Pharmacokinetics of the main components differing between Ligustri Lucidi Fructus and its wine-steamed product / 药学学报

A quantitative analysis method for ten principal components (phenylethanol, iridoids and triterpenes) of raw Ligustri Lucidi Fructus and its wine-steamed product was developed using liquid chromatography tandem triple quadrupole mass spectrometry (LC-QQQ-MS) to study their pharmacokinetic behavior in vivo. The results of methodological investigation were in accord with the criteria of biological analysis. After a single administration to rats of the water extracts of Ligustri Lucidi Fructus and its wine-steamed product, the plasma concentration of each component at different time points was measured and the pharmacokinetic parameters were determined. The AUC0-24 h and Cmax of the phenylethanol components (salidroside, tyrosol, hydroxytyrosol) were the greatest, suggesting that these components are the main pharmacological substances of Ligustri Lucidi Fructus. In addition, the tmax values of the eight major components were even lower with administration of the wine-steamed product, suggesting that these components are rapidly absorbed. However, the tmax values of specnuezhenide and oleanolic acid were greater with administration of the wine-steamed product, indicating that these two components were more slowly absorbed. A secondary peak phenomenon of tyrosol and hydroxytyrosol were observed in two sample groups, whereas the secondary peak phenomenon of salidroside occurred only with the wine-steamed product. This result suggests that the effect of wine-steamed product could persist for a long period. Meanwhile, the relative bioavailability of specnuezhenide and oleanolic acid was greater than 100% with administration of the wine-steamed product, consistent with the Traditional Chinese Medicine theory of the wine-steamed product being more effective than the raw material. The results reveal the different pharmacokinetic parameters and relative bioavailability of each component of Ligustri Lucidi Fructus and its wine-steamed product, and also demonstrate the variation and correlation of various components in vivo and in vitro, providing an experimental basis for the selection of quality control indexes, mechanisms of processing and the metabolic rule in vivo of Ligustri Lucidi Fructus. These experiments were approved by the Ethics Committee of Institute of Basic Theory for Chinese Medicine, China Academy of Chinese Medicine Science.

Qualitative and quantitative analysis on triterpenoids in Ligustri Lucidi Fructus / 中国中药杂志

Triterpenoids are one of the most active constituents in Ligustri Lucidi Fructus, but only oleanolic acid has been mostly studied. In recent years, a growing number of studies have shown that other triterpenes from Ligustri Lucidi Fructus also have various biological activities, so it is necessary to build up a detailed profile of the triterpenoids in Ligustri Lucidi Fructus. The chromatographic separation was performed on a C_(18) column(4.6 mm×250 mm, 5 μm) with mobile phase of methanol-acetonitrile-0.2% formic acid for gradient elution. The detection wavelength was set at 210 nm, with a flow rate of 0.5 mL·min~(-1), and the column temperature of 25 ℃. The HPLC fingerprint of triterpenoids in Ligustri Lucidi Fructus was built by testing 21 batches of samples from different sources. The structures of the total 15 common chromatographic peaks were elucidated with UHPLC-ESI-Orbitrap-MS/MS technique and six of them were identified as tormentic acid, pomolic acid, maslinic acid, botulin, oleanolic acid and ursolic acid by comparison to the reference substances. Under the same chromatographic condition, four main triterpenes(podocarboxylic acid, hawthorn acid, oleanolic acid and ursolic acid) were quantified and the results of system adaptability and methodology investigation all met the requirements of content determination. Meanwhile, with oleanolic acid(A) as the internal reference substance, quantitative analysis of multi-components by single marker(QAMS) method was used to analyze the above four components. The relative correction factor of oleanolic acid(B), hawthorn acid(C) and ursolic acid(D) to oleanolic acid was f_(B/A)=1.12, f_(C/A)=1.02 and f_(D/A)=0.88, respectively, and the relative retention values of these three to oleanolic acid was RRV_(B/A)=0.46, RRV_(C/A)=0.70 and RRV_(D/A)=1.03, respectively. The contents determined by two methods were similar. In conclusion, the method built in this paper is proved to be simple, reliable and specific for the simultaneous qualitative and quantitative analysis of the triterpenoids in Ligustri Lucidi Fructus, which can lay foundation for further assays of the triterpenoids in Ligustri Lucidi Fructus and the relative products.

