GmMATE100 Is Involved in the Import of Soyasaponins A and B into Vacuoles in Soybean Plants (Glycine max L.).

Triterpenoid saponins, synthesized via the mevalonic acid (MVA) pathway in the cytoplasm, provide protection against pathogens and pests in plants and health benefits for humans. However, the mechanisms by which triterpenoid saponins are transported between cellular compartments remain uncharacterized. Here, we characterize a tonoplast localized multidrug and toxic compound extrusion transporter, GmMATE100 (encoded by Glyma.18G143700), from soybean (Glycine max L.). GmMATE100 is co-expressed with soyasaponin biosynthetic genes, and its expression was induced by MeJA treatment, which also led to soyasaponin accumulation in soybean roots. GmMATE100 efficiently transports multiple type-B soyasaponins as well as type-A soyasaponins with low affinity from the cytosol to the vacuole in a yeast system. The GmMATE100 loss-of-function mutant showed a significant decrease in type-A and type-B soyasaponin contents in soybean roots. This study not only characterized the first soybean triterpenoid saponin transporter but also provided new knowledge for the rational engineering of soyasaponin content and composition in soybean plants to modulate their levels within crop environments.

Gut Microbiota Plays Essential Roles in Soyasaponin's Preventive Bioactivities against Steatohepatitis in the Methionine and Choline Deficient (MCD) Diet-Induced Non-Alcoholic Steatohepatitis (NASH) Mice.

SCOPE: Gut microbiota (GM) is involved in nonalcoholic steatohepatitis (NASH) development. Phytochemicals soyasaponins can prevent NASH possibly by modulating GM. This study aims to investigate the preventive bioactivities of soyasaponin monomers (SS-A1 and SS-Bb) against NASH and explores the mechanisms by targeting GM. METHODS AND RESULTS: Male C57BL/6 mice are fed with methionine and choline deficient (MCD) diet containing SS-A1 , SS-Bb, or not for 16 weeks. Antibiotics-treated pseudo germ-free (PGF) mice are fed with MCD diet containing SS-A1 , SS-Bb, or not for 8 weeks. GM is determined by 16S rRNA amplicon sequencing. Bile acids (BAs) are measured by UPLC-MS/MS. In NASH mice, SS-A1 and SS-Bb alleviate steatohepatitis and fibrosis, reduce ALT, AST, and LPS in serum, decrease TNF-α, IL-6, α-SMA, triglycerides, and cholesterol in liver. SS-A1 and SS-Bb decrease Firmicutes, Erysipelotrichaceae, unidentified-Clostridiales, Eggerthellaceae, Atopobiaceae, Aerococcus, Jeotgalicoccus, Gemella, Rikenella, increase Proteobacteria, Verrucomicrobia, Akkermansiaceae, Romboutsia, and Roseburia. SS-A1 and SS-Bb alter BAs composition in liver, serum, and feces, activate farnesoid X receptor (FXR) in liver and ileum, increase occludin and ZO-1 in intestine. However, GM clearance abrogates the preventive bioactivities of SS-A1 and SS-Bb against NASH. CONCLUSION: GM plays essential roles in soyasaponin's preventive bioactivities against steatohepatitis in MCD diet-induced NASH mice.

Disruption of a licorice cellulose synthase-derived glycosyltransferase gene demonstrates its in planta role in soyasaponin biosynthesis.

KEY MESSAGE: CRISPR-Cas9-mediated disruption of a licorice cellulose synthase-derived glycosyltransferase gene, GuCSyGT, demonstrated the in planta role of GuCSyGT as the enzyme catalyzing 3-O-glucuronosylation of triterpenoid aglycones in soyasaponin biosynthesis. Triterpenoid glycosides (saponins) are a large, structurally diverse group of specialized metabolites in plants, including the sweet saponin glycyrrhizin produced by licorice (Glycyrrhiza uralensis) and soyasaponins that occur widely in legumes, with various bioactivities. The triterpenoid saponin biosynthetic pathway involves the glycosylation of triterpenoid sapogenins (the non-sugar part of triterpenoid saponins) by glycosyltransferases (GTs), leading to diverse saponin structures. Previously, we identified a cellulose synthase-derived GT (CSyGT), as a newly discovered class of triterpenoid GT from G. uralensis. GuCSyGT expressed in yeast, which could transfer the sugar glucuronic acid to the C3 position of glycyrrhetinic acid and soyasapogenol B, which are the sapogenins of glycyrrhizin and soyasaponin I, respectively. This suggested that GuCSyGT is involved in the biosynthesis of glycyrrhizin and soyasaponin I. However, the in planta role of GuCSyGT in saponin biosynthesis remains unclear. In this study, we generated GuCSyGT-disrupted licorice hairy roots using CRISPR-Cas9-mediated genome editing and analyzed the saponin content. This revealed that soyasaponin I was completely absent in GuCSyGT-disrupted lines, demonstrating the in planta role of GuCSyGT in saponin biosynthesis.

