[Research on preparation process of andrographolide-glycyrrhizic acid polymeric micelles].

This study aimed to prepare andrographolide (AP)-loaded glycyrrhizic acid (GA) micelles (AP-GA)-PMs to enhance the solubility and anti-tumor effect of andrographolide. Firstly, andrographolide (AP) was used as the model drug and glycyrrhizic acid (GA) as carriers to prepare (AP-GA)-PMs. Then the preparation methods and the ratios of drug and carrier were screened and optimized based on particle size, encapsulation efficiency (EE) and loading capacity of micelles. Finally, the pharmaceutical characters and the inhibition rate on HepG2 cells were evaluated on the (AP-GA)-PMs prepared by optimal process. The results showed that the prepared micelles under the optimal process had a nanosize of (127.11±1.38) nm, zeta potential of (-24.01±0.55) mV, the entrapment efficiency rate of (92.01±4.02)% , the drug loading rate of (51.44±1.24)% and high storage stability at 4 °C in 30 d, with slow but highly stable in vitro release. Moreover, (AP-GA)-PMs with the IC50 value of 19.25 mg·L⁻¹ had a more synergistic and better anti-tumor effect in comparison with AP (IC50=122.40 mg·L⁻¹) on HepG2 cells (P<0.01). In conclusion, the (AP-GA)-PMs prepared with glycyrrhizic acid as a carrier had a small particle size, large drug loading capacity, and high stability, and could significantly improve the anti-tumor effects of AP.

Validated LC-MS/MS method for simultaneous determination of doxorubicin and curcumin in polymeric micelles in subcellular compartments of MCF-7/Adr cells by protein precipitation-ultrasonic breaking method.

A specific and sensitive LC-MS/MS with protein precipitation- ultrasonic breaking method has been developed and validated for simultaneous determination of doxorubicin (DOX) and curcumin (Cur) in DOX and Cur co-loaded hyaluronic acid-vitamin E succinatemicelles [(DOX + Cur)-polymeric micelles (PMs)] in subcellular compartments of resistant MCF-7/Adr cells. Sequential extraction of four subcellular protein fractions (cytosolic, membrane/organelle, nucleic and cytoskeleton) was performed directly from MCF-7/Adr cells after incubation with (DOX + Cur)-PMs. An ultrasonic breaking-methanol precipitation method was used for extraction of the fractions, and the micelle breaking efficiency with methanol was 98.1 and 97.6% for DOX and Cur, respectively. The analytes were analyzed using positive electrospray ionization coupled with multiple reaction monitoring. The calibration curves were linear over a concentration range of 0.5-400 ng/mL for DOX and 2-2000 ng/mL for Cur, and the recovery for the two analytes were >85% with negligible matrix effect. The intra-day and inter-day precision was <10.80% and relative error was within ±7.70%. The developed method was successfully applied for subcellular determination of DOX and Cur in MCF-7/Adr cells. Moreover, Cur and (DOX + Cur)-PMs had a marked promoting effect on the distribution of DOX in the nucleic protein fraction.

[Preparation characterization and antitumor activity in vitro of berberine hydrochloride polymeric micelles].

With polyethylene glycol vitamin E succinate (TPGS) as the carrier materials, and berberine hydrochloride ( BER) as model drug, we formed berberine hydrochloride (BER) -loaded TPGS nanomicells (BER-PMs) using filming-rehydration method to improve its solubility and in vitro anti-tumor effect. The transmission electron microscope (TEM) was used to observe the particle appearance; particle detector was used to detect the diameter and Zeta potential; and ultracentrifugation was utilized to determine the encapsulation efficiency (EE) and drug-loading (DD); dynamic dialysis method was used to study the in vitro release behavior of BER-PMs, and the anti-tumor activity against MCF-7 cells was determined by MTT method. Results showed that the average particle size of BER-PMs was (12.45 ± 1.46) nm; particle size was uniform and spherical; drug loading and encapsulation efficiency were (5.7 ± 0.22)% and (95.67 ± 5.35)%, respectively. Zeta potential was (-1.12 ± 0.23) mV; release rate within 24 h was 37.20% and 41.14% respectively in pH 7.4 and pH 6.5 phosphate buffer in vitro; compared with BER, BER-PMs can significantly inhibit MCF-7 cell proliferation (P < 0.05), promote cell apoptosis and improve the anti-tumor activity of BER in vitro. Therefore, the formed berberine hydrochloride micelle can more effectively promote the apoptosis of MCF-7 cell, and improve the drug's in vitro anti-tumor effect.

