The exploration of neuraminidase inhibitory activity on Fallopia denticulata, an ethnic herb in China.

This study was designed to explore the bioactive ingredients in the extracts of Fallopia denticulata (C.C. Huang) Holub, a medicinal plant grown in China, which exhibits the best neuraminidase (NA) inhibition activity. Three fractions of ethyl acetate, ethanol, and water were tested on NA inhibition assay, and the best one was conducted by ultra-performance liquid chromatography-time-of-flight mass spectrometry in the negative and positive modes to analyze the metabolic components. The results revealed the identification of the following 21 compounds: 3 organic acids, 11 flavonoids, 1 coumarin, and 6 others, such as ß-daucosterol, gallic acid, and syringic acid, of which 12 compounds were discovered for the first time in F. denticulata. In addition, we used the molecular docking technique to support the anti-NA activity of each compound in the best extract. The results confirmed that the two better bioactive compounds were (-)-epicatechin gallate and (+)-catechin. Therefore, F. denticulata could be used as a potential material for new anti-influenza drugs.

SP94 Peptide-Functionalized PEG-PLGA Nanoparticle Loading with Cryptotanshinone for Targeting Therapy of Hepatocellular Carcinoma.

To achieve improved drug delivery efficiency to hepatocellular carcinoma (HCC), biodegradable poly (ethylene glycol)-poly (lactic-co-glycolic acid) (PEG-PLGA) nanoparticles (NP), surface-modified with SP94 peptide, were designed for the efficient delivery of cryptotanshinone to the tumor for the treatment of HCC. Cryptotanshinone NP and SP94-NP were prepared by using nanoprecipitation. The physicochemical and pharmaceutical properties of the NP and SP94-NP were characterized, and the release kinetics suggested that both NP and SP94-NP provided continuous, slow release of cryptotanshinone for 48 h. The in vitro cellular experiment demonstrated that SP94-NP significantly enhanced the cellular uptake of cryptotanshinone and induced high cytotoxicity and cellular apoptosis of hepatocellular carcinoma (HepG2) cells. The in vivo detecting results of targeting effect using the Cy5.5 probe evidenced that SP94-NP showed an accumulation in tumor more efficiently than that of unconjugated ones. Meanwhile, SP94-NP exhibited the smallest tumor size than other groups and showed no toxicity to body. The results of this study provide a promising nanoplatform for the targeting of HCC.

[Optimization of Enzymatic Hydrolysis Conditions of Xiaoyao-pill Herb Residues by Cellulase].

Objective: To explore the enzymatic hydrolysis conditions of Xiaoyao-pill herb residues by cellulose. Methods: Based on the single factor test,an Box-Benhnken design was used to optimize the parameters, three factors, including cellulase dosage, enzymolysis time, and solid-liquid ratio were regarded as investigation factors,the yield of enzymatic hydrolysis as index, Xiaoyao-pill herb residues enzymatic hydrolysis catalyzed by cellulase, and a mathematical regression model was established. Results: The optimal parameters were obtained as follows,cellulase dosage was 6%,enzymolysis time was 5. 6 h and solid-liquid ratio was 1 ∶ 12( g / m L). Under this condition,the yield of enzymatic hydrolysis was 43. 89%. Conclusion: The results may provide new reference for further exploring Chinese herbal medicine efficiently.

New agar microspheres for the separation and purification of natural products.

A new type of agar chromatography media has been prepared with a yield over 80% using a water-in-oil emulsion technique. These microspheres have regular spherical shapes and particle diameters in the range 40-165 µm (average ∼90 µm). Cross-linking of the resulting agar microspheres with epichlorohydrin and 1,4-butanediol diglycidyl ether enhanced their mechanical and thermal stability. The alkaline conditions used during the cross-linking reaction also decreased the content of ionized sulfate groups of the polysaccharide, thus reducing the nonspecific adsorption of positively charged molecules. The cross-linked agar microspheres were functionalized with (i) branched poly(ethyleneimine) to obtain a stationary phase useful for the separation of proteins in an anion-exchange mode and (ii) with poly-ß-cyclodextrin enabling direct isolation and purification of puerarin from a crude extract of Radix puerariae. Using a 23.5 mL column loaded with 20 mg extract (0.85 mg/mL gel), puerarin with a purity of 96% was recovered with a yield of 86%.