Knockout of CAFFEOYL-COA 3-O-METHYLTRANSFERASE 6/6L enhances the S/G ratio of lignin monomers and disease resistance in Nicotiana tabacum.

Background: Nicotiana tabacum is an important economic crop, which is widely planted in the world. Lignin is very important for maintaining the physiological and stress-resistant functions of tobacco. However, higher lignin content will produce lignin gas, which is not conducive to the formation of tobacco quality. To date, how to precisely fine-tune lignin content or composition remains unclear. Results: Here, we annotated and screened 14 CCoAOMTs in Nicotiana tabacum and obtained homozygous double mutants of CCoAOMT6 and CCoAOMT6L through CRSIPR/Cas9 technology. The phenotype showed that the double mutants have better growth than the wild type whereas the S/G ratio increased and the total sugar decreased. Resistance against the pathogen test and the extract inhibition test showed that the transgenic tobacco has stronger resistance to tobacco bacterial wilt and brown spot disease, which are infected by Ralstonia solanacearum and Alternaria alternata, respectively. The combined analysis of metabolome and transcriptome in the leaves and roots suggested that the changes of phenylpropane and terpene metabolism are mainly responsible for these phenotypes. Furthermore, the molecular docking indicated that the upregulated metabolites, such as soyasaponin Bb, improve the disease resistance due to highly stable binding with tyrosyl-tRNA synthetase targets in Ralstonia solanacearum and Alternaria alternata. Conclusions: CAFFEOYL-COA 3-O-METHYLTRANSFERASE 6/6L can regulate the S/G ratio of lignin monomers and may affect tobacco bacterial wilt and brown spot disease resistance by disturbing phenylpropane and terpene metabolism in leaves and roots of Nicotiana tabacum, such as soyasaponin Bb.

A recently evolved BAHD acetyltransferase, responsible for bitter soyasaponin A production, is indispensable for soybean seed germination.

Soyasaponins are major small molecules that accumulate in soybean (Glycine max) seeds. Among them, type-A soyasaponins, fully acetylated at the terminal sugar of their C22 sugar chain, are responsible for the bitter taste of soybean-derived foods. However, the molecular basis for the acetylation of type-A soyasaponins remains unclear. Here, we identify and characterize GmSSAcT1, encoding a BADH-type soyasaponin acetyltransferase that catalyzes three or four consecutive acetylations on type-A soyasaponins in vitro and in planta. Phylogenetic analysis and biochemical assays suggest that GmSSAcT1 likely evolved from acyltransferases present in leguminous plants involved in isoflavonoid acylation. Loss-of-function mutants of GmSSAcT1 exhibited impaired seed germination, which attribute to the excessive accumulation of null-acetylated type-A soyasaponins. We conclude that GmSSAcT1 not only functions as a detoxification gene for high accumulation of type-A soyasaponins in soybean seeds but is also a promising target for breeding new soybean varieties with lower bitter soyasaponin content.

Cover Crop Cultivar, Species, and Functional Diversity is Reflected in Variable Root Exudation Composition.

Cover cropping has emerged as a sustainable alternative to traditional crop rotational practices, yet the effects of variable root exudation from cover crop species and cultivars within species remains unclear. Here, we assess the chemical heterogeneity of root exudates from 16 commonly used cover crop species as well as 3 distinct cultivars of hairy vetch. Plants were grown hydroponically and analyzed via nontargeted gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), and targeted LC-MS/MS to evaluate patterns in root exudate composition across species and functional plant type. Overall, root exudation profiles are heterogeneous across crop species and cultivars. Species within legumes stand out as a unique functional group of plants capable of producing distinct chemical environments rich with complex secondary metabolites, such as triterpenoid saponins (soyasaponins), isoflavonoids, and flavonoids.

Simultaneous determination of abrine, hypaphorine, schaftoside and soyasaponin Bb in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study after oral administration of Abrus cantoniensis Hance extract.

