Soyasaponin Ab Research Articles

Development of Extraction Method for Determination of Saponins in Soybean-Based Yoghurt Alternatives: Effect of Sample pH.

The number of plant-based dairy alternative products on the market is growing rapidly. In the case of soybean-based yoghurt alternatives, it is important to trace the content of saponins, the phytomicronutrients with a disputable health effect, which are likely to be responsible for the bitter off-taste of the products. We present a new sample extraction method followed by hydrophilic interaction liquid chromatography with mass spectrometric detection (HILIC-MS) for identifying and quantifying soyasaponins in soybean-based yoghurt alternatives. Soyasaponin Bb, soyasaponin Ba, soyasaponin Aa, and soyasaponin Ab were quantified using commercially available standard compounds and with asperosaponin VI as the internal standard. As the recoveries of soyasaponins were unacceptable in yoghurt alternatives at their natural acidic pH, the adjustment of pH was performed as one of the first steps in the sample extraction procedure to achieve the optimum solubility of soyasaponins. The validation of the method included the assessment of linearity, precision, limit of detection and limit of quantification (LOQ), recovery, and matrix effect. The average concentrations of soyasaponin Bb, soyasaponin Ba, soyasaponin Ab, and soyasaponin Aa in several measured soybean-based yoghurt alternatives utilising the developed method were 12.6 ± 1.2, 3.2 ± 0.7, 6.0 ± 2.4 mg/100 g, and below the LOQ, respectively. This method provides an efficient and relatively simple procedure for extracting soyasaponins from yoghurt alternatives followed by rapid quantification using HILIC-MS and could find a rightful application in the development of healthier and better-tasting dairy alternatives.

Novel colloidal associations of soyasaponins and lipid components (DPPC, cholesterol) as potential adjuvants for vaccines

Soyasaponins from soybean (Glycine max) represent promising new potent adjuvants for vaccine research because of their immunostimulating properties and weak hemolytic activity. In the present study, saponin microstructures of soyasaponins (soyasaponin Bb, soyasaponin Ab) with lipid components (cholesterol, DPPC (dipalmitoylphosphatidylcholine)) were designed by the lipid film method. In interaction studies between soyasaponins (soyasaponin Ab/Bb) and Langmuir monolayers (model membranes), composed of cholesterol and DPPC, marked interactions between soyasaponins and a pure cholesterol monolayer were observed. No interaction was detected for soyasaponins with a pure DPPC monolayer. The intercalation of soyasaponins in a mixed DPPC/cholesterol (3:1, w/w) monolayer was only observed for the monodesmosidic soyasaponin Bb whereas the second sugar chain of the bidesmosidic soyasaponin Ab impaired the access to the monolayer. Transmission electron microscopy was used for visualizing particle formation of soyasaponins and lipid components. Pseudo-binary systems (soyasaponin Ab/Bb, cholesterol) formed colloidal associations built up from ring-like subunits in the nanometer size range. In pseudo-ternary systems (soyasaponin, cholesterol, DPPC) soyasaponin Bb attacked the liposomal membrane by forming colloidal associations. Colloidal associations in pseudo-ternary systems with soyasaponin Ab, cholesterol and a phospholipid were only observed in the presence of PE (phosphatidylethanolamine) instead of DPPC. In an MTT assay with a HaCaT cell line (keratinocyte cell line) the cell viability was neither affected by the soyasaponins nor by the corresponding formulations. Both the pure soyasaponin solution and the saponin formulations may be promising adjuvant systems for the intradermal vaccine application. Furthermore, interaction studies between the model antigen ovalbumin and colloidal associations of saponins and cholesterol using MST (Microscale Thermophoresis) gave first indications of an antigen binding to colloidal associations. Ex vivo T-cell proliferation in the presence of soyasaponin Ab was confirmed.

Soyasaponin Ab alleviates postmenopausal obesity through browning of white adipose tissue

Abstract Postmenopausal obesity is becoming a serious global health concern. Soyasaponin Ab (SA) inhibits adipocyte differentiation; however, the preventive and therapeutic potential of SA in postmenopausal obesity has not been clearly established. Here, we investigated the anti-obesity effects of SA in postmenopausal obesity in vitro and in vivo. SA inhibited adipocytic differentiation of 3T3-L1 cells and reduced lipid accumulation and lipid droplet formation. Moreover, SA significantly decreased weight gain, adipocyte area, serum metabolic profiles in OVX mice. SA also inhibited the lipid metabolism-related factors but increased the thermoregulatory factors in both in vitro and in vivo. SA treatment further increased energy expenditure through beige fat activation. Overall, we found that SA effectively accelerated energy consumption by stimulating beige fat activation, even under estrogen-deficient conditions. Thus, SA treatment may be a promising strategy for the prevention of postmenopausal obesity by promoting weight loss, reducing fat accumulation, and improving obesity-related metabolic disorders.