Qualitative and quantitative analysis on non-triterpenoids in Ligustri Lucidi Fructus / 中国中药杂志

In order to improve the quality control level of Ligustri Lucidi Fructus(LLF) and to explore the changes of chemical components after processing,the HPLC method for fingerprint and simultaneous determination of the major polar components in LLF were established. The octadecylsilane bonded silica gel was used as the stationary phase,with acetonitrile as the mobile phase A and0. 2% formic acid as the mobile phase B in a gradient elution procedure at a flow rate of 1. 0 m L·min-1. The detection wavelength was set at 280 nm and the column temperature was 25 ℃. There were 22 common peaks,20 of which were selected from the fingerprint of LLF and its wine-steamed product,respectively,and 14 chromatographic peaks were identified with reference substances. With the same chromatographic conditions,seven components were quantitatively analyzed and the results of system adaptability and methodology investigation all met the requirements of content determination. Compared with the crude LLF,the content of 5-hydroxymethyl furfural and salidroside significantly increased in wine-steamed LLF,while the contents of iridoid glycosides generally decreased. The method provided a basis for quality control of LLF and its processed products as well as the related preparations.

Content change of main components in processed Ligustrum lucidum fruits of different processing time and their characteristic quality standard based on UPLC technology / 中国中药杂志

To investigate the dynamic change law of the main components in Ligustri Lucidi Fructus during the wine-steaming process and attempt to establish the characteristic quality standard of wine-steamed Ligustri Lucidi Fructus by determining the content of salidroside and specnuezhenide using Ultra Performance Liquid Chromatography (UPLC) technology at different processing time points (12, 15, 18, 21, 24 h). The chromatographic separation was performed on Waters Acquity UPLC®BEH C₁₈ column (2.1 mm×50 mm, 1.7 μm) with acetonitrile and 0.2% formic acid aqueous solution as the mobile phase for gradient elution; and the detection wavelength was set at 280 nm; the flow rate was 0.5 mL·min⁻¹, and the column temperature was set at 40 °C. The results showed that the two components were well separated in the above conditions. The salidroside and specnuezhenide showed a good linear relationship within the range of 10.19-326 ng and 49.53-1 585 ng, respectively. Their average recovery was 103.4% and 101.7% with RSD of 0.81% and 0.79%, respectively. With the extension of processing time, the content of specnuezhenide was decreased, while salidroside was gradually increased. For the 27 batches of Ligustri Lucidi Fructus, the content of salidroside was between 0.042 5% and 0.192 4%, and that of specnuezhenide was between 0.829 7% and 5.218 0%. While for the 25 batches of wine-steamed Ligustri Lucidi Fructus, the content of the first one was between 0.229 2% and 1.045 8%, and the latter one was between 0.743 8% and 3.645 4%. As compared with Ligustri Lucidi Fructus, the ratio of specnuezhenide to salidroside was significantly decreased in the wine-steamed Ligustri Lucidi Fructus. According to the experimental results, the quality standard of Ligustri Lucidi Fructus is tentatively fixed as follows: the content of specnuezhenide shall not be less than 0.80%, and the ratio of specnuezhenide content/salidroside content (Sp/Sa) should not be smaller than 15. As for the wine-steamed ones, the content of salidroside should not be less than 0.20%, and specnuezhenide content should not be less than 0.70%; Sp/Sa should not be greater than 8. The method established in this study is simple and reliable, which could be used for the content detection of salidroside and specnuezhenide in Ligustri Lucidi Fructus samples. The characteristic quality standard established in this study could be used to distinguish the Ligustri Lucidi Fructus and wine-steamed Ligustri Lucidi Fructus.

[Mongolian medicine cha gan beng ga regulated activity of biomarker PGC-1α].

