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1.
Biol Reprod ; 98(3): 408-421, 2018 03 01.
Article in English | MEDLINE | ID: mdl-29281003

ABSTRACT

With half a million babies born preterm each year in the USA and about 15 million worldwide, preterm birth (PTB) remains a global health issue. Preterm birth is a primary cause of infant morbidity and mortality and can impact lives long past infancy. The fact that there are numerous, and many currently unidentified, etiologies of PTB has hindered development of tools for risk evaluation and preventative therapies. Infection is estimated to be involved in nearly 40% of PTBs of known etiology; therefore, understanding how infection-mediated inflammation alters the cervical milieu and leads to preterm tissue biomechanical changes are questions of interest. Using RNA-seq, we identified enrichment of components involved in inflammasome activation and unique proteases in the mouse cervix during lipopolysaccharide (LPS)-mediated PTB and not physiologically at term before labor. Despite transcriptional induction of inflammasome components, there was no evidence of functional activation based on assessment of mature IL1B and IL18 proteins. The increased transcription of proteases that target both elastic fibers and collagen and concentration of myeloid-derived cells capable of protease synthesis in the cervical stroma support the structural disruption of elastic fibers as a functional output of protease activity. The recent demonstration that elastic fibers contribute to the biomechanical function of the pregnant cervix suggests their protease-induced disruption in the infection model of LPS-mediated PTB and may contribute to premature loss of mechanical competency and preterm delivery. Collectively, the transcriptomics and ultrastructural data provide new insights into the distinct mechanisms of premature cervical remodeling in response to infection.


Subject(s)
Cervix Uteri/metabolism , Lipopolysaccharides , Premature Birth/metabolism , Transcriptome , Animals , Female , Interleukin-18/metabolism , Interleukin-1beta/metabolism , Mice , Premature Birth/chemically induced , Premature Birth/genetics
2.
Exp Ther Med ; 13(3): 1032-1038, 2017 Mar.
Article in English | MEDLINE | ID: mdl-28450937

ABSTRACT

Although Radix Achyranthis Bidentatae (RAB) and Radix Cyathulae (RC) are from two different medicinal plants, they are both used as 'Niu-Xi', a widely used traditional Chinese medicine that is believed to stimulate menstruation and affect bone injury. Angiogenesis is actively involved in treating these illnesses. The aim of the present study was to investigate whether the whole extracts of RAB and RC possess pro-angiogenic effects. In order to examine this idea whole extracts of RAB and RC were extracted with boiling water followed by ethanol, respectively. Results from the MTT, wound healing and tube formation assays in human umbilical vein endothelial cells (HUVECs) in vitro revealed that the whole extracts of RAB and RC did not increase cell proliferation or tube formation, but enhanced cell migration. Their angiogenic effects were also confirmed in zebrafish in vivo via increasing the sprout numbers in the sub-intestinal vessel. As determined by quantitative polymerase chain reaction, the whole extracts of RAB and RC both regulated the expression of cell migration-related genes in zebrafish. It is concluded that the whole extracts of RAB and RC induced angiogenesis in HUVECs in vitro and in zebrafish in vivo via increasing cell migration.

3.
Article in English | MEDLINE | ID: mdl-26744789

ABSTRACT

Gallocatechin gallate (GCG) possesses multiple potential biological activities. However, the content of GCG in traditional green tea is too low which limits its in-depth pharmacological research and application. In the present study, a simple, efficient and environment-friendly chromatographic separation method was developed for preparative enrichment and separation of GCG from cocoa tea (Camellia ptilophylla) which contains high content of GCG. In the first step, the adsorption properties of selected resins were evaluated, and XAD-7HP resin was chosen by its adsorption and desorption properties for GCG. In order to maximize column efficiency for GCG collection, the operating parameters (e.g., flow rate, ethanol concentration, and bed height) were optimized. We found that the best combination was the feed concentration at 20mg/mL, flow rate at 0.75 BV/h and the ratio of diameter to bed heights as 1:12. Under these conditions, the purity of GCG was 45% with a recovery of 89%. In order to obtain pure target, a second step was established using column chromatography with sephadex LH-20 gel and 55% ethanol-water solution as eluent. After this step, the purity of the GCG was 91% with a recovery of 68% finally.


