Study of the inhibition effects on glutathione peroxidase immobilized on MNPs using a stopped-flow microfluidic system.

A stopped-flow microfluidic system to monitor glutathione peroxidase (GPx) activity and evaluate potential inhibitors of the enzyme has been developed based on the integration of the microfluidic chip in the reaction/detection zone. This integration supposes the physical alignment at the optimal location of the microfluidic channel, both the magnetically retained enzyme microreactor (MREµR) and the remote luminescence detection using a focused bifurcated fiber optic bundle (BFOB) connected to a conventional spectrofluorometer detector. The method is based on the coupling of two competitive oxidative chemical reactions, in which glutathione (GSH) and homovanillic acid (HVA) competed for their interaction with hydrogen peroxide in the presence of the magnetically retained GPx-MNPs. The biocatalytic reaction was followed by monitoring the fluorescence of the biphenyl-HVA dimer formed. The dynamic range of the calibration graph was 0.45-10 µmol L-1, expressed as GSH concentration with a detection limit of 0.1 µmol L-1 (r2 = 0.9954, n = 10, r = 3). The precision expressed as the relative standard deviation (RSD%) was between 0.5 and 3.9%. The stopped-flow microfluidic system showed a sampling frequency of 25 h-1. The method was applied to the study of GPx inhibition provided by three inhibitory compounds, two metallic ions Hg(II) and Cu(II) and t-butyl hydroperoxide, and their presence in liquid samples, as water, milk, and edible oil. Recovery values between 88.7 and 99.4% were achieved in all instances.

A rapid and sensitive LC-MS/MS method for the determination of vanillic acid in rat plasma with application to pharmacokinetic study.

Vanillic acid, a phenolic compound isolated from Angelica sinensis and green tea, exhibits excellent antioxidant and anti-inflammatory activities. In this study, a rapid and sensitive ultra-high-performance liquid chromatography tandem mass spectrometry method was established and validated for the determination of vanillic acid in rat plasma. Plasma samples were prepared by protein precipitation with acetonitrile. Chromatographic separation was performed on a Zorbax RRHD Eclipse Plus C18 column (2.1 × 100 mm, 1.8 µm) with gradient elution at a flow rate of 0.3 ml/min, using mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B). Vanillic acid and caffeic acid (internal standard, IS) were quantified by multiple reaction monitoring in negative ion mode. The method was fully validated according to the US Food and Drug Administration guidelines. The calibration curve was linear over the range of 2-1,000 ng/ml with a correlation coefficient of >0.99. The carryover, matrix effect, extraction recovery, dilution effect, intra- and interday precision and accuracy were within acceptable limits. The method was then applied to a pharmacokinetic study of vanillic acid in rats. After oral administration at doses of 2, 5 and 10 mg/kg, the plasma concentration reached peaks of 0.42 ± 0.09, 0.73 ± 0.21 and 0.92 ± 0.28 µg/ml at the time of 0.55-0.64 h, respectively. The oral bioavailability was calculated as 25.3-36.2% in rat plasma. The result provided pre-clinical information for further application of vanillic acid.

Phenolic Profiles of Ten Australian Faba Bean Varieties.

Although Australia is the largest exporter of faba bean globally, there is limited information available on the levels of bioactive compounds found in current commercial faba bean varieties grown in this country. This study profiled the phenolic acid and flavonoid composition of 10 Australian faba bean varieties, grown at two different locations. Phenolic profiling by HPLC-DAD revealed the most abundant flavonoid to be catechin, followed by rutin. For the phenolic acids, syringic acid was found in high concentrations (72.4-122.5 mg/kg), while protocatechuic, vanillic, p-hydroxybenzoic, chlorogenic, p-coumaric, and trans-ferulic acid were all found in low concentrations. The content of most individual phenolics varied significantly with the variety, while some effect of the growing location was also observed. This information could be used by food processors and plant breeders to maximise the potential health benefits of Australian-grown faba bean.

Ionic chitosan Schiff bases supported Pd(II) and Ru(II) complexes; production, characterization, and catalytic performance in Suzuki cross-coupling reactions.

