New alternatives to cosmetics preservation.

In recent years, there is a considerable interest in the development of preservative-free or self-preserving cosmetics. The aim of our work was to develop new cosmetic formulations by replacing chemical preservatives with ingredients with antimicrobial properties that are not legislated as preservatives according to Annex VI of Commission Directive 76/768/EEC. This paper describes the preservative efficacy of the well-known antimicrobial extracts of Lonicera caprifoleum and Lonicera japonica in combination with glyceryl caprylate and/or levulinic acid, p-anisic acid, and ethanol. We prepared a series of acidic (pH = 5.5) aqueous and O/W formulations, i.e., tonic lotion, shampoo, shower gel, conditioning cream, anticellulite cream, cleansing milk and peeling cream, containing (0.2% w/w) Lonicera extracts, alone in the case of tonic lotion and in combination with (1% w/w) glyceryl caprylate in the other products, and we performed challenge tests according to the European Pharmacopoeia procedures and criteria. Formulations such as shampoo, shower gel, and conditioning cream fulfilled criterion A, while tonic lotion, anticellulite cream, cleansing milk, and peeling cream fulfilled criterion B, in regard to contamination from A. niger. Furthermore, we evaluated the efficacy of the antimicrobial systems in two states of use: the intact product and after three weeks of consumer use. The results showed that A. niger was also detected during use by consumers in the products that satisfied only criterion B in challenge tests. The addition of antimicrobial fragrance ingredients such (< or = 0.3% w/w) levulinic acid or (0.1% w/w) p-anisic acid and/or (5% w/w) ethanol afforded products that met criterion A in challenge tests and were also microbiologically safe during use. The small quantity (5% w/w) of ethanol gave an important assistance in order to boost the self-preserving system and to produce stable and safe products.

Self-preserving cosmetics.

Preservatives are added to products for two reasons: first, to prevent microbial spoilage and therefore to prolong the shelf life of the product; second, to protect the consumer from a potential infection. Although chemical preservatives prevent microbial growth, their safety is questioned by a growing segment of consumers. Therefore, there is a considerable interest in the development of preservative-free or self-preserving cosmetics. In these formulations traditional/chemical preservatives have been replaced by other cosmetic ingredients with antimicrobial properties that are not legislated as preservatives according to the Annex VI of the Commission Directive 76/768/EEC and the amending directives (2003/15/EC, 2007/17/EC and 2007/22/EC). 'Hurdle Technology', a technology that has been used for the control of product safety in the food industry since 1970s, has also been applied for the production of self-preserving cosmetics. 'Hurdle Technology' is a term used to describe the intelligent combination of different preservation factors or hurdles to deteriorate the growth of microorganisms. Adherence to current good manufacturing practice, appropriate packaging, careful choice of the form of the emulsion, low water activity and low or high pH values are significant variables for the control of microbial growth in cosmetic formulations. This paper describes the application of the basic principles of 'Hurdle Technology' in the production of self-preserving cosmetics. Multifunctional antimicrobial ingredients and plant-derived essential oils and extracts that are used as alternative or natural preservatives and are not listed in Annex VI of the Cosmetic Directive are also reported.

Scanning electron microscopy study on nanoemulsions and solid lipid nanoparticles containing high amounts of ceramides.

Ceramides are the most important intercellular lipids of the stratum corneum, regulating the barrier function of the skin and participating as second signal messenger in stress-induced apoptosis. The high lipophilicity of ceramides presents a pharmacological problem. In order to overcome this problem two lipophilic delivery systems were used for the incorporation of the ceramides: (1) nanoemulsions (NE) and (2) solid lipid nanoparticles (SLN). The influence of the incorporation of ceramides on the particle shape, size and Polydispersity Index was investigated by photon correlation spectroscopy (PCS) and scanning electron microscopy (SEM). The results showed that NE can incorporate larger amounts of ceramides than SLN (up to 23.2% and 5% of lipid matrix, respectively) without any significant alteration on the morphology of the dispersed particles. The incorporation of higher amounts of ceramides into SLN, leads to anisometric platelet-like formations that are known to be caused by the transition of triglycerides from alpha- to beta-mesomorph. The results of this study can be useful for the design of appropriate delivery systems and for further pharmacological evaluations.