Subject(s)
Camellia/chemistry , Catechin/analogs & derivatives , Chromatography, High Pressure Liquid/methods , Dextrans/chemistry , Catechin/analysis , Catechin/chemistry , Catechin/isolation & purification , Plant Extracts/chemistry , Resins, Synthetic/chemistry
4.
Chin Med ; 10: 33, 2015.
Article in English | MEDLINE | ID: mdl-26566393

ABSTRACT

BACKGROUND: Deer tendons are believed more effective than cattle tendons in tonifying kidney yang (shen yang) and enhancing bone and tendons. This study aims to differentiate the two types of tendons by a loop-mediated isothermal amplification (LAMP) test and bone remodeling bioassays. METHODS: Internal control primers to detect both types of tendons and specific primers for deer tendons were designed according to a sequence analysis. The LAMP test was set up and the results were analyzed by conventional gel electrophoresis, real-time fluorescence observation, and colorimetric detection. Crude tendon extracts were prepared by water extraction to compare their effects on bone. The anti-osteoclastic effects were investigated on mouse pre-osteoclast Raw264.7 cells by cell viability determination and tartrate-resistant acid phosphatase staining. The osteogenic effects were examined using rat osteoblast-like UMR106 cells by evaluation of cell proliferation, alkaline phosphatase activity, and calcium deposition. The relative gene expressions of bone remodeling-related markers, including nuclear factor of activated T-cells cytoplasmic 1, tartrate-resistant acid phosphatase, cathepsin K, and osteoprotegerin/receptor activator of NF-κB ligand, were determined by real-time PCR. RESULTS: In the LAMP test, both deer and cattle tendons were detected in the control reactions, while only deer tendons were amplified by the specific LAMP test. In the bioassays, both tendons inhibited the viability and differentiation of pre-osteoclast Raw264.7 cells, and promoted the proliferation and mineralization of osteoblast-like UMR106 cells. The mRNA expressions of bone remodeling-related markers were consistent with the results of the bioassays. CONCLUSION: This study demonstrated that the isothermal LAMP test can distinguish between deer tendons and cattle tendons. Both types of tendons exhibited similar beneficial effects on bone remodeling according to the bioassay findings.

5.
Phytomedicine ; 21(11): 1256-63, 2014 Sep 25.
Article in English | MEDLINE | ID: mdl-25172787

ABSTRACT

AIM: Carthami Flos (CF) is a Chinese herb traditionally used for cardiovascular disease and bone injury in China with pharmacological effects on improving blood circulation. The aim of this study was to investigate the angiogenic potential of CF whole extract (extracted by boiling with water, followed by ethanol) and the underlying mechanisms in human microvascular endothelial cells (HMEC-1) in vitro and in transgenic TG(fli1:EGFP)(y1)/+(AB) zebrafish with transgenic endothelial cells expressing EGFP (Enhanced Green Fluorescent Protein) in vivo. METHODS: Effects of CF whole extract on cell proliferation, migration and tube formation in HMEC-1 cells in vitro were detected by MTT assay, wound healing assay and tube formation assay. Its angiogenic effect in zebrafish was investigated by monitoring the sprout number in the sub-intestinal vessel (SIV), and the underlying mechanisms were tested by quantitative real-time PCR. RESULTS: CF whole extract increased cell proliferation, migration and tube formation in vitro in HMEC-1 cells. Its angiogenic effect was also confirmed in vivo in zebrafish by increasing the sprout number in the SIV. As determined by quantitative real-time PCR, CF whole extract up-regulated the expression of angiogenesis-related genes in zebrafish, including angiogenic and its associated growth factors and receptors (e.g. IGF1, CTGF, NRP2, and VEGFR3), transcription factor (e.g. HIF1A), matrix degradation and endothelial cell migration-related factors (e.g. MMP2, MMP9, TIMP2, PLG and PLAU), cell adhesion molecules (e.g. ITGAV, ITGB3, beta-catenin and PECAM1), tubule formation factors (e.g. ANGPT1, TIE-2, PDGFR-B, CDH5, S1PR1, FGF2, Shh, and TGFRB1), and blood vessel maturation/formation factor (e.g. Ephrin B2). CONCLUSIONS: CF whole extract increased angiogenesis in HMEC-1 cells in vitro and in zebrafish in vivo with multiple mechanisms.


