Innovative orthogonal two-dimensional reversed-phase liquid chromatography × supercritical fluid chromatography with a phenyl/tetrazole stationary phase for the preparative isolation of diarylheptanoids.

In this investigation, we successfully isolated and purified natural diarylheptanoids using an orthogonal offline two-dimensional RPLC × SFC approach, employing only the phenyl/tetrazole stationary phase. First, a styrene-divinylbenzene matrix medium pretreatment liquid chromatography system effectively processed chlorophyll-containing plant extract solution with a recovery rate of 33.8 %, obviating the need for concentration steps. Subsequently, an offline two-dimensional RPLC × SFC employing only the phenyl/tetrazole stationary phase achieved a remarkable 96.38 % orthogonality and was established and utilized in the preparative separation and purification of natural products. Finally, the constructed single stationary phase highly orthogonal RPLC × SFC system was successfully applied in the preparative separation and purification of natural diarylheptanoids from the Saxifraga tangutica target fraction and yielded four diarylheptanoids with purities exceeding 95 %.

Exploring the hypolipidemic effects of bergenin from Saxifraga melanocentra Franch: mechanistic insights and potential for hyperlipidemia treatment.

OBJECTIVE: The goal of this study was to explore the hypolipidemic effects of bergenin extracted from Saxifraga melanocentra Franch (S. melanocentra), which is a frequently utilized Tibetan medicinal plant known for its diverse bioactivities. Establishing a quality control system for black stem saxifrage is crucial to ensure the rational utilization of its medicinal resources. METHODS: A one-step polyamide medium-pressure liquid chromatography technique was applied to isolate and prepare bergenin from a methanol extract of S. melanocentra. A zebrafish model of hyperlipidemia was used to investigate the potential hypolipidemic effects of bergenin. RESULTS: The results revealed that bergenin exhibited substantial hypo efficacy in vivo. Specifically, bergenin significantly reduced the levels of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-c) while simultaneously increasing high-density lipoprotein cholesterol (HDL-c) levels. At the molecular level, bergenin exerted its effects by inhibiting the expression of FASN, SREBF1, HMGCRα, RORα, LDLRα, IL-1ß, and TNF while promoting the expression of IL-4 at the transcriptional level. Molecular docking analysis further demonstrated the strong binding affinity of bergenin to proteins such as FASN, SREBF1, HMGCRα, RORα, LDLRα, IL-4, IL-1ß, and TNF. CONCLUSIONS: Findings indicate that bergenin modulates lipid metabolism by regulating lipid and cholesterol synthesis as well as inflammatory responses through signaling pathways associated with FASN, SREBF1, and RORα. These results position bergenin as a potential candidate for the treatment of hyperlipidemia.

Investigation of phylogenetic relationships within Saxifraga diversifolia complex (Saxifragaceae) based on restriction-site associated DNA sequence markers.

Subsect. Hirculoideae Engl. & Irmsch., belonging to Saxifraga sect. Ciliatae Haw., has high species richness. It can be divided into S. diversifolia, S. pseudohirculus, and S. sinomontana complexes based on morphological characteristics. The species with prominent leaf veins on the posterior leaf edge were placed in the S. diversifolia complex, which is mainly distributed on the eastern and southern margins of the Qinghai-Tibetan Plateau. In this study, 53 samples, representing 15 of the 33 described species in the S. diversifolia complex, were sequenced using the Restriction-site Associated DNA Sequence (RAD-seq) technique. A total of 111,938 high-quality SNP loci were screened to investigate the phylogenetic relationships within the S. diversifolia complex. The result of the neighbor-joining (NJ) tree shows that the S. diversifolia complex is a paraphyletic group. Despite of some inconsistencies as revealed by genetic structural analysis, clustering results of representative species reconstructed by both NJ and principal component analysis analyses support previous biogeographic and morphological evidences. In addition, long-distance gene flow events for 11 taxa were detected in the S. diversifolia complex, respectively from S. implicans 1 to S. implicans 2, S. diversifolia and S. maxionggouensis, and from S. maxionggouensis to S. nigroglandulifera. These findings may improve our comprehension of the phylogeny, classification, and evolution of the S. diversifolia complex.

Seven New Phenylhexanoids with Antioxidant Activity from Saxifraga umbellulata var. pectinata.