A novel drug-phospholipid complex loaded micelle for baohuoside I enhanced oral absorption: in vivo and in vivo evaluations.

Baohuoside I is an effective anti-cancer drug currently used for a variety of cancers in vitro. Unfortunately, baohuoside I has a very poor solubility in both water and in organic solvents. Besides that, it is subject to significant efflux. This work therefore aimed at evaluating the ability of mixed micelles to solubilize baohuoside I, increase permeability and inhibit efflux of baohuoside I to promote oral absorption. A novel (TPGS-baohuoside I-phospholipid complex) mixed micelles was formed by phospholipid complex and TPGS to increase the solubility, enhance permeability, and inhibit efflux of baohuoside I. Micelle formation was confirmed by differential scanning calorimetry and transmission electron microscopy. The average diameters and efflux ratio of mixed micelles decreased as the ratio of TPGS increased with a respective increase in solubility of baohuoside I. Nevertheless, a slow release of baohuoside I from loaded micelles was noted. The results showed that at a 1:9 ratio for baohuoside I-phospholipid complex: TPGS in mixed micelles, solubility of baohuoside I increased up to 88 fold while the efflux ratio decreased by 85%. Their smaller size and higher zeta potential values indicated that mixed micelles would be easily absorbed and more stable. The relative bioavailability of the micelles (AUC(0-∞)) compared with baohuoside I (AUC(0-∞)) was 533%, demonstrating great potential for clinical application. Hence, the novel micelles formed with phospholipid complex and TPGS considerably increased drug concentration in the media and enhanced absorption.

Target RNA expression omics approach to reveal the liver detoxification effect induced by Chinese medicine prescription Niu Huang Jie Du against realgar overexposure to mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Niu Huang Jie Du prescription (NHJD) is a traditional Chinese medicine (TCM) widely used in patients suffering from excessive inner fire toxin (Huo Du Nei Sheng) syndrome, such as sore throat, gingival swelling, and pain, mouth and tongue sores, etc. This formula contains realgar (As4S4) which is one of the 28 toxic medicinal materials promulgated by the Chinese Ministry of Health. Many studies reported its toxicity on the liver and kidney, and the detoxification effect of NHJD. However, its detoxification mechanism is still unclear. AIM OF THE STUDY: To clarify the detoxification mechanism of NHJD to realgar, this study evaluated the detoxification effect of NHJD on realgar exposure in mice, and analyzed differences in mRNA expression profiles in liver tissues and associated functional predictions. MATERIAL AND METHODS: ICR mice were administered with NHJD, realgar, and CMC-Na as blank control for 12 weeks, respectively. Liver injury was evaluated by histopathologic examination and liver mRNA gene were sequenced by Illumina. Differentially expressed gene, functionally enrichment and protein association network analysis were conducted. RESULTS: 43 genes were screened out, among which 15 genes in the realgar group were decreased, but the extent of the decline has been restored in the NHJD group. The remaining 28 genes have exactly the opposite trends. Functional module analysis revealed that those detoxification function-related genes were primarily for positive regulation of glutathione metabolism, P450 on the metabolism of exogenous compounds, oxidative stress and immune-related, etc. CONCLUSIONS: The results indicated that realgar mainly causes liver damage by changing the common enzymes of drug metabolism, especially the expression of genes related to CYPs, GSTs family, oxidative stress, and complement immunity, while the TCM prescription NHJD has a regulatory effect on the abnormal expression of corresponding genes. Our results will provide some clues for the detoxification mechanism of arsenic-containing TCM prescriptions.

[Research progress of chromanone derivatives from Calophyllum].