Taking the advantage of multifunctional characteristics of chitosan (CS), we have developed new scaffolds (imidazolium-vanillyl-chitosan Schiff bases (IVCSSBs)) for supporting Pd(II) and Ru(II) ions in catalyzing Suzuki coupling reactions. The structures of new materials were described based on their elemental, spectral, thermal, and microscopic analysis. The strong interactions between the binding sites of IVCSSB ligand (OH, H-C=N, and OCH3 groups) and Pd(II) ions resulted in the formation of an excellent heterogeneous catalyst (Pd(II)IVCSSB1) with amazing catalytic activity (up to 99%) and highly stable in the reaction medium. The reusability experiments for Pd(II)IVCSSB1 revealed that there is no appreciable decrease in its catalytic activity even after five consecutive operation runs. Furthermore, this heterogeneous catalyst showed an excellent selectivity toward the cross-coupling reaction where no homo-coupling byproducts were observed in the 1H NMR spectra of the obtained products. Consequently, the present ionic catalytic system may open a new window for a novel generation of ionic bio-based catalysts for organic transformations.

Protective Role of Vanillic Acid against Diethylnitrosamine- and 1,2-Dimethylhydrazine-Induced Hepatocarcinogenesis in Rats.

This study aimed to evaluate the cancer chemopreventive activity of vanillic acid (VA) in diethylnitrosamine- and 1,2-dimethylhydrazine-induced liver and colon carcinogenesis in rats. VA did not induce the formation of hepatic glutathione S-transferase placental form (GST-P) positive foci and colonic aberrant crypt foci, demonstrating no carcinogenic activity. VA (75 mg kg-1 body weight) could significantly reduce the number and areas of hepatic GST-P positive foci when administered before carcinogen injections, but no such effect was seen when it was administered after carcinogen injection. No protection was seen in the colon when VA was treated before or after carcinogen injection. Immunohistochemical studies demonstrated the decreased expression of proliferating cell nuclear antigen and the induction of apoptosis. Mechanistic studies showed that VA significantly induced the expression of GSTA-5 and Nrf-2 genes, which are associated with the detoxification system. Likewise, the antiproliferative effect was noticed by the reduction of Cyclin D1 expression. The apoptotic activity may be due to the upregulation of Caspase-3 and Bad levels and downregulation of the Bcl-2 level. These data suggest that VA exhibited significant protection against diethylnitrosamine- and 1,2-dimethylhydrazine-induced hepatocarcinogenesis, which might be related to the induction of the detoxifying enzyme, the reduction of proliferation and the induction of apoptosis.

Binding parameters and molecular dynamics of ß-lactoglobulin-vanillic acid complexation as a function of pH - Part A: Acidic pH.

Protein-phenolic compound interactions are commonly investigated with inappropriate linear equations for the analysis of binding strength and stoichiometry. This work utilises more appropriate protocols for the investigation of molecular interactions between vanillic acid and ß-lactoglobulin at pH 2.4, where the protein predominately exists as a monomer. Non-linear binding and Job plot analysis were conducted on fluorescence data to effectively determine the interaction's dissociation constant (KD, 2.93 × 10-5 M) and stoichiometry (1:1). Furthermore, spectroscopic techniques revealed statistically significant alterations to the conformational characteristics of ß-lactoglobulin upon complexation. Molecular dynamics (MD) simulations support a 1:1 interaction stoichiometry and reveal that the stabilisation of vanillic acid was dynamic in nature but mainly supported by four π-alkyl interactions and one hydrogen bond, located within the ß-barrel of the monomer. Water molecules, which are generally not accounted for in MD simulation analysis, were shown to be an important factor in the ligand stabilization via bridging interactions.

Towards a new understanding of the retro-aldol reaction for oxidative conversion of lignin to aromatic aldehydes and acids.

The retro-aldol reaction is one of the key steps involved in the oxidative conversion of lignin to aromatic aldehydes and acids. In principle, the retro-aldol reaction can proceed in the absence of oxygen. In this work, a new approach based on the influence of oxygen on the oxidation of lignin was investigated. In this approach, the duration of oxygen charged during the reaction was optimized to, for the first time, improve the yield of aromatic aldehydes and acids. The effect of reaction chemistry, time, temperature, and lignin feedstock plays a key role on the yield of aromatic aldehydes and acids. At 140 °C, oxidation of softwood Lignoboost kraft lignin for 40 min results in combined maximum yield of 5.17% w/w of vanillin and vanillic acid. In comparison, using the new approach in which oxygen was charged for only 20 min during the 40 min reaction improved this yield considerably to 6.95%. Further, yield improvement was obtained when applying this approach to different lignin feedstocks. Oxidation also increased the carboxyl content in lignin from 0.49 mmol/g to 1.41 mmol/g which represents a marked improvement. The current study provides new evidence showing that the oxidation reaction is a crucial pathway for lignin valorization.

