Anneslea fragrans Wall. ameliorates ulcerative colitis via inhibiting NF-κB and MAPK activation and mediating intestinal barrier integrity.

ETHNOPHARMACOLOGICAL RELEVANCE: Anneslea fragrans Wall. is traditionally used as a folk medicine in treating indigestion, fever, dysentery, diarrhea, and liver inflammation in China, Vietnam and Cambodia. However, its anti-inflammatory activity and mechanism under a safety therapeutic dose as well as the main chemical components have not yet been fully investigated. AIM OF THE STUDY: This study aimed to explore the therapeutic effect and possible molecular mechanisms of aqueous-methanol extract (AFE) of A. fragrans leaves on dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) mice and illustrate its potent anti-inflammatory chemical compounds. MATERIALS AND METHODS: The AFE was obtained and then analyzed by high performance liquid chromatography (HPLC). Phytochemical investigation on the AFE was carried out to isolate and characterize its major components. The acute toxicity test was performed to provide the safety information of AFE. Subsequently, the protective effect of AFE on DSS-induced UC was evaluated by physiological changes, histopathological and immunohistochemical analysis, and the expressions of antioxidant enzyme, pro-inflammatory cytokines and anti-inflammatory cytokines. The expressions of target proteins in nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) were determined by western blot analysis. The tight junction (TJ) proteins in colon tissue were performed by immunohistochemical technique for evaluating the intestinal barrier integrity. RESULTS: HPLC guided isolation of AFE resulted into two dihydrochalcones, which were elucidated as vacciniifolin (1) and confusoside (2). Acute toxicity evaluation revealed that median lethal dose (LD50) of AFE was greater than 5000 mg/kg. Furthermore, AFE significantly attenuated ulcerative colitis symptoms, suppressed myeloperoxidase activity, and increased the expression of superoxide dismutase and glutathione. AFE treatment could also reduce the levels of tumor necrosis factor-α, interleukin-1ß, and interleukin-6 and increase the levels of interleukin-4 and interleukin-10 in colon tissues and serum of DSS-induced UC mice. In addition, AFE significantly increased the expression of zonula occludens-1, occludin and claudin-1, and inhibited the phosphorylation of target protein of the NF-κB and MAPK signaling pathways in colon tissue. CONCLUSION: Dihydrochalcone glycosides are the major chemical constituents in AFE. AFE ameliorated DSS-induced UC in mice by inhibiting the inflammatory response via modulation of NF-κB and MAPK pathways and maintaining the intestinal barrier function, indicating that the plant A. fragrans could be used as a therapeutic candidate for ulcerative colitis.

A natural complex product Yaocha reduces uric acid level in a live zebrafish model.

INTRODUCTION: This study was aimed to assess uric acid (UA)-lowering effect and its possible mechanisms of a natural complex product Yaocha in a live zebrafish model. METHODS: The zebrafish high UA model was established by feeding 5 dpf zebrafish with both an uricase inhibitor potassium oxonate at 10 mM and an UA synthesis precursor xanthine sodium at 0.5 mM for 24 h. Yaocha was administered to the high UA zebrafish through soaking at 3 various concentrations, with allopurinol as a positive control. UA level, xanthine oxidase (XOD) activity, and mRNA expression of hypoxanthine guanine-phosphoribosyltransferases transferase (HPRT1) and organic anion transporter 1 (OAT1) were measured. RESULTS: Yaocha effectively reduced UA level and inhibited xanthine oxidase (XO) activity in the high UA zebrafish. Yaocha could be a potential therapeutics for hyperuricemia through up-regulating HPRT1 and OAT1 gene expression and suppressing XO activity. DISCUSSION: These results suggested that Yaocha hold a potential for high UA prevention and therapy, possibly through inhibiting UA production and promoting urate secretion and purine conversion.

Anticandidal Potential of Stem Bark Extract from Schima superba and the Identification of Its Major Anticandidal Compound.