The role of the anticancer drug vinorelbine in lipid bilayers using differential scanning calorimetry and molecular modeling.

Differential scanning calorimetry (DSC) has been employed to investigate the thermal changes caused by the anticancer alkaloid drug vinorelbine in dipalmytoylphosphatidylcholine (DPPC) bilayers. The total enthalpy change was increased by the presence of the drug molecule, indicating a partial interdigitation of the lipid alkyl chains. The presence of cholesterol in DPPC bilayers including vinorelbine induced an obstruction of the interdigitation, since cholesterol interrupts the upraise of enthalpy change. Vinorelbine's interdigitation ability and stabilizing properties with the active site of the receptor have been compared with those of similar in structure amphipathic and bulky alkaloid vinblastine. The obtained results may in part explain their similar mechanism of action but different bioactivity.

Labd-14-ene-8,13-diol (sclareol) induces cell cycle arrest and apoptosis in human breast cancer cells and enhances the activity of anticancer drugs.

Sclareol is a labdane-type diterpene that has demonstrated a significant cytotoxic activity against human leukemic cell lines. Here, we report the effect of sclareol against the human breast cancer cell lines MN1 and MDD2 derived from the parental cell line, MCF7. MN1 cells express functional p53, whereas MDD2 cells do not express p53. Flow cytometry analysis of the cell cycle indicated that sclareol was able to inhibit DNA synthesis induce arrest at the G(0/1) phase of the cycle apoptosis independent of p53. Sclareol-induced apoptosis was further assessed by detection of fragmented DNA in the cells. Furthermore, sclareol enhanced the activity of known anticancer drugs, doxorubicin, etoposide and cisplatinum, against MDD2 breast cancer cell line.

Antitumor activity of doxorubicin encapsulated in hexadecylphosphocholine (HePC) liposomes against human xenografts on Scid mice.

Doxorubicin was encapsulated into liposomes composed of hexadecylphosphocholine:egg yolk phosphatidylcholine:stearylamine (HePC.EPC:SA) 10:10.0.1 (molar ratio) (1) and EPC:SA 10:0.1 (molar ratio) (2). Liposomal formulations 1 and 2, as well as free doxorubicin and free HePC, were tested in vitro against HCT116 human colon cancer cell lines and peripheral blood mononuclear cells (PBMCs) obtained from healthy donors, using the sulphorodamine B assay. The activity of doxorubicin was retained or slightly improved when entrapped into liposomes 1 and 2, while liposomal formulation 1 incorporating doxorubicin was found to be less toxic against normal cells. The liposomes were tested in vivo against human colon cancer xenografts in scid mice. The antitumor activities of liposomes 1 and 2 were statistically similar to that of free doxorubicin, but their toxicity was significantly lower. Based on these results, the combination of HePC and doxorubicin in one liposomal formulation may be justified for further evaluation.

A molecular basis explanation of the dynamic and thermal effects of vinblastine sulfate upon dipalmitoylphosphatidylcholine bilayer membranes.

Differential scanning calorimetry has been employed to study the thermal effects of vinblastine sulfate upon aqueous, single and multiple bilayer dispersions of 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC). The calorimetric results summarized to an increase in the gel to liquid-crystalline phase transition enthalpy and the abolishment of the L(beta)' (gel phase) to P(beta)' (ripple phase) pretransition for the uni- and multilamellar dispersions, as well as an increase in the transition temperature T(m) and the transition cooperativity for single bilayer DPPC/vinblastine mixed vesicles, are consistent with an induced, partially interdigitated, gel phase. Computational analysis has been successfully applied to clarify the intermolecular effects and verify the feasibility of the proposed interdigitation for the vinblastine sulfate molecules and also for the ursodeoxycholic acid (UDCAH) and bromocylated taxanes, which have been shown to induce an interdigitated gel phase in DPPC bilayers.