Seven new phenylhexanoids, (S)-(+)-3,4-dihydroxy-11-methoxyphenylhex-9-one (1), (E) 3,4-dihydroxy-phenylhex-10-en-9-one (2), (E)-4-hydroxyphenylhex-10-en-9-one (3), (R)-(-)-3,4,11-trihydroxyphenylhex-9-one 11-O-ß-d-glucopyranoside (4), (R)-(-)-4,11-dihydroxyphenylhex-9-one 11-O-ß-d-glucopyranoside (5), phenylhex-4,9,11-triol 11-O-ß-d-glucopyranoside (6), and 9-O-acetyl-phenylhex-4,9,11-triol 11-O-ß-d-glucopyranoside (7), were isolated and identified from Tibetan medicine Saxifraga umbellulata var. pectinate. The antioxidant activities of these compounds were evaluated using the DPPH and ABTS radical scavenging experiments. In the ABTS experiment, compounds 1 (IC50 13.99 ± 2.53 µM) and 2 (IC50 13.11 ± 0.94 µM) exhibited significantly better antioxidant activity than L-ascorbic acid (IC50 23.51 ± 0.44 µM).

Rapid Virucidal Activity of Japanese Saxifraga Species-Derived Condensed Tannins against SARS-CoV-2, Influenza A Virus, and Human Norovirus Surrogate Viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), and norovirus are global threats to human health. The application of effective virucidal agents, which contribute to the inactivation of viruses on hands and environmental surfaces, is important to facilitate robust virus infection control measures. Naturally derived virucidal disinfectants have attracted attention owing to their safety and eco-friendly properties. In this study, we showed that multiple Japanese Saxifraga species-derived fractions demonstrated rapid, potent virucidal activity against the SARS-CoV-2 ancestral strain and multiple variant strains, IAV, and two human norovirus surrogates: feline calicivirus (FCV) and murine norovirus (MNV). Condensed tannins were identified as active chemical constituents that play a central role in the virucidal activities of these fractions. At a concentration of 25 µg/mL, the purified condensed tannin fraction Sst-2R induced significant reductions in the viral titers of the SARS-CoV-2 ancestral strain, IAV, and FCV (reductions of ≥3.13, ≥3.00, and 2.50 log10 50% tissue culture infective doses [TCID50]/mL, respectively) within 10 s of reaction time. Furthermore, at a concentration of 100 µg/mL, Sst-2R induced a reduction of 1.75 log10 TCID50/mL in the viral titers of MNV within 1 min. Western blotting and transmission electron microscopy analyses revealed that Sst-2R produced structural abnormalities in viral structural proteins and envelopes, resulting in the destruction of viral particles. Furthermore, Saxifraga species-derived fraction-containing cream showed virucidal activity against multiple viruses within 10 min. Our findings indicate that Saxifraga species-derived fractions containing condensed tannins can be used as disinfectants against multiple viruses on hands and environmental surfaces. IMPORTANCE SARS-CoV-2, IAV, and norovirus are highly contagious pathogens. The use of naturally derived components as novel virucidal/antiviral agents is currently attracting attention. We showed that fractions from extracts of Saxifraga species, in the form of a solution as well as a cream, exerted potent, rapid virucidal activities against SARS-CoV-2, IAV, and surrogates of human norovirus. Condensed tannins were found to play a central role in this activity. The in vitro cytotoxicity of the purified condensed tannin fraction at a concentration that exhibited some extent of virucidal activity was lower than that of 70% ethanol or 2,000 ppm sodium hypochlorite solution, which are popular virucidal disinfectants. Our study suggests that Saxifraga species-derived fractions containing condensed tannins can be used on hands and environmental surfaces as safe virucidal agents against multiple viruses.

Chloroplast phylogenomics and the taxonomy of Saxifraga section Ciliatae (Saxifragaceae).

