A two-step approach for systematic identification and quality evaluation of wild and introduced Anemone flaccida Fr. Schmidt (Di Wu) based on DNA barcode and UPLC-QTOF-MS/MS.

Herbal materials have both medicinal and commercial values. As such, accurate species and content identification and verification are necessary to ensure the safe and effective use for medical and commodity purposes. Herein, we introduce a two-step approach for systematic identification and quality evaluation of wild and introduced Anemone flaccida Fr. Schmidt (aka Di Wu) using DNA barcode and ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS). To begin, a precise and rapid identification method based on internal transcribed spacer 2 (ITS2) sequence was developed to ensure the authenticity of 'Di Wu' species. Next, the major active components were fully characterized utilizing a targeted profile of oleanane-type triterpenoid saponins, which was established via UPLC-QTOF-MS/MS. As a result, 34 oleanane-type triterpenoid saponins were identified or characterized in 'Di Wu.' The qualitative and relative quantitative analysis showed obvious differences between wild and introduced 'Di Wu.' Furthermore, dynamic changes in the contents of triterpenoid saponins throughout various harvesting periods were clearly explained and mid-April was identified as the appropriate harvest time. Moreover, results indicate that the contents of five main saponins (anhuienoside E, glycosideSt-I4a, hemsgiganoside B, flaccidoside II, and hederasaponin B) are more appropriate as a quality evaluation indicator than the current quality standard. The two-step approach provides a suitable strategy to evaluate the genuine quality of wild and introduced 'Di Wu,' and can be applied to the targeted analysis of other triterpenoid saponin analogues for quality evaluation. Graphical Abstract .

Structure Elucidation of Two New Norlignans from Anemone vitifolia and Their Anti-Inflammatory Activities.

Two new norlignans together with two known phenylpropanoids were isolated from the whole herb of Anemone vitifolia. All compounds were reported from this plant for the first time. The structures of these compounds were identified by comprehensive HR-ESI-MS, 1D and 2D NMR spectroscopic data analysis and comparison with literature data. Additionally, bioactivity study results showed that two new compounds have potential anti-inflammatory activity. The plausible biosynthetic pathway for these compounds were also speculated in this article.

Anticancer Potential of Raddeanin A, a Natural Triterpenoid Isolated from Anemone raddeana Regel.

Natural compounds extracted from plants have gained immense importance in the fight against cancer cells due to their lesser toxicity and potential therapeutic effects. Raddeanin A (RA), an oleanane type triterpenoid is a major compound isolated from Anemone raddeana Regel. As an anticancer agent, RA induces apoptosis, cell cycle arrest, inhibits invasion, migration and angiogenesis in malignant cell lines as well as in preclinical models. In this systemic review, the pharmacological effects of RA and its underlying molecular mechanisms were carefully analyzed and potential molecular targets have been highlighted. The apoptotic potential of RA can be mediated through the modulation of Bcl-2, Bax, caspase-3, caspase-8, caspase-9, cytochrome c and poly-ADP ribose polymerase (PARP) cleavage. PI3K/Akt signaling pathway serves as the major molecular target affected by RA. Furthermore, RA can block cell proliferation through inhibition of canonical Wnt/ß-catenin signaling pathway in colorectal cancer cells. RA can also alter the activation of NF-κB and STAT3 signaling pathways to suppress invasion and metastasis. RA has also exhibited promising anticancer potential against drug resistant cancer cells and can enhance the anticancer effects of several chemotherapeutic agents. Overall, RA may function as a promising compound in combating cancer, although further in-depth study is required under clinical settings to validate its efficacy in cancer patients.

In Vitro Anti-proliferative Activity and Mechanism of Action of Anemone nemorosa.

Anemone nemorosa is part of the Ranunculaceae genus Anemone (order Ranunculales) which comprises more than 150 species. Various parts of the plant have been used for the treatment of numerous medical conditions such as headaches, tertian agues, rheumatic gout, leprosy, lethargy, eye inflammation as well as malignant and corroding ulcers. The Anemone plants have been found to contain various medicinal compounds with anti-cancer, immunomodulatory, anti-inflammatory, anti-oxidant and anti-microbial activities. To date there has been no reported evidence of its use in the treatment of cancer. However, due to the reported abundance of saponins which usually exert anti-cancer activity via cell cycle arrest and the induction of apoptosis, we investigated the mode of cell death induced by an aqueous A. nemorosa extract by using HeLa cervical cancer cells. Cisplatin was used as a positive control. With a 50% inhibitory concentration (IC50) of 20.33 ± 2.480 µg/mL, treatment with A. nemorosa yielded a delay in the early mitosis phase of the cell cycle. Apoptosis was confirmed through fluorescent staining with annexin V-FITC. Apoptosis was more evident with A. nemorosa treatment compared to the positive control after 24 and 48 h. Tetramethylrhodamine ethyl ester staining showed a decrease in mitochondrial membrane potential at 24 and 48 h. The results obtained imply that A. nemorosa may have potential anti-proliferative properties.

