Protective effects of psoralen polymer lipid nanoparticles on doxorubicin - induced myocardial toxicity

Abstract Doxorubicin (DOX) induced myocardial toxicity may limit its therapeutic use in clinic. Psoralen (PSO), a major active tricyclic furocoumarin extracted from Psoralea corylifolia, is widely used as an antineoplastic agent in treatment of leukemia and other cancers. This study is aim to find the protective effect of psoralen polymer lipid nanoparticles (PSO-PLN) on doxorubicin-induced myocardial toxicity in mice. The model of myocardial toxicity induced by DOX was established. The experiment was divided into 6 groups: normal saline group, DOX + Sulfotanshinone Sodium, DOX + PSO-PLN (3 mg/kg), DOX + PSO-PLN (6 mg/kg), DOX + PSO-PLN (9 mg/ kg), DOX group. DOX alone treated mice lead to a significant decrease in the body weight, heart weight, and increase in the serum levels of lactate dehydrogenase (LDH), creatine kinase (CK) and malondialdehyde (MDA) markers of cardiotoxicity. However, DOX reduced glutathione (GSH) content and activities of antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GPX), were recovered by PSO-PLN. And PSO-PLN also decreased markers of cardiotoxicity in the serum. Western blotting data showed that the protective effects of PSO-PLN might be mediated via regulation of protein kinase A (PKA) and p38. Our study suggest that PSO-PLN possesses antioxidant activities, inactivating PKA and p38 effect, which in turn protect the heart from the DOX-induced cardiotoxicity.

Evaluation of the Absorption Behavior of Main Component Compounds of Salt-Fried Herb Ingredients in Qing'e Pills by Using Caco-2 Cell Model.

Qing'e Pills is a Chinese traditional herbal product, which is often used to strengthen muscles and bones in TCM (traditional Chinese Medicine) practice. Its two main component herbs, namely, Cortex Eucommiae and Fructus Psoraleae are both required to be salt-fried according to TCM theory. We have evaluated the effects of salt-frying treated herbs on Caco-2 cell uptake behavior for those active ingredients of Qing'e Pills. By investigating of various variables, including MTT, temperature, inhibitors, pH, salt concentration and herb processing methods, we tried to clarify whether the salt-processing on herbs was necessary or not. Results showed that, compared to other processing methods, the salt-frying process significantly (p < 0.01) enhanced the absorption of effective components of Qing'e Pills. The way that psoralen, isopsoralen, psoralenoside and geniposide acid entered Caco-2 cells at low concentrations was via passive diffusion. These components were not substrates of P-glycoprotein. It demonstrated that the salt-frying process not only enhanced the concentration of active components in herb extract, but also changed their absorption behaviors. Nevertheless, the mechanism of absorption behavior changing needs to be further investigated.

Characterization of Compounds in Psoralea corylifolia Using High-Performance Liquid Chromatography Diode Array Detection, Time-of-Flight Mass Spectrometry and Quadrupole Ion Trap Mass Spectrometry.

High-performance liquid chromatography with diode array detection (HPLC-DAD), time-of-flight mass spectrometry (HPLC-TOFMS) and quadrupole ion trap mass spectrometry (HPLC-QITMS) were used for separation and identification of multi-components in Psoralea corylifolia. Benefiting from combining the accurate mass measurement of HPLC-TOFMS to generate elemental compositions, the complementary multilevel structural information provided by HPLC-QITMS and the characteristic UV spectra obtained from HPLC-DAD, 24 components in P. corylifolia were identified. The five groups of isomers were differentiated based on the fragmentation behaviors in QITMS and UV spectra. It can be concluded that an effective method based on the combination of HPLC-DAD, HPLC-TOFMS and HPLC-QITMS for identification of chemical components in P. corylifolia was established. The results provide essential data for further pharmacological and clinical studies of P. corylifolia and facilitate the rapid quality control of the crude drug.