Subject(s)
Angiogenesis Inducing Agents/pharmacology , Carthamus/chemistry , Endothelial Cells/drug effects , Plant Extracts/pharmacology , Animals , Animals, Genetically Modified/embryology , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Drugs, Chinese Herbal/pharmacology , Embryo, Nonmammalian/drug effects , Humans , Zebrafish/embryology
6.
J Ethnopharmacol ; 154(3): 711-8, 2014 Jul 03.
Article in English | MEDLINE | ID: mdl-24815219

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: Er-Miao-San (EMS) is a traditional Chinese herbal formulation that contains combinations of Rhizoma Atractylodis (RA) and Cortex Phellodendri (CP). It exhibits analgesic and anti-inflammatory activities and have been used for the treatment of various "Bi Zheng" for thousand years in China. The aims of the present study were to investigate the anti-inflammatory activities of EMS and elucidate the underlying mechanisms with regard to its molecular basis of action for the best combination. MATERIALS AND METHODS: The anti-inflammatory effects of EMS were studied by using lipopolysaccharide (LPS)-stimulated activation of nitric oxide (NO) and pro-inflammatory cytokine production in mouse RAW264.7 macrophages. Expression of inducible NO synthase (iNOS), mitogen-activated protein kinases (MAPKs) phosphorylation, p65 phosphorylation, inhibitor-κBα (IκBα) degradation, and NF-κB DNA-binding activity were further investigated. RESULTS: The present study demonstrated that EMS could suppress the production of NO in LPS-stimulated RAW264.7 macrophages. However, CP and RA did not have significant inhibitory effect on them. EMS also inhibited the production of tumor necrosis factor-alpha, interleukin-1 beta and macrophage chemotactic protein-1. Further investigations showed EMS could suppress iNOs expression and p38 phosphorylation. EMS significantly decreased the content of IκBα, reduced the level of phosphorylated p65 and suppressed the NF-κB DNA-binding activity. All these results suggested the inhibitory effects of EMS on the production of inflammatory mediators through the inhibition of the NF-κB pathway. CONCLUSIONS: Our results indicated that EMS inhibited inflammatory events and iNOS expression in LPS-stimulated RAW264.7 cells through the inactivation of the MAPK and NF-κB pathway. This study gives scientific evidence validating the use of EMS in treatment of patients with "Bi Zheng" in clinical practice in traditional Chinese medicine.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Drugs, Chinese Herbal/chemistry , Inflammation Mediators/antagonists & inhibitors , Macrophages/drug effects , Mitogen-Activated Protein Kinases/antagonists & inhibitors , NF-kappa B/antagonists & inhibitors , Phellodendron/chemistry , Rhizome/chemistry , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/isolation & purification , Cells, Cultured , Cytokines/antagonists & inhibitors , Cytokines/biosynthesis , Inflammation Mediators/metabolism , Lipopolysaccharides/pharmacology , MAP Kinase Signaling System/drug effects , Macrophages/metabolism , Medicine, Chinese Traditional , Mice , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Nitric Oxide/antagonists & inhibitors , Nitric Oxide/biosynthesis
7.
J Nutr Biochem ; 25(3): 319-28, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24524904

ABSTRACT

Angiogenesis, the process of blood vessel formation, is critical to tumor growth. Ant-angiogenic strategies demonstrated importance in cancer therapy. Cocoa tea (Camellia ptilophylla), a naturally decaffeinated tea commonly consumed as a healthy drink in southern China, had recently been found to be a potential candidate for antiangiogenesis. A novel proanthocyanidin, GC-(4→8)-GCG, which consisted of gallocatechin and gallocatechin 3-O gallate moieties, was discovered and thought to be one of the effective candidates for antiangiogenesis. Hence, the present study aimed to evaluate the antiangiogenesis activities of GC-(4→8)-GCG in vitro and in vivo, and SU5416 was applied as a positive control. The inhibitory effects of GC-(4→8)-GCG on three important processes involved in angiogenesis, i.e., proliferation, migration and differentiation, were examined using human microvascular endothelial cell line HMEC-1 by MTT assay, scratch assay and tube formation assay, respectively. Using transgenic zebrafish embryos TG(fli1:EGFP)y1/+(AB) as an animal model of angiogenesis, the antiangiogenic effect of GC-(4→8)-GCG was further verified in vivo. Our results demonstrated that GC-(4→8)-GCG significantly inhibited migration (P<.001) and tubule formation (P<.001-.05) of HMEC-1 in dose-dependent manner. Regarding intracellular signal transduction, GC-(4→8)-GCG attenuated the phosphorylation of ERK, Akt and p38 dose-dependently in HMEC-1. In zebrafish embryo, the formation of new blood vessels was effectively inhibited by GC-(4→8)-GCG in a dose-dependent manner after 3 days of treatment (P<.001-.05). In conclusion, these results revealed that our novel proanthocyanidin, GC-(4→8)-GCG might be a potential and promising agent of natural resource to be further developed as an antiangiogenic agent.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Cacao/chemistry , Proanthocyanidins/pharmacology , Cell Line , Humans , In Vitro Techniques
8.
J Ethnopharmacol ; 151(1): 565-75, 2014.
Article in English | MEDLINE | ID: mdl-24247081