A comparative study of the effects of cholesterol and sclareol, a bioactive labdane type diterpene, on phospholipid bilayers.

Sclareol (labd-14-ene-8,13-diol) is a highly water-insoluble molecule that belongs to the labdane type diterpenes and is characterized as a biologically active molecule, due to its cytotoxic and cytostatic effects against human leukemic cell lines. A superimposition study between sclareol and cholesterol, based on their corresponding hydrophobic and polar molecular segments calculated from their lipophilic profiles, revealed their spatial similarities. This structural similarity between the two molecules prompted us to compare their effects on the structure and stability of phospholipid dipalmitoylphosphatidylcholine (DPPC) membranes. Differential scanning calorimetry (DSC) was applied to compare the thermal changes caused by either cholesterol or sclareol when are incorporated in DPPC bilayers. The results showed that sclareol is incorporated into phospholipid model membranes and mimics the thermal effects of cholesterol especially at concentrations up to X(sclareol)=9.1 mol%. These effects can be summarized as the abolition of pre-transition, lowering of the main phase transition and reduction of the enthalpy change (DeltaH) of the gel to liquid-crystalline phase transition of DPPC bilayers. At concentrations X> or =16.7 mol%, sclareol and cholesterol caused different heterogeneity in lipid bilayers or a reversible transition from a vesicular suspension to an extended peak bilayer network. This different fluidization, exerted by the two molecules at high concentration, may be related to their different stability and the z-average mean diameter of the liposomes they form. Small unilamellar vesicles, prepared by the thin film hydration method showed that DPPC bilayers containing a high concentration of sclareol in equimolar ratio sclareol:cholesterol were unstable, in contrast to the ones containing only cholesterol.

Antileishmanial and trypanocidal activities of new miltefosine liposomal formulations.

Liposomes composed of hexadecylphosphocholine/egg phosphatidylcholine/stearylamine (HePC/EPC/SA) 10:10:0.1, 10:10:0.5 and 10:10:1 (molar ratio) (1-3) were prepared and lyophilized. The liposomes were physicochemically characterized (size and zeta-potential) and they were found stable at 4 degrees C over a period of 4 weeks. In vitro, liposomes 1 and 2 were about twice more active than HePC against Leishmania donovani WT whereas liposomes 3 were about three times more active than HePC on HePC-resistant promastigotes. Although liposomes 1-3 were inactive on the in vitro intramacrophage amastigote model, the ability of the liposomes to accumulate within the liver where parasites are located justifies a further in vivo evaluation. We observed that liposome 1 was twice more active than HePC against Trypanosoma brucei brucei bloodstream forms maintained in vitro. In vivo results showed that liposomal HePC seemed to be less toxic than the free drug despite the absence of significant antitrypanosomal activity.

Development of simple thiol-reactive liposome formulations, one-step analysis and physicochemical characterization.

The aim of this study was to synthesize simple thiol-reactive conjugates from maleimide and lipoamines (stearylamine or oleylamine) and to develop a simple, fast and low-cost method for the preparation of lyophilized general-purpose thiol-reactive liposomes. A formulation of egg phosphatidylcholine-dipalmitoylphoshatidylglycerol (9:0.1 molar ratio) was developed and characterized. Freeze-drying methodology was established to produce a stock of liposomes and the physicochemical characteristics of the reconstituted liposomes were compared with those of the initial preparation. The physicochemical properties (size and zeta potential) of the new liposomal formulations were studied. High-performance thin-layer chromatography coupled to a flame ionization detector was applied for one-step analysis of the liposomal components and for determining the maleimide-lipoamine conjugates phospholipid molar ratio. The differences concerning the incorporation efficiency of the synthetic conjugates into liposomes were discussed on the basis of their conformational properties. The small difference in structure between the two thiol-reactive conjugates (i.e., the C18 alkyl chain double bond) causes a considerable difference in phospholipids packing of the resulting lipidic bilayers of the liposomes; the conformational bending of conjugate maleimide-oleylamine may contribute to the final architecture of liposomes.