Comprising ca. 200 species, Saxifraga sect. Ciliatae is the most species-rich section of Saxifraga s.str., whose center of diversity is in the Tibeto-Himalayan region. The infra-sectional classification of sect. Ciliatae is still in debate due to the high level of species richness, as well as remarkable variations of habitat, morphology, physiology and life cycles. Subdivisions of this section proposed in various taxonomic systems have not been adequately tested in previous phylogenetic studies, partly due to low taxonomic sampling density, but also to the use of few DNA markers. In order to achieve a more robust infra-sectional classification of sect. Ciliatae, complete chloroplast genomes of 94 taxa from this section were analyzed, of which 93 were newly sequenced, assembled and annotated. The length of the 94 plastomes of sect. Ciliatae taxa range from 143,479 to 159,938 bp, encoding 75 to 79 unique protein-coding genes (PCGs). Analyses of the 94 plastomes revealed high conservation in structural organization, gene arrangement, and gene content. Gene loss and changes of IR boundaries were detected but in extremely low frequency. The molecular phylogenetic tree from concatenated PCGs and complete chloroplast genome sequences exhibited high resolution and support values and confirms that sect. Ciliatae is monophyletic. Three well-supported clades were revealed within the section that agree relatively well with the subsectional taxonomy of Gornall (1987), but some minor modifications should be made. Firstly, the monotypic subsection Cinerascentes should be abandoned and its constituent species, S. cinerascens, assigned to subsect. Gemmiparae. Secondly, subsections Rosulares and Serpyllifoliae should be merged and become subsect. Rosulares. Section Ciliatae thus comprises: subsect. Hirculoideae Engl. & Irmsch.; subsect. Rosulares Gornall; subsect. Gemmiparae Engl. & Irmsch.; subsect. Flagellares (C. B. Clarke) Engl. & Irmsch. and subsect. Hemisphaericae (Engl. & Irmsch.) Gornall.

Homoploid hybrids are common but evolutionary dead ends, whereas polyploidy is not linked to hybridization in a group of Pyrenean saxifrages.

Hybridization and polyploidy are major forces in plant evolution. Homoploid hybridization can generate new species via hybrid speciation, or modify extant evolutionary lineages through introgression. Polyploidy enables instantaneous reproductive isolation from the parental lineage(s) and is often coupled with evolutionary innovations, especially when linked to hybridization. While allopolyploidy is a well-known and common mechanism of plant speciation, the evolutionary role of autopolyploidy might have been underestimated. Here, we studied the saxifrages of Saxifraga subsection Saxifraga in the Pyrenees, which easily hybridise and include polyploid populations of uncertain origin, as a model to unravel evolutionary consequences and origin of hybridization and polyploidy. Additionally, we investigate the phylogenetic relationship between the two subspecies of the endemic S. pubescens to ascertain whether they should rather be treated as different species. For these purposes, we combined ploidy-informed restriction associated DNA analyses, plastid DNA sequences and morphological data on a comprehensive population sample of seven species. Our results unravel multiple homoploid hybridization events at the diploid level between different species pairs, but with limited evolutionary impact. The ploidy-informed analyses reveal that all tetraploid populations detected in the present study belong to the widespread alpine species S. moschata. Although of autopolyploid origin, they are to some extent morphologically differentiated and underwent a different evolutionary pathway than their diploid parent. However, the high plastid DNA diversity and the internal structure within eastern and western population groups suggest multiple origins of the polyploids. Finally, our phylogenetic analyses show that S. pubescens and S. iratiana are clearly not sister lineages, and should consequently be considered as independent species.

Molecular and quantitative genetic variation within and between populations of the declining grassland species Saxifraga granulata.

Formerly common plant species are expected to be particularly susceptible to recent habitat fragmentation. We studied the population genetics of 19 recently fragmented Saxifraga granulata populations (max. distance 61 km) in Luxembourg and neighboring Germany using RAPD markers and a common garden experiment. We assessed (1) the relationships between plant fitness, quantitative genetic variation, molecular genetic variation, and population size; and (2) the relative importance of genetic drift and selection in shaping genetic variation. Molecular genetic diversity was high but did not correlate with population size, habitat conditions, or plant performance. Genetic differentiation was low (F ST = 0.079 ± 0.135), and there was no isolation by distance. Longevity, clonality, and the long-lived seed bank of S. granulata may have prevented strong genetic erosion and genetic differentiation among populations. However, genetic distinctness increased with decreasing genetic diversity indicating that random genetic drift occurred in the studied populations. Quantitative and molecular genetic variations were correlated, and their differentiation (Q ST vs. F ST) among S. granulata populations was similar, suggesting that mainly random processes have shaped the quantitative genetic differentiation among populations. However, pairwise quantitative genetic distances increased with geographic and climatic distances, even when adjusted for molecular genetic distances, indicating diversifying selection. Our results indicate that long-lived clonal species may be buffered at least temporarily against the negative effects of fragmentation. The relationship between quantitative genetic and geographic distance may be a more sensitive indicator of selection than Q ST-F ST differences.