ABSTRACT

ETHNOPHARMACOLOGICAL IMPORTANCE: The root of Rehmannia glutinosa (Rehmanniae Radix (RR)) is clinically used as a wound-healing agent in traditional Chinese medicine. Angiogenesis acts crucially in the pathogenesis of chronic wound healing. The present study investigated the angiogenesis effect and its underlying mechanism of RR through zebrafish sprout angiogenesis guided-fractionation. MATERIALS AND METHODS: The in vivo angiogenesis effect was studied by analyzing the number of ectopic sprouts formed upon sub-intestinal vessel of transgenic TG(fli1:EGFP)(y1)/+(AB) zebrafish embryos by fluorescence microscopy. Quantitative real-time PCR gene expression of the zebrafish embryos was further performed using a panel of 30 angiogenesis-associated genes designed for zebrafish sprout angiogenesis. Classical in vitro angiogenesis assays using human microvascular endothelial cells (HMEC-1) was accompanied. RESULTS: We demonstrated that among all RR sub-fractions tested, C1-1 treated-zebrafish embryos possessed the most potent angiogenesis activities (from 190 to 780 ng/ml, p<0.001) in sprout formation in the zebrafish model. Quantitative gene expression of the treated embryos demonstrated significant up-regulation in MMP-9 (p<0.05), ANGPT1 (p<0.05), EGFR (p<0.05), EPHB4 (p<0.01), and significant down-regulation in Ephrin B2 (p<0.05), Flt-1 (p<0.05) and Ets-1 (p<0.05). C1-1 treatment could also significantly (p<0.001-0.05) stimulate HMEC-1 cell migration in scratch assay. Significant increase (p<0.05) in mean tubule length was observed in the C1-1-treated HMEC-1 cells in the tubule formation assay. CONCLUSIONS: Our zebrafish sprout angiogenesis model-guided fractionation revealed that C1-1 possessed the most potent angiogenesis effect in RR. The design of the panel with 30 tailor-made angiogenesis-associated genes exhibited in zebrafish gene expression analysis showed that C1-1 could trigger differential expression of various angiogenesis-associated genes, such as VEGFR3 and MMP9, which played key role in angiogenesis. The pro-angiogenic activity of C1-1 was further confirmed in the translated study in motogenic and tubule-inducing effect using HMEC-1.


Subject(s)
Neovascularization, Physiologic/drug effects , Plant Roots/chemistry , Rehmannia/chemistry , Animals , Animals, Genetically Modified , Cell Line , Cell Proliferation/drug effects , Embryo, Nonmammalian/drug effects , Endothelial Cells/drug effects , Gene Expression Regulation/drug effects , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Zebrafish/embryology
9.
Wound Repair Regen ; 21(4): 579-87, 2013.
Article in English | MEDLINE | ID: mdl-23755905

ABSTRACT

The emergence of electric cell-substrate impedance sensing (ECIS) technology has provided new insight in advanced cell behavioral study by its nanometer sensitivity, precise electrical wounds generation, and high reproducibility that can be monitored in real time in a noninvasive way. However, little is known regarding pro-angiogenic agents in wound healing studies using endothelial cells evaluated with ECIS technology. Our previous studies showed a prominent wound healing effect of a two-herb formula (NF3) comprising of Astragali Radix and Rehmanniae Radix in a rat chronic wound model through actions including angiogenesis. Here we further investigated the angiogenic effect and its underlying molecular mechanism through proliferation, motility, and tubule formation of human vascular endothelial cells (HECV) using ECIS technology. It was first shown that HECV treated with NF3 had a higher resistance than that of control using ECIS cell attachment and cell migration model (p < 0.01). We further validated in a scratch assay that NF3 treatment significantly stimulated HECV cell migration (p < 0.01-0.05). Also, NF3-treated HECV were observed to develop into a significantly more branched tubular structure when compared with control (p < 0.05-0.01). Meanwhile, Western blot analysis of NF3-treated HECV revealed the activated expression of p-Akt, and mitogen-activated protein (MAP) kinases for p-ERK, p-p38, and p-JNK. We propose that the effect of NF3 in the promotion of endothelial cell migration and tubule formation could be mediated through pathways involving p-Akt and activated MAP kinases. Hence, we demonstrated the complexity of the angiogenic effect activated by NF3 molecularly and functionally. NF3 treatment could offer therapeutic value to chronic wound healing for its pro-angiogenic efficacy.