Plant-derived extracts are a promising source of new drugs. Schima superba is traditionally used in China for heat clearing, detoxification, and treatment of furuncles. In this study, the anticandidal properties and mechanism of action of S. superba (SSE) were explored using a stem bark extract. SSE possessed high polyphenol and saponin contents of 256.6 ± 5.1 and 357.8 ± 31.5 µg/mg, respectively. A clear inhibition zone was observed for C. albicans growth through the disc diffusion method and the 50% inhibition of C. albicans by SSE was 415.2 µg/mL. Transcriptomic analysis in C. albicans treated with different doses of SSE was conducted through RNA-seq. Average values of 6068 genes and 20,842,500 clean reads were identified from each sample. Among these samples, 1680 and 1956 genes were differentially expressed genes (DEGs) from the SSE treatments of 0.2 and 0.4 mg/mL, respectively. C. albicans growth was inhibited by the changes in gene expression associated with the cell wall and membrane composition including the regulation of chitin degradation and ergosterol biosynthesis. This result could be reflected in the irregularly wrinkled morphology of the ruptured cell as revealed through SEM analysis. ESI-MS and NMR analyses revealed that the major compound purified from SSE was sasanquasaponin III and the 50% inhibition of C. albicans was 93.1 µg/mL. In summary, the traditional Chinese medicine S. superba can be applied as an anticandidal agent in complementary and alternative medicine.

Sasanquasaponin ΙΙΙ from Schima crenata Korth induces autophagy through Akt/mTOR/p70S6K pathway and promotes apoptosis in human melanoma A375 cells.

BACKGROUND: Melanoma is a high fatality skin cancer which lacks effective drugs. Sasanquasaponin, an important sort of constituents in theaceae, has been demonstrated to have potent anti-tumor effect in breast cancer and hepatocellular carcinoma. As a sasanquasaponin, we speculate that Sasanquasaponin III (SQS III) isolated from Schima crenata Korth may also have anti-tumor activity. PURPOSE: This study aims to investigate whether SQS III has anti-melanoma activity and examine the underlying mechanisms of SQS III against melanoma. METHODS/STUDY DESIGNS: The anti-proliferative effect of SQS III was assessed by cells viability assay. Annexin V-FITC/PI double staining assay was utilized for detection of apoptosis. Mitochondrial membrane potential and reactive oxygen species (ROS) production were detected using JC-1 and DCFH-DA assay, respectively. Autophagy was monitored using transmission electron microscopy (TEM) and GFP-LC3 transfection fluorescence analysis. Autophagosome-lysosome fusion and lysosomal degradation were determined using a GFP-LC3 & LAMP1 co-localization assay and DQ-BSA staining. Proteins related to apoptosis and autophagy were analyzed by Western blotting. RESULTS: Our results demonstrated that the SQS III exhibited potent anti-cancer activity in A375 cells by inducing both apoptosis and autophagy. In melanoma cells treated with SQS III, caspases were activated and PARP was cleaved, proving the occurrence of apoptosis. Mechanistic studies indicated that the pro-apoptosis activity of SQS III was mediated by death receptor pathway and mitochondrial dysfunction which was induced by ROS accumulation and reversed by the ROS inhibitor N-acetyl-cysteine (NAC). In addition to triggering apoptosis, SQS III may also cause autophagy in melanoma cells. Our results demonstrated that SQS III induced up-regulated expression of GFP-LC3, autophagosome-lysosomal fusion and lysosomal degradation. Additionally, the ROS accumulation was also involved in the activation of autophagy. Meanwhile, it was also found that after SQS III treatment, the expression of LC3-II was up-regulated and the AKT/mTOR signaling pathway was inhibited. The autophagy inhibitor 3-MA converted cytotoxicity and apoptosis of SQS III in A375 cells, which indicated that autophagy promoted the SQS III-induced apoptosis. CONCLUSION: SQS III showed potent anti-cancer activity by inducing apoptosis and autophagy, which provides insights into its possible use as a therapy for melanoma.

Nitrogen and phosphorus stoichiometry of Schima superba under nitrogen deposition.

In this study, leaf nitrogen (N) and phosphorus (P) stoichiometry were used as indicators of nitrogen saturation and to assess ecosystem nutrient limitations. Schima superba, a representative and widely distributed dominant evergreen broadleaf tree species of the subtropical forests in southern China, was used for this purpose. A nutrient-addition experiment and a field survey were conducted to test the responses of trees from different provenances to N deposition. The relationships between leaf N and P stoichiometry and biomass, nutrient limitation, and soil N:P were analyzed. There was a relationship between leaf N, P, N:P, soil N:P and plant dry biomass. A threshold leaf N:P ratio (16.3) divided the five provenances into different nutrient-limitation classes that were related to the soil N:P ratio or N deposition. The leaf N:P ratio provided an indication of P limitation. A higher soil P level reduced the N deposition effect on plant growth. The leaf N:P ratio of individuals from different provenances can be used as a predictor of nutrient limitation, and this was related to the soil N:P ratio.