NMR-Based Metabolomic Analyses to Identify the Effect of Harvesting Frequencies on the Leaf Metabolite Profile of a Moringa oleifera Cultivar Grown in an Open Hydroponic System.

Moringa oleifera Lam. is one of the world's most useful medicinal plants. Different parts of the M. oleifera tree contain a rich profile of important minerals, proteins, vitamins, and various important bioactive compounds. However, there are differences in the phytochemical composition of the medicinal plant's raw materials due to seasonal variation, cultivation practices, and post-harvest processing. The main objective of this study was therefore to determine the effect of harvesting frequencies on selected bioactive compounds of a M. oleifera cultivar (PKM1) grown in a hydroponic system under a shade net structure. Three harvesting frequency treatments were applied in the study, with the plants harvested at every 30 days (high frequency), 60 days (intermediate frequency), and 90 days (low frequency) respectively. 1H-NMR was used for data acquisition, and multivariate data analysis by means of principal component analysis (PCA), partial least square discriminatory analysis (PLS-DA), and orthogonal partial least square discriminatory analysis (OPLS-DA) were applied to determine the changes in the leaf metabolite profile, and also to identify the spectral features contributing to the separation of samples. Targeted metabolite analysis was used to match the NMR peaks of the compounds with the NMR chemical shifts of the contribution plot. The contribution plot showed that the increase in concentration of some compounds in aliphatic, sugar and aromatic regions contributed to the separation of the samples. The results revealed that intermediate and low harvesting frequencies resulted in a change in the leaf metabolite profile. Compounds such as chlorogenic acid, ferulic acid, vanillic acid, wogonin, esculetin, niazirin, and gamma-aminobutyric acid (GABA) showed an increase under intermediate and low harvesting frequencies. These results provide insight into the effect of harvesting frequencies on the metabolite profile and associated medicinal activity of M. oleifera.

Feeding strategies to optimize vanillin production by Amycolatopsis sp. ATCC 39116.

The growing consumer demand for natural products led to an increasing interest in vanillin production by biotechnological routes. In this work, the biotechnological vanillin production by Amycolatopsis sp. ATCC 39116 is studied using ferulic acid as precursor, aiming to achieve maximized vanillin productivities. During biotech-vanillin production, the effects of glucose, vanillin and ferulic acid concentrations in the broth proved to be relevant for vanillin productivity. Concerning glucose, its presence in the broth during the production phase avoids vanillin conversion to vanillic acid and, consequently, increases vanillin production. To avoid the accumulation of vanillin up to a toxic concentration level, a multiple-pulse-feeding strategy is implemented, with intercalated vanillin removal from the broth and biomass recovery. This strategy turned out fruitful, leading to 0.46 g L-1 h-1 volumetric productivity of vanillin of and a production yield of 0.69 gvanillin gferulic acid-1, which are among the highest values reported in the literature for non-modified bacteria.

Vanillic acid as phospholipase A2 and proteases inhibitor: In vitro and computational analyses.

Enzymatic inhibition by natural compounds may represent a valuable adjuvant in snakebite serum therapy. The objective in this work was to evaluate possible in vitro interactions between vanillic acid and enzymes from Bothrops spp. and Crotalus durissus terrificus venoms, and also suggest a theory as how they interact based on molecular docking. Vanillic acid inhibited the phospholipase activity induced by Bothrops alternatus (∼25% inhibition); the caseinolytic activity induced by Bothrops atrox (∼30%), Bothrops jararacussu (∼44%), and C. d. terrificus (∼33%); the fibrinogenolysis induced by B. jararacussu, B. atrox, and C. d. terrificus (100%); the serine protease activity induced by Bothrops moojeni (∼45%) and Bothrops jararaca (∼66%); the hemolytic activity induced by B. moojeni (∼26%); the thrombolysis activity induced by B. atrox (∼30%) and B. jararacussu (∼20%); and the thrombotic activity induced by C. d. terrificus (∼8%). The compound was also capable of delaying the coagulation time in citrated plasma by 60, 35, and 75 Sec, when incubated with B. moojeni, B. atrox, and B. jararaca, respectively. The results obtained expand the possibilities for future pharmaceutical use of vanillic acid, considering the high homology degree among human and snake venom phospholipases A2 and proteases (involved in chronic inflammatory diseases). Also, this compound can be used as adjuvant to improve currently available treatments for ophidism victims.

