Favorable effect of creams with skimmed donkey milk encapsulated in nanoliposomes on skin physiology.

The use of donkey milk has a long history in external dermal application for cosmetic and medical purposes. The encapsulation of different drugs into nanoliposomes and nutrients may have many benefits for the delivery of the targeted substance. The aim of the present study is to test creams with nanoliposomes encapsulated with skimmed donkey milk, reported in our registered patent, compared with effect of the placebo cream and the untreated skin. To investigate the effect of formulated cream, we have measured the electrical capacitance, transepidermal water loss and the skin pH value. The study included 15 healthy volunteers (11 women and 4 men). The patented creams exhibited satisfactory moisturizing properties very soon after application, while hydration was reached earlier in the spots treated with night cream. Transepidermal water loss slightly decreased only after 4 weeks of treatment in each of the spots assessed. The pH value was similar after each of the treatments. Tested creams may enhance the moisture of skin very soon after application, in this way potentially enable deeper permeation of phospholipids and essential proteins, without changing the pH. It may also contribute to additional anti-aging effects.

Nanoliposome-encapsulated ellagic acid prevents cyclophosphamide-induced rat liver damage.

In this study, we aimed to evaluate whether the encapsulation of ellagic acid (EA) into nanoliposomes would improve its potential in preventing cyclophosphamide-induced liver damage. Stability and antioxidative potential of free and encapsulated EA were determined. Experimental study conducted in vivo included ten groups of rats treated with cyclophosphamide and ellagic acid in its free and encapsulated form during 5 days. The protective effect of EA in its free and encapsulated form was determined based on serum liver function, liver tissue antioxidative capacities, and oxidative tissue damage parameters. Also, tissue morphological changes following cyclophosphamide administration were studied using standard histopathological and immunohistochemical analyses. The encapsulation of EA significantly prevented its degradation and improved its antioxidant properties in in vitro conditions. In in vivo experiments in both forms of EA were found to prevent rat liver damage induced by cyclophosphamide estimated through the changes in serum liver-damage parameters and tissue antioxidant capacities, as well as based on oxidatively modified lipids and proteins. Also, changes in morphology of liver cells and the expressions of Bcl-2, HIF-1α, and CD15 molecules in livers of animals of different experimental groups are in accordance with the obtained biochemical parameters. Thus, the encapsulation process might be effective in preventing EA from different environmental influences and could significantly increase its hepatoprotective potential. The encapsulation could prevent ellagic acid degradation and might deliver this potent compound to its target tissue in significantly larger quantities than when it is administered in its free form.

Wild Bilberry (Vaccinium myrtillus L., Ericaceae) from Montenegro as a Source of Antioxidants for Use in the Production of Nutraceuticals.

The aim of this study was to establish correlation of chemical composition and antioxidant activity of bilberry plants from Montenegro. Total phenolic, tannin, flavonoid, procyanidin and anthocyanin contents were determined in fruits and leaves extracts using spectrophotometric methods, while the measurements of metal content was carried out in an Inductively Coupled Atomic Emission Spectrometer. Qualitative and quantitative analyses of major phenolics were achieved by HPLC. In the investigated extracts, the most abundant phenolic was chlorogenic acid, followed by protocatechuic acid, while resveratrol, isoquercetin, quecetin and hyperoside were also present in significant quantities. Antioxidant potential was evaluated using two in vitro assays-FRAP and DPPH-being in the accordance with the cyclic voltammetry tests, performed as well. The results revealed that all the investigated extracts were rich in phenolic and essential mineral constituents, with significant antioxidant activity, depending on the polyphenolic and mineral contents, which was confirmed by principal component analysis.

Standardized extract of wild apple fruit in alkyl-polyglucoside-based cosmetic cream - estimation of stability, safety, antioxidant activity and efficiency.

