Hunan Academy of Chinese Medicine

Changsha, China

Hunan Academy of Chinese Medicine

Changsha, China
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Huang H.-S.,Hunan Academy of Chinese Medicine | Gong W.,Beijing Institute of Pharmacology and Toxicology | Zhang H.,Beijing Institute of Pharmacology and Toxicology | Zheng X.-L.,Shanghai Asiapioneer Pharmaceuticals | Mei X.-G.,Beijing Institute of Pharmacology and Toxicology
Chinese Pharmaceutical Journal | Year: 2010

OBJECTIVE: To prepare vinorelbine tartrate(VRT) thermo-sensitive liposomes and establish methods for determining its VRT content and entrapment efficiency. METHODS: VRT thermo-sensitive liposomes were prepared by pH-gradients method. VRT contents were determined by HPLC. Liposomes and free drug were separated by mini-column centrifugation. The influential factors of VRT elution including sephadex type, elution solution, and column height and elution volume were investigated. RESULTS: Mean particle size of VRT thermo-sensitive liposomes was (94.6 ± 1.6) nm. VRT content was (1.83 ± 0.03) mg·mL -1. The entrapment efficiencies of three batches of VRT thermo-sensitive liposomes were (95.75 ± 1.42)% , (91.12 ± 1.21)% and (94.10 ± 2.03)%, and the elution recoveries were (98.3 ± 2.42)%, (93.5 ± 1.83)% and (96.6 ± 1.65)%. Sephadex G - 25 was chosen to prepare mini-column. Elution solution were PBS, 4 cm of column height was optimal, and one of gradients-elution-volumes was ascertained. CONCLUSION: The preparation technology of VRT liposomes was stable, drug loading and entrapping capacity of VRT liposomes were well. The methods for determination of VRT content and entrapment efficiency were simple, rapid and accurate. These studies can provide bases for further development of intravenous injection VRT thermo-sensitive liposomes.


Hu X.,Central South University | Chen L.,Hunan Academy of Chinese Medicine | Shi S.,Central South University | Cai P.,Hunan Academy of Chinese Medicine | And 2 more authors.
Journal of Pharmaceutical and Biomedical Analysis | Year: 2016

Lonicerae macranthoides with strong antioxidant activity is commonly used in traditional Chinese medicine and folk tea/beverage. However, detailed information about its antioxidant activity and bioactive compounds is limited. Then at first, we comparatively evaluated total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activities of water extract, petroleum ether, ethyl acetate and n-butanol fractions of L. macranthoides. Ethyl acetate fraction exhibited the highest level of TPC (207.38 mg GAE/g DW), TFC (53.06 mg RE/g DW) and the best DPPH scavenge activity and reducing power. n-Butanol fraction showed the best ABTS+ and O2 - scavenging activities. Interestingly, water extract, ethyl acetate and n-butanol fractions showed stronger antioxidant activities than positive control, butylated hydroxytoluene (BHT). After that, thirty-one antioxidant phenolic compounds, including twenty-two phenolic acids and nine flavonoids, were screened by DPPH-HPLC experiment and then identified using HPLC-DAD-QTOF-MS/MS. It is noted that twenty-one compounds (1, 3-4, 6-17, 19, 23, 26, 28-29, and 31), as far as was known, were discovered from L. macranthoide for the first time, and eleven of them (3-4, 10-17, and 23) were reported in Lonicera species for the first time. Results indicated that L. macranthoides could serve as promising source of rich antioxidants in foods, beverages and medicines for health promotion. © 2016.


Yang X.-M.,Hunan Academy of Chinese Medicine | Wang X.-H.,Hunan Academy of Chinese Medicine | Chen L.-F.,Hunan Academy of Chinese Medicine | Wang X.-Q.,Hunan Academy of Chinese Medicine
Zhongguo Zhongyao Zazhi | Year: 2012