Content of Phenolic Compounds in Meadow Vegetation and Soil Depending on the Isolation Method.

The aim of this paper was to determine the effect of the hydrolysis method on the amounts of phenolic compounds in the plant material in soil and, as a consequence, on the parameters to determine the degree of lignins transformation in soils. The study included the plant material (hay, sward, and roots) and soil-Albic Brunic Arenosol (horizon A, AE, and Bsv) samples. Phenolic compounds were isolated at two stages by applying acid hydrolysis followed by alkaline re-hydrolysis. The quantitative and qualitative analysis of phenolic compounds was performed with high-performance liquid chromatography with a DAD. The content of phenolic compounds in the extracts depended on the hydrolysis method and it was determined by the type of the research material. The amounts of phenolic compounds contained in the alkaline hydrolysates accounted for 55.7% (soil, horizon Bsv)-454% (roots) of their content in acid hydrolysates. In the extracts from acid hydrolysates, chlorogenic and p-hydroxybenzoic acids were dominant. In the alkaline extracts from the plant material, the highest content was recorded for p-coumaric and ferulic acids, and in the extracts from soil, ferulic and chlorogenic acids. A combination of acid and alkaline hydrolysis ensures the best extraction efficiency of insoluble-bound forms of polyphenols from plant and soil material.

Dietary Change Enables Robust Growth-Coupling of Heterologous Methyltransferase Activity in Yeast.

Genetic modifications of living organisms and proteins are made possible by a catalogue of molecular and synthetic biology tools, yet proper screening assays for genetic variants of interest continue to lag behind. Synthetic growth-coupling (GC) of enzyme activities offers a simple, inexpensive way to track such improvements. In this follow-up study we present the optimization of a recently established GC design for screening of heterologous methyltransferases (MTases) and related pathways in the yeast Saccharomyces cerevisiae. Specifically, upon testing different media compositions and genetic backgrounds, improved GC of different heterologous MTase activities is obtained. Furthermore, we demonstrate the strength of the system by screening a library of catechol O-MTase variants converting protocatechuic acid into vanillic acid. We demonstrated high correlation (R2 = 0.775) between vanillic acid and cell density as a proxy for MTase activity. We envision that the improved MTase GC can aid evolution-guided optimization of biobased production processes for methylated compounds with yeast in the future.

Iron acquisition system of Sphingobium sp. strain SYK-6, a degrader of lignin-derived aromatic compounds.

Iron, an essential element for all organisms, acts as a cofactor of enzymes in bacterial degradation of recalcitrant aromatic compounds. The bacterial family, Sphingomonadaceae comprises various degraders of recalcitrant aromatic compounds; however, little is known about their iron acquisition system. Here, we investigated the iron acquisition system in a model bacterium capable of degrading lignin-derived aromatics, Sphingobium sp. strain SYK-6. Analyses of SYK-6 mutants revealed that FiuA (SLG_34550), a TonB-dependent receptor (TBDR), was the major outer membrane iron transporter. Three other TBDRs encoded by SLG_04340, SLG_04380, and SLG_10860 also participated in iron uptake, and tonB2 (SLG_34540), one of the six tonB comprising the Ton complex which enables TBDR-mediated transport was critical for iron uptake. The ferrous iron transporter FeoB (SLG_36840) played an important role in iron uptake across the inner membrane. The promoter activities of most of the iron uptake genes were induced under iron-limited conditions, and their regulation is controlled by SLG_29410 encoding the ferric uptake regulator, Fur. Although feoB, among all the iron uptake genes identified is highly conserved in Sphingomonad strains, the outer membrane transporters seem to be diversified. Elucidation of the iron acquisition system promises better understanding of the bacterial degradation mechanisms of aromatic compounds.

Polycaprolactone/polyvinylpyrrolidone coaxial electrospun fibers containing veratric acid-loaded chitosan nanoparticles for bone regeneration.