Comparative Analysis of Chloroplast Genomes within Saxifraga (Saxifragaceae) Takes Insights into Their Genomic Evolution and Adaption to the High-Elevation Environment.

Saxifraga species are widely distributed in alpine and arctic regions in the Northern hemisphere. Highly morphological diversity within this genus brings great difficulties for species identification, and their typical highland living properties make it interesting how they adapt to the extreme environment. Here, we newly generated the chloroplast (cp) genomes of two Saxifraga species and compared them with another five Saxifraga cp genomes to understand the characteristics of cp genomes and their potential roles in highland adaptation. The genome size, structure, gene content, GC content, and codon usage pattern were found to be highly similar. Cp genomes ranged from 146,549 bp to 151,066 bp in length, most of which comprised 130 predicted genes. Yet, due to the expansion of IR regions, the second copy of rps19 in Saxifraga stolonifera was uniquely kept. Through sequence divergence analysis, we identified seven hypervariable regions and detected some signatures of regularity associated with genetic distance. We also identified 52 to 89 SSRs and some long repeats among seven Saxifraga species. Both ML and BI phylogenetic analyses confirmed that seven Saxifraga species formed a monophyletic clade in the Saxifragaceae family, and their intragenus relationship was also well supported. Additionally, the ndhI and ycf1 genes were considered under positive selection in species inhabiting relatively high altitudes. Given the conditions of intense light and low CO2 concentration in the highland, the products of these two genes might participate in the adaptation to the extreme environment.

Screening and Isolation of Potential Anti-Inflammatory Compounds from Saxifraga atrata via Affinity Ultrafiltration-HPLC and Multi-Target Molecular Docking Analyses.

In this study, a 100 g sample of Saxifraga atrata was processed to separate 1.3 g of 11-O-(4'-O-methylgalloyl)-bergenin (Fr1) after 1 cycle of MCI GEL® CHP20P medium pressure liquid chromatography using methanol/water. Subsequently, COX-2 affinity ultrafiltration coupled with reversed-phase liquid chromatography was successfully used to screen for potential COX-2 ligands in this target fraction (Fr1). After 20 reversed-phase liquid chromatography runs, 74.1 mg of >99% pure 11-O-(4'-O-methylgalloyl)-bergenin (Fr11) was obtained. In addition, the anti-inflammatory activity of 11-O-(4'-O-methylgalloyl)-bergenin was further validated through molecular docking analyses which suggested it was capable of binding strongly to ALOX15, iNOS, ERBB2, SELE, and NF-κB. As such, the AA metabolism, MAPK, and NF-κB signaling pathways were hypothesized to be the main pathways through which 11-O-(4'-O-methylgalloyl)-bergenin regulates inflammatory responses, potentially functioning by reducing pro-inflammatory cytokine production, blocking pro-inflammatory factor binding to cognate receptors and inhibiting the expression of key proteins. In summary, affinity ultrafiltration-HPLC coupling technology can rapidly screen for multi-target bioactive components and when combined with molecular docking analyses, this approach can further elucidate the pharmacological mechanisms of action for these compounds, providing valuable information to guide the further development of new multi-target drugs derived from natural products.

Targeted isolation of 1,1-diphenyl-2-picrylhydrazyl inhibitors from Saxifraga atrata and their antioxidant activities.

Saxifraga atrata is an important traditional Tibetan medicine used to treat cough and pneumonia, and has tremendous medicinal potential. In this study, we devised a technique to separate 1,1-diphenyl-2-picrylhydrazyl inhibitors from a methanol extract of S. atrata. The material was first processed using MCI GEL CHP20P medium-pressure liquid chromatography, yielding 1.1 g of the target fraction Fr2. Subsequently, online hydrophilic interaction liquid chromatography-1,1-diphenyl-2-picrylhydrazyl assay was used to identify prospective 1,1-diphenyl-2-picrylhydrazyl inhibitors, and two 1,1-diphenyl-2-picrylhydrazyl inhibitor fractions (Fr24 and Fr25) were identified from Fr2. Then, medium-pressure preparation was continued using an XIon column to separate two 1,1-diphenyl-2-picrylhydrazyl inhibitor fractions (Fr24 and Fr25). The target compound was concentrated in fractions Fr24 and Fr25 using reverse-phase liquid chromatography during further separation procedures. Finally, the purity, structure, and 1,1-diphenyl-2-picrylhydrazyl inhibitory activity of the isolated 1,1-diphenyl-2-picrylhydrazyl inhibitors were determined. Two 1,1-diphenyl-2-picrylhydrazyl inhibitors (adenosine with the half maximal inhibitory concentration of 66.87 ± 14.33 µM and (-)-4-O-(E)-caffeoyl-l-threonic acid with the half maximal inhibitory concentration of 59.06 ± 5.02 µM) were isolated with purities exceeding 95%. The results showed that this technology is effective in the targeted separation of antioxidants from natural products.