Subject(s)
Angiogenesis Inducing Agents/pharmacology , Drugs, Chinese Herbal/pharmacology , Endothelial Cells/drug effects , MAP Kinase Signaling System/drug effects , Oncogene Protein v-akt/drug effects , Rehmannia , Astragalus Plant , Astragalus propinquus , Cell Adhesion/drug effects , Cell Line , Cell Movement/drug effects , Cell Survival/drug effects , Electric Impedance , Humans , Neovascularization, Physiologic/drug effects , Oncogene Protein v-akt/metabolism , Phosphorylation/drug effects , Signal Transduction/drug effects , Wound Healing/drug effects
10.
J Ethnopharmacol ; 143(3): 867-75, 2012 Oct 11.
Article in English | MEDLINE | ID: mdl-23034094

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Rehmannia glutinosa (RR) is commonly used to reduce inflammation in various traditional Chinese herbal formulae; however, little is known regarding its active component(s). AIM OF STUDY: The objective of the present study was to examine the active component(s) responsible for the anti-inflammatory activity of RR via anti-nitric oxide production assay-guided fractionation; and the underlying anti-inflammatory mechanism of action of such component(s) was further investigated. MATERIALS AND METHODS: Anti-nitric oxide (NO) activities with lipopolysaccharides (LPS)-stimulated RAW264.7 murine macrophages was used as screening platform. Gene, protein and inflammatory mediators' expression were also studied using real-time PCR, western blotting and ELISA, respectively. RESULTS: Using anti-NO assay-guided fractionation, sub-fraction C3 (from 31.25 to 62.5 µg/ml, p=0.001 to 0.01) possessed 100-fold more potent anti-inflammatory effect than that of the aqueous extract of RR. Characterization of C3 showed that the anti-inflammatory effect could be partly due to the presence of rehmapicrogenin, which could significantly inhibit NO production (p<0.001). C3 was further demonstrated in blocking inflammation by inhibiting gene (p<0.001) and protein expression of inducible NO synthase (iNOS) dose-dependently. Besides, C3 also significantly inhibited the production of prostaglandin E(2) (p<0.001 to 0.01), IL-6 (p<0.001 to 0.05) and COX-2 (p<0.05). CONCLUSIONS: Rehmapicrogenin was, for the first time, shown to possess nitric oxide inhibitory activities. Bioassay-guided fractionation demonstrated that rehmapicrogenin-containing subfraction C3 exhibited potent anti-inflammatory effect by inhibiting iNOS, COX-2 and IL-6, while rehmapicrogenin was only partially responsible for the anti-inflammatory effect of RR.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Drugs, Chinese Herbal/pharmacology , Plant Extracts/pharmacology , Rehmannia , Animals , Biological Assay , Cell Line , Cyclooxygenase 2 Inhibitors/pharmacology , Dinoprostone/metabolism , Interleukin-6/antagonists & inhibitors , Interleukin-6/metabolism , Lipopolysaccharides , Mice , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/antagonists & inhibitors , Plant Roots
11.
J Ethnopharmacol ; 141(1): 250-6, 2012 May 07.
Article in English | MEDLINE | ID: mdl-22366433

