Quillaja saponin variants with central glycosidic linkage modifications exhibit distinct conformations and adjuvant activities

Immunological adjuvants such as the saponin natural product QS-21 help stimulate the immune response to co-administered antigens and have become increasingly important in the development of prophylactic and therapeutic vaccines. However, clinical use of QS-21 is encumbered by chemical instability, dose-limiting toxicity, and low-yielding purification from the natural source. Previous studies of structure–activity relationships in the four structural domains of QS-21 have led to simplified, chemically stable variants that retain potent adjuvant activity and low toxicity in mouse vaccination models. However, modification of the central glycosyl ester linkage has not yet been explored. Herein, we describe the design, synthesis, immunologic evaluation, and molecular dynamics analysis of a series of novel QS-21 variants with different linker lengths, stereochemistry, and flexibility to investigate the role of this linkage in saponin adjuvant activity and conformation. Despite relatively conservative structural modifications, these variants exhibit striking differences in in vivo adjuvant activity that correlate with specific conformational preferences. These results highlight the junction of the triterpene and linear oligosaccharide domains as playing a critical role in the immunoadjuvant activity of the Quillaja saponins and also suggest a mechanism of action involving interaction with a discrete macromolecular target, in contrast to the non-specific mechanisms of emulsion-based adjuvants.     

Purification of saponins from leaves of Panax notoginseng using preparative two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography

Saponins are widely distributed in the plant kingdom and have been shown to be active components of many medicinal herbs. In this study, a two-dimensional purification method based on reversed-phase liquid chromatography coupled with hydrophilic interaction liquid chromatography was successfully applied to purify saponins from leaves of Panax notoginseng. Nine saponin reference standards were used to test the separation modes and columns. The standards could not be resolved using C₁₈ columns owing to their limited polar selectivity. However, they were completely separated on a XAmide column in hydrophilic interaction liquid chromatography mode, including two pairs of standards that were coeluted on a C₁₈ column. The elution order of the standards on the two columns was sufficiently different, with a correlation coefficient between retention times on the C₁₈ and XAmide columns of 0.0126, indicating good column orthogonality. Therefore, the first-dimension preparation was performed on a C₁₈ column, followed by a XAmide column that was used to separate the fractions in the second dimension. Fifty-four fractions were prepared in the first dimension, with 25 fractions rich in saponins. Eight saponins, including two pairs of isomeric saponins and one new saponin, were isolated and identified from three representative fractions. This procedure was shown to be an effective approach for the preparative isolation and purification of saponins from leaves of P. notoginseng. Moreover, this method could possibly be employed in the purification of low-content and novel active saponins from natural products.

Comparative chemical diversity and antioxidant activities of three species of Akebia herbal medicines

Akebia stem has long been used extensively as a rare Chinese herbal medicine. The three most significant Akebia medicinal species are Akebia quinata (Thunb.) Decne. (A. quinata), Akebia trifoliata (Thunb.) Koidz. (A. trifoliata), and Akebia trifoliata (Thunb.) Koidz. var. Australis (Diels) Rehd. (A. trifoliata. var). They have significant therapeutic effects and are widely used in the pharmaceutical and cosmetics industries. Only a few studies compared their chemical differences and antioxidant activities. To better demonstrate each species' characteristics and antioxidant properties, the ultra-performance liquid chromatography coupled with quadrupole Orbitrap mass spectrometry (UPLC-Q-Orbitrap/MS)-based metabolomics was applied to investigate the chemome diversity of three Akebia species. Their antioxidant activities were evaluated by DPPH and ABTS assays. In total, 65 different metabolites were identified, including 5 phenolic acids, 2 phenylpropanoids, 4 lignan glycosides, and 54 triterpenoid saponins. The different aglycone types of triterpenoid saponins were found to be the component differences between the three Akebia species. The chemical composition of A. trifoliata and A. trifoliata. var is similar. The 2-(3,4-dihydroxyphenyl)-ethyl-O-β-d-glucopyranoside has been found only in A. quinata. In contrast, the triterpenoid saponins akemisaponin B, akemisaponin D, oleanolic-acid-3-O-arabinopyranosyl-28-O-glucopyranosyl-glucopyranosyl-rhamnopyranosyl-arabinopyranosyl, akemisaponin C and saponin P_j1 have been found A. trifoliata and A. trifoliata. var. As a result, these six compounds can be considered marker compounds that distinguish three Akebia species. The antioxidant activities results indicated that the antioxidants of three Akebia species were the same in different antioxidative test systems. A. trifoliata (IC₅₀: 2.28–6.97 mg·mL^?1) and A. trifoliata. var (IC₅₀: 2.09–6.87 mg·mL^?1) showed 2–3 times higher antioxidant activity than A. quinata (IC₅₀: 5.56–11.21 mg·mL^?1). This study reveals the antioxidant activity differences of three Akebia species, laying a foundation for further development and utilization. This type of study can lead to the identification of a compound that, with further work and more extensive studies, has the potential to be used as a biomarker, in this case to distinguish different medicinal species.     