OBJECTIVE: To investigate the regulation of Cha Gan Beng Ga on the activity of biomarker PGC-1α in vivo and in vitro, and lay the foundation for studying the efficacy result of Cha Gan Beng Ga on xenograft tumor model and extracting active constituents. METHOD: (1) The coarse powder of Cha Gan Beng Ga was extracted with 70% ethanol solution through heating and refluxing, and finally was used to freeze dry powder. (2) 50 mg x kg(-1) of freeze-dried power was orally administrated to KM and C57BL/6J mice once daily, lasting for 5 consecutive days; different concentrations of extracted materials was given to non-small cell lung cells A549. (3) The expression level of PGC-1α mRNA was quantitatively determined in lung tissue of mice and non-small cell lung cells A549. RESULT: The expression levels of PGC-1α in lung tissue of different mice strains had an increasing tendency. Furthermore, the expression levels of PGC-1α in non-small cell lung cells A549 also had an increasing tendency, showing dose and time-dependent relationships. CONCLUSION: Mongolian Medicine Cha Gan Beng Ga could induce the over-expression of PGC-1α mRNA in lung tissue of mice and in non-small cell lung cells A549. The present results will lay foundation for studying the efficacy result of antitumor and active constitutes in future.

Applications of digital holographic microscopy in therapeutic evaluation of Chinese herbal medicines.

Therapeutic use of Chinese herbal medicines (CHMs) is a new approach to treat neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. The detection of soma volume and neurite outgrowth of living neurons is a highly relevant biomarker related to various application fields, including therapy efficacy and drug safety evaluation. Through the use of digital holographic microscopy (DHM), we may evaluate the therapeutic effect of CHMs in curing neurodegeneration. Panax ginseng has been used in traditional Chinese herbal medicine for centuries. In this study, DHM is applied to monitor the three-dimensional morphology change of retinoic acid-induced human neuroblastoma SH-SY5Y cells during Panax ginseng treatment. We demonstrate the capability of DHM to detect noninvasively SH-SY5Y cell apoptosis and rescue through the measurement of neuronal volume and neurite outgrowth regulation without any labeling reagent. Through DHM, we observed the phase images of the rapidly shrinking cells with decreasing soma volume and shortening neurite outgrowth during glutamate treatments. Then shrinkage in glutamate-induced cells is significantly alleviated during Panax ginseng treatment. The results through DHM are consistent with the result from MTT assay for assessing cell viability during Panax ginseng treatment. Thus, we suggest that application of DHM for measuring soma volume and neurite outgrowth of living neurons may be one appropriate therapeutic evaluation for CHMs.

Risk of treatment-related mortality with sorafenib in patients with cancer.

BACKGROUND: Fatal adverse events (FAEs) have been reported with sorafenib, a vascular endothelial growth factor receptor kinase inhibitor (VEGFR TKI). We here performed an up-to-date and detailed meta-analysis to determine the overall risk of FAEs associated with sorafenib. METHODS: Databases, including PubMed, Embase and Web of Science, and abstracts presented at the American Society of Clinical Oncology annual meetings were searched to identify relevant studies. Eligible studies included randomized controlled trials evaluating sorafenib effects in patients with all malignancies. Summary incidence rates, relative risks (RRs), and 95% confidence intervals (CIs) were calculated for FAEs. In addition, subgroup analyses were performed according to tumor type and therapy regimen. RESULTS: 13 trials recruiting 5,546 patients were included in our analysis. The overall incidence of FAEs with sorafenib was 1.99% (95%CI, 0.98-4.02%). Patients treated with sorafenib had a significantly increased risk of FAEs compared with patients treated with control medication, with an RR of 1.77 (95%CI 1.25-2.52, P=0.001). Risk varied with tumour type, but appeared independent of therapy regimen. A significantly increased risk of FAEs was observed in patients with lung cancer (RR 2.26; 95% CI 1.03-4.99; P= 0.043) and renal cancer (RR 1.84; 95% CI 1.15-2.94; P= 0.011). The most common causes of FAEs were hemorrhage (8.6%) and thrombus or embolism (4.9%). CONCLUSIONS: It is important for health care practitioners to be aware of the risks of FAEs associated with sorafenib, especially in patients with renal and lung cancer.