[Research progress of studies on chemical constituents and biologic activities of Anemone species].

Anemone is an important genus which was distributed widely and used to folk medicines in China. It is rich of pentacyclic triterpenoid saponins,and more than 100 kinds of pentacyclic triterpenoid saponins had been isolated and identified. Anemone has been used to treat punch injury and rheumatoid arthritis. This article reviews the latest research progress of Anemone decoction from two aspects: chemical constituents and pharmacological. It will provide reference for further research and development of Anemone.

Raddeanin A, a natural triterpenoid saponin compound, exerts anticancer effect on human osteosarcoma via the ROS/JNK and NF-κB signal pathway.

Osteosarcoma (OS) is the most frequent and high mortality primary bone tumor in the adolescent. And it is well-known for poor prognosis due to high incidence of metastasis. Raddeanin A (RA), an active component of Anemone raddeana Regel, showed potential anti-cancer activities. However, the anti-tumor effect and molecular mechanism(s) of RA on osteosarcoma are still unclear. The present research is the first in vitro and in vivo investigate systematically anticancer of RA on human osteosarcoma. Our study demonstrated that RA induced mitochondria-dependent apoptosis in osteosarcoma cell lines and markedly suppressed the metastasis of osteosarcoma cells in vitro. And, RA treatment markedly inhibits tumor growth in vivo. Further mechanism study demonstrated that RA caused a significant enhance reactive oxygen species (ROS) level to stimulate phosphorylation of JNK. Moreover, RA led to decrease of p-IκBα level in the cytosol and reduction of p65 level in the nucleus, which was associated with the inhibition of NF-κB transcriptional activity. When NF-κB signaling was inhibited by siRNA targeting p65, a significant increase in cell apoptosis activity was observed. In addition, non-toxic RA concentrations (0.25, 0.5 and 1 µM) inhibited the migration and invasion of OS by suppressing MMP-2/9 expression associated with NF-κB-dependent transcription in vitro. The silencing of p65 increased the sensitivity of the osteosarcoma cells to RA suppressed migration and invasion. These findings suggest RA induces apoptosis and inhibits metastasis in OS cells, involved in provoking ROS/JNK and inhibiting NF-κB signaling pathways. Therefore, it may be a potential anti-metastatic and anti-proliferative therapeutic agent for human osteosarcoma.

Triterpenoid Saponins from Anemone rivularis var. Flore-Minore and Their Anti-Proliferative Activity on HSC-T6 Cells.

Five previously undescribed triterpenoid saponins (1-5), along with eight known ones (6-13), were isolated from the whole plants of Anemone rivularis var. flore-minore. Their structures were clarified by extensive spectroscopic data and chemical evidence. For the first time, the lupane-type saponins (3 and 12) were reported from the Anemone genus. The anti-proliferative activity of all isolated saponins was evaluated on hepatic stellate cells (HSC-T6). Saponins 12 and 13, which possess more monosaccharides than the others, displayed potent anti-proliferative activity, with IC50 values of 18.21 and 15.56 µM, respectively.

Anti-Proliferative Effect of Triterpenoidal Glycosides from the Roots of Anemone vitifolia through a Pro-Apoptotic Way.

A cytotoxicity-guided phytochemical investigation of Anemone vitifolia roots led to the isolation of six oleanane saponins (1-6), which were reported from the species for the first time. Their structures were determined by comparing its MS and NMR data with those in literature. Compounds 1-4 showed significant inhibitory effects on the proliferation of hepatocellular carcinoma HepG2 cells with IC50 values ranging from 2.0 to 8.5 µM, compared to positive control methotrexate with IC50 value of 15.8 µM. Flow cytometry analysis revealed that compounds 1-4 exerted anti-proliferative effects through a pro-apoptotic way of hepatocellular carcinoma cells.

Chemical constituents from rhizome of Anemone amurensis.

