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Jikan Mingmu Drops (JMD), a traditional Tibetan medicine containing six herbs, has been used to treat dry eye syndrome (DES) in individuals with diabetes mellitus. However, the activity of JMD ameliorates DES with diabetes mellitus has not been previously examined. The aim of the study is to investigate the molecular mechanism of JMD on db/db mice. The main chemical constituents of JMD were analyzed by high-performance liquid chromatography and gas chromatography-mass spectrometry. DES was then induced in db/db mice by applying 0.2% benzalkonium chloride to the ocular surface for 7 days. Eye drops containing JMD (0.25, 0.5, or 1 g/mL) or vehicle subsequently were administered three times daily for another 7 days, and the therapeutic effects were evaluated by phenol red thread tear and sodium fluorescein tests. Conjunctival specimens were subjected to hematoxylin and eosin staining and periodic acid-Schiff staining to examine pathological changes and number of goblet cells. ELISA was performed to assess the levels of various inflammatory cytokines. JMD contains hydroxysafflor yellow A, magnoflorine, jatrorrhizine hydrochloride, palmatine hydrochloride, berberine hydrochloride, gallic acid, ellagic acid, tauroursodeoxycholic acid, camphor, isoborneol, borneol, trans-cinnamic acid, and muscone. JMD treatment significantly increased the tear volume, decreased the corneal fluorescein staining score, restored the morphology and structure of conjunctival epithelial cells, and markedly downregulated the levels of interleukin (IL)-6, IL-17α, IL-1β, tumor necrosis factor-α, and vascular endothelial growth factor in the conjunctiva. Further data showed that these protective effects were accompanied by inhibition of inflammation in a dose-dependent manner. Amelioration of DES in db/db mice with diabetes mellitus by treatment with Tibetan medicine formula JMD maybe related to its anti-inflammatory effects.
A method that involved the combination of pH-zone-refining counter-current chromatography and semipreparative reversed-phase liquid chromatography has been established for the preparative separation of alkaloids from Hypecoum leptocarpum. From 1.2 g of crude sample, 31 mg N-feruloyltyramine, 27 mg oxohydrastinine, 47 mg hydroprotopine, 25 mg leptopidine, and 18 mg hypecocarpine have been obtained. The structure of the new compound, hypecocarpine, is confirmed based on the analysis of spectroscopic data, including NMR, UV, and IR spectroscopy and positive electrospray ionization mass spectrometry. The known chemical structures were characterized on the basis of (1) H and (13) C NMR spectroscopy. The purities of the five alkaloids are all over 92.7% as determined by high-performance liquid chromatography. The alkaloids' cytotoxicity in breast cancer cells is assessed by using a Cell Counting Kit assay and their inhibitory effect on fatty acid synthase expression is assessed by a Western blot assay. These results suggest that leptopidine could suppress growth and induce cytotoxicity in breast cancer cells and that the cytotoxicity of leptopidine may be related to its inhibitory effect on fatty acid synthase expression.
Naturally occurring oligostilbenes are receiving more attention because they exhibit several beneficial effects for health, including hepatoprotective, antitumor, anti-adipogenic, antioxidant, antiaging, anti-inflammatory, anti-microbial, antiviral, immunosuppressive and neuroprotective activities. Thus, they could be of some potentially therapeutic values for several diseases. In this study, we adopted the alkaline extraction-acid precipitation (AEAP) method for extraction of oligostilbenes from the seed kernel of Iris lactea Then, the high-speed counter-current chromatography (HSCCC) was used for preparative isolation and purification of oligostilbenes from the AEAP extracts. Finally, three oligostilbenes, namely vitisin D (73 mg), ampelopsin B (25 mg) and cis-vitisin A (16 mg), were successfully fractionated by HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (2:5:3:6, v/v/v/v) from 300 mg of the AEAP extracts in ∼ 190 min. The purities of the three isolated oligostilbenes were all over 95.0% as analyzed by high performance liquid chromatography. They all were isolated from I. lacteal for the first time.The method of AEAP for the preparation of the oligostilbene-enriched crude sample was simple, and the HSCCC technique for the isolation and purification of oligostilbenes was efficient.