Two new ent-kaurane diterpenes from the stems of Eurya chinensis.

Two new ent-kaurane diterpenes (1-2), together with five known analogs, were isolated from the stems of Eurya chinensis. The structures of new compounds were established by extensive analysis of mass spectrometric and 1D and 2D NMR spectroscopic data. Compound 3 exhibited noticeable anti-inflammatory activity as denoted by inhibiting LPS-induced nitric oxide (NO) production in RAW264.7 cells with an IC50 value of 7.82 µM. Compound 4 showed potent cytotoxic activity against human cancer cell lines NCI-H46, HepG2 and SW480 with IC50 values ranging from 7.45 to 8.54 µM.

Synthesis and structure-activity relationship of theasapogenol galactosides against Magnaporthe oryzae.

Camellia oleifera is expected to provide alternative aglycone to synthesize some saponins similar to that from Schima superba with inhibitory activity against Magnaporthe oryzae. Eight theasapogenol galactosides were synthesized via protection of adjacent hydroxyl groups by a benzylidene for regioselective glycosylation in the multi-hydroxyl sapogenin. Water soluble galactose chain connected far from liposoluble end was a key group in inhibiting the growth of M. oryzea unless theasapogenol was modified by two galactosyl groups or by one galactosyl group and one benzylidene group. The amphoteric characteristics of saponin such as saccharide group number, distance between bipolar groups play an important role in inhibiting mycelium growth of M. oryzae.

Effects of dissolved organic matter derived from forest leaf litter on biodegradation of phenanthrene in aqueous phase.

Dissolved organic matter (DOM) released from forest leaf litter is potentially effective for the degradation of polycyclic aromatic hydrocarbons (PAHs), yet the inherent mechanism remains insufficiently elucidated. In this study, we investigated the effects of DOM derived from Pinus elliottii and Schima superba leaf litter on the degradation of phenanthrene by the phenanthrene degrading bacterium Sphingobium sp. Phe-1. DOM from different origins and at a large range of concentrations enhanced the degradation rate of phenanthrene. DOM derived from P. elliottii leaf litter decomposed for 12 months used at a concentration of 100mg/L yielded the highest degradation rate (16.9% in 36h) and shortened the degradation time from 48h to 24h. Changes in the composition of DOM during degradation as measured by EEMs-FRI showed that proteins and tyrosine in the DOM supplied readily available nutrients that stimulated biological activity of Phe-1, increasing its growth rate and catechol 2,3-dioxygenase activity. Simultaneously, fulvic acid and humic acid in the DOM enhanced phenanthrene bioavailability by increasing the solubility and mass transfer of phenanthrene, enhancing the uptake kinetics of Phe-1, and increasing the bacteria's direct access to DOM-associated phenanthrene. Humic acid was co-metabolized by Phe-1, resulting in further stimulation of phenanthrene degradation.

Phenylacylphenol derivatives with anti-melanogenic activity from Stewartia pseudocamellia.

Three new phenylacylphenol derivatives, stewartianol (1), deoxystewartianol-4'-O-arabinoglucoside (2), and stewartianol-3-O-glucoside (3), along with nine known compounds, methylesculin (4), fraxoside (5), fraxetin (6), scopletin (7), (+)-dihydromyricetin (8), (+)-taxifolin-7-O-ß-D-glucose (9), (+)-taxifolin (10), (+)-dihydrokaempferol-7-O-ß-D-glucose (11), and 3-acetyl-ursolic acid (12), were isolated from the twigs of Stewartia pseudocamellia; commonly used as folk medicine in Korea. The structures of the isolated compounds were identified using spectroscopic analysis, including 1D, 2D NMR, MS and compared with published data. The compounds were tested for their anti-melanogenic activity in cultured murine B16 melanoma cells. Stewartianol (1) and stewartianol-3-O-glucoside (3) showed an inhibitory effect significantly on melanogenesis in a concentration-dependent manner.