A simple and sensitive liquid chromatography tandem mass spectrometry method was established and validated for the quantitative determination of abrine, hypaphorine, schaftoside and soyasaponin Bb in rat plasma. After preparation by protein precipitation with acetonitrile, the analytes and internal standard were separated on a Waters CORTECS T3 column using acetonitrile containing 0.1% formic acid and 0.1% formic acid in water as mobile phase by gradient elution in 2 min. The method showed excellent linearity over the range of 5-500 ng/ml with acceptable intra- and inter-day precision, accuracy, matrix effect and recovery. The stability assay indicated that the four analytes were stable during the analysis process. The method was applied to a pharmacokinetic study of Abrus cantoniensis Hance in rats. The result suggested that after oral administration, the four analytes were quickly absorbed into the plasma. The dose-normalized exposure of hypaphorine was the highest with a long elimination half-life (t1/2 9.83 h), followed by abrine and schaftoside with t1/2 values of 1.07 and 1.15 h. The dose normalized exposure of soyasaponin Bb was the lowest, which is possibily due to the high polarity and poor permeability. This study provides a basis for elucidating the material foundation of A. cantoniensis Hance.

Activation of bitter taste receptors by saponins and alkaloids identified in faba beans (Vicia faba L. minor).

Despite their interests, faba beans are characterised by bitterness but little is known about its compounds that activate the 25 human bitter receptors (TAS2Rs). This study aimed to determine the bitter molecules in faba beans, especially saponins and alkaloids. These molecules were quantified by UHPLC-HRMS in flour, starch and protein fractions of 3 faba bean cultivars. The fractions from the low-alkaloid cultivar and the protein fractions exhibited higher saponin content. Vicine and convicine were highly correlated with bitter perception. The bitterness of soyasaponin ßb and alkaloids was studied using a cellular approach. Soyasaponin ßb activated 11 TAS2Rs, including TAS2R42 whereas vicine activated only TAS2R16. The high vicine content should explain the faba bean bitterness considering that concentration of soyasaponin ßb was low. This research provides a better understanding of the bitter molecules in faba beans. Selection of low-alkaloid ingredients or alkaloid removal treatments could improve the faba bean flavour.

Soyasaponin I alleviates hypertensive intracerebral hemorrhage by inhibiting the renin-angiotensin-aldosterone system.

BACKGROUND: Hypertensive intracerebral hemorrhage (HICH) is a life-threatening disease and lacks effective treatments. Previous studies have confirmed that metabolic profiles altered after ischemic stroke, but how brain metabolism changes after HICH was unclear. This study aimed to explore the metabolic profiles after HICH and the therapeutic effects of soyasaponin I on HICH. METHODS: HICH model was established first. Hematoxylin and eosin staining was used to estimate the pathological changes after HICH. Western blot and Evans blue extravasation assay were applied to determine the integrity of the blood-brain barrier (BBB). Enzyme-linked immunosorbent assay was used to detect the activation of the renin-angiotensin-aldosterone system (RAAS). Next, liquid chromatography-mass spectrometry-untargeted metabolomics was utilized to analyze the metabolic profiles of brain tissues after HICH. Finally, soyasaponin I was administered to HICH rats, and the severity of HICH and activation of the RAAS were further assessed. RESULTS: We successfully constructed HICH model. HICH significantly impaired BBB integrity and activated RAAS. HICH increased PE(14:0/24:1(15Z)), arachidonoyl serinol, PS(18:0/22:6(4Z, 7Z, 10Z, 13Z, 16Z, and 19Z)), PS(20:1(11Z)/20:5(5Z, 8Z, 11Z, 14Z, and 17Z)), glucose 1-phosphate, etc., in the brain, whereas decreased creatine, tripamide, D-N-(carboxyacetyl)alanine, N-acetylaspartate, N-acetylaspartylglutamic acid, and so on in the hemorrhagic hemisphere. Cerebral soyasaponin I was found to be downregulated after HICH and supplementation of soyasaponin I inactivated the RAAS and alleviated HICH. CONCLUSION: The metabolic profiles of the brains changed after HICH. Soyasaponin I alleviated HICH via inhibiting the RAAS and may serve as an effective drug for the treatment of HICH in the future.

Anti-inflammatory Oleanane-Type Triterpenoids Produced by Nonomuraea sp. MYH522 through Microbial Transformation.