• HPLC-DAD-APCI/MS was set up for analysis of flavonoid aglycones in the RBP. • The method is capable of providing higher sensitivity and repeatability. • Four methods were applied and assessed for extraction of flavonoids from RBP. • The highest extraction efficiency of flavonoids from RBP was achieved by MAE. • MAE is of short extraction time, low solvent consumption and homogeneous conditions.<br>For identification and quantification of flavonoid aglycones in rape bee pollen (RBP) collected from the Qinghai-Tibetan Plateau, a high-performance liquid chromatography (HPLC) separation method with diode array detector (DAD) and atmospheric pressure chemical ionization/mass spectrometric (APCI/MS) detection and four extraction methods (i.e. microwave-assisted extraction, Soxhlet extraction, cold-soaked extraction, and heat reflux extraction) were developed in this study. The identification of flavonoid aglycones was based on retention time and mass spectra by comparison with standards. Results demonstrated that this method showed excellent reproducibility and correlation coefficient, and offered the detection limits of 0.77-15.50 pmol at signal-to-noise ratio of 3. Quercetin and kaempferol were presented in RBP, and microwave-assisted extraction (MAE) was superior to the other three methods in terms of efficiency, convenience and high content of quercetin (1.37 ± 0.059 mg/g) and kaempferol (23.44 ± 0.544 mg/g). Our work indicated that: 1) the proposed HPLC-DAD-APCI/MS was an accurate and precise analysis method to identify and quantify the flavonoid aglycones in RBP; and 2) MAE was efficient to extract flavonoids from RBP with short extraction time, low solvent consumption, and homogeneous extraction conditions.
High-speed counter-current chromatography (HSCCC) was successfully applied for the first time to isolate and purify four cis-trans isomers of coumaroylspermidine analogs from Safflower. HSCCC separation was achieved with a two-phase solvent system composed of chloroform-methanol-water (1:1:1, v/v/v) with the upper phase as the mobile phase. In a single run, a total of 1.3mg of N(1), N(5), N(10)-(E)-tri-p-coumaroylspermidine (EEE), 4.4mg of N(1)(E)-N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine (EZE), 7.2mg of N(1)(Z)-N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine (ZZE), and 11.5mg of N(1),N(5),N(10)-(Z)-tri-p-coumaroylspermidine (ZZZ) were obtained from 100mg of crude sample. High Performance Liquid Chromatography (HPLC) analysis showed that the purities of these four components are 95.5%, 98.1%, 97.5% and 96.2%, respectively. The chemical structures were identified by ESI-MS, (1)H NMR and (13)C NMR.
OBJECTIVES: To investigate the protective effect of Herpetospermum pedunculosum (H. pedunculosum) seed oil against carbon tetrachloride (CCl4)-induced liver damage.METHODS: This experimental study was conducted at the Northwest Institute of Plateau Biology, Chinese Academy of Sciences, and Yantai University, China from November 2012 to May 2013. The H. pedunculosum seed oil was extracted using supercritical carbon dioxide. The antioxidant activities of H. pedunculosum seed oil were assayed in vitro by 2,2-diphenyl-1-picrylhydrazyl assay, lipid peroxidation assay, and antihemolytic assay. Adult Sprague Dawley rats were randomly divided into 6 groups (10 rats/group) including control, CCl4, CCl4+bifendate, and CCl4+H. pedunculosum seed oil (3 different doses) groups.
RESULTS: The CCl4-induced liver lesions include hepatocyte necrosis, ballooning degeneration, calcification, and fibrosis. Moreover, CCl4 damage results in an obvious increase of serum triglycerides, high-density lipoprotein, low-density lipoprotein, malondialdehyde, total bilirubin, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activity. In addition, CCl4 also significantly decreased the activities of superoxide dismutase (SOD). By contrast, H. pedunculosum seed oil administration significantly ameliorated the CCl4-induced liver lesions, lowered the serum levels of hepatic enzyme markers, and increased the activities of SOD.
CONCLUSION: The results of this study show that H. pedunculosum seed oil can be proposed to protect the liver against CCl4-induced oxidative damage in rats, and the hepatoprotective effect might be correlated with its potent antioxidant and free radical scavenging effect.
A method of using high-speed counter-current chromatography (HSCCC) and semi-preparative reversed-phase liquid chromatography (semi-preparative RPLC) to preparatively separate flavone glucosides and lignan from the crude extracts of <i>Caragana korshinskii</i> has been established for the first time in this study. Five flavone glucosides, including 9 mg of kaempferol 3-O-{β-<i>d</i>-glucopyranosyl(1→2)-[α-<i>l</i>-rhamonopyranosyl(1 → 6)]-β-<i>d</i>-galactopyranoside}, 21 mg of kaempferol 3-O-α-<i>l</i>-rhamnopyranosyl(1→6)-β-<i>d</i>-galactopyranoside-7-O-α-<i>l</i>-rhamnopyranoside, 34 mg of kaempferol 3-O-β-<i>d</i>-galactopyranoside-7-O-α-<i>l</i>-rhamnopyranoside, 27 mg of kaempferol 3-O-β-<i>d</i>-glucopyranosyl-7-O-α-<i>l</i>-rhamnopyranoside, and 14 mg of calycosin 7-O-β-<i>d</i>-glucopyranoside, and one lignan, 16 mg of alangilignoside B, were successfully isolated from 1.8 g of the crude sample through the combination of HSCCC with a two-phase solvent system composed of ethyl acetate-<i>n</i>-butanol-0.5 % formic acid (4:1:5, <i>v/v/v</i>) and semi-preparative RPLC with a mobile phase of methanol-water (20:80, <i>v/v</i>). The purities of the six compounds are all over 95 % as determined by HPLC and the structures are confirmed by the analysis of UV, <sup>1</sup>H-NMR, and <sup>13</sup>C-NMR and compared with published data.