Phytochemical investigation of the 70% EtOH extract of the rhizome of Anemone amurensis led to the isolation of two new oleanane-type triterpenoid saponins 1 and 2. Their structures were elucidated on the basis of chemical and spectral analysis, including 1D, 2D NMR data, and HR-ESI-MS. Compounds 1 and 2 were tested for cytotoxicities against two human cancer cell lines (A549 and Hep-G2). Compound 2 showed potent cytotoxicity with IC50 values of 38.53 and 66.17 µM, respectively, while compound 1 with IC50 > 100 µM.

Total saponin from Anemone flaccida Fr. Schmidt abrogates osteoclast differentiation and bone resorption via the inhibition of RANKL-induced NF-κB, JNK and p38 MAPKs activation.

Osteoclasts, bone-specialized multinucleated cells, are responsible for bone destructive diseases such as rheumatoid arthritis and osteoporosis. Natural plant-derived products have received substantial attention given their potential therapeutic and preventive activities against bone destructive diseases. In the present study, we investigated the effects of total saponin (TS) from Anemone flaccida Fr. Schmidt, on receptor activator of nuclear factor-κB ligand (RANKL)-induced in vitro osteoclast differentiation. We observed that TS concentration-dependently inhibited RANKL-induced osteoclast formation from RAW 264.7 cell and bone marrow-derived macrophages (BMMs), as well as decreased extent of actin ring formation and lacunar resorption. The RANKL-stimulated expression of osteoclast-related transcription factors were also diminished by TS. Moreover, TS blocked the RANKL-triggered TRAF6 expression, phosphorylation of mitogen-activated protein kinases (MAPKs) and IκB-α, and inhibited NF-κB p65 DNA binding activity. Furthermore, TS almost abrogated the nuclear factor of activated T cells (NFATc1) and c-Fos expression. Taken together, our results demonstrated that TS suppresses RANKL-induced osteoclast differentiation and inflammatory bone loss via the down-regulation of TRAF6 level, suppression of JNK and p38 MAPKs and NF-κB activation, and subsequent decreased expression of c-Fos and NFATc1. Therefore, TS may be a potential agent and needs to be more evaluated in vivo or in clinical trials to become a therapeutic for lytic bone diseases.

Two new triterpenoid saponins from the aerial parts of Anemone taipaiensis.

Phytochemical study on the aerial parts of Anemone taipaiensis for the first time led to the isolation of two new oleanane-type triterpenoid saponins 1 and 2, together with four known saponins (3-6). Their structures were elucidated by extensive spectroscopic analysis and chemical evidences. Saponins 2-4 exhibited cytotoxicity against human glioblastoma U251MG cell line with IC50 values ranging from 1.56 to 80.62 µM.

Two new triterpenoid saponins from rhizome of Anemone amurensis.

Two new triterpenoid saponins were isolated from the 70% ethanol extract of the rhizome of Anemone amurensis, they are oleanolic acid 28-O-ß-d-glucopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 4)-ß-d-glucopyranosyl-(1 → 6)-ß-d-glucopyranosyl ester (1) and 23,27-dihydroxy oleanolic acid 3-O-α-l-arabinopyranoside (2). The structures of 1 and 2 were elucidated on the basis of chemical and spectral analysis, including 1D and 2D NMR data and HR-ESI-MS. Compounds 1 and 2 were tested for cytotoxicities against three human cancer cell lines (A549, Hep-G2, and MCF-7). Compound 1 showed potent cytotoxicity with IC50 values of 34.76, 41.17, and 28.92 µM, respectively, while compound 2 with IC50>100 µM.

Synthesis and biological evaluation of Raddeanin A, a triterpene saponin isolated from Anemone raddeana.

First, Raddeanin A, a cytotoxic oleanane-type triterpenoid saponin isolated from Anemone raddeana REGEL, was synthesized. Stepwise glycosylation was adopted in the synthesis from oleanolic acid, employing arabinosyl, glucosyl and rhamnosyl trichloroacetimidate as donors. The chemical structure of Raddeanin A was confirmed by means of (1)H-NMR, (13)C-NMR, IR, MS and elemental analysis, which elucidated the structure to be 3-O-α-L-rhamnopyranosyl-(1→2)-ß-D-glucopyranosyl-(1→2)-α-L-arabinopyranoside oleanolic acid. Biological activity tests showed that in the range of low concentrations, Raddeanin A displayed moderate inhibitory activity against histone deacetylases (HDACs), indicating that the HDACs' inhibitory activity of Raddeanin A may contribute to its cytotoxicity.

Triterpenoid saponins from the rhizomes of Anemone flaccida and their inhibitory activities on LPS-induced NO production in macrophage RAW264.7 cells.