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: Astragali Radix (AR) and Rehmanniae Radix (RR) are two traditional Chinese medicines widely used in China for treating diabetes mellitus and its complications, such as diabetic foot ulcer. AIM OF STUDY: In our previous study, a herbal formula NF3 comprising AR and RR in the ratio of 2:1 was found effective in enhancing diabetic wound healing in rats through the actions of tissue regeneration, angiogenesis promotion and inflammation inhibition. The aims of the present study were to investigate the herb-herb interaction (or the possible synergistic effect) between AR and RR in NF3 to promote diabetic wound healing and to identify the principal herb in the formula by evaluating the potencies of individual AR and RR in different mechanistic studies. MATERIALS AND METHODS: A chemically induced diabetic foot ulcer rat model was used to examine the wound healing effect of NF3 and its individual herbs AR and RR. For mechanistic studies, murine macrophage cell (RAW 264.7) inflammation, human fibroblast (Hs27) proliferation and human endothelial cell (HMEC-1) migration assays were adopted to investigate the anti-inflammatory, granulation formation and angiogenesis-promoting activities of the herbal extracts, respectively. RESULTS: In the foot ulcer animal model, neither AR nor RR at clinical relevant dose (0.98g/kg) promoted diabetic wound healing. However, when they were used in combination as NF3, synergistic interaction was demonstrated, of which NF3 could significantly reduce the wound area of rats when compared to water group (p<0.01). For anti-inflammation and granulation formation, AR was more effective than RR in inhibiting lipopolysaccharide (LPS)-induced nitric oxide production from RAW 264.7 cells and promoting Hs27 fibroblast proliferation. In the aspect of angiogenesis promotion, only NF3 promoted cell migration of HMEC-1 cells. CONCLUSIONS: AR plays a preeminent role in the anti-inflammatory and fibroblast-proliferating activities of NF3. The inclusion of RR, however, is crucial for NF3 to exert its overall wound-healing as well as the underlying angiogenesis-promoting effects. The results of present study justified the combined usage of AR and RR in the ratio of 2:1 as NF3 to treat diabetic foot ulcer and illustrated that AR is the principal herb in this herbal formula.


Subject(s)
Angiogenesis Inducing Agents/pharmacology , Anti-Inflammatory Agents/pharmacology , Astragalus Plant , Diabetic Foot/drug therapy , Drugs, Chinese Herbal/pharmacology , Rehmannia , Wound Healing/drug effects , Animals , Astragalus Plant/chemistry , Astragalus propinquus , Cell Line , Cell Movement/drug effects , Cell Proliferation/drug effects , Diabetes Mellitus, Experimental/complications , Diabetic Foot/etiology , Diabetic Foot/immunology , Diabetic Foot/pathology , Dose-Response Relationship, Drug , Drug Synergism , Endothelial Cells/drug effects , Endothelial Cells/pathology , Female , Fibroblasts/drug effects , Fibroblasts/pathology , Humans , Inflammation Mediators/metabolism , Macrophages/drug effects , Macrophages/immunology , Mice , Neovascularization, Physiologic/drug effects , Nitric Oxide/metabolism , Phytotherapy , Plants, Medicinal , Rats , Rats, Wistar , Rehmannia/chemistry , Time Factors
12.
J Tradit Complement Med ; 2(3): 227-34, 2012 Jul.
Article in English | MEDLINE | ID: mdl-24716137

ABSTRACT

This study aimed to investigate and compare the fibroblast-proliferating activities of different Rehmanniae Radix (RR) samples and its chemical components using human normal fibroblast cells Hs27. Those active components were quantified in differently treated RR samples using UPLC so as to correlate activity with component content. Our results showed that dried RR aqueous extract exhibited the most potent fibroblast-proliferating activity. Stronger effect was observed when ethanol with heating was applied in the extraction process. Stachyose and verbascoside were demonstrated for their first time to exhibit significant stimulatory effects on fibroblast proliferation. However, the proliferating effect of dried RR extract did not correlate with the stachyose content, and verbascoside was not responsible for the fibroblast proliferative effect of RR since it was undetectable in all samples. In conclusion, stachyose only contributed in part to the activity of RR, suggesting that other active components might be present and yet to be found.