Ether phospholipid-AZT conjugates possessing anti-HIV and antitumor cell activity. Synthesis, conformational analysis, and study of their thermal effects on membrane bilayers.

The 1-O-hexadecyl-2-O-methyl-sn-glyceryl phosphodiester AZT 4 and hexadecyl-phosphodiester AZT 5 derivatives were synthesized and found to be active against HIV-1, HIV-2, and tumor cell proliferation. Compared to AZT, compound 4 possessed ca. 10-fold lower anti-HIV activity and ca. 10-fold higher anti-tumor cell activity. Compound 5 was 10-fold less potent than compound 4 in both biological tests. In an attempt to correlate biological activity of compounds 4 and 5 with structure, their conformational and thermal effects on membrane bilayers were compared using a combination of NMR spectroscopy, computational analysis, and Differential Scanning Calorimetry. The obtained results showed that compound 4 adopts a compact conformation in which the alkyl chain, the 2-methoxyglyceryl functionality, and the methyl group of thymine are in spatial proximity, while analogue 5 possesses a less compact conformation of the nucleoside base and the alkyl chain. The presence of the 2-methoxyglyceryl group in compound 4 may augment its potency by inducing a turn of the alkyl chain stabilized by hydrophobic interactions. The DSC scans show that conjugate 4 affects less effectively the thermotropic properties of model membrane bilayers than compound 5. This may be attributed to the fact that compound 4 is incorporated in a compact conformation and does not perturb significantly the trans:gauche isomerization of the membrane phospholipids. In contrast, conjugate 5 may enter with a less compact conformation and perturb more the membrane bilayers.

Cytotoxic and antiproliferative effects of heptaacetyltiliroside on human leukemic cell lines.

The peracetylated derivative of kaempferol-3-O-beta-D-(6''-E-p-coumaroyl) glycopyranoside (tiliroside) (1a) was tested for its cytotoxic and cytostatic activity against several human leukemic cell lines. The significant cytotoxic activity of this derivative, prompted to an additional examination on some of the cell lines used. The effect on the uptake of [3H]thymidine as a marker of DNA synthesis and on the cell proliferation, was investigated as well as the morphology of the cells and the kind of death induced, using the Wright-Giemsa dye and horizontal agarose-gel electrophoresis. Flow cytometric experiments of 1a on some leukemic cell lines was also performed. Compound 1a showed a significant antiproliferative effect as soon as 1 h of continuous incubation at all cell lines tested. Cells were killed, through the process of apoptosis and the appearance of the apoptotic signs was time and dose-dependent, while from the flow cytometric experiments, a synchronisation (through a delay probably in the G(0/1) phase) of the cells seems to take place.

Labdane type diterpenes down-regulate the expression of c-Myc protein, but not of Bcl-2, in human leukemia T-cells undergoing apoptosis.

Sclareol (1) and ent-3beta-hydroxy-13-epi-manoyl oxide (2) belong to the labdane type diterpenes. They were isolated from the leaves and from the fruits of Cistus creticus subsp. creticus, and were found to be active against human leukemic cell lines. Compound 2 was converted to its thiomidazolide derivative (3). Compounds 1 and 3 were found to induce apoptotic cell death in human T-cell leukemia lines and to interfere with their cell cycle, arresting cells at G(0/1) phase. Apoptosis can involve the activation and/or suppression of critical genes such as c-myc whose reduction or its inappropriate expression can be associated with induction of cell death and bcl-2 whose activation prevents apoptosis in the latter case. In order to detect any concomitant effect (1 and 3) upon c-myc and bcl-2 oncogene expression, we performed Western blot analysis to determine the levels of expression of these two genes upon treatment with the above compounds. Western blot analysis showed that of c-myc proto-oncogene levels were markedly reduced before massive apoptosis ensued in H33AJ-JA1 and MOLT3 cells, while bcl-2 expression remained unaffected. Thus, induction of apoptosis due to compounds 1 and 3 in these T-cell leukemic cell lines is preceded by c-myc down regulation and furthermore sustained bcl-2 expression does not rescue cells from apoptosis under the conditions used.