Bellisosides A-F, six novel acylated triterpenoid saponins from Bellis perennis (compositae)

Six new acylated triterpenoid saponins, bellisosides A-F (1-6), were isolated from the roots of Bellis perennis (Compositae) together with a known saponin, bellissaponin BS2 (7). These saponins which bear polygalacic acid as sapogenin, are bisdesmosidic glycosides. The structures were elucidated on the basis of chemical and physicochemical evidence. The cytotoxic activities of 1-7 against HL-60 human promyelocytic leukemia cells are also reported.     

Rapid annotation and structural characterization of saponins in the active fraction of Albizia julibrissin by high‐performance liquid chromatography coupled with quadrupole time of flight mass spectrometry based on accurate mass database

The purified active fraction of Albizia julibrissin saponin was proved to be a promising adjuvant candidate for vaccine. In this study, a simple, convenient, and practical strategy was established for characterizing the saponins in this purified active fraction. The personal accurate mass database including chemical structure, molecular formula, and theoretical mass was first constructed by collecting 110 reported known saponins from genus Albizia species. The raw data was obtained by high‐performance liquid chromatography coupled with quadrupole time of flight mass spectrometry. The potential compounds were extracted from raw data, and matched with the accurate mass databases. A series of saponin compounds were predicted and their chemical structures were characterized by interpreting the tandem mass spectrometry data. A total of 29 saponins including 10 new compounds and 5 first found saponins from A. julibrissin were successfully characterized in this purified active fraction using this new strategy.     

Cytotoxic Triterpenoid Saponins Acetylated with Monoterpenoid Acid from Albizia julibrissin

Three new triterpenoid saponins, named julibroside J₁₆ ( 2 ), julibroside J₁₇ ( 3 ), and julibroside J₂₁ ( 4 ), each of which possesses an oleanane triterpenoid aglycone of acacic acid, two monoterpenoid acids, and nine sugar moieties, together with one known saponin, julibroside II ( 1 ), were isolated from the stem bark of Albizia julibrissin by chromatographic methods. Their structures were established by chemical and spectroscopic means. Saponins 1, 2 , and 4 showed inhibitory activities against Bel 7402 human cancer cell line in vitro.     

Evaluation of the Saponin Content in Panax vietnamensis Acclimatized to Lam Dong Province by HPLC–UV/CAD

Panax vietnamensis, or Vietnamese ginseng (VG), an endemic Panax species in Vietnam, possesses a unique saponin profile and interesting biological activities. This plant is presently in danger of extinction due to over-exploitation, resulting in many preservation efforts towards the geographical acclimatization of VG. Yet, no information on the saponin content of the acclimatized VG, an important quality indicator, is available. Here, we analyzed the saponin content in the underground parts of two- to five-year-old VG plants acclimatized to Lam Dong province. Nine characteristic saponins, including notoginsenoside-R1, ginsenoside-Rg1, -Rb1, -Rd, majonoside-R1, -R2 vina-ginsenoside-R2, -R11, and pseudoginsenoside-RT4, were simultaneously determined by HPLC coupled with UV and with a charged aerosol detector (CAD). Analyzing the results illustrated that the detection of characteristic ocotillol-type saponins in VG by CAD presented a superior capacity compared with that of UV, thus implying a preferential choice of CAD for the analysis of VG. The quantitative results indicating the saponin content in the underground parts of VG showed an increasing tendency from two to five years old, with the root and the rhizome exhibiting different saponin accumulation patterns. This is the first study that reveals the preliminary success of VG acclimatization and thereby encourages the continuing efforts to develop this valuable saponin-rich plant.     