Two Olean-27-carboxylic Acid-Type Triterpenoids as Chemical Markers Isolated from Saxifraga umbellulata.

Two olean-27-carboxylic acid-type triterpenoids (1 and 2) were isolated from Saxifraga umbellulata (Saxifragaceae), representing the first case in the chemical discoveries of genus Saxifraga. Compound 1 was determined to be a new compound named 'Saxifragic acid' based on the comprehensive spectroscopic and X-ray crystallographic analyses. Compound 2 (deacetylated saxifragic acid) is a known compound reported before, but its absolute configuration through X-ray crystallographic analyses was first described here. In addition, their cytotoxicity against five digestive human cancer cell lines (BGC-823, GBC-SD, CCC-9810, HT-29, and HepG2) and hepatoprotective activity against CCl4 -induced L-o2 cell injury in vitro were evaluated. Interestingly, UPLC-QTOFMS analysis showed that these two compounds could be used as chemical markers to discriminate between S. umbellulata and S. tangutica, both of which are used for the treatment of hepatobiliary diseases in traditional Tibetan medicine.

Preparative isolation of antioxidative gallic acid derivatives from Saxifraga tangutica using a class separation method based on medium-pressure liquid chromatography and reversed-phase liquid chromatography.

Saxifraga tangutica is widely used as a medicinal herb to treat hepatic diseases. Here, we developed a class separation method to separate gallic acid derivatives 1,1-diphenyl-2-picrylhydrazyl inhibitors from the methanol extract of Saxifraga tangutica. Firstly, an MCI GEL CHP20P medium-pressure liquid chromatography was used to pretreat the crude extract from Saxifraga tangutica (500 g) and the target sample (fraction Fr1, 1.7 g) was obtained. Then, an online reversed-phase liquid chromatography-1,1-diphenyl-2-picrylhydrazyl assay was employed for recognizing potential 1,1-diphenyl-2-picrylhydrazyl inhibitors and six 1,1-diphenyl-2-picrylhydrazyl inhibitors fractions were recognized from fraction Fr1. Subsequently, the six 1,1-diphenyl-2-picrylhydrazyl inhibitors fractions were isolated via a ReproSil-Pur C18 AQ preparative column. During the separation process, the hydrophilic liquid chromatography was used to enrich the target compounds (Fr1-3-1-1 and Fr1-3-1-2) from the fraction Fr1-3, which were hardly isolated only by one step reversed-phase liquid chromatography. Finally, six gallic acid derivatives were obtained and identified as gallic acid (Fr1-1-1), gallic acid 3-O-ß-D-glucoside (Fr1-1-2), protocatechuic acid (Fr1-2), 4-O-galloyl-(-)-shikimic acid (Fr1-3-1-1), 5-O-galloyl-(-)-shikimic acid (Fr1-3-1-2), and 3-O-galloyl-shikimic acid (Fr1-4), respectively. Thus, the present study indicated that this method was highly efficient for the preparative separation of gallic acid derivatives 1,1-diphenyl-2-picrylhydrazyl inhibitors from natural products.

Phylogeographic analysis of Saxifraga fortunei complex (Saxifragaceae) reveals multiple origins of morphological and ecological variations in the Japanese Archipelago.

Phenotypic polymorphism within a species is a notable phenomenon in evolutionary biology to understand the process of adaptive speciation and other historical events. The Saxifraga fortunei complex is a widespread herb found in East Asia. It includes several ecotypic taxa corresponding to their habitat environments. The distribution of the various ecotypes in a limited area of the Japanese Archipelago makes the species a suitable model to investigate the impact of population demographic history and natural selection on lineage diversification. Here, Sanger-based sequencing was used to estimate the divergence timeframe between populations of the Eurasian continent and Japan. Genome-wide SNPs obtained by ddRAD sequencing were used to investigate the phylogeographic origins of ecotypic taxa. The phylogenetic analyses revealed the divergence of the Japanese population from the continental population in the late Miocene. Two distinct regional clades of North and South Japan were identified; phenotypic diversification was evident only in the southern clade. The South Japan clades displayed a historical distribution expansion from north to south. The phenotypic variations appeared to have generated during the expansion. The ecotypic boundaries were incongruent with the genetic grouping. We propose that morphological and ecological specialization in Japanese populations was repeatedly generated by local natural selection.