Anti-metastatic effect and mechanisms of Wenshen Zhuanggu Formula in human breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Wenshen Zhuanggu Formula (WSZG), a traditional Chinese medicine (TCM) empirical prescription, has been used to treat the patients with breast cancer bone metastasis as an adjuvant in clinical practice. To explore the anti-metastatic activity and potential mechanisms of WSZG-containing serum (WSZG-CS) on highly bone-metastatic human breast cancer MDA-MB-231BO cells. MATERIALS AND METHODS: MDA-MB-231BO cells were cultured alone or co-cultured with bone marrow-derived mesenchymal stem cells (BMSCs). Invasion assays were carried out in Matrigel-coated Transwell chambers. CC chemokine 5 (CCL5) and interleukin (IL)-17B secretion levels were detected by ELISA. CCR5 and IL-17BR protein expression levels were determined by immunocytochemistry and Western blot analysis. RESULTS: Compared with control serum, WSZG-CS significantly inhibited BMSC induced MDA-MB-231BO breast cancer cell invasion, reduced CCL5 and IL-17B levels in co-culture supernatants, and downregulated CCR5 and IL-17BR protein expression in breast cancer cells co-cultured with BMSCs. CONCLUSIONS: WSZG-CS exerts an anti-metastatic activity against MDA-MB-231BO breast cancer cells, due to its ability to mitigate the interaction between BMSCs and breast cancer cells mediated via the CCL5/CCR5 and IL-17B/IL-17BR signaling pathways.

Psoralea corylifolia L. seed extract ameliorates streptozotocin-induced diabetes in mice by inhibition of oxidative stress.

Pancreatic beta-cell death is known to be the cause of deficient insulin production in diabetes mellitus. Oxidative stress is one of the major causes of beta-cell death. In this study, we investigated the effects of Psoralea corylifolia L. seed (PCS) extract on beta-cell death. Oral administration of PCS extract resulted in a significant improvement of hyperglycemia in streptozotocin-induced diabetic mice. PCS extract treatment improved glucose tolerance and increased serum insulin levels. To study the mechanisms involved, we investigated the effects of PCS extract on H2O2-induced apoptosis in INS-1 cells. Treatment with PCS extract inhibited cell death. PCS extract treatment decreased reactive oxygen species level and activated antioxidative enzymes. Among the major components of PCS extract, psoralen and isopsoralen (coumarins), but not bakuchiol, showed preventive effects against H2O2-induced beta-cell death. These findings indicate that PCS extract may be a potential pharmacological agent to protect against pancreatic beta-cell damage caused by oxidative stress associated with diabetes.

High-performance thin layer chromatographic quantification of bioactive psoralen and daidzein in leaves of Ficus carica L.

This study was undertaken to quantify psoralen and daidzein by high-performance thin layer chromatography (HPTLC). The methanolic extract of 10 mg mL(-1) concentration solution was prepared for HPTLC quantification of psoralen and daidzein. HPTLC aluminium-backed plates coated with 0.2 mm layers of silica gel 60 F(254) were used as the stationary phase. The working standard solution of psoralen and daidzein was applied along with the test sample solution by means of Camag Linomat IV sample applicator. R (f) values of psoralen and daidzein were found to be 0.60 and 0.88, whilst as their percentage values in methanolic extract were found to be 3.02% and 5.64% (w/w), respectively. A simple quantitative estimation method of psoralen and daidzein by HPTLC is reported that can be used for the quality control of marketed preparations containing Ficus carica. However, further study is warranted to isolate and quantify active constituents present in the leaves of F. carica by sophisticated techniques.

Preparation and evaluation of glyceryl monooleate-coated hollow-bioadhesive microspheres for gastroretentive drug delivery.

The purpose of this study was to produce hollow and bioadhesive microspheres to lengthen drug retention time in the stomach. In these microspheres, ethylcellulose was used as the matrix, Eudragit EPO was employed to modulate the release rate, and glyceryl monooleate (GMO) was the bioadhesive polymer in situ. The morphological characteristics of the microspheres were defined using scanning electron microscopy. The in vitro release test showed that the release rate of drug from the microspheres was pH-dependent, and was not influenced by the GMO coating film. The prepared microspheres demonstrated strong mucoadhesive properties with good buoyancy both in vitro and in vivo. Pharmacokinetic analysis indicated that the elimination half-life time of the hollow-bioadhesive microspheres was prolonged, and that the elimination rate was decreased. In conclusion, the hollow-bioadhesive synergic drug delivery system may be advantageous in the treatment of stomach diseases.

In vitro isolation, elicitation of psoralen in callus cultures of Psoralea corylifolia and cloning of psoralen synthase gene.

