than 50% cytotoxicity at 0.625 and 1 mg/mL, respectively. The antiviral activity against SARS-CoV-2 and PV-1 did not indicate that triterpene acids had any inhibitory capacity in the sub-toxic concentration range.

One new triterpene glycoside from Cyclocarya paliurus.

One previously undescribed triterpene glycoside (1) and two known compounds were isolated from the leaves of Cyclocarya paliurus (2-3). Their structures were elucidated based on methods of spectroscopic analysis and NMR data comparison with those in the literature. Compound 1 showed a moderate inhibitory effect on melanogenesis with an IC50 value of 282.3 µM, with the positive drug arbutin showing an IC50 value of 168.5 µM.

Oleanolic Acid Dimers with Potential Application in Medicine-Design, Synthesis, Physico-Chemical Characteristics, Cytotoxic and Antioxidant Activity.

The present work aimed to obtain a set of oleanolic acid derivatives with a high level of cytotoxic and antioxidant activities and a low level of toxicity by applying an economical method. Oleanolic acid was alkylated with α,ω-dihalogenoalkane/α,ω-dihalogenoalkene to obtain 14 derivatives of dimer structure. All of the newly obtained compounds were subjected to QSAR computational analysis to evaluate the probability of the occurrence of different types of pharmacological activities depending on the structure of the analysed compound. All dimers were tested for cytotoxicity activity and antioxidant potential. The cytotoxicity was tested on the SKBR-3, SKOV-3, PC-3, and U-87 cancer cell lines with the application of the MTT assay. The HDF cell line was applied to evaluate the tested compounds' Selectivity Index. The antioxidant test was performed with a DPPH assay. Almost all triterpene dimers showed a high level of cytotoxic activity towards selected cancer cell lines, with an IC50 value below 10 µM. The synthesised derivatives of oleanolic acid exhibited varying degrees of antioxidant activity, surpassing that of the natural compound in several instances. Employing the DPPH assay, compounds 2a, 2b, and 2f emerged as promising candidates, demonstrating significantly higher Trolox equivalents and highlighting their potential for pharmaceutical and nutraceutical applications. Joining two oleanolic acid residues through their C-17 carboxyl group using α,ω-dihalogenoalkanes/α,ω-dihalogenoalkenes resulted in the synthesis of highly potent cytotoxic agents with favourable SIs and high levels of antioxidant activity.

The Composition of Triterpene Glycosides in the Sea Cucumber Psolus peronii: Anticancer Activity of the Glycosides against Three Human Breast Cancer Cell Lines and Quantitative Structure-Activity Relationships (QSAR).

Eight sulfated triterpene glycosides, peronioside A (1) and psolusosides A (2), B (3), G (4), I (5), L (6), N (7) and P (8), were isolated from the sea cucumber Psolus peronii. Peronioside A (1) is a new glycoside, while compounds 2-8 were found previously in Psolus fabricii, indicating the phylogenetic and systematic closeness of these species of sea cucumbers. The activity of 1-8 against human erythrocytes and their cytotoxicity against the breast cancer cell lines MCF-7, T-47D and triple-negative MDA-MB-231 were tested. The most active against cancer cell compounds, psolusosides A (2) and L (6), which were not cytotoxic to the non-transformed cells of the mammary gland, were chosen to study the inhibition of the migration, formation and growth of colonies of the cancer cell lines. Glycoside 2 effectively inhibited the growth of colonies and the migration of the MDA-MB-231 cell line. Compound 6 blocked the growth of colonies of T-47D cells and showed a pronounced antimigration effect on MDA-MB-231 cells. The quantitative structure-activity relationships (QSAR) indicated the strong impact on the activity of the form and size of the molecules, which is connected to the length and architecture of the carbohydrate chain, the distribution of charge on the molecules' surface and various aspects of hydrogen bond formation, depending on the quantity and positions of the sulfate groups. The QSAR calculations were in good accordance with the observed SAR tendencies.

Antitumor activity and transcriptome sequencing (RNA-seq) analyses of hepatocellular carcinoma cells in response to exposure triterpene-nucleoside conjugates.