The anti-osteoporotic effect of velvet antler polypeptides from Cervus elaphus Linnaeus in ovariectomized rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The deer velvet antler is well known for its traditional medicinal value, and is widely used in the clinic. It is recorded in the Compendium of Materia Medica that the deer velvet antler replenishes vital essence and strengthens the bone. AIM OF THE STUDY: The goal of this study was to investigate the anti-osteoporotic effect of total velvet antler polypeptides from Cervus elaphus Linnaeus (TVAPL) on ovariectomized rats (OVX), and their possible mechanism of the action. MATERIALS AND METHODS: Wistar rats were divided into five groups: sham-operated group, OVX group, and OVX rats treated with 20, 40, or 60 mk/kg TVAPL for 12 weeks. Calcium and phosphorus levels, bone weight coefficient (BWC), bone mineral density (BMD), and bone mineral content (BMC) were evaluated. The MTT assay was used to measure the activities of interleukin-1 (IL-1) and interleukin-6 (IL-6). In addition, cartilage cells and osteoblast-like cells were exposed to TVAPL, natural velvet antler polypeptides (nVAP), and synthetic velvet antler polypeptides (sVAP), to determine their effects on cell proliferation using the tritiated thymidine incorporation assay. Finally, the enzyme-linked immunosorbent assay was used to determine the effects of nVAP and sVAP on cytokines related to bone metabolism. RESULTS: The administration of TVAPL for 12 weeks significantly reversed osteoporosis in OVX rats, thereby improving the BWC, BMD, BMC, and bone microarchitecture. IL-1 and IL-6 were significantly activated in the OVX group, and their activation was inhibited by TVAPL. In addition, nVAP and sVAP promoted the proliferation of cartilage and osteoblast-like cells (p<0.01 or p<0.001), and inhibited the secretion of IL-1α from THP-1 monocytic cells in vitro. CONCLUSION: These results suggest that TVAPL are effective in preventing bone loss in OVX rats. The effect of TVAPL on osteoporosis is due to inhibition of IL-1 and IL-6 by nVAP, and promotion of mitosis. sVAP has similar bioactivity as nVAP. Thus, both TVAPL and sVAP may be potential therapeutic agents for the treatment of postmenopausal osteoporosis.

[Effect of emodin on induction of apoptosis in jurkat cells and its possible mechanisms].

The aim of study was to investigate the effect of a traditional Chinese medicine, emodin, on proliferation and apoptosis in T lymphocytic leukemic cell line Jurkat and its mechanisms. Cell proliferation inhibition was detected by MTT assay. Cell apoptosis was measured by DNA ladder and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The expressions of related proteins and caspase family members were determined by Western blot. The results showed that emodin inhibited proliferation in Jurkat cells, with an IC50 about 20 micromol/L and induced cell apoptosis in both time-and dose-dependent manners. The expressions of proliferation-related protein C-MYC, hTERT and apoptosis-related protein BCL-2 were down-regulated in a time dependent manner after the treatment with emodin. The expressions of procaspase-3, -8 and -9 all decreased while activated caspase-3 and PARP expressions were up-regulated. It is concluded that emodin can remarkably inhibit cell proliferation and induce apoptosis in Jurkat cells. The down-regulation of proliferation-related proteins C-MYC, hTERT and apoptosis-related protein BCL-2 expressions and activation of caspase cascade may be involved in the process of apoptosis.

[Design and application of a new type minor smoke warming moxibustion cup].

OBJECTIVE: To design a new type minor smoke warming moxibustion cup for convenient use of both the physicians and the patients. METHODS: The double-deck minor smoke warming moxibustion cup is fixed on the part receiving moxibustion by vacuum adsorption; the filtration device on the upper can filtrate and adsorb the harmful substance in the moxa-smoke, and the device with a double-temperature control on the lower can sensitively regulate the moxibustion temperature. RESULTS: This new type minor smoke warming moxibustion cup has the advantages of minor smoke discharge, convenient fixation, sensitive regulation of temperature, saving moxibustion material, lasting action, safety, besides the advantages of traditional moxibustion. CONCLUSION: The new type minor smoke warming moxibustion cup can use for treatment and prevention of diseases, suitable to clinical treatment and family health care.