Veratric acid (3,4-dimethoxy benzoic acid) (VA) is a hydrophobic phenolic phytocompound possessing therapeutic potential, but it has not been reported as actuating bone regeneration to date. Furthermore, delivery of hydrophobic compounds is often impeded in the body, thus depreciating their bioavailability. In this study, VA was found to have osteogenic potential and its sustained delivery was facilitated through a nanoparticle-embedded coaxial electrospinning technique. Polycaprolactone/polyvinylpyrrolidone (PCL/PVP) coaxial fibers were electrospun, encasing VA-loaded chitosan nanoparticles (CHS-NP). The fibers showed commendable physiochemical and material properties and were biocompatible with mouse mesenchymal stem cells (mMSCs). When mMSCs were grown on coaxial fibers, VA promoted these cells towards osteoblast differentiation as was reflected by calcium deposits. The mRNA expression of Runx2, an important bone transcriptional regulator, and other differentiation markers such as alkaline phosphatase, collagen type I, and osteocalcin were found to be upregulated in mMSCs grown on the PCL/PVP/CHS-NP-VA fibers. Overall, the study portrays the delivery of the phytocompound, VA, in a sustained manner to promote bone regeneration.

Evaluation of process involved in the production of aromatic compounds in Gram-negative bacteria isolated from vanilla (Vanilla planifolia ex. Andrews) beans.

AIM: The present investigation was aimed at isolating and identifying bacterial strains from cured vanilla beans. Additionally, the study focused on evaluating bacterial processes pertaining to the aromatic compounds production (ACP). METHODS AND RESULTS: Three bacteria were isolated from Vanilla planifolia beans, previously subjected to the curing process. According to morphological, biochemical and 16S rRNA analysis, the strains were identified as Citrobacter sp., Enterobacter sp. and Pseudomonas sp. The polygalacturonase activity (PGA) was determined using the drop, cup-plate and DNS methods. Aromatic compounds production was analysed by cup-plate method using FA as substrate and quantified by high performance liquid chromatography (ppm), the functional groups of vanillic acid (VA) were identified by FT-IR and the aromatic compounds (AC) resistance was determined and reported as minimum inhibitory concentration. Citrobacter sp., Enterobacter sp. and Pseudomonas showed PGA (70·31 ± 364, 76·07 ± 12·47 and 51 ± 10·92 U ml-1 respectively), were producers of VA (3·23 ± 0·49, 324 ± 41 and 265·99 ± 11·61 ppm respectively) and were resistant to AC. CONCLUSIONS: The Gram-negative bacteria isolated from V. planifolia beans were responsible for ACP. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first evidence for the role of Gram-negative bacterial isolates from cured Mexican V. planifolia beans in the process related to ACP.

N-Vanillylnonanamide, a natural product from capsicum oleoresin, as potential inhibitor of collagen fibrillation.

Inhibition of collagen fibrillation by small molecules is of growing interest to develop therapeutics for the illnesses related to excess deposition of collagen. In this context, we have studied the inhibitory effect of N-Vanillylnonanamide (NVA), a natural product from capsicum oleoresin and an analog of capsaicin (a known inhibitor of collagen fibrillation), on collagen self-assembly that leads to fibrillation in vitro. Commercially, capsaicin was found to be expensive than NVA. Therefore, it would be an advantage economically if NVA could display a similar/better inhibitory activity compared to capsaicin. The conventional turbidity measurements indicate that NVA completely inhibits collagen fibrillation at body temperature (37 °C) and its inhibition were concentration-dependent. The inhibition efficiency was observed to reduce at room temperature (25 °C). NVA protects the triple helical structure of collagen while it increases the thermal stability of collagen compared to collagen alone. Fluorescence results suggest that NVA binds in both telopeptide and triple helical regions of collagen and thereby prevents collagen self-assembly. The present results thus indicate that NVA is a potential inhibitor and, economically, it could be a better choice as a therapeutic agent compared to capsaicin in evolving treatment for disorders associated with excessive collagen deposition.

Novel textile dye obtained through transformation of 2-amino-3-methoxybenzoic acid by free and immobilised laccase from a Pleurotus ostreatus strain.