The effect of sclareol on growth and cell cycle progression of human leukemic cell lines.

Sclareol, a labdane-type diterpene, was tested for cytotoxic effect against a panel of established human leukemic cell lines. The compound showed an IC50 lower than 20 microg/ml in most cell lines tested, while it was higher for resting peripheral blood mononuclear leukocytes (PBML). Furthermore, the compound was tested for cytostatic activity against four of the leukemic cell lines used. At a concentration of 20 microg/ml the compound showed a significant cytostatic effect as soon as 4 h after continuous incubation against two from B and two from T lineage cell lines. The morphology and the kind of death induced from sclareol in three cell lines, was also investigated. The effect of sclareol on the cell cycle progression of two cell lines, using flow cytometry, was examined. The results show that sclareol kills cell lines, through the process of apoptosis. The appearance of the apoptotic signs is time and dose dependent. From the flow cytometry experiments, a delay of the cell population on G0/1 seems to take place. This is the first report, that a labdane type diterpene kills tumor cells via a phase specific mechanism which induces apoptosis.

CYTOTOXIC ACTIVITY OF KAEMPFEROL GLYCOSIDES AGAINST HUMAN LEUKAEMIC CELL LINES IN VITRO.

Two kaempferol coumaroyl glycosides (i.e. platanoside and tiliroside) isolated from the methanolic extract of Platanus orientalis L. buds, were examined for their in vitro cytotoxic activity against a panel of human leukaemic cell lines. Platanoside (1) exhibited cytotoxic activity against most of the cell lines tested, while tiliroside (2) was active against two of the nine tested cell lines. Compound 1, was examined for its effect on the uptake of [(3)H]thymidine as a marker of DNA synthesis. Kaempferol was used as a control. 2000 Academic Press@p$hr Copyright 2000 Academic Press.

Encapsulation of vinblastine into new liposome formulations prepared from triticum (wheat germ) lipids and its activity against human leukemic cell lines.

Liposomes prepared from lipids isolated from Triticum sp. (wheat germ) were used to investigate the percentage of Vinblastine encapsulation and its retention into liposomes. The wheat germ total lipids (TL) were extracted by the Bligh-Dyer method and the lipid classes have been isolated using chromatographic techniques. The type of lipids and their percentage content have been examined by TLC coupled with an FID (latroscan). Two liposomal formulations, i.e., I and II, with encapsulated vinblastine, and formulation III (empty liposomes) have been prepared by thin film hydration method. The cytotoxic/cytostatic activity of these liposomal formulations have been examined against nine human leukemic cell lines. The results showed that the percentage content of vinblastine into liposomes I and II depended on the lipid composition and it was greater into formulation II (> 90%). The retention of the drug into liposomes was studied and found to be time-dependent at 37 degrees C. For the cytotoxic/cytostatic activity, the parameters GI50, TGI, LC50 were estimated according to the instructions given by the NCI. The results show that formulation III (empty liposomes), exhibited a growth inhibiting activity, against the most tested cell lines. Formulation II showed mean of LC50 at 124.6 nM, mean of TGI at 71.6 nM and mean of GI50 at 30.8 nM.

Cytotoxic activity and antiproliferative effects of a new semi-synthetic derivative of Ent-3 beta-hydroxy-13-epi-manoyl oxide on human leukemic cell lines.

Ent-3 beta-hydroxy-13-epi-manoyl oxide (1), was converted to its thiomidazolide derivative (2) which was tested for its cytotoxic activity against a panel of established human leukemic cell lines. Compound 2, exhibited cytotoxic activity against 13 of the cell lines tested. Additionally, compound 2 was examined for its effect on the uptake of [3H]-thymidine as a marker of DNA synthesis and on cell proliferation. The morphology of the cells and the kind of death induced, was investigated. Flow cytometry experiments on a leukemic cell line was also performed. The results show that the semi-synthetic compound, showed a significant antiproliferative effect and kills cells through the process of apoptosis. The appearance of the apoptotic signs was time and dose dependent. From the flow cytometry experiments, a synchronisation through a delay of the cells in G0/1, phase seems to take place.