Effect of emodin on induction of apoptosis in jurkat cells and its possible mechanisms / 中国实验血液学杂志

The aim of study was to investigate the effect of a traditional Chinese medicine, emodin, on proliferation and apoptosis in T lymphocytic leukemic cell line Jurkat and its mechanisms. Cell proliferation inhibition was detected by MTT assay. Cell apoptosis was measured by DNA ladder and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The expressions of related proteins and caspase family members were determined by Western blot. The results showed that emodin inhibited proliferation in Jurkat cells, with an IC50 about 20 micromol/L and induced cell apoptosis in both time-and dose-dependent manners. The expressions of proliferation-related protein C-MYC, hTERT and apoptosis-related protein BCL-2 were down-regulated in a time dependent manner after the treatment with emodin. The expressions of procaspase-3, -8 and -9 all decreased while activated caspase-3 and PARP expressions were up-regulated. It is concluded that emodin can remarkably inhibit cell proliferation and induce apoptosis in Jurkat cells. The down-regulation of proliferation-related proteins C-MYC, hTERT and apoptosis-related protein BCL-2 expressions and activation of caspase cascade may be involved in the process of apoptosis.

Design and application of a new type minor smoke warming moxibustion cup / 中国针灸

<p><b>OBJECTIVE</b>To design a new type minor smoke warming moxibustion cup for convenient use of both the physicians and the patients.</p><p><b>METHODS</b>The double-deck minor smoke warming moxibustion cup is fixed on the part receiving moxibustion by vacuum adsorption; the filtration device on the upper can filtrate and adsorb the harmful substance in the moxa-smoke, and the device with a double-temperature control on the lower can sensitively regulate the moxibustion temperature.</p><p><b>RESULTS</b>This new type minor smoke warming moxibustion cup has the advantages of minor smoke discharge, convenient fixation, sensitive regulation of temperature, saving moxibustion material, lasting action, safety, besides the advantages of traditional moxibustion.</p><p><b>CONCLUSION</b>The new type minor smoke warming moxibustion cup can use for treatment and prevention of diseases, suitable to clinical treatment and family health care.</p>

[Multiple central clinical studies on the needling method for regulating wei and strengthening brain for treatment of insomnia].

OBJECTIVE: To observe the clinical therapeutic effect of the needling method for regulating wei and strengthening brain on insomnia. METHODS: Two hundred cases of insomnia were randomly divided into a test group and a control group, 100 cases in each group. The test group were treated with the needling method for regulating wei and strengthening brain with Baihui (GV 20), Dazhui (GV 14), Shenmai (BL 62), Zhaohai (KI 6) and ear points Yuanzhong, Shenmen selected; and in the control group, Sishencong (EX-HN 1), Shenmen (HT 7) and Sanyinjiao (SP 6) were selected. Acupuncture was given once daily for 15 days. Pittsburgh sleep quality index (PSQI) was used for scoring before and after treatment. RESULTS: The total effective rate was 89.0% in the test group and 65.0% in the control group with a very significant difference between the two groups (P<0.01); the difference of PSQI scores before and after was -9.15+/-5.68 in the test group and -5.64+/-5.73 in the control group, with a very significant difference before and after treatment in the two groups (P<0.01). CONCLUSION: The therapeutic effect of the needling method for regulating wei and strengthening brain on insomnia is better than that of normal needling method.

Multiple central clinical studies on the needling method for regulating wei and strengthening brain for treatment of insomnia / 中国针灸

<p><b>OBJECTIVE</b>To observe the clinical therapeutic effect of the needling method for regulating wei and strengthening brain on insomnia.</p><p><b>METHODS</b>Two hundred cases of insomnia were randomly divided into a test group and a control group, 100 cases in each group. The test group were treated with the needling method for regulating wei and strengthening brain with Baihui (GV 20), Dazhui (GV 14), Shenmai (BL 62), Zhaohai (KI 6) and ear points Yuanzhong, Shenmen selected; and in the control group, Sishencong (EX-HN 1), Shenmen (HT 7) and Sanyinjiao (SP 6) were selected. Acupuncture was given once daily for 15 days. Pittsburgh sleep quality index (PSQI) was used for scoring before and after treatment.</p><p><b>RESULTS</b>The total effective rate was 89.0% in the test group and 65.0% in the control group with a very significant difference between the two groups (P<0.01); the difference of PSQI scores before and after was -9.15+/-5.68 in the test group and -5.64+/-5.73 in the control group, with a very significant difference before and after treatment in the two groups (P<0.01).</p><p><b>CONCLUSION</b>The therapeutic effect of the needling method for regulating wei and strengthening brain on insomnia is better than that of normal needling method.</p>

[Design and application of a cone-shaped warming-acupuncture-moxibustion apparatus].