Simultaneous characterisation of fifty coumarins from the roots of Angelica dahurica by off-line two-dimensional high-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry.

INTRODUCTION: The root of Angelica dahurica is a traditional Chinese medicine that used for the treatment of headache, toothache, abscess, furunculosis and acne. Coumarins were the major bioactive constituents of A. dahurica, hence it is worthwhile developing a method to simultaneously characterise them, especially those in trace amounts. OBJECTIVE: To develop an efficient method for the simultaneous characterisation of coumarins in A. dahurica. METHODS: A method using off-line two-dimensional high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (off-line 2D-HPLC-ESI/MS(n) ) was developed. RESULTS: In total 50 coumarins, including 32 linear furanocoumarins, 16 bifuranocoumarins and two non-furanocoumarins, were identified from the roots of A. dahurica. The possible MS fragmentations of these coumarins are also proposed. CONCLUSION: The method described here allows rapid and convenient identification of the coumarins in A. dahurica, and may be applied to other herbal medicines containing linear furanocoumarins.

Comparison of hyaluronic acid-based micelles and polyethylene glycol-based micelles on reversal of multidrug resistance and enhanced anticancer efficacy in vitro and in vivo.

Polyethylene glycol (PEG)-based block copolymer micelles and hyaluronic acid (HA)-based grafted copolymer micelles have been widely investigated in chemotherapy. In this study, to evaluate the differences among HA-based grafted polymer micelles, PEG-based block polymer micelles and the mixed of these two micelles in enhancing antitumor effects and overcoming MDR, two amphiphilic vitamin E succinate (VES) derivatives, HA VES (HA-g-VES) and PEG 2000 VES (TPGS2k), were applied as nanocarriers to prepare HA-VES micelles (HA-PMs), TPGS2k micelles (TPGS2k-PMs) and the mixed micelles (HA/TPGS2k-PMs) for the co-delivery of doxorubicin (DOX) and curcumin (Cur). With the addition of TPGS2k, the particle size of HA/TPGS2k-PMs (153.37 ± 1.00 nm) was smaller than that of HA-PMs (223.83 ± 1.84) but significantly larger than that of TPGS2k-PMs (about 20 nm). The loading efficiency of HA/TPGS2k-PMs was 7.10%, which was lower than HA-PMs (8.31 ± 0.15%) but higher than TPGS2k-PMs (4.38 ± 0.24%). In vitro, HA/TPGS2k-PMs and TPGS2k-PMs exhibited higher cytotoxicity and reversal MDR effects than HA-PMs in MCF-7/Adr cells. However, HA/TPGS2k-PMs, HA-PMs and TPGS2k-PMs all significantly improved the tumor biodistribution, the antitumor effects and reduced the side effects of DOX in 4T1-tumor-bearing mice, but these three micelles displayed no differences in vivo. Therefore, EPR passive targeting effects caused by PEGylated micelles and CD44 active targeting effects caused by HA-based micelles have no significant variance in the delivery of antitumor drugs by i.v.

Apically targeted oral micelles exhibit highly efficient intestinal uptake and oral absorption.