Calophyllum plants have great pharmaceutical value. Some species of this genus are used in folk medicine to treat and cure diseases, such as toothache, rheumatism, diarrhea, chronic ulcers, skin infections, and wounds. The genus is known to be rich in chromanone derivatives, and some of these compounds have antiviral, antifungal, antibacterial, cytotoxic, and other activities. In order to come to a more complete understanding of chromanone derivatives and gain new bioactive constituents from Calophyllum, chemical structures, pharmacological activities, and biogenesis of chromanone derivatives from Calophyllum have been discussed in this review.

Dopamine-polyethyleneimine co-deposition cellulose filter paper for α-Glucosidase immobilization and enzyme inhibitor screening.

In this work, cellulose filter paper (CFP), which is inexpensive and commercially available, was used as the carrier, and the immobilized α-glucosidase was obtained by two steps: firstly, the surface of CFP was modified by polydopamine/polyethyleneimine (PDA/PEI) co-deposition method to obtain CFP-PDA/PEI with a uniform coating of rich positive charge; subsequently, α-glucosidase was immobilized on the CFP-PDA/PEI by electrostatic adsorption. The free enzyme and immobilized enzyme have the same optimal temperature (70℃) and pH (8.0), and their Km is similar, which is 2.2 and 2.8, respectively. These results show that the immobilization process does not change the properties of the enzyme greatly. The immobilized enzyme still maintains 75.6% of its initial activity after 10 repeated uses, showing good reusability. The excellent repeatability (RSD = 2.2%, n = 5) and the verification of competitive inhibitor (acarbose) illustrates the reliability of the immobilized enzymes for enzyme inhibitor screening. Finally, combined with CE, a screening method based immobilized α-glucosidase was proposed and applied to screen the α-glucosidase inhibitory from 10 kinds of Traditional Chinese medicines (TCMs) in vitro. The results indicated that the method was a very effective tool for screening potential α-glucosidase inhibitors from TCMs.

Wax-matrix tablet for time-dependent colon-specific delivery system of sophora flavescens Aiton: preparation and in vivo evaluation.

A wax-matrix time-dependent colon-specific tablet (WM-TDCS) was studied. Wax-matrix tablet core consisting of semi-synthetic glycerides, as a wax polymeric expanding agent, carboxymethyl starch sodium (CMS-Na), and NaCl was prepared, and Sophora flavescens Aiton (ASF, extracts of traditional Chinese medicine) was used as model drug. The wax-matrix ASF tablets core was coated with Eudragit NE 30 D as the inner coating materials and with Opadry OY-P-7171 as the outer coating materials. The in vitro release behaviors of the coated tablets were examined and then in vivo absorption kinetics of the coated tablets in dogs was further investigated. The volume of the tablet core was markedly increased at 37 degrees C because of the expand effect of polymer semi-synthetic glycerides and CMS-Na. The drug release from WM-TDCS was more stable than TDCS in vitro and in vivo. The lag time of ASF release was also controlled by the thickness of the inner coating layer. In vivo evaluation demonstrated that in vivo lag time of absorption was in a good agreement with in vitro lag time of release. ASF wax-matrix tablets coated with Eudragit NE 30 D and Opadry OY-P-7171 using the regular coating technique could be designed to achieve a lag time of 3 h in the small intestinal tract.

Preparation of micro-cell membrane chromatographic columns with polyvinyl alcohol-modified polyether ether ketone tube as cellular membrane carrier.

Cell membrane chromatography is a promising technique for screening active components from complex matrices. Unfortunately, the large consumption of cells and low resolutions of analytes limit the applications of this method. Herein, we report polyether ether ketone tube as a novel cellular membrane carrier for cell membrane chromatography. Its inner surface is firstly coated by polyvinyl alcohol and then cell membranes are physically adsorbed onto the polyvinyl alcohol layer. To verify this approach, osteoclast and osteoblast micro-column were prepared and characterized by calcitonin and verapamil, respectively. Comparing with common cell membrane chromatographic column, the micro-cell membrane chromatographic columns showed about 1000-fold decrease of cell consumption and satisfactory retention behavior. The developed column was applied to screen potential active components from Cortex Phellodendri Chinensis. A total of 18 components in Cortex Phellodendri Chinensis extract were observed as having retention property of osteoclast micro-cell membrane chromatographic column, while 10 components retained on osteoblast micro-cell membrane chromatographic column. The results of in vitro assay showed that berberine, obacunoic acid and phellodendrine had an obvious inhibitory effect on osteoclast differentiation and function. Berberine and tetrahydropalmatine increased the osteoblast proliferations and mineralized nodules density. This cell membrane/polyvinyl alcohol column can be applied to various biological chromatography models.