Eleven oleanane-type triterpenoids named soyasapogenols B1-B11 have been obtained unexpectedly from a marine actinomycete Nonomuraea sp. MYH522. Their structures have been determined by extensive analysis of spectroscopic experiments and X-ray crystallographic data. Soyasapogenols B1-B11 exhibit subtle differences in the positions and degrees of oxidation on an oleanane skeleton. The feeding experiment suggested that soyasapogenols might be derived from soyasaponin Bb through microbial-mediated conversion. The biotransformation pathways from soyasaponin Bb to five oleanane-type triterpenoids and six A-ring cleaved analogues were proposed. The assumed biotransformation involves an array of reactions including regio- and stereo-selective oxidation. These compounds alleviated the 5,6-dimethylxanthenone-4-acetic acid-induced inflammation in Raw264.7 cells via the stimulator of interferon genes/TBK1/NF-κB signaling pathway. The present work provided an efficient approach for rapid diversification of soyasaponins and for developing food supplements with potent anti-inflammatory effects.

Saponin Biosynthesis in Pulses.

Pulses are a group of leguminous crops that are harvested solely for their dry seeds. As the demand for plant-based proteins grows, pulses are becoming important food crops worldwide. In addition to being a rich source of nutrients, pulses also contain saponins that are traditionally considered anti-nutrients, and impart bitterness and astringency. Saponins are plant secondary metabolites with great structural and functional diversity. Given their diverse functional properties and biological activities, both undesirable and beneficial, saponins have received growing attention. It can be expected that redirecting metabolic fluxes to control the saponin levels and produce desired saponins would be an effective approach to improve the nutritional and sensory quality of the pulses. However, little effort has been made toward understanding saponin biosynthesis in pulses, and, thus there exist sizable knowledge gaps regarding its pathway and regulatory network. In this paper, we summarize the research progress made on saponin biosynthesis in pulses. Additionally, phylogenetic relationships of putative biosynthetic enzymes among multiple pulse species provide a glimpse of the evolutionary routes and functional diversification of saponin biosynthetic enzymes. The review will help us to advance our understanding of saponin biosynthesis and aid in the development of molecular and biotechnological tools for the systematic optimization of metabolic fluxes, in order to produce the desired saponins in pulses.

Screening for Astragalus hamosus Triterpenoid Saponins Using HPTLC Methods: Prior Identification of Azukisaponin Isomers.

Due to their particular structural characteristics, the extraction and isolation of saponins from plants present a serious challenge. In this study, specific extraction protocols were first implemented to extract the secondary metabolites from Astragalus hamosus and, more precisely, the saponins. Subsequent purification of the extracts was based on a single chromatographic technique, high-performance thin-layer chromatography, applying two development systems: a one-step system that separated molecules according to their polarity and a multiple development system that made it possible to detect the triterpenoid saponins, azukisaponin or soyasapogenol at a retarded Rf of 0.2. The difficulties of detecting the Astragalus hamosus saponins encountered during the extraction and purification of the extracts have been highlighted and the strategy carried out to isolate the saponins has been discussed.

Metabolic profiling of soyasaponin Bb in rat plasma, urine, bile, and feces after intragastric administration.

Soyasaponin Bb is one of the bioactive oleanolic acid-type triterpenoid saponins mainly isolated from soybean. It possesses significant antithrombosis, hypolipidemic, anticancer, and antioxidant activities. However, the metabolic profiles of soyasaponin Bb are still unknown. The present study investigated the metabolites of soyasaponin Bb in plasma, bile, urine, and feces samples after intragastric administration using ultra-performance liquid chromatography coupled with quadrupole-time-of-flight-mass spectrometry, and its possible metabolic pathways were subsequently proposed. Using the metabolite profiling strategy, 11 metabolites were first identified from urine, plasma, bile, and feces of rats after intragastric administration of soyasaponin Bb. Hydroxylation and hydrolysis were the major metabolic pathways of soyasaponin Bb in rat. The results expand our knowledge of the metabolism of soyasaponin Bb, which could provide valuable information for better comprehension of future pharmacological research.

Anti-Colon Cancer Activity of Dietary Phytochemical Soyasaponin I and the Induction of Metabolic Shifts in HCT116.

Dietary phytochemicals play an important role in the prevention and treatment of colon cancer. It is reported that group B of soyasaponin, derived from dietary pulses, has anti-colonic effects on some colon cancer cell lines. However, it is uncertain which specific soybean saponins play a role. In our study, as one of the group B soyasaponin, the anti-colon cancer activity of soyasaponins I (SsI) was screened, and we found that it had the inhibitory effect of proliferation on colon cancer cell lines HCT116 (IC50 = 161.4 µM) and LoVo (IC50 = 180.5 µM), but no effect on HT29 between 0-200 µM. Then, nine potential targets of SsI on colon cancer were obtained by network pharmacology analysis. A total of 45 differential metabolites were identified by metabolomics analysis, and the KEGG pathway was mainly enriched in the pathways related to the absorption and metabolism of amino acids. Finally, molecular docking analysis predicted that SsI might dock with the protein of DNMT1, ERK1. The results indicated that the effect of SsI on HCT116 might be exerted by influencing amino acid metabolism and the estrogen signaling pathway. This study may provide the possibility for the application of SsI against colon cancer.