Fifteen betulonic/betulinic acid conjugated with nucleoside derivatives were synthesized to enhance antitumor potency and water solubility. Among these, the methylated betulonic acid-azidothymidine compound (8c) exhibited a broad-spectrum of antitumor activity against three tested tumor cell lines, including SMMC-7721 (IC50 = 5.02 µM), KYSE-150 (IC50 = 5.68 µM), and SW620 (IC50 = 4.61 µM) and along with lower toxicity (TC50 > 100 µM) estimated by zebrafish embryos assay. Compared to betulinic acid (<0.05 µg/mL), compound 8c showed approximately 40-fold higher water solubility (1.98 µg/mL). In SMMC-7721 cells, compound 8c induced autophagy and apoptosis as its concentration increased. Transcriptomic sequencing analysis was used to understand the potential impacts of the underlying mechanism of 8c on SMMC-7721 cells. Transcriptomic studies indicated that compound 8c could activate autophagy by inhibiting the PI3K/AKT pathway in SMMC-7721 cells. Furthermore, in the xenograft mice study, compound 8c significantly slowed down the tumor growth, as potent as paclitaxel treated group. In conclusion, methylated betulonic acid-azidothymidine compound (8c) not only increases water solubility, but also enhances the potency against hepatocellular carcinoma cells by inducing autophagy and apoptosis, and suppressing the PI3K/Akt/mTOR signaling pathway.

Oleanolic acid-3-glucoside, a synthetic oleanane-type saponin, ameliorates methylmercury-induced dysfunction of synaptic transmission in mice.

Methylmercury (MeHg) is widely distributed in nature and is known to cause neurotoxic effects. This study aimed to examine the anti-MeHg activity of oleanolic acid-3-glucoside (OA3Glu), a synthetic oleanane-type saponin derivative, by evaluating its effects on motor function, pathology, and electrophysiological properties in a mouse model of MeHg poisoning. Mice were orally administered 2 or 4 mg·kg-1·d-1 MeHg with or without 100 µg·kg-1·d-1 OA3Glu 5x/week for four weeks. Motor function was evaluated using beam-walking and dynamic weight-bearing (DWB) tests. High-dose MeHg exposure significantly increased the frequency of stepping off the hind leg while crossing the beam in the beam-walking test, and increased weight on forelegs when moving freely in the DWB test. OA3Glu treatment alleviated motor abnormality caused by high-dose MeHg exposure in both motor function tests. Additionally, OA3Glu treatment reduced the number of contracted Purkinje cells frequently observed in the cerebellum of MeHg-treated groups, although cerebrum histology was similar in all experimental groups. The synaptic potential amplitude in the cerebellum decreased as MeHg exposure increased, which was restored by OA3Glu treatment. Even in the cerebrum, where the effects of MeHg were not observed, the amplitude of the field potential was suppressed with increasing MeHg exposure but was restored with OA3Glu treatment. Taken together, the study findings suggest that OA3Glu improves neurotransmission and movement disorders associated with MeHg exposure via protection of Purkinje cells in the cerebellum while ameliorating pre/post-synaptic deficits in the cerebral cortex in which no changes were observed at the tissue level, potentially providing a treatment to mitigate MeHg toxicity.

Phytochemistry and Biological Activities of Agrostemma Genus-A Review.

The family Caryophyllaceae comprises more than 2600 species spread widely across all the continents. Their economic importance is mainly as ornamentals (carnation) and as weeds in agriculture. Some species have been used traditionally (and some are still) in herbal medicine or as emulsifiers in food processing. These applications are based on the high content of triterpenoid saponins. Typical for this family are also ribosome-inactivating proteins (RIPs), which are potentially highly toxic. Agrostemma githago L. (common corncockle) was historically considered a serious toxicological hazard owing to cereal grain contamination by its seeds. Notwithstanding, it was also recommended as a drug by various herbalists. In this review, the literature was searched in the PubMed, Google Scholar, and Scopus databases for papers focused on the chemical composition and bioactivity of the two accepted species of the Agrostemma genus. This systematic review adhered to the Preferred Reporting Items for Systematic Reviews and MetaAnalysis (PRISMA) guidelines. Current research reports the cytotoxicity against neoplastic cells; the protection against oxidative stress; the suppression of Leishmania major culture growth; the inhibition of protein synthesis; and the antiviral, anti-angiogenic, and antihypercholesterolemic activities of common corncockle. The future prospects of using A. githago saponins as adjuvants in drug formulations and enhancing the cytotoxicity of RIPs are also discussed.