Objective: To study the effects of dihydromyricetin(DMY) on tumor necrosis factor (TNF-α) and NF-κB p65 cells of the recurrent aphthous ulcer (RAU) rat. Method: Sixty of Sprague Dawley (SD) rats are randomly divided into 6 groups. The rat RAU models was established by injection of immunogen composed of the homogenate supernate of homogeneousoral oral mucosa from SD rats and Freund's complete adjuvant (FCA) into rat backs subcutaneously once every two weeks for 5 times, and the only FCA injected as normal control. DMY (50, 100, 200 mg·kg-1) and licorzine (67.5 mg·kg-1) were given intragastrically once daily for 7 days on the day of the last immunogen injection, respectively. Water was given instead of drugs in normal and model control groups. The blood was got from the fundus oculi vein of rats on the day after last administration, the serum was separated. Then the rats were put to death with the cervical dislocation and decollated on the ice stage. Two sides of rat buccal mucosal tissue were cut. One side of them was put into 4% neutral formalin and another was added into 10 times of phosphate buffer to homogenize it homogenate. The oral mucosa ulcer occurrence of rats was observed by the histopathology. The content of TNF-α in serum and oral mucosa was assayed with ELISA; the expression of NF-κB cells was determined by the immunohistochemisty and macrophagus was determined by azure-feosin-dyeing in oral mucosa tissue. The expression of TNF-α mRNA in serum and oral mucosa was detected by reverse transcription polymerase chain reaction. Result: In RAU rats, oral mucosa ulcer occurred, the content of TNF-α raised and the expression of TNF-α mRNA increased in serum and oral mucosa, the expression of positive NF-κB p65 cells and the amount of macrophages went up in oral mucosa. DMY and licorzine significantly reduced occurrence of oral mucosa ulcer in RAU rats, lowered content of TNF-α and the expression of TNF-α mRNA in serum and oral mucosa, reduced expression of positive NF-κB p65 cells and the amount of macrophages. Conclusion: It is considered that DMY could inhibited occurrence of oral mucosa ulcer in RAU rats. One principle of it's effects could be that DMY controlled NF-κB p65 regulation on transcription and release of TNF-α mRNA in macrophages in oral mucosa ulcer tissue and lead to fall of TNF-α content in oral mucosa tissue causing role of anti-oral mucosa ulcer.


Liang X.,Hunan Academy of Chinese Medicine | Zhang Y.,Central South University | Chen W.,Hunan Academy of Chinese Medicine | Cai P.,Hunan Academy of Chinese Medicine | And 3 more authors.
Journal of Chromatography A | Year: 2015

A challenge in coupling high-speed counter-current chromatography (HSCCC) online with high performance liquid chromatography (HPLC) for purity analysis was their time incompatibility. Consequently, HSCCC-HPLC was conducted by either controlling HPLC analysis time and HSCCC flow rate or using stop-and-go scheme. For natural products containing compounds with a wide range of polarities, the former would optimize experimental conditions, while the latter required more time. Here, a novel HSCCC-HPLC-diode array detector-mass spectrometry (HSCCC-HPLC-DAD-MS) was developed for undisrupted purification, analysis and identification of multi-compounds from natural products. Two six-port injection valves and a six-port switching valve were used as interface for collecting key HSCCC effluents alternatively for HPLC-DAD-MS analysis and identification. The ethyl acetate extract of Malus doumeri was performed on the hyphenated system to verify its efficacy. Five main flavonoids, 3-hydroxyphloridzin (1), phloridzin (2), 4',6'-dihydroxyhydrochalcone-2'- O-β- d-glucopyranoside (3, first found in M. doumeri), phloretin (4), and chrysin (5), were purified with purities over 99% by extrusion elution and/or stepwise elution mode in two-step HSCCC, and 25. mM ammonium acetate solution was selected instead of water to depress emulsification in the first HSCCC. The online system shortened manipulation time largely compared with off-line analysis procedure and stop-and-go scheme. The results indicated that the present method could serve as a simple, rapid and effective way to achieve target compounds with high purity from natural products. © 2015 Elsevier B.V.


Peng M.-J.,JiShou University | Shi S.-Y.,Central South University | Chen L.,Hunan Academy of Chinese Medicine | Zhang S.-H.,Hunan Academy of Chinese Medicine | And 2 more authors.
Analytical and Bioanalytical Chemistry | Year: 2016