A method of using high-speed counter-current chromatography (HSCCC) for preparative isolation and purification of oligostilbenes from the ethanol extracts of seed kernel of Iris lactea Pall. var. chinensis (Fisch.) Koidz was established in this study. Four oligostilbenes were successfully separated and purified by HSCCC with two sets of two-phase solvent system, n-hexane-ethyl acetate-methanol-water (3:6:4.2:5.5, v/v/v/v) in the head-to-tail elution mode for the first separation to mainly isolate vitisin A (58 mg), ɛ-viniferin (76 mg) and peak II (43 mg) from 300 mg of the crude ethanol extracts, and then light petroleum-ethyl acetate-methanol-water (5:5:3:6, v/v/v/v) in the tail-to-head elution mode for the second separation to isolate vitisin B (52 mg) and vitisin C (11 mg) from 100mg of peak II. The purities of the isolated four oligostilbenes were all over 95.0% as determined by HPLC. Vitisin A, vitisin B and vitisin C, resveratrol tetramers, were isolated from Iris lactea for the first time. The preparation of crude sample was simple and the HSCCC method for the isolation and purification of four oligostilbenes was rapid, efficient and economical.
Two novel organic amide alkaloids, 4-[(<i>E</i>)-<i>p</i>-coumaroylamino]butan-1-ol (<b>1</b>) and 4-[(<i>Z</i>)-<i>p</i>-coumaroylamino]butan-1-ol (<b>2</b>), together with a rare pyridoindole alkaloid, hippophamide (<b>3</b>), were isolated from the seed residue of <i>Hippophae rhamnoides</i> Linn. subsp. <i>sinensis</i> Rousi. Their structures were determined by spectroscopic means. The results show that compounds <b>1</b> and <b>2</b> are (<i>E</i>/<i>Z</i>)<i>-</i>isomers, compound <b>3</b>, a pyridoindole alkaloid concerted with <i>γ</i>-lactam ring.
A new triterpenoid, namely myricarin C (compound 1), together with three known compounds myricarin A (compound 2) and myricarin B (compound 3), 3α-hydroxy-D-friedoolean-14-en-28-oic acid (compound 4), was isolated from the overground part of Myricaria squamosa. Compound 2 and compound 3 existed in the solution by the form of cis-trans isomers. Their structures were elucidated by means of extensive spectroscopic methods, including 1D-NMR, 2D-NMR, and HR-ESI-MS. The antioxidant properties of all compounds were calculated based on the DPPH radical scavenging activities. Results showed that myricarin A and myricarin C had general antioxidant activities with EC50 values of 40.90 μg/ml, 42.22 μg/ml, respectively, compared to the control, rutin (5.17 μg/ml). The EC50 values of myricarin B was 195.81 μg/ml. Compound 4 had no antioxidant activities.
AIM: The objective of this study was to evaluate the effects of seed oil of Caragana korshinskii Kom. against Trichophyton mentagrophytes on an in vivo guinea pig model of dermatophytosis.METHODS: The skin of albino guinea pigs was infected with T. mentagrophytes, and the animals were divided into five groups: negative control (NC group), positive control (PC group), vehicle control, CK50% group (received topical 50% seed oil of C.korshinskii), and CK100% group (received topical 100% seed oil of C.korshinskii). Evaluation of clinical efficacy was performed 72 h after the completion of a 10-day treatment regimen. Skin biopsy samples were processed for histopathological examination.
RESULTS: The infected untreated control guinea pigs showed patches of hair loss and ulcerated or scaly skin. Lower clinical scores indicate improved efficacy compared with NC. The lesion scores significantly declined in the CK50%, CK100%, and PC groups in comparison with the NC group. The CK50% group (45.31%) and the CK100% group (75%) showed clinical efficacy compared with the PC group (78.13%). In addition, no fungal elements, inflammation, or tissue destruction was observed in any of the PAS-stained sections of the infected skin in the groups treated with CK100% or 1% terbinafine.
CONCLUSION: Seed oil of C.korshinskii demonstrated high antifungal efficacy in experimental dermatophytosis.