INTRODUCTION: Polymeric micelles (PMs) hold promise for improving solubility and oral absorption of poorly soluble drugs. Unfortunately, the oral absorption of PMs is also limited by intestinal epithelium. To improve the oral delivery efficiency of micelles, transporter-mediated micelles could enhance the transport efficiency across the epithelial barrier, and they have attracted more attention. METHODS: Peptide transporter 1 (PepT1)-mediated micelles (Val-PMs/Phe-PMs) were designed by grafting valine (or phenylalanine) onto the surface of curcumin (Cur)-loaded-D-α-tocopheryl polyethylene glycol 1000 succinate micelles (TP-PMs). The oral absorption mechanism and oral bioavailability were further investigated in vitro and in vivo. RESULTS: The cellular study showed that Val-PMs/Phe-PMs had a high PepT1 affinity, resulting in a higher drug uptake and transcellular transport than TP-PMs. In rats, Val-PMs/Phe-PMs exhibited higher intestinal accumulation in the apical side of the intestinal epithelium than TP-PMs, promoting drug diffusion across epithelial barrier. The oral bioavailability of Cur was significantly improved by Val-PMs/Phe-PMs, which was about 10.50- and 3.40-fold greater than that of Cur-Sol and TP-PMs, respectively. CONCLUSION: PepT-1-mediated micelles, using PepT1 as a target on intestinal epithelium, have unique functions with intestine and prove promising for oral delivery of poorly water-soluble drugs.

[Studies on chemical constituents from root of Clematis hexapetala].

OBJECTIVE: To study the chemical constituents from the roots and rhizomes of Clematis hexapetala. METHOD: The compounds were separated by means of solvent extraction, repeated chromatography with silica gel and HPLC. The structures were determined by spectral analysis. RESULT: Nine compounds were isolated as friedelin (1) , anemonin (2) , beta-sitosterol (3) , palmitic acid (4) , vanillic acid (5) , isolariciresinol (6) , 5-hydroxumethyl-5H-furan-2-one (7) , n-nonane (8) , daucosterol (9). CONCLUSION: All the compounds were isolated from the plant for the first time.

[Isolation and purification of total tubeimosides from Bolbostemma paniculatum by macroporous resin].

OBJECTIVE: To select the optimum purification process of all tubeimosides from Bolbostemma paniculatum by macroporous resin. METHOD: Static absorption test and deabsorption test were carried out to screen the best macroporous resin for all tubeimosides. The single factor test, orthogonal experimental designs and variance analysis were applied to optimize the manipulation parameters of macroporous resin. RESULT: The macroporous resin NKA possessed the strongest absorption ability to all tubeimosides among the 9 resins studied, in addition to an easy de-absorption property. The optimum absorption conditions were A2B3C1, namely concentration of feed 30 mg x mL(-1), diameter: height of the chromatography column 1:7, the weight ratio of raw material and resins 1:2. CONCLUSION: The above experimental results can provide experimental basis for large-scale purification process of all tubeimosides.

[Study on the pretreatment of the series of HPD macroporous adsorption resin].

OBJECTIVE: To develop a method for the pretreatment of the series of HPD macroporous adsorption resin (MAR). METHOD: The effect of 2% NaOH to pretreatment of the series of HPD MAR was studied by gas chromatography and UV spectrometery. RESULT: The eluting effect by ethanol was better after the MAR was marinated and eluted by 2% NaOH. CONCLUSION: The pretreatment of the series of HPD MAR was: after marinated and eluted by 2% NaOH, the series of HPD MAR were eluted by ethanol at 2 BV x h(-1) at 60 degrees C for 3-4 bed volumes (BV). At then the UV absorbance of eluate was 0.2-0.5. Benzene was not detected and the other residues less than 10 mg x L(-1) in eluted-MAR by GC. And the MARs match to the standard of medicine.

Synergistically Improved Anti-tumor Efficacy by Co-delivery Doxorubicin and Curcumin Polymeric Micelles.