Transformation of 2-amino-3-methoxybenzoic acid into novel and eco-friendly orange dye (N15) was performed using native and immobilised laccase (LAC) from Pleurotus ostreatus strain. A several parameters affecting laccase-mediated transformation efficiency included the selection of type and pH value of buffer, reaction temperature, substrate and laccase concentration as well as the type of carrier and LAC storage conditions were evaluated. The optimal conditions for N15 dye synthesis were 40 mM sodium-tartrate buffer pH 5.5 containing 3 mM of the substrate, efficiently transformed by 2 U of free laccase per 1 mmol of the substrate. Laccase was immobilised on porous Purolite® carriers, which had never been tested as a support for oxidoreductases. Immobilised laccase, characterised by a high immobilisation yield, was obtained by adsorption of laccase on a porous acrylic carrier with octadecyl groups (C18) incubated in optimum conditions of 40 mM phosphate buffer pH 7.0 containing 1 mg of laccase per 1 g of the carrier (wet mass). The immobilised LAC showed the highest storage stability for 21 days and higher thermostability at 40 ℃ and 60 ℃ in comparison to its native form. The N15 dye showed good dyeing properties towards natural fibres, and the dyed fibre demonstrated resistance to different physicochemical factors during use, which was confirmed by commercial quality tests. The N15 dye is a phenazine, i.e. a heterogenic compound containing amino-, methoxy-, and three carboxyl functional groups with the molecular weight of approximately 449.37 U.

Clerodendrum petasites S. Moore: The therapeutic potential of phytochemicals, hispidulin, vanillic acid, verbascoside, and apigenin.

Clerodendrum petasites S. Moore has been prescribed in Thai traditional medicine for over 30 years for the treatment of ailments including asthma, inflammation, fever, cough, vomiting, and skin disorders. The phytochemicals from this plant have been identified as phenolic acids, flavones, flavone glycosides, glycosides, phenylpropanoid, and diterpenoid. The pharmacological activities both in vitro and in vivo have mostly been reported from crude extracts and not from pure compounds. This review, therefore, brings together information on the specific phytochemicals found in C. petasites in order to provide a guide to the natural bioactive compounds that are potentially used in medicines together with mechanisms underlying their pharmacological uses. All relevant information was searched for the terms of plant name, naturally-occurring compounds, and traditional uses from reliable databases, such as PubMed, Science Direct and Google Scholar, along with Thai traditional medicine textbooks. There was no specific timeline set for the search and this review selected to report only mechanisms studied by using standard compounds for their biological activities. Four dominant compounds comprising hispidulin, vanillic acid, verbascoside, and apigenin, have robust evidence to support their medical effects. Hispidulin was discovered to be possibly responsible for the treatment of cancer, osteolytic bone diseases, and neurological diseases. Other compounds were also found to tentatively support the uses in inflammation and neurological diseases. C. petasites extracts may provide an option as complimentary medicine, and or for the pharmacological development of new drugs derived from the phytochemicals found within.

Anti-osteoporotic effects of syringic acid and vanilic acid in the extracts of waste beds after mushroom cultivation.

In recent years, the number of patients with osteoporosis has increased as population grows older. Therefore, the chemoprevention of osteoporosis by better nutrition is important. White-rot fungi degrades milled wood lignin for growth and development. This degradation results in the formation of phenolic compounds such as syringic acid (SA) and vanillic acid (VA). In the artificial culture of edible mushrooms using a mushroom bed, the disposal of waste beds after mushroom cultivation is an important issue. The present study investigated the presence and amount of both SA and VA in the discarded waste beds after mushroom cultivation. The extracts from waste beds after cultivation of shiitake mushrooms, Lentinula edodes; buna shimeji, Hypsizygus marmoreus; maitake, Grifola frondosa; king trumpet mushrooms, Pleurotus eryngii; and butterscotch mushrooms, Pholiota microspora were analyzed using high performance liquid chromatography. Although the content of SA and VA was considerably different among the mushrooms, SA and VA were present in extracts obtained from all the waste beds. We also demonstrated that SA and VA exert their anti-osteoporotic effect independently of the estrogen receptor-mediated pathway using murine monocytic RAW264.7 cells, ovariectomized mice, and human breast cancer MCF-7 cells. Thus, these results suggest that the extracts are effective sources of SA and VA, which are effective in preventing osteoporosis.