Screening and analysis of bioactive compounds from natural products is challenging work due to their complexity. This study presents the first report on hyphenation of solid-phase ligand-fishing using immobilized xanthine oxidase microcolumn (IXOM) and high-performance liquid chromatography–diode array detector–tandem mass spectrometry (HPLC–DAD–MS/MS) for screening and identification of XO inhibitors from complex mixtures. Solid-phase ligand-fishing system was hyphenated with the HPLC system via four-port switching valve and a six-port injection valve as an interface for transferring effluents from IXOM to HPLC, and collecting chromatograms from LFMC (ligand-fishing microextraction column) and C18 column in a run by only one DAD. Mixtures containing allopurinol (positive control) and tryptophane (negative control) were analyzed in order to verify the specificity and reproducibility of the approach. Subsequently, the newly developed system was applied to screening and identification of XO inhibitors from L. macranthoides and its human microsomal metabolites. Six prototype compounds (3-caffeoylquinic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid) and three metabolites (3-caffeoyl-epi-quinic acid, 5-caffeoyl-epi-quinic acid, 4-caffeoyl-epi-quinic acid) with XO binding affinities were identified. The XO inhibition activities of six prototype compounds were evaluated and confirmed using in vitro enzymatic assay. With the online system developed here, we present a feasible, selective, and effective strategy for rapid screening and identification of enzyme inhibitors from complex mixtures. © 2016, Springer-Verlag Berlin Heidelberg.


Zhang S.-H.,Hunan Academy of Chinese Medicine | Hu X.,Central South University | Shi S.-Y.,Central South University | Huang L.-Q.,Chinese Academy of Sciences | And 3 more authors.
Analytical and Bioanalytical Chemistry | Year: 2016

A major challenge of profiling chlorogenic acids (CGA) in natural products is to effectively detect unknown or minor isomeric compounds. Here, we developed an effective strategy, typical ultraviolet (UV) spectra in combination with diagnostic mass fragmentation analysis based on HPLC–DAD–QTOF-MS/MS, to comprehensively profile CGA in the buds of Lonicera macranthoides. First, three CGA UV patterns were obtained by UV spectra screening. Second, 13 types of CGA classified by molecular weights were found by thorough analysis of CGA peaks using high-resolution MS. Third, selected ion monitoring (SIM) was carried out for each type of CGA to avoid overlooking of minor ones. Fourth, MS/MS spectra of each CGA were investigated. Then 70 CGA were identified by matching their UV spectra, accurate mass signals and fragmentation patterns with standards or previously reported compounds, including six caffeoylquinic acids (CQA), six diCQA, one triCQA, three caffeoylshikimic acids (CSA), six diCSA, one triCSA, three p-coumaroylquinic acids (pCoQA), four p-coumaroylcaffeoylquinic acids (pCoCQA), four feruloylquinic acids (FQA), five methyl caffeoylquinates (MCQ), three ethyl caffeoylquinates (ECQ), three dimethoxycinnamoylquinic acids (DQA), six caffeoylferuloylquinic acids (CFQA), six methyl dicaffeoylquinates (MdiCQ), four FQA glycosides (FQAG), six MCQ glycosides (MCQG), and three ethyl dicaffeoylquinates (EdiCQ). Forty-five of them were discovered from Lonicera species for the first time, and it is noted that CGA profiles were investigated for the first time in L. macranthoides. Results indicated that the developed method was a useful approach to explore unknown and minor isomeric compounds from complex natural products. [Figure not available: see fulltext.] © 2016 Springer-Verlag Berlin Heidelberg


PubMed | Hunan Academy of Chinese Medicine and Central South University
Type: | Journal: Journal of pharmaceutical and biomedical analysis | Year: 2016

Lonicerae macranthoides with strong antioxidant activity is commonly used in traditional Chinese medicine and folk tea/beverage. However, detailed information about its antioxidant activity and bioactive compounds is limited. Then at first, we comparatively evaluated total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activities of water extract, petroleum ether, ethyl acetate and n-butanol fractions of L. macranthoides. Ethyl acetate fraction exhibited the highest level of TPC (207.38 mg GAE/g DW), TFC (53.06 mg RE/g DW) and the best DPPH scavenge activity and reducing power. n-Butanol fraction showed the best ABTS(+) and O2(-) scavenging activities. Interestingly, water extract, ethyl acetate and n-butanol fractions showed stronger antioxidant activities than positive control, butylated hydroxytoluene (BHT). After that, thirty-one antioxidant phenolic compounds, including twenty-two phenolic acids and nine flavonoids, were screened by DPPH-HPLC experiment and then identified using HPLC-DAD-QTOF-MS/MS. It is noted that twenty-one compounds (1, 3-4, 6-17, 19, 23, 26, 28-29, and 31), as far as was known, were discovered from L. macranthoide for the first time, and eleven of them (3-4, 10-17, and 23) were reported in Lonicera species for the first time. Results indicated that L. macranthoides could serve as promising source of rich antioxidants in foods, beverages and medicines for health promotion.