A new ursane-type triterpenoid saponin, flaccidoside IV (1), and three new oleanane-type triterpenoid saponins, flaccidosides V-VII (2-4), along with 17 known saponins (5-21), were isolated from the rhizomes of Anemone flaccida. The structures of the new triterpenoid saponins were determined based on spectroscopic analyses and chemical methods. All the isolated saponins were tested for their inhibitory activities on lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages, and several bisdesmosidic oleanane-type triterpenoid saponins (2, 7, and 10) showed significant inhibitory activities, which indicated they had potential anti-inflammatory activities under their noncytotoxic concentrations in vitro.

Cytotoxic oleanane-type triterpenoid saponins from the Rhizomes of Anemone rivularis var. flore-minore.

Phytochemical investigation of the n-BuOH extract of the rhizomes of Anemone rivularis var. flore-minore led to the isolation of five new oleanane-type triterpenoid saponins 1-5, together with five known saponins 6-10. Their structures were determined by the extensive use of 1D and 2D NMR experiments, along with ESIMS analyses and acid hydrolysis. The aglycone of 4 and 5 was determined as 21α-hydroxyoleanolic acid, which was reported in this genus for the first time. The cytotoxicity of these compounds was evaluated against four human cancer cell line, including HL-60 (promyelocytic leukemia), HepG2 (hepatocellular carcinoma), A549 (lung carcinoma) and HeLa (cervical carcinoma). The monodesmosidic saponins 6-8 exhibited cytotoxic activity toward all tested cancer cell lines, with IC50 values in the 7.25-22.38 µM range.

Microscopic investigation and pharmacognostic evaluation of leaves of Anemone rupicola Camb.

There is almost no scientific literature available on the phytochemistry and pharmacognosy of this plant so the basic aim of the study is to analyze pharmacognostic features of dried leaves of Anemone rupicola Camb. Morphology was studied by using the features like margins, shape, length, width, surface and so forth. For anatomical examination leaves were preserved in formalin acetic alcohol (FAA) and cutting the thin section of leaves and petiole with razor blade. Macroscopic characters of powdered drug were evaluated through organoleptic method. Qualitative phytochemical analysis was done by adopting the standard procedures. The antimicrobial activity of methanolic extract of Anemone rupicola Camb. was performed by Agar well diffusion method. The powder microscopic study showed epidermal cells, trichomes, various type of tracheids, pitted vessels, and sclerenchymatous fibers. Transverse cutting of leaves and petiole revealed the presence of different cells such as epidermis, palisade parenchyma, spongy parenchyma cells, aerenchyma, collenchyma, trichomes, and vascular bundles. The investigation of phytochemicals in distilled water, methanol and in ethanol solvent indicated the presence of various secondary compounds such as phenols, saponins, glycosides, alkaloids, tannins, cardiac glycosides, terpenoids, coumarins, and flavonoids. The methanolic extract of Anemone rupicola Camb. has potential as an antimicrobial agent. However, further studies are needed to confirm its efficacy and safety. This study can be used as powerful tool for the identification and authentication of this plant. For the purity and quality control these results will be helpful tool. This will also shed light into new areas where researchers can intervene in developing new drugs for future use. RESEARCH HIGHLIGHTS: Morphological Features of the Anemone rupicola Anatomical examination of leaves of Anemeone rupicola Microscopic features of powdered drug of leaves of Anemone rupicola Qualitative Phytochemical analysis of the leaves of Anemone rupicola.

Raddeanin A induces human gastric cancer cells apoptosis and inhibits their invasion in vitro.

Raddeanin A is one of the triterpenoid saponins in herbal medicine Anemone raddeana Regel which was reported to suppress the growth of liver and lung cancer cells. However, little was known about its effect on gastric cancer (GC) cells. This study aimed to investigate its inhibitory effect on three kinds of different differentiation stage GC cells (BGC-823, SGC-7901 and MKN-28) in vitro and the possible mechanisms. Proliferation assay and flow cytometry demonstrated Raddeanin A's dose-dependent inhibitory effect and determined its induction of cells apoptosis, respectively. Transwell assay, wounding heal assay and cell matrix adhesion assay showed that Raddeanin A significantly inhibited the abilities of the invasion, migration and adhesion of the BGC-823 cells. Moreover, quantitative real time PCR and Western blot analysis found that Raddeanin A increased Bax expression while reduced Bcl-2, Bcl-xL and Survivin expressions and significantly activated caspase-3, caspase-8, caspase-9 and poly-ADP ribose polymerase (PARP). Besides, Raddeanin A could also up-regulate the expression of reversion inducing cysteine rich protein with Kazal motifs (RECK), E-cadherin (E-cad) and down-regulate the expression of matrix metalloproteinases-2 (MMP-2), MMP-9, MMP-14 and Rhoc. In conclusion, Raddeanin A inhibits proliferation of human GC cells, induces their apoptosis and inhibits the abilities of invasion, migration and adhesion, exhibiting potential to become antitumor drug.