13.
J Ethnopharmacol ; 137(3): 1323-7, 2011 Oct 11.
Article in English | MEDLINE | ID: mdl-21843616

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Rehmannia glutinosa (RR) is commonly used as a wound-healing agent in various traditional Chinese herbal formulae; while angiogenesis is one of the crucial aspects in wound-healing. AIM OF THE STUDY: The objective of the present study was to investigate the angiogenesis effects of RR aqueous crude extract and its active component(s) using zebrafish model. MATERIALS AND METHODS: The in vivo angiogenesis effect was studied using transgenic TG(fli1:EGFP)(y1)/+(AB) zebrafish embryos by observing the capillary sprouts formation in sub-intestinal vessel (SIV) of zebrafish embryos after 72 h post-fertilization under fluorescence microscopy. RESULTS: Our results indicated that RR aqueous crude extract (250 µg/ml) exhibited significant angiogenesis effect, with an increase in capillary sprouts formation in SIV. Following sequential solvent partition of the RR aqueous crude extract with dichloromethane, ethyl acetate and n-butanol successively, the dichloromethane fraction (DCM) was found to have the most sprouts formation in the SIV region. Subjected to column chromatography, DCM fraction was further fractionated into six sub-fractions and among these tested, the sub-fraction C2 exhibited the most potent angiogenesis effect. The major component, C2A, was isolated and identified as norviburtinal using nuclear magnetic resonance (NMR) and mass spectrometry (MS). The compound norviburtinal (at 50 µg/ml) was shown to possess significant angiogenesis effect in zebrafish model (p < 0.001). CONCLUSIONS: Norviburtinal was, for the first time, found in the extract of RR and possessed novel angiogenesis effect. Bioassay-guided fractionation suggested that norviburtinal was not the only active component responsible for the angiogenesis effect of RR.


Subject(s)
Angiogenesis Inducing Agents/pharmacology , Biological Assay , Embryo, Nonmammalian/drug effects , Neovascularization, Physiologic/drug effects , Plant Extracts/pharmacology , Rehmannia , Terpenes/pharmacology , Zebrafish/embryology , 1-Butanol/chemistry , Acetates/chemistry , Angiogenesis Inducing Agents/chemistry , Angiogenesis Inducing Agents/isolation & purification , Animals , Animals, Genetically Modified , Chemical Fractionation , Chromatography , Embryo Culture Techniques , Embryo, Nonmammalian/blood supply , Green Fluorescent Proteins/genetics , Humans , Magnetic Resonance Spectroscopy , Mass Spectrometry , Methylene Chloride/chemistry , Microscopy, Fluorescence , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Roots , Plants, Medicinal , Rehmannia/chemistry , Solvents/chemistry , Terpenes/chemistry , Terpenes/isolation & purification , Vascular Endothelial Growth Factor A/pharmacology , Zebrafish/genetics
14.
J Ethnopharmacol ; 134(3): 831-8, 2011 Apr 12.
Article in English | MEDLINE | ID: mdl-21291991

ABSTRACT

ETHNOPHARMACOLOGICAL RELEVANCE: The herbs Radix Astragali (RA) and Radix Rehmanniae (RR) have long been used in traditional Chinese Medicine and serve as the principal herbs in treating diabetic foot ulcer. AIM OF STUDY: Diabetic complications, such as foot ulcer, impose major public health burdens worldwide. In our previous clinical studies, two Chinese medicine formulae F1 and F2 have achieved over 80% limb salvage. A simplified 2-herb formula (NF3) comprising of RA and RR in the ratio of 2:1 was used for further study. NF3 was examined for the ulcer healing effect in diabetic rats, and its potential mechanisms of action in fibroblast proliferation, angiogenesis and anti-inflammation in vitro. MATERIALS AND METHODS: A chemically induced diabetic foot ulcer rat model was used for studying the wound healing effect. In the in vitro mechanistic studies, human fibroblast cells (Hs27), human umbilical vein endothelial cells (HUVEC) and mouse macrophage cells (RAW264.7) were assessed for tissue regeneration, angiogenesis and anti-inflammatory activities, respectively. RESULTS: Our in vivo results demonstrated a significant reduction of wound area at day 8 in NF3 (0.98g/kg) group as compared to control (p<0.01). NF3 could significantly stimulate Hs27 proliferation in a dose dependent manner (p<0.05). Besides, NF3 could significantly increase the cell migration and tube formation (p<0.05-0.001) of HUVEC in the angiogenesis study. Furthermore, significant inhibition of nitric oxide production (p<0.01) was found in NF3-treated macrophage cells, suggesting its anti-inflammatory activity. CONCLUSIONS: Our study presents for the first time scientific evidence towards the efficacy of the two-herb formula NF3 in enhancing diabetic wound healing through the actions of tissue regeneration, angiogenesis and anti-inflammation.


Subject(s)
Diabetes Mellitus, Experimental/complications , Herbal Medicine , Wound Healing , Animals , Anti-Inflammatory Agents , Cell Line , Female , Fibroblasts/cytology , Humans , Mice , Neovascularization, Physiologic , Rats , Rats, Wistar
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