Molecular cloning and functional characterization of 2,3-oxidosqualene cyclases from Artemisia argyi.

Artemisia argyi is a traditional medicinal and edible plant, generating various triterpenoids with pharmacological activities, such as anti-virus, anti-cancer, and anti-oxidant. The 2,3-oxidosqualene cyclase family of A. argyi offers novel insights into the triterpenoid pathway, which might contribute to the medicinal value of its tissue extracts. Nevertheless, the biosynthesis of active triterpenoids in Artemisia argyi is still uncertain. In this study, four putative OSC (2,3-oxidosqualene cyclase) genes (AaOSC1-4) were first isolated and identified from A. argyi. Through the yeast heterologous expression system, three AaOSCs were characterized for the biosynthesis of diverse triterpenoids including cycloartenol, ß-amyrin, (3S,13R)-malabarica-14(27),17,21-trien-3ß-ol, and dammara-20,24-dien-3ß-ol. AaOSC1 was a multifunctional dammara-20,24-dien-3ß-ol synthase, which yielded 8 different triterpenoids, including tricyclic, and tetracyclic products. AaOSC2 and AaOSC3 were cycloartenol, and ß-amyrin synthases, respectively. As a result, these findings provide a deeper understanding of the biosynthesis pathway of triterpenes in A. argyi.

Optimized extraction, enrichment, identification and hypoglycemic effects of triterpenoid acids from Hippophae rhamnoides L pomace.

The Hippophae rhamnoides L. pomace was generated in the production process for juice, wine of food industry. To expand the application of pomace, the extraction process optimization, enrichment and identification of triterpene acids were performed in this study. The extraction yield was 14.87% under optimal ultrasound-assisted extraction techniques performed via response surface methodology. The extract was subsequently purified to obtain the triterpenoid acid enrichment fraction (TPF) with the content of 75.23% ± 1.45%. 13 triterpenoid acids were identified via UPLC-Triple-TOF MS/MS and further semi-quantified through comparison with triterpenoid acid standards. TPF exhibited a strong inhibitory effect on α-glucosidase with IC50 value of 5.027 ± 0.375 µg/mL, as determined via enzyme inhibition experiment and molecular docking. Additionally, the TPF significantly reduced postprandial glucose levels, as revealed via carbohydrate tolerance tests, as well as ameliorate serum lipid profiles. Therefore, pomace may be a promising resource of functional food components with therapeutic and commercial values.

Symplosaponins A-D: New acylated oleanane-type triterpene saponins from Symplocos cochinchinensis and their hepatoprotective effect.

Four new acylated oleanane-type triterpene saponins, symplosaponins A-D (1-4) were successfully isolated from the leaves of Symplocos cochinchinensis (Lour.) S. Moore, alongside with five known compounds (5-9), 2-methoxy-4-prop-1-enylphenyl-1-O-ß-D-apiofuranosyl-(1 â†’ 6)-ß-D-glucopyranoside (5), and 1-[O-ß-d-xylopyranosyl-(1 â†’ 6)-O-ß-d-glucopyranosyl]-2,6-dimethoxy-4-propenyl-phenol (6), 6-O-p-coumaroylsucrose (7), arillatose B (8), and (-)-secoisolariciresinol-O-ß-D-glucopyranoside (9). The structures of these compounds were elucidated through spectroscopic methods, comparison with existing data, and chemical methods. Furthermore, all compounds were assessed for their impact on hepatocellular viability using the Resazurin reduction assay. These investigations aimed to explore the potential hepatoprotective properties of isolated compounds. As a result, 1-[O-ß-d-xylopyranosyl-(1 â†’ 6)-O-ß-d-glucopyranosyl]-2,6-dimethoxy-4-propenyl-phenol (6) and (-)-secoisolariciresinol-O-ß-D-glucopyranoside (9) demonstrated statistically significant hepatoprotective activity in a concentration-dependent manner.

Mutational Analysis of RIP Type I Dianthin-30 Suggests a Role for Arg24 in Endocytosis.