Bioactive oleanane-type saponins from the rhizomes of Anemone taipaiensis.

Investigation of the n-BuOH extract of the rhizomes of Anemone taipaiensis led to the isolation of five new oleanane-type triterpenoid saponins (1-5), together with seven known saponins (6-12). Their structures were determined by the extensive use of (1)D and (2)D NMR experiments along with ESIMS analyses and acid hydrolysis. The aglycone of 1, 2 and 4 was determined as siaresinolic acid, which was reported in this genus for the first time. The cytotoxicities of the saponins 1-12, prosapogenins 4a, 5a, 10a-12a and sapogenins siaresinolic acid (SA), oleanolic acid (OA), hederagenin (HE) were evaluated against five human cancer cell lines, including HepG2, HL-60, A549, HeLa and U87MG. The monodesmosidic saponins 6-8, 5a, 10a-12a and sapogenins SA, OA, HE exhibited cytotoxic activity toward all cancer cell lines, with IC50 values ranging from 2.25 to 57.28 µM. Remarkably, the bisdesmosidic saponins 1-4 and 9 showed selective cytotoxicity against the U87MG cells.

[Study on triterpenoid saponins in the rhizome of Anemone hofengensis].

OBJECTIVE: To study the triterpenoid saponins in the rhizome of Anemone hofengensis. METHODS: The constituents were separated with various chromatographic techniques and their structures were elucidated by physicochemical properties and spectral data. RESULTS: Five compounds were isolated and identified as 3-O-alpha-L-rhamnopyranosyl-(1 --> 2)-alpha-L-arabino-pyranosyl-oleanolic acid (1), 3-O-alpha-L-rhamnopyranosyl-(1 --> 2)-beta-D-glucopyranosyl-(1 --> 2)-alpha-L-rhamnopyranosyl-oleanolic acid 28-O-alpha-L-rhamnopyranosyl-(1 --> 4) -beta-D-glucopyranosyl-(1 --> 6)-beta-D-glucopyranoside (2), 3-O-alpha-L-rhamnopyranosyl-(1 --> 2) [beta-D-glucopyranosyl-(1 --> 4)]-alpha-L-rhamnopyranosyl-oleanolic acid-28-O-beta-D-glucopyranosyl-(1 --> 6)-beta-D-gluco-pyranoside (3), 3-O-beta-D-glucopyranosyl-(1 --> 2)-beta-D-xylopyranosyl-oleanolic acid 28-O-beta-D-glucopyranosyl-(1 --> 6)-beta-D-glucopyranoside (4), oleanolic acid-28-O-alpha-L-rhamnopyra-nosyl-(1 --> 4)-beta-D-glucopyranosyl-(1 --> 6)-beta-D-glucopyranoside (5). CONCLUSION: Compound 1 - 5 are isolated from this plant for the first time.

Alcohol extracts from Anemone flaccida Fr. Schmidt treat rheumatoid arthritis via inhibition of synovial hyperplasia and angiogenesis.

Anemone flaccida Fr. Schmidt, a Traditional Chinese Medicine, has been used in the treatment of rheumatoid arthritis (RA) for numerous years. However, the specific mechanisms remain to be elucidated. Thus, the present study aimed to investigate the main chemical constituents and potential mechanisms of Anemone flaccida Fr. Schmidt. The ethanol extract obtained from Anemone flaccida Fr. Schmidt (EAF) was analyzed using mass spectrometry to determine the main components and the therapeutic effects of EAF on RA were verified using a collagen­induced arthritis (CIA) rat model. Results of the present study demonstrated that synovial hyperplasia and pannus of the model rats were significantly improved following EAF treatment. Moreover, the protein expression levels of VEGF and CD31­labeled neovascularization were significantly reduced in the synovium of CIA rats following treatment with EAF, compared with those of the untreated model group. Subsequently, in vitro experiments were carried out to verify the impact of EAF on synovial proliferation and angiogenesis. Results of the western blot analysis revealed that EAF inhibited the PI3K signaling pathway in endothelial cells, which is associated with anti­angiogenesis. In conclusion, results of the present study demonstrated the therapeutic effects of Anemone flaccida Fr. Schmidt on RA and preliminarily revealed the mechanisms of this drug in the treatment of RA.