Germinated soy germ with increased soyasaponin Ab improves BMP-2-induced bone formation and protects against in vivo bone loss in osteoporosis

Osteoporosis is frequently induced following menopause, and bone fractures result in serious problems including skeletal deformity, pain, and increased mortality. Therefore, safe and effective therapeutic agents are needed for osteoporosis. This study aimed to clarify the bone protecting effects of germinated soy germ extracts (GSGE) and their mode of action. GSGE increased expression of alkaline phosphatase (ALP) and osteocalcin (OCL) by stimulating the expression of runt-related transcription factor 2 (Runx2) and osterix (Osx) through activation of Smad signaling molecules. Furthermore, germination of soy germ increased levels of nutritional components, especially soyasaponin Ab. The anabolic activity of soyasaponin Ab in GSGE was also evaluated. GSGE and soyasaponin Ab significantly protected against ovariectomy (OVX)-induced bone loss and improved bone-specific alkaline phosphatase (BALP) level in mouse serum. These in vitro and in vivo study results demonstrated that GSGE and soyasaponin Ab have potential as therapeutic candidate agents for bone protection in postmenopausal osteoporosis.

Soyasaponin Ab protects against oxidative stress in HepG2 cells via Nrf2/HO-1/NQO1 signaling pathways

Soyasaponin Ab (SA) is a typical oleanane triterpene saponin isolated from soybean and represents effects of antioxidation and anti-inflammation. In this study, we found the expressions of some phase II detoxifying enzymes were stimulated by SA via the signaling pathway of nuclear factor erythroid 2-related factor 2 (Nrf2) in HepG2 cells. Luciferase assay results demonstrated that SA promoted the Nrf2 transcription via binding to the antioxidant response element. In addition, a combining of luciferase assay and Western blotting suggested that SA activated ERK1/2 signaling pathways, and made Nrf2 translocated from cytoplasm to nucleus. SA also upregulated some Nrf2-related antioxidant enzymes such as heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase-1 (NQO1), and glutamate cysteine ligase catalytic subunit (GCLC). Inhibition of si-Nrf2 resisted expressions of SA-induced antioxidant genes. Collectively, these results suggest that SA exerted its protection against oxidative stress via the Nrf2/HO-1/NQO1 signaling pathways.

Soyasaponin Ab inhibits lipopolysaccharide-induced acute lung injury in mice

Soyasaponin Ab (SA) has been reported to have anti-inflammatory effect. However, the effects of SA on lipopolysaccharide (LPS)-induced acute lung injury (ALI) have not been reported. The aim of this study was to investigate the anti-inflammatory effects of SA on LPS-induced ALI and clarify the possible mechanism. The mice were stimulated with LPS to induce ALI. SA was given 1h after LPS treatment. 12h later, lung tissues were collected to assess pathological changes and edema. Bronchoalveolar lavage fluid (BALF) was collected to assess inflammatory cytokines and nitric oxide (NO) production. In vitro, mice alveolar macrophages were used to investigate the anti-inflammatory mechanism of SA. Our results showed that SA attenuated LPS-induced lung pathological changes, edema, the expression of cycloxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in lung tissues, as well as TNF-α, IL-6, IL-1β, and NO production in mice. Meanwhile, SA up-regulated the activities of superoxide dismutase (SOD) and catalase decreased by LPS in mice. SA also inhibited LPS-induced TNF-α, IL-6 and IL-1β production as well as NF-κB activation in alveolar macrophages. Furthermore, SA could activate Liver X Receptor Alpha (LXRα) and knockdown of LXRα by RNAi abrogated the anti-inflammatory effects of SA. In conclusion, the current study demonstrated that SA exhibited protective effects against LPS-induced acute lung injury and the possible mechanism was involved in activating LXRα, thereby inhibiting LPS-induced inflammatory response.