Analytical and semipreparative separation of 25 (R/S)‐spirostanol saponin diastereomers using supercritical fluid chromatography

Spirostanol saponins are a class of steroidal saponins with many pharmacological activities. The structural complexity of the spirostanol saponins presents a daunting challenge in separating their 25 R/S diastereomers. Using two CHIRALPAK IC columns coupled in series, six 25 (R/S)‐spirostanol saponin diastereomers from the Trigonella foenum‐graecum L. seed were successfully separated using supercritical fluid chromatography (SFC) for the first time. In addition, three 25 (R/S)‐spirostanol saponin diastereomers were isolated into their respective individual isomers. The structures of the isolated isomers were unambiguously confirmed by NMR analysis. The SFC method development strategy and its associated underlying principles presented in this paper are generally applicable. SFC is a viable addition to the natural product research toolbox, especially for stereoselective analysis and purification.     

Ligand fishing from Dioscorea nipponica extract using human serum albumin functionalized magnetic nanoparticles

Dioscorea nipponica and the preparations made from it have been used for long to prevent and treat coronary heart disease in traditional Chinese medicine. A group of steroidal saponins present in the plant are believed to be the active ingredients. It has been a challenge to study the individual saponins separately due to the similarities in their chemical and physical properties. In this work, human serum albumin (HSA) functionalized magnetic nanoparticles (MNPs) were used to isolate and identify saponin ligands that bind to HSA from D. nipponica extract. Electrospray ionization mass spectrometry (ESI-MS) was used for compound identification and semi-quantification. Three saponins, i.e. dioscin, gracillin, and pseudo-protodioscin showed affinity to HSA-MNPs and thus isolated effectively from the extract. The other two saponins detected in the extract (i.e. protodioscin and 26-O-β-d-glucopyranosyl-3β,20α,26-triol-25(R)-Δ^5,22-dienofurostan-3-O-α-l-rhamnopyranosyl (1→2)-[α-l-rhamnopyranosyl (1 → 4)]-β-d-glucopyranoside) exhibited no affinity at all. Among the three saponins fished out, dioscin bound to HSA much stronger than gracillin and pseudo-protodioscin did. The results indicated that affinity interaction between HSA immobilized on MNPs and small molecule compounds were highly dependent on chemical structures and, potentially, medicinal usefulness. The present work demonstrates a facile and effective way to isolate and identify ligands of receptors from medicinal plants.     

Simultaneous Quantification of Seven Main Triterpenoid Saponins in Radix et Rhizoma Clematidis by LC–ELSD

Chinese clematis is a Chinese medicine with anti-inflammatory and anti-rheumatoid properties. Studies showed the triterpenoid saponins of this herb represent the major active components with therapeutic activities for the treatment of inflammatory diseases. The LC–ELSD method was developed to quantitatively analyze seven major triterpenoid saponins in 22 samples from different habitats. The linearity, sensitivity, precision, and accuracy were investigated. It showed the great variation of the contents of each saponin. We concluded clematichinenoside AR is the major saponin in all the samples. This method can be used to effectively evaluate and monitor the quality of Chinese clematis in clinical use.     

Bioactive Saponins and Glycosides. Part 29

Three new acylated oleanane‐type triterpene saponins, theasaponins A₆ ( 1 ), A₇ ( 2 ), and B₅ ( 3 ), were isolated from the saponin fraction of the seeds of the Japanese tea plant Camellia sinensis together with the known constituent foliatheasaponin III ( 4 ). The structures of the glycosides 1 – 3 were elucidated on the basis of spectroscopic, chemical, and physico‐chemical evidence.     

The substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from Curvularia lunata