Psoralen, an important furanocoumarin occurring abundantly in seeds of Psoralea corylifolia is used as an anticancerous compound against leukemia and other cancer cell lines. Evaluation and isolation of psoralen from the calluses derived from different plant parts, viz. cotyledons, nodes, leaves and roots have been done in the present case for the first time. Amongst all, a maximum of 1934.75 µg/g f.w. of psoralen was recorded in callus derived from cotyledons, followed by 1875.50 and 1465.75 µg/g f.w. of psoralen in node and leaf derived calluses, respectively. Amount of psoralen enhanced further when cotyledonary calluses were exposed to different concentrations of organic elicitors (yeast extract, proline, inositol, casein hydrolyzate (CH), glycine, glutamine and sucrose) and precursors of psoralen (umbelliferone, cinnamic acid and NADPH). Isolation of psoralen was done using methanol as solvent through column chromatography and TLC. FT-IR and NMR further characterized and confirmed the structure of psoralen. In addition, the putative gene, psoralen synthase involved in psoralen synthesis pathway has been isolated, cloned and sequenced which comprised 1237 bp length. BLAST analysis of the gene sequence of psoralen synthase revealed that its nucleotide sequence showed 93% homology with psoralen synthase isolated from Ammi majus. This is the first report of isolation, cloning and characterization of psoralen synthase from Psoralea corylifolia.

[High-performance liquid chromatography for determination of psoralene, bergapten and apigenin in Ficus hirta Vahl].

OBJECTIVE: To establish a high-performance liquid chromatography (HPLC)-based method for determining the contents of psoralene, bergapten and apigenin in Ficus hirta Vahl. METHODS: A Hypersil C18 column (250 mm × 4.6 mm, 5 µm) was used with the mobile phase of methanol-water (60:40), flow rate of 1.0 ml/min, detection wavelength of 268.7 nm and column temperature of 30 degrees celsius. RESULTS: The calibration curve was linear within the range of 10.0-30.0 µg/ml for psoralene (r=0.9998), 15.0-45.0 µg/ml for bergapten (r=0.9998) and 5.0-15.0 µg/ml for apigenin (r=0.9992). The average recovery of psoralene was 99.7% (RSD=1.99%), that of bergapten was 99.9% (RSD=1.71%) and that of apigenin was 100.3% (RSD=1.78%). CONCLUSION: The method is simple, economic and accurate with good reproducibility for the contents of psoralene, bergapten and apigenin.

Production, characterization, in vitro and ex vivo studies of babchi oil-encapsulated nanostructured solid lipid carriers produced by a hot aqueous titration method.

An aqueous dispersion of solid fat nanoparticles of babchi oil (BOSLN) was prepared by means of the hot water titration method. Surface morphology was determined by HR-TEM which revealed a fairly spherical shape of the formulations. Further they were evaluated for in vitro drug release characteristics and ex vivo skin permeation profile, zeta potential and particle diameter, rheological measures and droplet size distribution. Highest values for steady state flux (Jss), permeability coefficient (Kp) and enhancement ratio (Er) were observed for formulation, BOSLN3 comprised of oil [10% v/v; BO (3.33%), CAT (6.67%)], Tween 80 (9.25% v/v), transcutol-P (28.75% v/v) and distilled water (53% v/v). These results suggest that the studied SLN might be promising vehicles for babchi oil in the management of psoriasis.

Determination of psoralen and plumbagin from its polyherbal oil formulations by an HPTLC densitometric method.

Many polyherbal oil formulations in Indian and Chinese traditional systems of medicine used for control of skin diseases contain seeds of Psoralea corylifolia and roots of Plumbago zeylanica L. Psoralen and plumbagin are the reliable markers for Ps. corylifolia and Pl. zeylanica, respectively. However, no attempt is made to standardize the polyherbal oil formulations containing Ps. corylifolia and Pl. zeylanica in terms of their active ingredients or marker compounds. In this paper, a simple, rapid, and sensitive HPTLC method is described for the first time to identify and quantify psoralen and plumbagin from such polyherbal oil formulations. The methanolic extract of oil formulations was used for analysis of markers. Psoralen gives a sharp UV absorbance peak at 302 nm and plumbagin at 275 nm. Good resolution of psoralen (Rf = 0.37) and plumbagin (Rf = 0.77) was attained using toluene-ethyl acetate (7.5 + 2.5, v/v) mobile phase. The method was validated in terms of calibration curve, limits of detection and quantification, precision, accuracy, and robustness following a standard protocol. Polyherbal oil formulations were analyzed with reasonable accuracy, and no matrix interference was observed. The method developed can be used for marker-based quality assurance of oil formulations containing Ps. corylifolia and Pl. zeylanica as one active ingredient.