Accumulation of vinblastine into transfersomes and liposomes in response to a transmembrane ammonium sulfate gradient and their cytotoxic/cytostatic activity in vitro.

Vinblastine was encapsulated into liposomes composed from lipids dimiristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC), with cholesterol and transfersomes with sodium cholate prepared by the thin film hydration method. The percentage of vinblastine encapsulation, the stability of transfersomes and liposomes and the rate of release of encapsulated vinblastine at 37 degrees C were studied. The results showed that encapsulation of vinblastine into liposomes was higher than 98% at a drug/phospholipid molar ratio from 0.17 to 0.18, while encapsulation of vinblastine into transfersomes varied from 50% to 80% at a drug/phospholipid molar ratio from 0.05 to 0.09. The retention of drug in liposomes and in transfersomes was found to be time/dependent. The retention of drug in transfersomes compared to the liposomes was reduced due to the presence of sodium cholate which caused destabilization and reduced the main phase transition temperature Tm of the PC bilayers. The cytotoxic/cytostatic activity of the two liposome formulations and the two transfersome formulations with or without encapsulated vinblastine were examined against nine human cell lines and the parameters GI50, TGI, LC50 were estimated according to the NCI protocol. Free DPPC/sodium cholate liposomes found to exhibit strong antiproliferative activity in contrast to the other three free liposomal formulations (DPPC/cholesterol, DMPC/cholesterol, DMPC/sodium cholate). On the other hand, vinblastine encapsulated into the liposomes found to exhibit 20-fold less activity on average, in the three parameters calculate compare to the free vinblastine.

Effect of nitrogen concentration of the nutrient solution on the volatile constituents of leaves of Salvia fruticosa Mill. in solution culture.

Essential oils from hydroponically cultivated Salvia fruticosa were analyzed by GC-MS techniques. Three different levels of nitrogen (100, 150, and 200 mg/L) were used in the nutrient solution for the cultivation, using the nutrient film technique. A total of 79 compounds were identified, and qualitative and quantitative differences have been observed between the samples collected at full bloom (flowering stage) and at the end of the seed formation stage. 1,8-Cineole, beta-caryophyllene, and viridiflorol were the predominant constituents in most cases. 13-epi-Manool was identified by using GC parameters and mass spectrum fragmentation pattern, whereas labd-7,13-dien-15-ol, a labdane type diterpene, was identified for the first time in the genus Salvia, using GC parameters and an authentic sample. The results obtained from GC-MS analyses were submitted to chemometric analysis.

Encapsulation of naturally occurring flavonoids into liposomes: physicochemical properties and biological activity against human cancer cell lines.

Liposomes consisting of egg phosphatidylcholine were prepared by a thin-film hydration method followed by sonication and were used to investigate the percentage encapsulation of four flavonoids (quercetin, rutin, isoscutellarein and isoscutellarein diglycoside). The lipid recovery and the flavonoid-to-lipid molar ratio were measured using high-performance thin-layer chromatography/flame ionization detection and UV-vis spectroscopy. Differential scanning calorimetry was used to study the effect of the flavonoids on the phase transition temperature and on the enthalpy of the main phase transition of dipalmitoylphosphatidylcholine bilayers, and their ability to influence the membrane fluidity. The final liposomal formulation incorporating flavonoids, as well as free flavonoids, were tested for their activity against human cancer cell lines using the sulforhodamine B assay. The results showed that the encapsulation efficiency varied from 95% (0.21 flavonoid-to-lipid molar ratio) to 37.5% (0.09 flavonoid-to-lipid molar ratio) for isoscutellarein and its glycoside, respectively. The differential scanning calorimetry data showed close thermal and dynamic effects depending on the structure of the flavonoids, and suggest that there is a relationship between flavonoid molecular structure and the interaction with model membranes. Liposomal isoscutellarein showed improved growth inhibiting activity against all cell lines tested in comparison with that of its free form, which was inactive (>100 microM).