In previous work, we studied and reported that an enzyme from Curvularia lunata 3.4381 had the novel specificity to hydrolyze the terminal rhamnosyl at C-3 position of steroidal saponin and obtained four transformed products; the enzyme was purified and ascertained as glucoamylase (EC 3.2.1.3 GA). In this work, the enzyme exhibiting steroidal saponin-rhamnosidase activity was systematically studied on 21 steroidal saponins and 6 ginsenosides. The results showed that the α-1,2-linked end-rhamnosyl residues at C-3 position of steroidal saponins could be hydrolyzed to corresponding secondary steroidal saponins, among which 18 compounds were isolated and identified, including 3 new secondary compounds. For the furostanosides having glucosyl residues at the C-26 position, hydrolysis occurred first at end-rhamnosyl at C-3 position to produce secondary furostanosides. The reaction of hydrolyzing glucosyl at C-26 position depended considerably on longer reaction times yielding the corresponding secondary spirostanosides (without rhamnosyl and glucosyl residues). The enzyme had the strict specificity for the terminal α-1,2-linked rhamnosyl residues of linear chain, or the terminal α-1,2-linked rhamnosyl residues with branched chain of 1,4-linked glycosyl residues of sugar chain at C-3 position of steroidal saponins, it was not specific for different aglycones, different glycons, and the number of glycon of sugar chain of steroidal saponin. The end-rhamnosyl of ginsenosides and p-nitrophenyl-α-l-rhamnopyranoside (pNPR) could not be hydrolyzed by the enzyme from C. lunata.     

Two new triterpenoid saponins from Lonicera macranthoides

Two new triterpenoid saponins named lonimacranthoideⅣ(1) and lonimacranthoideⅤ(2) were isolated from the flower buds of Lonicera macranthoides Hand.-Mazz.(Caprifoliaceae).They have hederagenin as aglycone.LonimacranthoideⅣ(1) is a rare chlorogenic acid ester acylated on the C-23 of hederagenin.LonimacranthoideⅣis a new sulfated triterpenoid saponin.The structures of the saponins were established by chemical and spectral methods.     

Surface shear rheology of saponin adsorption layers

Saponins are a wide class of natural surfactants, with molecules containing a rigid hydrophobic group (triterpenoid or steroid), connected via glycoside bonds to hydrophilic oligosaccharide chains. These surfactants are very good foam stabiliziers and emulsifiers, and show a range of nontrivial biological activities. The molecular mechanisms behind these unusual properties are unknown, and, therefore, the saponins have attracted significant research interest in recent years. In our previous study (Stanimirova et al. Langmuir2011, 27, 12486-12498), we showed that the triterpenoid saponins extracted from Quillaja saponaria plant (Quillaja saponins) formed adsorption layers with unusually high surface dilatational elasticity, 280 ± 30 mN/m. In this Article, we study the shear rheological properties of the adsorption layers of Quillaja saponins. In addition, we study the surface shear rheological properties of Yucca saponins, which are of steroid type. The experimental results show that the adsorption layers of Yucca saponins exhibit purely viscous rheological response, even at the lowest shear stress applied, whereas the adsorption layers of Quillaja saponins behave like a viscoelastic two-dimensional body. For Quillaja saponins, a single master curve describes the data for the viscoelastic creep compliance versus deformation time, up to a certain critical value of the applied shear stress. Above this value, the layer compliance increases, and the adsorption layers eventually transform into viscous ones. The experimental creep-recovery curves for the viscoelastic layers are fitted very well by compound Voigt rheological model. The obtained results are discussed from the viewpoint of the layer structure and the possible molecular mechanisms, governing the rheological response of the saponin adsorption layers.     

Impact of the Hydrolysis and Methanolysis of Bidesmosidic Chenopodium quinoa Saponins on Their Hemolytic Activity

Saponins are specific metabolites abundantly present in plants and several marine animals. Their high cytotoxicity is associated with their membranolytic properties, i.e., their propensity to disrupt cell membranes upon incorporation. As such, saponins are highly attractive for numerous applications, provided the relation between their molecular structures and their biological activities is understood at the molecular level. In the present investigation, we focused on the bidesmosidic saponins extracted from the quinoa husk, whose saccharidic chains are appended on the aglycone via two different linkages, a glycosidic bond, and an ester function. The later position is sensitive to chemical modifications, such as hydrolysis and methanolysis. We prepared and characterized three sets of saponins using mass spectrometry: (i) bidesmosidic saponins directly extracted from the ground husk, (ii) monodesmosidic saponins with a carboxylic acid group, and (iii) monodesmosidic saponins with a methyl ester function. The impact of the structural modifications on the membranolytic activity of the saponins was assayed based on the determination of their hemolytic activity. The natural bidesmosidic saponins do not present any hemolytic activity even at the highest tested concentration (500 µg·mL⁻¹). Hydrolyzed saponins already degrade erythrocytes at 20 µg·mL⁻¹, whereas 100 µg·mL⁻¹ of transesterified saponins is needed to induce detectable activity. The observation that monodesmosidic saponins, hydrolyzed or transesterified, are much more active against erythrocytes than the bidesmosidic ones confirms that bidesmosidic saponins are likely to be the dormant form of saponins in plants. Additionally, the observation that negatively charged saponins, i.e., the hydrolyzed ones, are more hemolytic than the neutral ones could be related to the red blood cell membrane structure.     