Evaluation of cytotoxicity and immune modulatory activities of soyasaponin Ab: An in vitro and in vivo study

To improve the immune efficacy of protein subunit vaccines, novel adjuvants are needed to elicit a suitable protective immune response and to promote long term immunologic memory. In this work, soyasaponin Ab, a major constituent among group A soyasaponins in soybeans was purified and prepared from soy hypocotyls. The immunomodulatory effects of soyasaponin Ab both in vitro and in vivo were investigated, and its pro-immunomodulatory molecular mechanism was also studied. For in vitro assays, with mouse macrophage cell line RAW264.7 as the studying model, both cytotoxicity and immune stimulatory activity were investigated to evaluate the potential of soyasaponin Ab as the vaccine adjuvant. The results indicated that soyasaponin Ab could be significantly safer than Quillaja saponins (QS). Soyasaponin Ab showed no toxicities over the tested concentration ranges compared to QS. Soyasaponin Ab was proved able to promote releases of inflammatory cytokines like TNFα and IL-1β in a dose-dependent manner. Furthermore, NF-κB signalling was also activated by soyasaponin Ab effectively. In addition, with TLR4 gene expression of RAW264.7 cell inhibited by RNA interference, immune stimulatory effects by soyasaponin Ab dropped down significantly. On the other hand, the in vivo experiment results showed that anti-ovalbumin (OVA) IgG, IgG1, IgG2a, IgG2b were significantly enhanced by the soyasaponin Ab and QS groups (p<0.05 or p<0.01). The results suggested that compared to QS, soyasaponin Ab may represent a viable candidate for effective vaccine adjuvant. TLR4 receptor dependent pathway may be involved in immune stimulatory effects of soyasaponin Ab.

Rapid characterisation and comparison of saponin profiles in the seeds of Korean Leguminous species using ultra performance liquid chromatography with photodiode array detector and electrospray ionisation/mass spectrometry (UPLC–PDA–ESI/MS) analysis

The present work was reported on investigation of saponin profiles in nine different legume seeds, including soybean, adzuki bean, cowpea, common bean, scarlet runner bean, lentil, chick pea, hyacinth bean, and broad bean using ultra performance liquid chromatography with photodiode array detector and electrospray ionisation/mass spectrometry (UPLC–PDA–ESI/MS) technique. A total of twenty saponins were characterised under rapid and simple conditions within 15min by the 80% methanol extracts of all species. Their chemical structures were elucidated as soyasaponin Ab (1), soyasaponin Ba (2), soyasaponin Bb (3), soyasaponin Bc (4), soyasaponin Bd (5), soyasaponin αg (6), soyasaponin βg (7), soyasaponin βa (8), soyasaponin γg (9), soyasaponin γa (10), azukisaponin VI (11), azukisaponin IV (12), azukisaponin II (13), AzII (14), AzIV (15), lablaboside E (16), lablaboside F (17), lablaboside D (18), chikusetusaponin IVa (19), and lablab saponin I (20). The individual and total saponin compositions exhibited remarkable differences in all legume seeds. In particular, soyasaponin βa (8) was detected the predominant composition in soybean, cowpea, and lentil with various concentrations. Interestingly, soybean, adzuki bean, common bean, and scarlet runner bean had high saponin contents, while chick pea and broad bean showed low contents.

Efficient protocol for isolation and purification of different soyasaponins from soy hypocotyls

Soyasaponins are naturally occurring triterpenoid glycosides associated with many biological activities. The aim of the present study was to develop an effective method for isolation and purification of differently glycosylated, acetylated, and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-conjugated soyasaponins from soy hypocotyls. Both gel filtration using Sephadex LH-20 chromatography (Amersham Pharmacia Biotech AB; elution phase: methanol, flow rate: 3.0 mL/min, sample loading: 60 mg) and high-speed countercurrent chromatography (stationary phase: n-butanol-acetic acid (5.0%, v/v), mobile phase: water flow rate: 3.0 mL/min, sample loading: 100 mg) could effectively fractionate isoflavones and soyasaponins from the crude extract with yield of soyasaponin complexes 20.5 mg and 22.3 mg, respectively. After fractionation, the soyasaponin complexes could be purified further using preparative HPLC to separate individuals. A total of nine soyasaponins, triacetyl soyasaponin Ab (yield 1.55%, HPLC purity >98%), Aa (2.68%, >99%), Ab (18.53%, >98%), Ae (0.85%, >98%), Ba (0.63%, >91%), Af (1.12%, >85%), Bb (3.45%, >98%) and Be (0.59%, >76.8%) were obtained. DDMP-conjugated groups, αg (2.06%, >85%), βg (7.59%, >85%), and γg (0.29%, >85%) that were very labile even in mild conditions, were also collected. The method described here can be used as an effective protocol to separate different soyasaponins occurring in the original sample.