Principles of Biomedical Agriculture Applied to the Plant Family Theaceae To Identify Novel Interventions for Cancer Prevention and Control.

Plant materials from the family Theaceae have been used for over a thousand years as integral components within the food systems of many globally distributed cultures and to treat a variety of human ailments. These markedly different uses remain of considerable interest in the 21st century. This perspective draws heavily from the agricultural and biomedical literature published using plant materials from the genus Camellia. Our objective is to provide a rationale and framework for broadening the scope of investigation of genera and species within Theaceae beyond Camellia sinensis to accelerate the development of a new generation of Theaceae-based pharmaceuticals/nutraceuticals and the more general enhancement of the food supply with Theaceae-containing products that affect the development of chronic diseases such as cancer. This will require a concerted effort to systematically capitalize on the rapidly growing knowledge of germplasm resources within Theaceae using metabolomic profiling in combination with in vivo and in vitro approaches. The successful translation of this research into products that affect human health will be facilitated by recognition of the agronomic factors that are critical in making hot water infusions generically referred to as tea as well as food products containing ground leaf powders.

Triterpenoid saponins from the root bark of Schima superba and their cytotoxic activity on B16 melanoma cell line.

Eight new oleanane-type triterpenoid saponins, schisusaponins A-H, along with eight known triterpenoid saponins, were isolated from the root bark of Schima superb (Theaceae). Their structures were elucidated on the basis of extensive spectroscopic analyses and chemical methods. The cytotoxicity of the new compounds against B16 melanoma cells was assessed. Among the isolated new saponins, schisusaponins C and E showed more potent effects (with IC50 values of 10.08 and 10.89 µM) than vinblastine (with an IC50 value of 19.48 µM).

Characteristic odor components of essential oils from Eurya japonica.

The chemical compositions of essential oils from the flower and aerial parts (i.e., leaf and branch) of Eurya japonica were determined and quantified using gas chromatography-mass spectrometry (GC-MS). A total of 87 and 50 compounds were detected in the oils from the flower and aerial parts, respectively. The main compounds of the flower oil were linalool (14.0%), (9Z)-tricosene (12.0%), and nonanal (7.4%). In the oil from the aerial parts, linalool (37.7%), α-terpineol (13.5%), and geraniol (9.6%) were detected. In the oils from the flower and aerial parts, 13 and 8 aroma-active compounds were identified by GC-olfactometry (GC-O) analysis, respectively. The key aroma-active compounds of the flower oil were heptanal [fatty, green, flavor dilution (FD) = 128, odor activity value (OAV) = 346], nonanal (sweet, citrus, FD = 128, OAV = 491), and eugenol (sweet, spicy, FD = 64, OAV = 62): in the oil from the aerial parts, the key aroma-active compounds were linalool (sweet, citrus, FD = 64, OAV = 95), (E)-ß-damascenone (sweet, FD = 256, OAV = 4000), and (E)-ß-ionone (floral, violet, FD = 128, OAV = 120). This study revealed that nonanal and eugenol impart the sweet, citrus, and spicy odor of the flower oil, while (E)-ß-damascenone and (E)-ß-ionone contribute the floral and sweet odor of the oil from the aerial parts.

The stimulatory effects of Stewartia koreana extract on the proliferation and migration of fibroblasts and the wound healing activity of the extract in mice.

Stewartia koreana (S. koreana) has been used in the treatment of inflammatory diseases, such as acute gastroenteritis and aches, in Korean folk medicine and has been reported to have a number of biological activities, such as anti-inflammatory activity and the promotion of angiogenesis. In this study, we aimed to determine the effects of S. koreana extract (SKE) and its components on dermal fibroblast growth and migration, and to investigate the wound healing activity of the extract in mice. In vitro experiments revealed that the numbers of SKE-treated cells increased by approximately 2.5-­ and 3.7-fold with 50 and 100 µg/ml of SKE, respectively. 5-bromo-2'-deoxy-uridine (BrdU) incorporation was also increased in the SKE-treated cells by 2.3-fold. SKE promoted the migration of human skin fibroblasts and, among the isolated compounds, hyperin increased the proliferation and migration of the fibroblasts to almost the same degree as SKE. Western blot analysis demonstrated that SKE stimulated the MEK/ERK1/2 and PI3K/Akt signaling pathways. In in vivo experiments, the SKE-treated wound lesions of mice decreased by approximately 7% in diameter after 2 days of treatment with SKE compared with the wound lesions on the 1st day of the experiment. On the 9th day of treatment, the diameter of the lesions was further reduced by approximately 83% in the SKE-treated wound areas compared with the wound areas on the 1st day of treatment. Our results demonstrate that methanol extracts of S. koreana leaves promote the proliferation and migration of skin fibroblasts and possess effective wound healing activity through the activation of the MEK/ERK1/2 and PI3K/Akt signaling pathways. Hyperin was identified as an active compound responsible for the stimulation of fibroblast growth and migration.