Comparative analysis of spatial genetic structures in sympatric populations of two riparian plants, Saxifraga acerifolia and Saxifraga fortunei.

PREMISE: The genetic structure between plant populations is facilitated by the spatial population arrangement and limited dispersal of seed and pollen. Saxifraga acerifolia, a local endemic species in Japan, is a habitat specialist that is confined to waterfalls in riparian environments. Its sister species, Saxifraga fortunei, is a generalist that is widely distributed along riverbanks. Here, we examined sympatric populations of the two Saxifraga species to test whether the differences in habitat preference and colonization process influenced regional and local genetic structures. METHODS: To reveal genetic structures, we examined chloroplast microsatellite variations and genome-wide nucleotide polymorphisms obtained by genotyping by sequencing. We also estimated the gene flow among and within populations and performed landscape genetic analyses to evaluate seed and pollen movement and the extent of genetic isolation related to geographic distance and/or habitat differences. RESULTS: We found strong genetic structure in the specialist S. acerifolia, even on a small spatial scale (<1 km part); each population on a different waterfall in one river system had a completely different predominant haplotype. By contrast, the generalist S. fortunei showed no clear genetic differentiation. CONCLUSIONS: Our findings suggest that the level of genetic isolation was increased in S. acerifolia by the spatially fragmented habitat and limited seed and pollen dispersal over waterfalls. Habitat differentiation between the sister taxa could have contributed to the different patterns of gene flow and then shaped the contrasting genetic structures.

Inhibitory effect of strawberry geranium (Saxifraga stolonifera) on Toll-like receptor 2-mediated inflammatory response in human skin keratinocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Strawberry geranium (Saxifraga stolonifera [L.] Meeb) has traditionally been used as a drug to treat skin disorders in Japan. However, little is known about its physiological effects on skin keratinocytes. AIM OF THE STUDY: We investigated the anti-inflammatory effects of a strawberry geranium extract (SGE) on human skin keratinocytes. MATERIALS AND METHODS: The human keratinocyte cell line, HaCaT, was treated with SGE, and then stimulated with tumor necrosis factor (TNF)-α. The expression of 207 genes related to the innate immune system was analyzed using DNA microarrays. The effect of SGE on the target proteins in primary human epidermal keratinocytes was confirmed by quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The mechanisms of action and active components involved in the suppressive effect of SGE were evaluated by fractionation and a transcription assay. RESULTS: The microarray analysis revealed that SGE primarily suppressed Toll-like receptor (TLR)2 expression through procyanidin B2 3,3'-di-O-gallate, without TLR2 downregulation, in TNF-α-stimulated HaCaT cells. SGE suppressed TLR2 expression and interleukin (IL)-8 production induced by TLR2 ligands in primary human epidermal keratinocytes and HaCaT cells. Multiple components downregulating TLR2 expression suppressed the Sp1 activity. CONCLUSIONS: We identified a novel physiological function of SGE, which suppresses TLR2 expression and TLR2-mediated inflammation in human skin keratinocytes. This study provides significant insights into the anti-inflammatory effect of SGE in human skin.

Large-scale preparative isolation of bergenin standard substance from Saxifraga atrata using polyamide coupled with MCI GEL® CHP20P as stationary phases in medium pressure chromatography.

In this study, polyamide and MCI GEL® CHP20P were employed as stationary phases in medium pressure chromatography (MPC) for the efficient preparative separation of bergenin from Saxifraga atrata. Ethanol-water, methanol-water, and acetonitrile-water mobile phases all showed good enrichment capacity for bergenin fraction when polyamide was used as a stationary phase. After 5 cycles of polyamide MPC using acetonitrile/water, 1.2 g of bergenin fraction was isolated from 180 g Saxifraga atrata herb. Further purification of this fraction was conducted using MCI GEL® CHP20P styrene-divinylbenzene beads. The bergenin fraction was separated into two fractions, and after three runs of MPC, 714.2 mg of bergenin with purity above 99% was obtained. The results demonstrate that the combination of polyamide and styrene-divinylbenzene MPC can be utilized for preparative isolation of compounds from natural products with high yield and purity.