Soyasaponin Ab Ameliorates Colitis by Inhibiting the Binding of Lipopolysaccharide (LPS) to Toll-like Receptor (TLR)4 on Macrophages

Many clinical studies have shown that daily intake of soybean [ Glycine max (L.) Merr., Fabacease] or its foods may reduce the risk of osteoporosis, heart attack, hyperlipidemia, coronary heart disease, cardiovascular and chronic renal diseases, and cancers, including prostate, colon, and breast cancers. Of the soy constituents, soyasaponins exhibit anti-aging, antioxidant, apoptotic, and anti-inflammatory effects. However, the anti-inflammatory effect of soyasaponin Ab has not been thoroughly studied. Therefore, we investigated its anti-inflammatory effects in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitic mice and lipopolysaccharide (LPS)-stimulated peritoneal macrophages. Soyasaponin Ab inhibited colon shortening, myeloperoxidase activity, the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), and activation of the transcription factor nuclear factor-κB (NF-κB). Soyasaponin Ab (1, 2, 5, and 10 μM) inhibited the production of NO (IC(50) = 1.6 ± 0.1 μM) and prostaglandin E(2) (IC(50) = 2.0 ± 0.1 ng/mL), the expression of tumor necrosis factor (TNF)-α (IC(50) = 1.3 ± 0.1 ng/mL), interleukin (IL)-1β (IC(50) = 1.5 ± 0.1 pg/mL), and toll-like receptor (TLR)4, and the phosphorylation of interleukin-1 receptor-associated kinase (IRAK)-1 in LPS-stimulated peritoneal macrophages. Soyasaponin Ab weakly inhibited the phosphorylation of ERK, JNK, and p38. Soyasaponin Ab significantly reduced the binding of Alexa-Fluor-594-conjugated LPS to peritoneal macrophages. Soyasaponin Ab did not affect TLR4 expression or LPS-induced NF-κB activation in TLR4 siRNA-treated peritoneal macrophages (knockdown efficiency of TLR4 > 94%). On the basis of these findings, soyasaponin Ab may ameliorate colitis by inhibiting the binding of LPS to TLR4 on macrophages.

Liquid chromatography/mass spectrometry-based structural analysis of soyasaponin Ab metabolites by human fecal microflora

Soyasaponin Ab, a major constituent of soybean by human intestinal microflora, was anaerobically incubated with fecal suspensions from ten individuals for 48 h and its metabolites were measured by LC-MS/MS analysis. Ten metabolites were detected. The spectra of the parental constituent soyasaponin Ab showed a peak at m/z 1435 [M-H](-) ion and those of its nine metabolites showed peaks at m/z 1310.0 [M-3C(2)H(2)O-H](-) ion, m/z 1267.9 [M-4C(2)H(2)O-H](-) ion, m/z 1105.3 [M-Glc-4C(2)H(2)O-H](-) ion, m/z 973.2 [M-Glc-Ara-4C(2)H(2)O-H](-) ion, m/z 943.4 [M-2Glc-4C(2)H(2)O-H](-) ion, m/z 811.1 [M-2Glc-Ara-4C(2)H(2)O-H](-) ion, m/z 781.2 [M-2Glc-Gal-4C(2)H(2)O-H](-) ion, m/z 649.0 [M-2Glc-Gal-Ara-4C(2)H(2)O-H](-) ion, and m/z 458.8 [Soyasapogenol A+H](+) ion. Metabolic activity varied significantly between individuals. The metabolic pathway was classified into two groups: the first group potently produced soyasapogenol A and the second group accumulated soyasapogenol A 3-beta-D-glucuronide.

Soyasaponins Resist Extrusion Cooking and Are Not Degraded during Gut Passage in Atlantic Salmon (Salmo salar L.)