The discovery of novel SNPs associated with group A soyasaponin biosynthesis from Korea soybean core collection.

Soyasaponin is a type of glycoside such as steroids, steroidal alkaloids or triterpenes, which enhance the body immunity. In order to efficiently identify genes and markers related to the soyasaponin, we used a 180K Axiom® SoyaSNP array and whole genome resequencing data from the Korean soybean core collection. As a result of conducting GWAS for group A soyasaponin (Aa and Ab derivatives), 16 significant common markers associated with Aa and Ab derivatives were mapped to chromosome 7, and three candidate genes including Glyma.07g254600 were detected. The functional haplotypes for candidate genes showed that Aa and Ab contents were mainly determined by alleles of AX-90322128, the marker of Glyma.07g254600. In addition, 14 novel SNPs variants closely associated with Aa and Ab derivatives were discovered for Glyma.07g254600. Therefore, the results of this study that identified soyasaponin-associated markers and useful genes utilizing various genomic information could provide insight into functional soybean breeding.

Mechanism of interactions between soyasaponins and soybean 7S/11S proteins.

In this study, the proteins glycinin (11S) and ß-conglycinin (7S) were mixed with soyasaponin (Ssa) Ab/Bb to form a composite system. We used fluorescence and synchronous fluorescence spectra to demonstrate the changes in the surrounding environment and the structure of the proteins. Dynamic interface behavior analysis showed the possible interface behavior induced by the composite system. The interactions between Ssa and the proteins, along with the mode of action, were analyzed by molecular docking. The interactions between Ssa and soy protein increased with the change in concentration. The interactions between the two proteins were mediated by tryptophan (Trp) and primarily involved hydrogen bonds, which changed the microenvironment and loosened the protein structure. These results helped in understanding the mechanism underlying the interactions between Ssa Ab/Bb and 7S/11S. Furthermore, these results highlighted the theoretical fundamentals for the future applications of composite systems as surfactants in the food industry.

Soyasaponin Ag inhibits triple-negative breast cancer progression via targeting the DUSP6/MAPK signaling.

INTRODUCTION: Soyasaponins are triterpenoid glycosides discovered in soybean and have anti-cancer properties. Soyasaponin A was reported to repress estrogen-insensitive breast cancer cell proliferation. This study intends to explore the role of one isomer of soyasaponin A, i.e. soyasaponin Ag (Ssa Ag), in triple-negative breast cancer (TNBC) development. MATERIAL AND METHODS: Bioinformatic databases were used to predict DUSP6 expression in breast cancer (BC) as well as the correlation between the expression of DUSP6 (or MAPK1, MAPK14) with the prognosis of patients with BC. The expression of DUSP6/MAPK signaling-related genes (DUSP6, MAPK1, and MAPK14) in TNBC cell lines was assessed via Western blot analysis and RT-qPCR. Levels of cell apoptosis proteins (Bax and Bcl-2) in TNBC cells were assessed via Western blot analysis. CCK-8 assay, colony formation assay, and flow cytometry analysis were conducted for the measurement of TNBC cell growth and apoptosis. In vivo xenograft assay was employed for investigating the biological influence of Ssa Ag on tumor growth. RESULTS: The poor prognosis of BC patients was linked to the aberrant expression of DUSP6/MAPK pathway genes. Low expression of DUSP6 or high expression of MAPK1 (or MAPK14) was correlated to poor prognosis. DUSP6 was downregulated while MAPK1 and MAPK14 were upregulated in TNBC cells versus normal cells. Ssa Ag upregulated DUSP6 expression while downregulated MAPK1 and MAPK14 expression, inhibiting the MAPK signaling pathway. Additionally, Ssa Ag promoted in vitro TNBC cell apoptosis and restrained cell growth, and repressed in vivo tumor growth. CONCLUSIONS: Ssa Ag inhibited TNBC progression via upregulating DUSP6 and inactivating the MAPK signaling pathway.