Anti-microtubule activity of tubeimoside I and its colchicine binding site of tubulin.

BACKGROUND: Tubeimoside I (TBMS1) was isolated from the tubers of Bolbostemma paniculatum (Maxim.) Franquet. TBMS1 shows potent anti-tumor activity. The present study was conducted to investigate the anti-microtubule role of TBMS1 and its binding site of tubulin. METHODS: Cell growth inhibition was measured by MTT after treatment with TBMS1. Uptake kinetics of TBMS1 by human nasopharyngeal carcinoma CNE-2Z cell line (CNE-2Z) was assayed by HPLC. Microtubule protein (MTP) was prepared from porcine brain through two cycles of polymerization-depolymerization in a high molarity buffer. Inhibition of MTP polymerization induced by TBMS1 was determined by a turbidity measurement and a sedimentation assay; the interactions of TBMS1 with tubulin within CNE-2Z cells were investigated by immunofluorescence microscopy and immunoblotting. TBMS1 was tested for its ability to inhibit binding of known tubulin ligands through competitive binding assay. RESULTS: TBMS1 displayed growth inhibitory activity against CNE-2Z cells with IC(50) value of 16.7 microM for 72 h. HPLC analysis of TBMS1 uptake by CNE-2Z cells displayed the initial slow TBMS1 uptake and then gradually reaching an maximum uptake near 18 h. CNE-2Z cells treated with TBMS1 (25 microM, 3 h) were sufficient to cause the microtubular network disruption. Immunoblot analysis showed that the proportion of cytosolic tubulin of cells treated with TBMS1 increased in a time- and concentration-dependent manner. TBMS1 did not inhibit the binding of vinblastine to tubulin. Colchicine binding to tubulin was inhibited in the presence of TBMS1. CONCLUSIONS: TBMS1 is an anti-microtubule agent, and its binding site of tubulin is the colchicine binding site of tubulin.

[Optimization of matrix formulation of effective parts cataplasm of Pogostemon cablin by uniform design].

OBJECTIVE: To optimize the matrix formulation of the effective part Cataplasm of Pogostemon Cablin. METHOD: The optimal preparation prescription was selected by U17 (17(11)) uniform design,and the tacking strength, cohesive strength and transdermal speed constant were used as test indexes. The equations of three test indexes were established by SPSS. With analysis of the contribution of factors by SPSS regression, the optimal matrix formulation was acquired. RESULT: The optimal matrix formulation is carbopol U10-NoveriteTM7s-glycerine-sorbitol-kaolin-citric acid-aluminum trichloride (1.0:5.0:20:2.0:2.0:0.25:0.2). CONCLUSION: The matrix has good adhesive property, proper drug release rate, desirable hemocompatibility with the extractions of Pogostemon cablin.

[Optimization for formulation matrix proportion of xiangyu cataplasm by uniform design].

OBJECTIVE: To optimize the matrix formulation of cataplasm used to cure infantile diarrhea. METHODS: The optimum proportion of matrix for the preparation technology process of cataplasm was selected by uniform design and SPSS regression analysis. A check-up for adhibition , peeling strength, nonflowing, content of cream was founded. RESULTS: The best matrix's prescription gelatin: CMC-Na: PANA: kaolin: aluminum trichloride: citric acid: PVP K-30: PEG400: trimethylene glycol: tween-80 was 0.25 : 0.1 : 0.2 : 1.5 : 0.4 : 0.6 : 0.8 : 2 : 1 : 0.5. CONCLUSION: The preparation technique of cataplasm is feasible, and its quality is steerable, it is a safe and effective transdermal-drug delivery system.