Identification of psoralen loaded PLGA microspheres in rat skin by light microscopy.

Drug delivery systems involving the use of polymers are widely studied and discovery of biocompatible polymers has become the focus of research in this area. Psoralen loaded poly(DL-lactide-co-glycolide) (PLGA) microspheres to be used in PUVA therapy (psoralen and UVA irradiation (ultraviolet A, 320-400 nm) of psoriasis were identified in paraffin sections by histological analysis. The psoralen loaded PLGA microspheres were prepared using the solvent evaporation technique. They were spherical and possessed an external smooth surface as observed by scanning electron microscopy (SEM) analysis. This study describes a modification in the routine preparation of microsphere samples for examination by light microscopy. The changes involved fixative agents and/or stains allowing the identification of microspheres containing a non-fluorescent material. The preservation and identification of microspheres in tissues for histological processing in paraffin was greatly improved by these modifications as proven by our results.

Rapid densitometric method for simultaneous analysis of umbelliferone, psoralen, and eugenol in herbal raw materials using HPTLC.

The term 'phenolics' refers to a vast array of biologically active compounds ubiquitous in plants, many of which have been used in traditional medicine for thousands of years. Umbelliferone, psoralen, and eugenol are widely occurring phenolic compounds of plant origin, for which many biological activities against chronic diseases have been reported. A simple HPTLC method has been developed for the simultaneous quantification of umbelliferone, psoralen, and eugenol. These three compounds were quantified in the dried fruit pulp of Aegle marmelos and in the fruit of Trachyspermum ammi and Foeniculam vulgare. The technique enables rapid and sensitive simultaneous analysis in different samples. The method was validated for precision, repeatability, and accuracy in accordance with ICH guidelines. The accuracy of the method was checked by a recovery study conducted at three different levels and the average percentage recovery was found to be 98.88% for umbelliferone, 100.104% for psoralen, and 99.33% for eugenol. The proposed HPTLC method for the simultaneous quantification of umbelliferone, psoralen, and eugenol was found to be simple, precise, specific, sensitive, and accurate. It can be used for routine quality control of herbal raw materials as well as formulations containing any or all of these compounds.

Chemical fingerprint and quantitative analysis of fructus psoraleae by high-performance liquid chromatography.

Fructus Psoraleae, a widely used traditional Chinese medicine, is well known as a health supplement ingredient. In our study, an improved and comprehensive HPLC fingerprint of Fructus Psoraleae was established. Two important new benzofuran glycosides, psoralenoside and isopsoralenoside, were identified as characteristic constituents for the first time. HPLC separation was performed on an RP-C8 column. The mobile phase was acetonitrile and 0.1% acetic acid solution with linear gradient change of acetonitrile from 10 to 82% in 40 min. The flow rate was 1.0 mL/min, and the detection wavelength was set at 310 nm. The HPLC chromatograms of twenty-six samples from different regions of China showed a similar pattern. Twelve peaks were selected as characteristic peaks and further identified as psoralenoside, isopsoralenoside, psoralen, isopsoralen, bavachromene, corylifolin, corylin, psoralidin, isobavachalcone, bavachinin, corylifol A, and bakuchiol, respectively. Nine of them were simultaneously quantitatively analyzed for the first time. A more comprehensive analytical method was established for the fingerprint of Fructus Psoraleae. It is very useful for authentication and quality assessment of the crude drug.

A method for quantitative determination of furanocoumarins in capsules and tablets of phytochemical preparations.

A method for sample preparation and analysis by high-performance liquid chromatography with UV detection (HPLC-UV) was developed for analysis of psoralen, bergapten and 5-[3-(4,5-dihydro-5,5-dimethyl-4-oxo-2-furanyl)-butoxy]-7H-furo[3-2-g][1]benzopyran-7-one in capsules and tablets employed in Brazil for certain illnesses. The linearity, accuracy, the inter- and intra-day precision of the procedure were evaluated. Analytical curves for furanocoumarins were linear in the range of 1.0-50.0 microg/ml. The recoveries of the furanocoumarins in the products analyzed were 97.3-99.5%, and the percent coefficient of variation for the quantitative analysis of the furanocoumarins in the analyses was under 5%. For inter-equipment study gas chromatography (GC) was employed.

[Comparation of constitutions between raw Psoralea corylifolia L. and processed one].