The comparison of DPPH-scavenging capacity and anti-inflammatory effects of phenolic compounds isolated from the stems of Stewartia koreana Nakai.

The Stewartia koreana Nakai (SK) had been used in oriental traditional medicine as a remedy for acute gastroenteritis, liver diseases, quadriplegia and pain. The antioxidant activity guided isolation 80% methyl extract from stems of SK yielded eight phenolic compounds. We evaluated the anti-oxidative and anti-inflammatory effects of these compounds via assays of 1,1-diphenyl-2-picrylhydazyl (DPPH) radicals and inhibition of nitric oxide (NO) production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells. The results demonstrated that syringaresinol (6) exhibited significant DPPH radical-scavenging activity and inhibitory effects on NO production compared with its positive controls, ascorbic acid and L-NMMA, respectively.

Antioxidant lignans and chromone glycosides from Eurya japonica.

Four new 8,8',7,2'-lignans, (+)-ovafolinin B-9'-O-ß-d-glucopyranoside (1), (-)-ovafolinin B-9'-O-ß-d-glucopyranoside (2), (+)-ovafolinin E-9'-O-ß-d-glucopyranoside (3), and (-)-ovafolinin E-9'-O-ß-d-glucopyranoside (4), two neolignans, eusiderin N (5) and (7S,8R)-3,5,5'-trimethoxy-4',7-epoxy-8,3'-neolignan-9,9'-diol-4-O-ß-d-xylopyranoside (6), and two new chromone glycosides, 5,7-dihydroxy-4H-chromen-4-one-3-O-ß-d-glucopyranoside (7) and 5,7-dihydroxy-4H-chromen-4-one-3-O-ß-d-xylopyranoside (8), together with 25 known compounds, were isolated from the stems of Eurya japonica. Structural elucidation of compounds 1-8 was established by spectroscopic methods, especially 2D NMR techniques, electronic circular dichroism data, and comparison with reported data. The isolates were evaluated for antioxidant and anti-NO production activities. Compounds 1, 2, 12-20, and 29 (ED50 23.40 µM for 1) demonstrated potent antioxidant activity compared to the positive control α-tocopherol (ED50 27.21 µM). On the other hand, compounds 1, 2, 7-9, 12-20, and 32 showed only weak anti-NO production activity when compared to the positive control quercetin.

Three new orcinol-conjugated hydrolysable tannins from the leaves of Cleyera japonica.

Three new orcinol (3-hydroxy-5-methylphenol)-conjugated hydrolysable tannins, together with two known compounds were isolated from the leaves of Cleyera japonica (CJ), and have been tentatively named cleyeratannin A (1), cleyeratannin B (2) and cleyeratannin C (3). The chemical structures of these compounds were elucidated using 1 dimensional (1D)/2D NMR and high resolution FAB-MS, and the absolute configuration was confirmed by circular dichroism (CD). To evaluate their anti-oxidative activities, 1,1-diphenyl-2-picrylhydrazyl (DPPH)/free radical scavenging activity and nitroblue tetrazolium (NBT)/superoxide anion scavenging activity were determined.

Cytotoxic triterpenoid saponins from the stems of Gordonia longicarpa.