P-gp mediated drug efflux has been recognized as a major obstacle limiting the success of cancer chemotherapy. To overcome this issue, doxorubicin (DOX) and curcumin (Cur; P-gp inhibitor and apoptosis inhibitor) co-encapsulated pegylated polymeric micelles ((DOX+Cur)-PMs) were designed, prepared and characterized to simultaneously deliver chemotherapeutic drug and multidrug resistance (MDR) modulator to tumor sites. The (DOX+Cur)-PMs were spherical nano-size particle, with a loading content of 6.83%, and high colloidal stability. Co-delivery micelles exhibited excellent cytotoxicity by reversing MDR, promoting cellular uptake and enhancing cellular apoptosis in MCF7/Adr cells. The tumor growth inhibitory effect of (DOX+Cur)-PMs in 4T1-bearing mice was more effective compared with the combination solution of DOX and Cur and even DOX-PMs. In conclusion, simultaneous delivery of DOX and Cur by (DOX+Cur)-PMs has been demonstrated to be a promising approach for overcoming MDR and improving antitumor efficacy.

[Study on liposome preparation technique of Bupleurum chinense volatile oil for injection].

OBJECTIVE: To study the preparation technique of the liposome of Bupleurum chinense volatile oil. METHOD: The composition, entrapment ratio of volatile oil, availability of freeze-drying and the changes of finger prints were studied. RESULT: Entrapment ratio reached 81.9%. After freeze-dried, the remaining ratio of the oil entrapped in the liposome reached 88.6%. CONCLUSION: It is practicable to make B. chinense injection into freeze-dried liposome powder.

Correlation study between molecular structure of sesquiterpene lactones and the selective adsorption performance of molecularly imprinted polymers.

We preliminarily report that the structure of template molecules and target components correlates with the selective adsorption performance of molecularly imprinted polymer (MIPs) in sesquiterpene lactones. Template molecules involved three categories of sesquiterpene lactones with distinct ring systems: 5-mem lactone ring atractylenolide III, 7-mem lactone ring dehydrocostus lactone, and 10-mem lactone ring costunolide lactone, of which the conformations were verified by variable-temperature (1)H NMR spectroscopy. Reciprocal MIPs were prepared by precipitation polymerization and employed as selective sorbents in the columns of solid phase extraction (SPE). These columns were further used for enriching the mixed adsorption solution of sesquiterpene lactone ingredients and reference components. Finally, the extract of Radix Aucklandiae, a Chinese medicine herb, was used to verify the efficiency of this method. Our results demonstrate that the steric conformational stability of molecules is associated with the selective adsorption of their corresponding MIPs. We have further observed that the maximum adsorption capacity occurs when the target molecule conformation is consistent with that of the template molecule. The addition of more hydrophilic groups correlates with weaker adsorption of MIPs. Our findings provide important information to help guide the selection of appropriate template molecules for synthesis of MIPs with specific adsorption.

[Determination of organic residues in macroporous adsorption resins by gas chromatography].

A method has been developed for the simultaneous determination of benzene, toluene, xylene, styrene, decane, diethylbenzene, undecane, divinylbenzene, dodecane and naphthalene residues in macroporous adsorption resins (MARs). The organic residues were extracted from resin samples by ultrasonic extraction with dichloromethane and analyzed by gas chromatography. The chromatographic conditions were as follows: a DB-624 capillary column (30 m x 0.32 mm i.d., 1.8 microm); carrier gas, nitrogen with a flow rate of 2.5 mL/min; column temperature, programmed from 40 degrees C to 200 degrees C at a rate of 14 degrees C /min and kept at 200 degrees C for 1 min; flame ionization detector temperature, 250 degrees C; injector temperature, 220 degrees C; splitless injection, 1 microL. All the 10 organic residues were separated well in 12 min. The recoveries for spiked standards (n = 3) were 73.8% - 107.9%. The relative standard deviations were 1.3% - 4.4%. The limits of detection were 0.007 - 0.03 mg/L. This method is sensitive, accurate and quick. Nine commercial MARs and their pretreated samples were assayed, and the results show that the contents of organic residues varied greatly in two kinds of samples. And the pretreated MARs can be used safely in the production of traditional Chinese medicines.