An Improved Method to Resolve Plant Saponins and Sugars by TLC

Thin-layer chromatography (TLC) plays an important role in the initial selection of mutants having a unique seed saponin composition from the germplasm collections of the subgenus Soja. In the conventional TLC procedure, the dehydrated free sugars are retained just below the major saponins and interrupt the identification of some minor saponin constituents. To resolve this problem, we developed an efficient and reliable method to move sugars from the saponin area on TLC. A developing chamber was saturated with the lower phase of chloroform:methanol:water (65:35:10, v/v) for 2?h and the TLC plates were developed in it for 50?min. Plates were then dried at 100?°C for 10?min to evaporate the excess mobile phase and developed again with 10?% H₂SO₄ for 15?min. While sulfuric acid migrates over the surface of SiO₂, sugar molecules are dehydrated and hydrophilic interactions between free sugars and SiO₂ are strongly reduced. Thus, the positions of dehydrated sugars were shifted to above the saponin area on the TLC plate. This resulted in easy recognition of the saponin composition without any discrimination. This amended protocol would be applicable to all TLC analyses in which the target components should be separate from the interrupting sugar molecules.     

Comparison of Silica-, C18−, and NH2−HPLC Columns for the Separation of Neutral Steroid Saponins from Dioscorea Plants

Abstract

Six neutral steroid saponins including dioscin and gracillin from the tubers of Dioscorea plants and their peracetates were chromatographed by silica-, C₁₈?, and NH₂?column high-performance liquid chromatography (HPLC). The eluent containing water was used in the silica-column HPLC for the separation of these saponins. These HPLC systems complement each other and allow separation of the saponins. The number of carbohydrate units in the saponin molecule plays the most important role for the separations. The more carbohydrate units in the molecule, the more polar is the saponin or its peracetate. Separation of saponins containing the same number of carbohydrate units can be improved by using their peracetates.     

Modulation of Triterpene Saponin Production: In Vitro Cultures,Elicitation, and Metabolic Engineering

Saponins are secondary metabolites that are widely distributed in the plant kingdom and are often the active components in medicinal herbs. Hence, saponins have a potential for the pharmaceutical industry as antibacterial, virucidal, anti-inflammatory, and anti-leishmanial drugs. However, their commercial application is often hindered because of practical problems, such as low and variable yields and limited availability of natural resources. In vitro cultures provide an alternative to avoid problems associated with field production; they offer a system in which plants are clonally propagated and yield is not affected by environmental changes. Additionally, treatment of in vitro cultures with elicitors such as methyl jasmonate may increase the production of saponins up to six times. In vitro cultures are amenable to metabolic engineering by targeting specific genes to enhance saponin production or drive production towards one specific class of saponins. Hitherto, this approach is not yet fully explored because only a limited number of saponin biosynthesis genes are identified. In this paper, we review recent studies on in vitro cultures of saponin-producing plants. The effect of elicitation on saponin production and saponin biosynthesis genes is discussed. Finally, recent research efforts on metabolic engineering of saponins will also be presented.     

Synthesis and structure–activity relationship study of cytotoxic lupane-type 3β-O-monodesmosidic saponins with an extended C-28 side chain

A concise synthesis of lupane triterpenes with an elongated carbon chain at the C-28 position, as well as saponins containing d-mannose, l-arabinose, and l-rhamnose moieties at the C-3 position is described. The overall synthesis of the new triterpenes involved seven linear steps starting from natural betulin: selective protection of a hydroxyl group, oxidation, elongation of the carbon chain by Grignard reaction, and deoxygenation. O-Glycosides were obtained by glycosylation of triterpenes with classical Schmidt's donors. Additionally, all new compounds were evaluated in vitro for their cytotoxic activities. Several triterpenes and the corresponding saponins exhibited an interesting cytotoxic activity profile against human cancer cell lines. The therapeutical index of active triterpenes is very high, since almost none of them were cytotoxic for normal BJ fibroblasts. These results open the way to the synthesis of various lupane-type saponin derivatives as potentially bioactive compounds.