[Preparation and evaluation in vivo of Scutellaria thermosensitive gel].

OBJECTIVE: To study scutellaria thermosensitive gel in situ. METHOD: The phase transition temperature of gel with various concentration polymer solutions was studied by using stirring method and the viscosity of gel was monitored under different temperatures. Eye irritation experiments were performed with rabbit. The duration of residence time in rabbit eyes of scutellaria thermosensitive gel was observed with 2% fluorescein. RESULT: 20% poloxmar407 and 10% poloxmar 188 were suitable to scutellaria thermosen-sitive gel in situ. The results suggested that the scutellaria thermosensitive gel in situ based on poloxmar were nonirritant and retention time on the surface of eye was (150 +/- 8) min. CONCLUSION: The scutellaria thermosensitive gel in situ forms gel in eye and has longer release time than that of eye drops.

Biomimetic Gastrointestinal Tract Functions for Metal Absorption Assessment in Edible Plants: Comparison to In Vivo Absorption.

A biomimetic gastrointestinal tract, including in vitro digestion and biomimetic biomembrane extraction, has been proposed for absorption assessment of metals from edible plants. However, its validity is still unknown. Herein, two species of edible plants, Anoectochilus roxburghii and Radix astragali, were selected and digested in a bionic mouth, stomach, and intestine, and then trace metals (Cr, Mn, Fe, Ni, Cu, Zn, Se, Sr, As, and Pb) were transformed to their final metal species. To check model predictability, in vitro and in vivo metal absorption were imitated and tested by monolayer liposome extraction and rat stomach or single-pass duodenal intestine, respectively. A strong correlation was established between in vivo and in vitro metal absorption ratios, with 0.89 > R2 > 0.66, and a significant relationship (p < 0.05) was exhibited for stomach, intestine, two plant species, and 10 metal species. Our biomimetic system could be used as low-cost alternatives to animal and clinical studies for multi-metal absorption.

Nanoemulsion for improving solubility and permeability of Vitex agnus-castus extract: formulation and in vitro evaluation using PAMPA and Caco-2 approaches.

The purpose of this study was to develop new formulation for an improved oral delivery of Vitex agnus-castus (VAC) extract. After the optimization and validation of analytical method for quali-quantitative characterization of extract, nanoemulsion (NE) was selected as lipid-based nanocarrier. The composition of extract-loaded NE resulted in triacetin as oil phase, labrasol as surfactant, cremophor EL as co-surfactant and water. NE contains until 60 mg/mL of extract. It was characterized by DLS and TEM analyses and its droplets appear dark with an average diameter of 11.82 ± 0.125 nm and a polydispersity index (PdI) of 0.117 ± 0.019. The aqueous solubility of the extract was improved about 10 times: the extract is completely soluble in the NE at the concentration of 60 mg/mL, while its solubility in water results less than 6 mg. The passive intestinal permeation was tested by using parallel artificial membrane permeation assay (PAMPA) and the permeation across Caco-2 cells after preliminary cytotoxicity studies were also evaluated. NE shows a good solubilizing effect of the constituents of the extract, compared with aqueous solution. The total amount of constituents permeated from NE to acceptor compartment is greater than that permeated from saturated aqueous solution. Caco-2 test confirmed PAMPA results and they revealed that NE was successful in increasing the permeation of VAC extract. This formulation could improve oral bioavailability of extract due to enhanced solubility and permeability of phytocomplex.

Biopharmaceutical studies on crude drug preparations. I: Permeation of paeonol in a decoction and dry extract of Paeonia suffruticosa root cortex using an absorption simulator.

The permeation behavior of paeonol, one active constituent of a crude Chinese drug called Moutan Cortex, through an artificial gastric lipid barrier was examined in vitro using an absorption simulator in order to clarify how the administered form influenced the bioavailability of paeonol from a crude preparation. Paeonol concentrations were determined by a high-performance liquid chromatographic method developed during this study. Paeonol showed greater permeation from artificial gastric juice into artificial plasma when it was applied as a decoction or freeze-dried extract of Moutan Cortex than when applied as purified paeonol alone.