Saponin-mediated endosomal escape is a mechanism that increases the cytotoxicity of type I ribosome-inactivating proteins (type I RIPs). In order to actualize their cytotoxicity, type I RIPs must be released into the cytosol after endocytosis. Without release from the endosomes, type I RIPs are largely degraded and cannot exert their cytotoxic effects. Certain triterpene saponins are able to induce the endosomal escape of these type I RIPs, thus increasing their cytotoxicity. However, the molecular mechanism underlying the endosomal escape enhancement of type I RIPs by triterpene saponins has not been fully elucidated. In this report, we investigate the involvement of the basic amino acid residues of dianthin-30, a type I RIP isolated from the plant Dianthus caryophyllus L., in endosomal escape enhancement using alanine scanning. Therefore, we designed 19 alanine mutants of dianthin-30. Each mutant was combined with SO1861, a triterpene saponin isolated from the roots of Saponaria officinalis L., and subjected to a cytotoxicity screening in Neuro-2A cells. Cytotoxic screening revealed that dianthin-30 mutants with lysine substitutions did not impair the endosomal escape enhancement. There was one particular mutant dianthin, Arg24Ala, that exhibited significantly reduced synergistic cytotoxicity in three mammalian cell lines. However, this reduction was not based on an altered interaction with SO1861. It was, rather, due to the impaired endocytosis of dianthin Arg24Ala into the cells.

Four Unidentified Compounds Isolated from the Stem Barks of Aphanamixis polystachya and Their NO Production Inhibition in LPS Activated RAW 264.7 Cells.

Phytochemical study on the methanol extract of the stem barks of Aphanamixis polystachya led to the isolation of four previously undescribed (1-4) and ten known compounds (5-14). Their chemical structures were elucidated to be 11-methoxysawaranospiroride C (1), 6α,9S,10,13-tetrahydroxymegastigmane-3-one (2), 11-hydroxyaphanamixin B (3), (2Z,6E,13E)-2,6,13-triene-11,15-dihydroxyphytanic acid (4), cinnacasside D (5), cinnacasside E (6), vilsonol F (7), (3S,5R,6S,7E,9R)-3,5,6,9-tetrahydroxy-7-en-megastigmane (8), (3S,5R,6R,7E,9R)-3,6,9,10-tetrahydroxy-7-en-megastigmane (9), citroside A (10), threo-1-(3,4,5-trimethoxyphenyl)-1,2,3-propanetriol (11), 3,4,5-trimethoxyphenyl-1-O-ß-D-glucopyranoside (12), p-coumaric acid (13), ferulic acid (14) by HR-ESI-MS, ECD, 1D-, and 2D-NMR spectra. Compounds 1, 3, 4, and 9 showed NO production inhibitory activity in LPS activated RAW 264.7 cells with IC50 values of 42.0, 67.9, 20.5, and 78.6 µM, respectively, while the remaining compounds were inactive with IC50 values over 100 µM.

Histochemical and ultrastructural localization of triterpene saponins in Medicago truncatula.

In the Medicago genus, saponins are complex mixtures of triterpene pentacyclic glycosides extensively studied for their different and economically relevant biological and pharmaceutical properties. This research is aimed at determining for the first time the tissue and cellular localization of triterpene saponins in vegetative organs of Medicago truncatula, a model plant species for legumes, by histochemistry and transmission electron microscopy. The results showed that saponins are present mainly in the palisade mesophyll layer of leaves, whereas in stems they are mostly located in the primary phloem and the subepidermal cells of cortical parenchyma. In root tissue, saponins occur in the secondary phloem region. Transmission electron microscopy revealed prominent saponin accumulation within the leaf and stem chloroplasts, while in the roots the saponins are found in the vesicular structures. Our results demonstrate the feasibility of using histochemistry and transmission electron microscopy to localize M. truncatula saponins at tissue and cellular levels and provide important information for further studies on biosynthesis and regulation of valuable bioactive saponins on agronomic relevant Medicago spp., such as alfalfa (Medicago sativa L.). RESEARCH HIGHLIGHTS: The Medicago genus represents a valuable rich source of saponins, one of the most interesting groups of secondary plant metabolites, which possess relevant biological and pharmacological properties. Plant tissue and cellular localization of saponins is of great importance to better understand their biological functions, biosynthetic pathway, and regulatory mechanisms. We elucidate the localization of saponins in Medicago truncatula with histochemical and transmission electron microscopy studies.