OBJECTIVE: The aim of this study was in vitro and in vivo characterization of cosmetic cream with 6% of wild apple fruit water extract, containing 3.5% of alpha-hydroxyacids-AHAs and polyphenolic compounds, stabilized by biodegradable alkyl-polyglucoside emulsifiers. METHODS: In vitro characterization of cream included organoleptic and physico-chemical (pH values and electrical conductivity) analysis, antioxidant activity-AA estimation (using DPPH test) during 180 days of storage at 22±2°C and determination of cosmetic active substances content-AHAs (using HPLC analysis). In vivo estimation of skin irritation potential after creams application under occlusion during 24h was investigated employing the biophysical methods on 12 healthy volunteers; hydration efficiency, transepidermal water loss-TEWL, skin pH, erythema-EI and melanin index-MI on 10 healthy volunteers after 28 days of cream application and bleaching efficiency on 10 healthy volunteers after 7 days of cream application after artificially induced skin hyperpigmentation. RESULTS: Investigated cosmetic cream was pale beige color, odorless, semi-solid consistency and homogeneous, with pH values of 6.53±0.14, electrical conductivity above 50 µS/cm and AA of 24.96%RSC after preparation and these characteristics were stable during investigated period. In vivo measurements revealed absence of skin irritation after cream application under occlusion, which was part of it's safety profile. Increase of skin hydration after 14 days of cream application was 17.28±12.23 and after 28 days 21.19±7.59. In addition, in performed experiment TEWL and skin pH values during cream application remained unchanged. Cream application after artificial hyperpigmentation induced decrease of MI (∆MI after 7 days was -45.30±18.55) CONCLUSION: Formulated cosmetic cream with 6% of standardized wild apple fruit water extract and stabilized by biodegradable alkyl-polyglucoside emulsifiers demonstrated good stability, acceptable level of in vitro antioxidant activity, absence of skin irritation after cream application under occlusion and positive effects of cream on human skin after application (hydrating and bleaching effects). All mentioned properties make the cream suitable for possible usage as cosmetic product for preventing the skin damages caused by oxidative stress, for moisturizing the dry skin and bleaching of skin hyperpigmentation. This article is protected by copyright. All rights reserved.

Usnea barbata CO2-supercritical extract in alkyl polyglucoside-based emulsion system: contribution of Confocal Raman imaging to the formulation development of a natural product.

Topical treatment of skin infections is often limited by drawbacks related to both antimicrobial agents and their vehicles. In addition, considering the growing promotion of natural therapeutic products, our objective was to develop and evaluate naturally-based emulsion system, as prospective topical formulation for skin infections-treatment. Therefore, alkyl polyglucoside surfactants were used for stabilization of a vehicle serving as potential carrier for supercritical CO2-extract of Usnea barbata, lichen with well-documented antimicrobial activity, incorporated using two protocols and three concentrations. Comprehensive physicochemical characterization suggested possible involvement of extract's particles in stabilization of the investigated system. Raman spectral imaging served as the key method in disclosing extract's particles potential to participate in the microstructure of the tested emulsion system via three mechanisms: (1) particle-particle aggregation, (2) adsorption at the oil-water interface and (3) hydrophobic particle-surfactant interactions. Stated extract-vehicle interaction proved to be correlated to the preparation procedure and extract concentration on one hand and to affect the physicochemical and biopharmaceutical features of investigated system, on the other hand. Thereafter, formulation with the best preliminary stability and liberation profile was selected for further efficiency and in vivo skin irritation potential evaluation, implying pertinent in vitro antimicrobial activity against G+ bacteria and overall satisfying preliminary safety profile.

Comparative Study of the Biological Activity of Allantoin and Aqueous Extract of the Comfrey Root.