OBJECTIVE: To study the processing material foundation of Psoralea corylfolia L. and compare the content of psoralen, isopsoralen and microelement between raw Psoralea corylifolia L. and processed one. METHODS: The content of psoralen, isopsoralen and microelement were obtained by HPLC-gradient elution and ICP-AES. RESULTS: The content of psoralen, isopsoralen and microelement Mn, Ca, Mg, Fe, Zn were raising, but the change of that of microelement Cu was not obvious. CONCLUSION: The content of effective composition of Psoralea corylifolia L. is raised by Lei-Gong method and the biological activity is improved simultaneously.

[The preparation and the in vitro release of OANO-1 microspheres].

OBJECTIVE: To prepare OANO-1 microspheres and test their release in vitro. METHOD: OANO-1 microspheres were made by W/O/W-liquid drying process. The surface morphology of the microspheres was observed by SEM. The mean diameter and the size distribution of microspheres, the drug loading and the incorporation efficiency were examined. The release of OANO-1 microspheres in vitro was examined by small cup method. The accumulated release percent of OANO-1 microspheres was examined. RESULT: The OANO-1 microspheres were regular in their morphology. The average particle size was 8.59 microm with over 90% of the microspheres being in the range of 1-12 microm. The drug loading and the incorporation efficiency were 48.39% and 19.32% respectively. The accumulated release percent of OANO-1 microspheres was 78.4% after 108 h. The release half-life t1/2 was 40.8 h and Higuchi equation was Y = 0.1326 X - 0.4782, r = 0.9951. CONCLUSION: The preparation of OANO-1 microspheres was well. The release in vitro of OANO-1 microspheres showed significant sustained release.

Fingerprint analysis of Psoralea corylifolia L. by HPLC and LC-MS.

High-performance liquid chromatography (HPLC) was developed for fingerprint analysis of Psoralea corylifolia. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MSn) technique was first employed to identify the components of the fingerprint. The samples were separated with an Alltima C18 column (250 mm x 4.6 mm, 5 microm) by linear gradient elution using water-acetic acid (A; 100:0.1, v/v) and acetonitrile (B; 0 min, 40%; 15 min, 50%; 35 min, 60%; 45 min, 70%; 55 min, 80%; and maintained for 5 min) as mobile phase at a flow rate of 1.0 ml/min and detector wavelength at 245 nm. A standard procedure was developed for HPLC fingerprint analysis. Average chromatogram of 10 batches of P. corylifolia L. from Sichuan and Henan Provinces, PR China, which has been considered as the original and genuine herbal medicine for a long time, was first established as the characteristic fingerprint. There are 12 common peaks in this fingerprint. Ten of these common peaks were identified by MS data. This profile was then used to identify and assess the differences among the herb grown in various areas of China. The HPLC fingerprint analysis is specific and may serve for quality identification and comprehensive evaluation of P. corylifolia.

Genetic variation in primary metabolites of Pastinaca sativa; can herbivores act as selective agents?

Although insect herbivory has been shown to act as a selective agent on plant secondary metabolism, whether primary metabolites contribute to resistance and can respond to selection by herbivores remains untested. In the wild parsnip (Pastinaca sativa), its principal herbivore, Depressaria pastinacella, acts as a selective agent on furanocoumarin resistance factors. In this study, we determined whether webworms can, by causing differential reductions in fitness, act as selective agents on parsnip primary metabolites. Estimates of narrow-sense heritabilities were significantly different from zero for C18 fatty acids in buds and developing fruits, fructose and sorbitol in buds, fructose, myo-inositol, bergapten, and psoralen in fruits. Wild parsnips protected from webworms by insecticide produced 2.5 times as much seed biomass as unsprayed plants; that webworms accounted for this difference in plant fitness was indicated by a significant negative relationship between reproductive effort and an index of webworm damage. Only a handful of metabolites influenced resistance to webworms; these included osthol, sorbitol, and protein in developing fruits as well as previously documented furanocoumarins. Osthol, a coumarinic compound, enhanced resistance, as did protein content, while sorbitol lowered resistance. Other primary metabolites may affect resistance to webworms, but their effect was context-dependent, that is, their effect depended on concentrations of other metabolites (epistasis). Susceptible plant phenotypes were found to have average chemical compositions. Although there was genetic variation in some of the primary metabolites in parsnips, the epistatic nature of their involvement in resistance and the lack of genetic variation in some suggest that selection on them from webworms will be either inconsistent or ineffective.