To design a warming acupuncture-moxibustion apparatus suitable to warming acupuncture therapy and easily attaching to the filiform needle handle. The apparatus consists of a container for moxa, a tray and regulative clips. The container for moxa is for store of moxa-wool without bottom and it can prevent moxa fire and ash from dropping and burning the skin of the patient or the clothes; the tray with holes on it can serve as a support of the moxa in the container, and can control the moxa burning and heat radiation; the clips can adjust the distance between the cone and the skin. With the advantages of handy material, easy installation, lower cost, and convenient operation, the apparatus is worthy of clinical application and popularization.

Design and application of a cone-shaped warming-acupuncture-moxibustion apparatus / 中国针灸

To design a warming acupuncture-moxibustion apparatus suitable to warming acupuncture therapy and easily attaching to the filiform needle handle. The apparatus consists of a container for moxa, a tray and regulative clips. The container for moxa is for store of moxa-wool without bottom and it can prevent moxa fire and ash from dropping and burning the skin of the patient or the clothes; the tray with holes on it can serve as a support of the moxa in the container, and can control the moxa burning and heat radiation; the clips can adjust the distance between the cone and the skin. With the advantages of handy material, easy installation, lower cost, and convenient operation, the apparatus is worthy of clinical application and popularization.

[Study on extraction technology for extract and flavonoids in Chrysanthemum morifolium by orthogonal design].

OBJECTIVE: To ascertain extraction technology condition for extract and flavonoids from Chrysanthum morifoliwn. METHOD: The optimizing ultrasonic extraction condition on the basis of extractive yield and flavonoids were determined by orthogonal design. RESULT: The order of factors which affected the flavonoid extraction was extraction times > ethanol concentration > ultrasonic time > solvent quantity. CONCLUSION: The optimum ultrasonic extractions are A2B3C3D3. Compared with traditional extraction, ultraction method is timesaving, simple to operate, stable and need not be heated.

[Study on ultrasonic extraction techniques of flavonoids in Chrysanthemum morifolium].

OBJECTIVE: To optimize the ultrasonic extraction condition for flavonoids from Chrysanthemum morifolium. METHOD: The extraction rate of flavonoids optimized condition (ethanol concentration, ultrasonic time, solvent quantity and extraction times) was determined by orthogonal design. UV-Spectrophotometry was used for the determination. RESULT: The order of factors to affect the flavonoid extraction was ethanol concentration > extraction times > solvent quantity > ultrasonic time. CONCLUSION: The optimum ultrasonic extractions were: A2 B2 C3 D3. Compared with traditional extraction, ultraction method can save time, be easy to operate, improve extraction rates and need no heating.

Study on extraction technology for extract and flavonoids in Chrysanthemum morifolium by orthogonal design / 中国中药杂志

<p><b>OBJECTIVE</b>To ascertain extraction technology condition for extract and flavonoids from Chrysanthum morifoliwn.</p><p><b>METHOD</b>The optimizing ultrasonic extraction condition on the basis of extractive yield and flavonoids were determined by orthogonal design.</p><p><b>RESULT</b>The order of factors which affected the flavonoid extraction was extraction times > ethanol concentration > ultrasonic time > solvent quantity.</p><p><b>CONCLUSION</b>The optimum ultrasonic extractions are A2B3C3D3. Compared with traditional extraction, ultraction method is timesaving, simple to operate, stable and need not be heated.</p>

Study on ultrasonic extraction techniques of flavonoids in Chrysanthemum morifolium / 中国中药杂志

<p><b>OBJECTIVE</b>To optimize the ultrasonic extraction condition for flavonoids from Chrysanthemum morifolium.</p><p><b>METHOD</b>The extraction rate of flavonoids optimized condition (ethanol concentration, ultrasonic time, solvent quantity and extraction times) was determined by orthogonal design. UV-Spectrophotometry was used for the determination.</p><p><b>RESULT</b>The order of factors to affect the flavonoid extraction was ethanol concentration > extraction times > solvent quantity > ultrasonic time.</p><p><b>CONCLUSION</b>The optimum ultrasonic extractions were: A2 B2 C3 D3. Compared with traditional extraction, ultraction method can save time, be easy to operate, improve extraction rates and need no heating.</p>