Bridging the Gaps between Plant and Human Health: A Systematic Review of Soyasaponins.

Saponins, prominent secondary plant metabolites, are recognized for their roles in plant defense and medicinal benefits. Soyasaponins, commonly derived from legumes, are a class of triterpenoid saponins that demonstrate significant potential for plant and human health applications. Previous research and reviews largely emphasize human health effects of soyasaponins. However, the biological effects of soyasaponins and their implications for plants in the context of human health have not been well-discussed. This review provides comprehensive discussions on the biological roles of soyasaponins in plant defense and rhizosphere microbial interactions; biosynthetic regulation and compound production; immunological effects and potential for therapeutics; and soyasaponin acquisition attributed to processing effects, bioavailability, and biotransformation processes based on recent soyasaponin research. Given the multifaceted biological effects elicited by soyasaponins, further research warrants an integrated approach to understand molecular mechanisms of regulations in their production as well as their applications in plant and human health.

Soyasaponin A2 Alleviates Steatohepatitis Possibly through Regulating Bile Acids and Gut Microbiota in the Methionine and Choline-Deficient (MCD) Diet-induced Nonalcoholic Steatohepatitis (NASH) Mice.

SCOPE: Nonalcoholic steatohepatitis (NASH) is a chronic progressive disease with complex pathogenesis of which the bile acids (BAs) and gut microbiota are involved. Soyasaponins (SS) exhibits many health-promoting effects including hepatoprotection, but its prevention against NASH is unclear. This study aims to investigate the preventive bioactivities of SS monomer (SS-A2 ) against NASH and further clarify its mechanism by targeting the BAs and gut microbiota. METHODS AND RESULTS: The methionine and choline deficient (MCD) diet-fed male C57BL/6 mice were intervened with obeticholic acid or SS-A2 for 16 weeks. Hepatic pathology is assessed by hematoxylin-eosin and Masson's trichrome staining. BAs in serum, liver, and colon are measured by ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-TQMS). Gut microbiota in caecum are determined by 16S rDNA amplicon sequencing. In the MCD diet-induced NASH mice, SS-A2 significantly reduces hepatic steatosis, lobular inflammation, ballooning, nonalcoholic fatty liver disease activity score (NAS) scores, and fibrosis, decreases Erysipelotrichaceae (Faecalibaculum) and Lactobacillaceae (Lactobacillus) and increases Desulfovibrionaceae (Desulfovibrio). Moreover, SS-A2 reduces serum BAs accumulation and promotes fecal BAs excretion. SS-A2 changes the BAs profiles in both liver and serum and specifically increases the taurohyodeoxycholic acid (THDCA) level. Faecalibaculum is negatively correlated with serum THDCA. CONCLUSION: SS-A2 alleviates steatohepatitis possibly through regulating BAs and gut microbiota in the MCD diet-induced NASH mice.

Phytochemical and pharmacological appraisal of the aerial parts of Lotus corniculatus L. growing in Egypt.

Lotus corniculatus L. (Fabaceae) is widely grown in Egypt. It has a great history of folkloric medicinal uses. All fractions of aerial parts of L. corniculatus L. showed significant antioxidant and immunostimulant activities and could strongly induce lymphoproliferation. However, the light petrol fraction had antifungal activity against C. neoformans with IC50 value (<8 µg/mL) and exhibited strongest in-vitro antiprotozoal activity against protozoan parasites belonging to the genera Trypanosoma with IC50 value (0.98 µg/mL) and Plasmodium (with 100% inhibition using a sample concentration of 15866.7 ng/mL). This is the first study of the immunostimulant and antiprotozoal activities of genus Lotus. By this approach, it was possible to isolate eight compounds (-)-7,2'-dihydroxy-4'-methoxyisoflavan (vestitol) (1), kaempferol (2), kaempferol 3-O-α-L-rhamnoside (afzelin) (3), kaempferol 3, 7-O-α-L-dirhamnoside (kaempferitin) (4), kaempferol-3-O-[ß-D-xylopyranosyl (1″'→2″)-ß-D-galactopyranoside] (5), 3-O-[ß-D-glucuronopyranosyl] soyasapogenol B (6), kaempferol-3-O-[ß-D-xylopyranosyl (1″'→2″)-ß-D-galactopyranoside]-7-O-α-L-rhamnopyranoside (7) and 3-O-[α-L-rhamnopyranosyl (1″'→2″)-ß-D-galactopyranosyl-(1″→2')-ß-D-glucuronopyranosyl] soyasapogenol B (soyasaponin І) (8).