The stability of soyasaponins in fish feed formulations was investigated. The level of soyasaponin Ab, Bb, Bc, Ba-2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (Ba-DDMP), Bb-DDMP, and Bc-DDMP was quantified in 15 samples of defatted soybean meal, two full fat soybean meals, and two soybean protein concentrates by reverse phase high-performance liquid chromatography. The total level of saponins in the 15 samples of commercial defatted soybean meal ranged from 4.8-6.8 micromol/g (5.1-7.0 g/kg). The two full fat meals contained 4.4 and 4.7 micromol/g whereas no saponins could be detected in the alcohol-extracted soybean protein concentrates. Fifteen batches of fish feed containing 20% defatted soybean meal were produced by twin-screw extrusion from the 15 different samples of defatted soybean meal. Extrusion did not reduce the total level of group B saponins, but the ratio between DDMP-conjugated group B saponins and non-DDMP-conjugated group B saponins was slightly reduced. A soybean-containing diet was fed to seawater adapted Atlantic salmon for 9 weeks. Yttrium oxide was included in the feed as an inert marker in order to estimate the disappearance of saponins during gut passage. High levels of intact non-DDMP-conjugated group B soyasaponins were found in feces whereas only low levels of DDMP-conjugated saponins could be detected. The overall disappearance of saponins was close to zero, and the concentration of intact saponins in dry feces reached levels several fold higher than dietary levels. The present work demonstrates that non-DDMP-conjugated group B soyasaponins resist extrusion cooking and remain intact during gut passage in Atlantic salmon. The latter is contrary to earlier findings in endothermic animals.

Preparative chromatographic purification and surfactant properties of individual soyasaponins from soy hypocotyls

The amphipathic character of soyasaponins and their consequent biological and technological properties are well-recognised. However, mainly due to the absence of purified compounds, no data are available on the amphiphilic surfactant properties of individual soyasaponins. In this study we developed a preparative method for the purification of the main soyasaponins species from soy germ. Reversed-phase chromatography (Source 15 RPC) gave a good resolution of the various soyasaponins, and was used for the purification of non- and fully-acetylated soyasaponin Ab, DDMP-conjugated and unconjugated soyasaponins Ba and Bb. For these compounds, the critical micelle concentration (CMC), the minimal attainable surface tension ( γ CMC) and the surface density ( Γ max) were determined using the Wilhelmy plate method. The order of CMC values was as follows: soyasaponin Bb < Ba < βg < αg < Ab < non-acetylated Ab, and the CMC was found to range from 0.56 and to 3.2 g/L. It was concluded that the number of sugar side chains, the presence of acetyl groups and the presence of a DDMP-group are the main factors influencing the CMC of soyasaponins.

Prevention of Hydrogen Peroxide Damage by Soybean Saponins to Mouse Fibroblasts

Soybean saponin is one of several soybean glycosides. Recently, new soybean saponins which possess the DDMP (2,5-dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one) moiety have been detected. Soyasaponin beta g of these new soybean saponins was shown to inhibit hydrogen peroxide damage to mouse fibroblast cells. Not only the new saponin but also soyasaponin I, which has lost the DDMP moiety from soyasaponin beta g, was tested and showed higher inhibition in this assay. In order to investigate the relationship between the effect and chemical structure, glycyrrhizin, which has a similar chemical structure to soyasaponin I, was also tested and exhibited the same effect as soyasaponins beta g and I. Soyasaponin Ab, a major authentic bisdesmosidic saponin that exists only in the soybean hypocotyl, was also tested and exhibited the highest inhibition of hydrogen peroxide damage among the four saponins. It was concluded that water-soluble soybean saponins protected the cells from damage by hydrogen peroxide.

Chemiluminescence of soybean saponins in the presence of active oxygen species

Natural active oxygen scavengers (Y; catalytic compounds) exhibit chemiluminescence (CL) in the presence of an X (active oxygen compounds) and a Z (receptive compounds). The CL derived from soyasaponins Ab and βg, major components of group A and DDMP saponins present at high level in the hypocotyl of soybean seeds, was compared with (−)-epigallocatechin (EGC) as the most effective Y and acetaldehyde (MeCHO) as the most effective Z in the presence of H 2O 2 as X. We have found that soyasaponin Ab and soyasaponin βg are Y equivalent to EGC and Z equivalent to MeCHO, respectively. In the presence of soyasaponin Ab as Y, CL of soyasaponin βg showed about 100-fold than that of MeCHO as Z. These results suggest that group A and DDMP saponins play an important role in the scavenging function of active oxygen.