Nine new triterpenoid saponins named longicarposides A-I (1-9), together with three known saponins (10-12), were isolated from the stems of Gordonia longicarpa. The structures of the saponins were elucidated by a combination of 1D and 2D NMR techniques, mass spectrometry, and chemical methods. They were characterized to be oleanane-type saponins with sugar moieties linked to C-3 of the aglycone. Cytotoxic activities of these saponins were evaluated against five human tumor cell lines (HCT-8, Bel-7402, BGC-823, A549, and A2780) by using the MTT in vitro assay. Compounds 5, 7, 8, 10, and 11 exhibited potent cytotoxic activity with IC50 values of 1.42-8.42 µM, while 6, 9, and 12 showed selective cytotoxic activity toward the tested cell lines.

Triterpenoid glycosides from the stems of Gordonia kwangsiensis.

Eleven oleanane-type triterpenoid glycosides, named gordonsaponins A-K, were isolated from the stems of Gordonia kwangsiensis. Their structures were elucidated by spectroscopic and chemical methods. The cytotoxic activities of all eleven were evaluated against five human tumor cell lines (HCT-8, Bel-7402, BGC-823, A549, and A2780), with only one having activity against all tested cell lines, with IC50 values ranging from 0.1 to 2.41 µM.

Inhibitory effects of a spinasterol glycoside on lipopolysaccharide-induced production of nitric oxide and proinflammatory cytokines via down-regulating MAP kinase pathways and NF-κB activation in RAW264.7 macrophage cells.

Extracts from the leaves of Stewartia koreana are known to exhibit strong anti-inflammatory activity. Investigation of bioactive compounds from S. koreana has led to the isolation of 3-O-ß-d-glucopyanosylspinasterol (spinasterol-Glc), a spinasterol glycoside. In the present study, we examined the effects of spinasterol-Glc on production of nitric oxide (NO) and proinflammatory cytokines in LPS-treated RAW264.7 macrophage cells and in mouse models. Our results showed that spinasterol-Glc inhibited the production of NO and proinflammatory cytokines such as TNF-α, IL-6 and IL-1ß in dose-dependent manners in LPS-treated RAW264.7 cells. Spinasterol-Glc inhibited the expression of iNOS and the proinflammatory cytokine genes. Spinasterol-Glc also inhibited phosphorylation of IκB-α and IKKα/ß as well as translocation of NF-κB to the nucleus. We demonstrated that spinasterol-Glc reduced transcription of the NF-κB minimal promoter and NF-κB DNA binding activity. Administration of the spinasterol-Glc significantly decreased the plasma levels of these inflammatory mediators including TNF-α, IL-6 and IL-1ß in LPS-injected mice and improved survival of septic mice with lethal endotoxemia. These results suggest that spinasterol-Glc has effective inhibitory effects on production of inflammatory mediators via inhibition of MAP kinases/NF-κB activities, and can be used as a potential anti-inflammatory agent for the prevention and treatment of inflammatory diseases.

Dihydrochalcone glucosides and antioxidant activity from the roots of Anneslea fragrans var. lanceolata.

Bioassay-guided fractionation of the roots of Anneslea fragrans var. lanceolata led to the isolation of four dihydrochalcone glucosides, davidigenin-2'-O-(6″-O-4″'-hydroxybenzoyl)-ß-glucoside (1), davidigenin-2'-O-(2″-O-4″'-hydroxybenzoyl)-ß-glucoside (2), davidigenin-2'-O-(3″-O-4″'-hydroxybenzoyl)-ß-glucoside (3), and davidigenin-2'-O-(6″-O-syringoyl)-ß-glucoside (4), and 13 known compounds. The structures were identified by means of spectroscopic analysis. Davidigenin-2'-O-(6″-O-syringoyl)-ß-glucoside (4), 1-O-3,4-dimethoxy-5-hydroxyphenyl-6-O-(3,5-di-O-methylgalloyl)-ß-glucopyranoside (5), lyoniresinol (10), and syringic acid (13) showed ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] cation radical scavenging activity, with SC(50) values of 52.6 ± 5.5, 26.0 ± 0.7, 6.0 ± 0.2, and 27.5 ± 0.6 µg/mL in 20 min, respectively. Lyoniresinol (10), isofraxidin (12), and syringic acid (13) also showed DPPH [1,1-diphenyl-2-picrylhydrazyl] radical scavenging activity, with SC(50) values of 8.4 ± 1.8, 51.6 ± 2.2, and 4.3 ± 0.7 µg/mL in 30 min, respectively.