This study investigates the biological activity of pure allantoin (PA) and aqueous extract of the comfrey (Symphytum officinale L.) root (AECR) standardized to the allantoin content. Cell viability and proliferation of epithelial (MDCK) and fibroblastic (L929) cell line were studied by using MTT test. Anti-irritant potential was determined by measuring electrical capacitance, erythema index (EI) and transepidermal water loss of artificially irritated skin of young healthy volunteers, 3 and 7 days after application of creams and gels with PA or AECR. Pure allantoin showed mild inhibitory effect on proliferation of both cell lines at concentrations 40 and 100 µg/ml, but more pronounced on MDCK cells. Aqueous extract of the comfrey root effect on cell proliferation in concentrations higher than 40 µg/ml was significantly stimulatory for L929 but inhibitory for MDCK cells. Pharmaceutical preparations that contained AECR showed better anti-irritant potential compared with PA. Creams showed better effect on hydration and EI compared with the gels that contained the same components. Our results indicate that the biological activity of the comfrey root extract cannot be attributed only to allantoin but is also likely the result of the interaction of different compounds present in AECR. Topical preparations that contain comfrey extract may have a great application in the treatment of skin irritation.

Anti-inflammatory, gastroprotective, and cytotoxic effects of Sideritis scardica extracts.

Sideritis scardica Griseb. (ironwort, mountain tea), an endemic plant of the Balkan Peninsula, has been used in traditional medicine in the treatment of gastrointestinal complaints, inflammation, and rheumatic disorders. This study aimed to evaluate its gastroprotective and anti-inflammatory activities. Besides, continuously increasing interest in assessing the role of the plant active constituents preventing the risk of cancer was a reason to make a detailed examination of the investigated ethanol, diethyl ether, ethyl acetate, and N-butanol extracts regarding cytotoxicity. Oral administration of the investigated extracts caused a dose-dependent anti-inflammatory effect in a model of carrageenan-induced rat paw edema. Gastroprotective activity of the extracts was investigated using an ethanol-induced acute stress ulcer in rats. The cytotoxic activity of plant extracts was assessed on PBMC, B16, and HL-60 cells and compared to the cytotoxicity of phenolic compounds identified in extracts. Apoptotic and necrotic cell death were analyzed by double staining with fluoresceinisothiocyanate (FITC)-conjugated annexin V and PI. The developed HPLC method enabled qualitative fingerprint analysis of phenolic compounds in the investigated extracts. Compared to the effect of the positive control, the anti-inflammatory drug indomethacine (4 mg/kg), which produced a 50 % decrease in inflammation, diethyl ether and N-butanol extracts exhibited about the same effect in doses of 200 and 100 mg/kg (53.6 and 48.7 %; 48.4 and 49.9 %, respectively). All investigated extracts produced dose-dependent gastroprotective activity with the efficacy comparable to that of the reference drug ranitidine. The diethyl ether extract showed significant dose-dependent cytotoxicity on B16 cells and HL-60 cells, decreasing cell growth to 51.3 % and 77.5 % of control, respectively, when used at 100 µg/mL. It seems that phenolic compounds (apigenin, luteolin, and their corresponding glycosides) are responsible for the diethyl ether extract cytotoxic effect. It also appears that induction of oxidative stress might be involved in its cytotoxicity, since B16 and HL-60 cells increased their ROS production in response to treatment with diethyl ether extract. Neither of the tested extracts nor any phenolic compounds showed significant cytotoxic effect to human PBMC. These results demonstrated the potent anti-inflammatory and gastroprotective activities, as well as the promising cytotoxicity.

Chemical and antimicrobial evaluation of supercritical and conventional Sideritis scardica Griseb., Lamiaceae extracts.