PubMed | Chinese Academy of Sciences, Hunan Academy of Chinese Medicine and Central South University
Type: Journal Article | Journal: Analytical and bioanalytical chemistry | Year: 2016

A major challenge of profiling chlorogenic acids (CGA) in natural products is to effectively detect unknown or minor isomeric compounds. Here, we developed an effective strategy, typical ultraviolet (UV) spectra in combination with diagnostic mass fragmentation analysis based on HPLC-DAD-QTOF-MS/MS, to comprehensively profile CGA in the buds of Lonicera macranthoides. First, three CGA UV patterns were obtained by UV spectra screening. Second, 13 types of CGA classified by molecular weights were found by thorough analysis of CGA peaks using high-resolution MS. Third, selected ion monitoring (SIM) was carried out for each type of CGA to avoid overlooking of minor ones. Fourth, MS/MS spectra of each CGA were investigated. Then 70 CGA were identified by matching their UV spectra, accurate mass signals and fragmentation patterns with standards or previously reported compounds, including six caffeoylquinic acids (CQA), six diCQA, one triCQA, three caffeoylshikimic acids (CSA), six diCSA, one triCSA, three p-coumaroylquinic acids (pCoQA), four p-coumaroylcaffeoylquinic acids (pCoCQA), four feruloylquinic acids (FQA), five methyl caffeoylquinates (MCQ), three ethyl caffeoylquinates (ECQ), three dimethoxycinnamoylquinic acids (DQA), six caffeoylferuloylquinic acids (CFQA), six methyl dicaffeoylquinates (MdiCQ), four FQA glycosides (FQAG), six MCQ glycosides (MCQG), and three ethyl dicaffeoylquinates (EdiCQ). Forty-five of them were discovered from Lonicera species for the first time, and it is noted that CGA profiles were investigated for the first time in L. macranthoides. Results indicated that the developed method was a useful approach to explore unknown and minor isomeric compounds from complex natural products.


A challenge in coupling high-speed counter-current chromatography (HSCCC) online with high performance liquid chromatography (HPLC) for purity analysis was their time incompatibility. Consequently, HSCCC-HPLC was conducted by either controlling HPLC analysis time and HSCCC flow rate or using stop-and-go scheme. For natural products containing compounds with a wide range of polarities, the former would optimize experimental conditions, while the latter required more time. Here, a novel HSCCC-HPLC-diode array detector-mass spectrometry (HSCCC-HPLC-DAD-MS) was developed for undisrupted purification, analysis and identification of multi-compounds from natural products. Two six-port injection valves and a six-port switching valve were used as interface for collecting key HSCCC effluents alternatively for HPLC-DAD-MS analysis and identification. The ethyl acetate extract of Malus doumeri was performed on the hyphenated system to verify its efficacy. Five main flavonoids, 3-hydroxyphloridzin (1), phloridzin (2), 4,6-dihydroxyhydrochalcone-2-O--D-glucopyranoside (3, first found in M. doumeri), phloretin (4), and chrysin (5), were purified with purities over 99% by extrusion elution and/or stepwise elution mode in two-step HSCCC, and 25mM ammonium acetate solution was selected instead of water to depress emulsification in the first HSCCC. The online system shortened manipulation time largely compared with off-line analysis procedure and stop-and-go scheme. The results indicated that the present method could serve as a simple, rapid and effective way to achieve target compounds with high purity from natural products.


Due to the complexity of natural products, efficient identification of bioactive compounds, especially for minor compounds, would require a huge effort. Here, we developed an effective strategy based on combining major constituents knockout with high-performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry (HPLC-DAD-QTOF-MS/MS) to comprehensively identify minor antioxidants in Malus doumeri, one of the longest known and most used tonic plant in Taiwan. First, five major compounds (I-V) in M. doumeri were knocked out by two-step stepwise high-speed countercurrent chromatography (HSCCC). Second, minor antioxidants were screened by 1,1-diphenyl-2-picrylhydrazyl radical-HPLC (DPPH-HPLC) assay. Third, structures of thirty minor antioxidants, including 11 dihydrochalcones, 4 flavanones, 3 flavonols, 2 flavones, 3 aurones and 7 phenolic acids, were unambiguously or tentatively identified by matching their characteristic UV spectra, accurate mass signals and key diagnostic fragment ions with standards or previously reported compounds. Twenty-six of them, as far as was known, were discovered from M. doumeri for the first time. The results indicated that the proposed method was a useful approach to explore minor bioactive compounds from complex natural products.

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