Screening of bioactive compounds in Radix Salviae Miltiorrhizae with liposomes and cell membranes using HPLC.

A new method employing HPLC, LC-MS, liposomes and cell membranes for the screening of bioactive compounds in traditional Chinese medicines (TCMs) has been proposed. We hypothesized that exposure of the TCM extracts to liposome membranes or cell membranes should decrease the concentration of membrane-permeable compounds in the solution. Using this approach, the permeability of the compounds in Radix Salviae Miltiorrhizae was investigated. By comparing chromatograms of samples prepared both before and after interaction with liposome membranes, erythrocyte membranes and cardiac myocyte membranes, 12 permeable compounds of Radix Salviae Miltiorrhizae were identified, and they were proven to be biologically active, with the exception of vanillic acid. There was a good correlation between the liposome model and the erythrocyte membranes, which was determined by comparing the binding degree of the permeable compounds (n=0.9059). Additionally, it was found that dihydrotanshinone I, cryptotanshinone, tanshinone I, tanshinone IIA combined specifically with cardiac myocyte membranes, which might indicate a useful approach for revealing the cardiovascular effects of Radix Salviae Miltiorrhizae. Based on these results, this method could be a novel approach for identifying potentially bioactive components in other TCMs.

Microsphere resin chromatography combined with microbial biotransformation for the separation and purification of salvianolic acid B in aqueous extract of roots of Salvia multiorrihza Bunge.

Salvianolic acid B was separated and purified from Salvia miltiorrhiza Bunge (danshen) by microbial transformation together with chromatography of microsphere resin. The aqueous extract of danshen was transformed by Fusarium graminearum in a bioreactor containing phosphate buffer (PBS), in which rosmarinic acid was transformed into danshensu and caffeic acid and the yield of salvianolic acid B was higher than 85%. After biotransformation, salvianolic acid B was purified by microsphere resin. A parallel test for making a comparison of microsphere resin chromatography between elution by methanol water solution and water was done. The purity of salvianolic acid B was up to 95% at the yield of 62% when impurities and salvianolic acid B were eluted by 45% and 55% methanol solution respectively. The purity of salvianolic acid B was up to 99% at the yield of 90% when distilled water was used to elute the impurities and salvianolic acid B. The total yield of salvianolic acid B was up to 75% at the purity over 99% while biotransformation combined with microsphere resin chromatography by water elution. Microbial biotransformation together with water elution of microsphere resin supplied an efficient method to eliminate the micromolecular impurities and a possible method to purify water-soluble compounds in traditional Chinese medicine.

Structural transformation of lignan compounds in rat gastrointestinal tract; II. Serum concentration of lignans and their metabolites.

Serum concentrations of arctiin, tracheloside, and their metabolites formed in the gastrointestinal tract were investigated in the rat. Arctiin or tracheloside was not detected in the serum after oral administration (200 mg/kg). In regard to their metabolites, each metabolite 1 (AM1, TM1), their genuine genins, appeared in the serum, and the serum concentration of arctiin metabolite 1 (AM1) reached its peak at 4 h and that of tracheloside metabolite 1 (TM1) reached its peak at 8 h. On the other hand, both metabolites 2 (AM2, TM2), which each possess a catechol moiety as reported previously, were not found in the serum. Now, we have studied the detection of their metabolites in the rat large intestinal contents after oral administration. It was revealed that all metabolites reported previously were certainly formed in rat gastrointestinal tract in vivo. Thus, we presumed a possibility that metabolite 2 was converted into metabolite 1 through C-3" methylation by catechol-O-methyltransferase (COMT) in rat liver. Each metabolite 2 was incubated with rat liver cytosol in the presence of S-adenosyl-L-methionine. It was proved that metabolite 2 was rapidly converted into metabolite 1 within 3 min. We suggest that arctiin or tracheloside was transformed to at least two metabolites in the gastrointestinal tract, and after absorption from the intestine, metabolite 2 was converted into metabolite 1 through methylation by COMT in the liver, and arctiin and tracheloside existed as metabolite 1, the genuine genin, in the blood stream.