Targeted isolation of 1,1-diphenyl-2-picrylhydrazyl inhibitors from Saxifraga atrata using medium- and high- pressure liquid chromatography combined with online high performance liquid chromatography-1,1-diphenyl-2- picrylhydrazyl detection.

Traditional Tibetan medicine (TTM) is a valuable source of novel therapeutic lead molecules inspired by natural products (NPs). The health benefits of Saxifraga atrata are well documented in TTM, but reports on its chemical composition are limited, most likely due to the complicated purification process. Herein, target separation and identification of 4 main radical scavenging compounds from the methanolic extract of S. atrata was were performed using medium- and high-pressure liquid chromatography coupled with online HPLC-DPPH detection. The sample was pretreated using medium pressure liquid chromatography with MCI GELⓇ CHP20P styrene-divinylbenzene beads as a stationary phase, yielding 1.4 g of the target DPPH inhibitors (Fr4, 11.9% recovery). The compounds were further purified and isolated using HPLC on RP-C18 (ReproSil-Pur C18 AQ) followed by HILIC (Click XIon) column separation, resulting in 2.8 mg of fraction Fr4-1-1, 6.8 mg of fraction Fr4-2, 244.9 mg of the Fr4-3-1 sample, and 38.3 mg of Fr4-4-1. The structure and purity of the target compounds were determined, and four compounds (ethyl gallate, 11-O-galloylbergenin, rutin and isoquercitrin) were isolated with >95% purity. The developed methodology is efficient for targeted isolation of high-purity radical scavengers from NP extracts and could be used for rapid identification and isolation of DPPH inhibitors from various NPs.

Saxifraga spinulosa-Derived Components Rapidly Inactivate Multiple Viruses Including SARS-CoV-2.

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), and norovirus (NV) are highly contagious pathogens that threaten human health. Here we focused on the antiviral potential of the medicinal herb, Saxifraga spinulosa (SS). Water-soluble extracts of SS were prepared, and their virus-inactivating activity was evaluated against the human virus pathogens SARS-CoV-2 and IAV; we also examined virucidal activity against feline calicivirus and murine norovirus, which are surrogates for human NV. Among our findings, we found that SS-derived gallocatechin gallate compounds were capable of inactivating all viruses tested. Interestingly, a pyrogallol-enriched fraction (Fr 1C) inactivated all viruses more rapidly and effectively than did any of the component compounds used alone. We found that 25 µg/mL of Fr 1C inactivated >99.6% of SARS-CoV-2 within 10 s (reduction of ≥2.33 log10 TCID50/mL). Fr 1C resulted in the disruption of viral genomes and proteins as determined by gel electrophoresis, electron microscopy, and reverse transcription-PCR. Taken together, our results reveal the potential of Fr 1C for development as a novel antiviral disinfectant.

Preparative isolation of arylbutanoid-type phenol [(-)-rhododendrin] with peak tailing on conventional C18 column using middle chromatogram isolated gel column coupled with reversed-phase liquid chromatography.

Reversed-phase liquid chromatography coupled with middle chromatogram isolated gel column was employed for the efficient preparative separation of the arylbutanoid-type phenol [(-)-rhododendrin] from Saxifraga tangutica. Universal C18 (XTerra C18) and XCharge C18 columns were compared for (-)-rhododendrin fraction analysis and preparation. Although tailing and overloading occurred on the XTerra C18 column, the positively charged reversed-phase C18 column (XCharge C18) overcame these drawbacks, allowing for favorable separation resolution, even when loading at a on a preparative scale (3.69 mg per injection). The general separation process was as follows. First, 365.0 mg of crude (-)-rhododendrin was enriched from 165 g Saxifraga tangutica extract via a middle chromatogram isolated gel column. Second, separation was performed on an XTerra C18 preparative column, from which 73.8 mg of the target fraction was easily obtained. Finally, the 24.0 mg tailing peak of (-)-rhododendrin on XTerra C18 column was selectively purified on the XCharge C18 analytical column. These results demonstrate that the tailing nonalkaloid peaks can be effectively used for preparative isolation on XCharge C18 columns.