Sideritis scardica Griseb., Lamiaceae (ironwort, mountain tea), an endemic plant of the Balkan Peninsula, has been used in traditional medicine in the treatment of antimicrobial infections, gastrointestinal complaints, inflammation and rheumatic disorders. This study reports a comparison between conventional (hydrodistillation HD and solvent extraction SE) and alternative (supercritical carbon dioxide SC CO2) extraction methods regarding the qualitative and quantitative composition of the obtained extracts as analyzed by GC and GC-MS techniques and their anitimicrobial activity. Different types of extracts were tested, the essential oil EO obtained by HD, EO-CO2 and AO-CO2 obtained by SC CO2 at different preasures 10 and 30 MPa, at 40 °C, respectively, and the fractions A, B, C and D obtained by successive solvent extraction (SE) A: ethanol, B: diethyl ether, C: ethyl acetate and D: n-butanol). While EO was characterized by the presence of the high percentage of oxygenated monoterpenes and sesquiterpenes (30.01 and 25.54%, respectively), the rest of the investigated samples were the most abundant in fatty acids and their esters and diterpenes (from 16.72 to 71.07% for fatty acids and their esters, and from 23.30 to 72.76%, for diterpenes). Microbial susceptibility tests revealed the strong to moderate activity of all investigated extracts against the tested microorganisms (MIC from 40 to 2,560 µg/mL). Although differences in the chemical compositions determined by GC and GC-MS analysis were established, the displayed antimicrobial activity was similar for the all investigated extracts.

Preparation of novel apigenin-enriched, liposomal and non-liposomal, antiinflammatory topical formulations as substitutes for corticosteroid therapy.

Two oil-in-water formulations, containing equal amounts of apigenin-enriched chamomile flower extracts, for potential use as topical antiinflammatory agents, were prepared and their physicochemical properties evaluated. A pilot clinical study was then carried out to assess patient acceptability and efficacy. The creams were either non-liposomal or liposomal. The liposomal formulations were more viscous, thus producing superior release characteristics in vitro. The clinical study also showed that the liposomal creams were, as antiinflammatory agents, slightly more effective in vivo than the non-liposomal formulations. These results suggest that there is scope for the further development of even more effective and safer alternatives to corticosteroids.

Estimation of dermatological application of creams with St. John's Wort oil extracts.

Oleum Hyperici, the oil extract of St. John's Wort (SJW), is one of the oldest folk remedies, traditionally used in the topical treatment of wounds, bruises, ulcers, cuts, burns, hemorrhoids and also as an antiseptic. Considering the advantageous characteristics of emulsion applications, in the present study we have formulated three O/W creams containing 15% (w/v) of SJW oil extract as an active ingredient. The aim was to estimate dermatological application of the prepared creams for the abovementioned indications. The extracts were prepared according to the prescriptions from traditional medicine, however with different vegetable oils used as an extractant, namely: Olive, palm and sunflower oil. The investigated O/W creams demonstrated significant antiinflammatory effects in an in vivo double-blind randomized study, using a sodium lauryl sulphate test. Both skin parameters assessed in the study (electrical capacitance and erythema index), were restored to the baseline value after a seven-day treatment with the tested creams. Almost all investigated SJW oil extracts and corresponding creams displayed the same antimicrobial activity against the most of the investigated microorganisms with obtained minimal inhibitory concentrations values of 1,280 µg/mL, 2,560 µg/mL or >2,560 µg/mL.

Towards a modern approach to traditional use: in vitro and in vivo evaluation of Alchemilla vulgaris L. gel wound healing potential.

ETHNOPHARMACOLOGICAL RELEVANCE: Alchemilla vulgaris is an important remedy in European folk medicine, known for its astringent and anti-inflammatory properties; it is traditionally used to heal gynecological and gastrointestinal diseases. Despite its folkloric use in wound healing, there is a lack of scientific data to support this therapeutic application. AIM OF THE STUDY: To analyze the wound healing potential of different solvent A. vulgaris extracts per se and after incorporation into hydrogels as topical vehicles, using two complementary methods - in vitro wound healing assay with L929 fibroblasts and in vivo assessment of skin barrier repair potential. Besides scientific justification of the traditional usage, we aimed to ephasize the importance of a proper vehicle for herbal extracts. The wound healing activity has been connected to the chemical profile of the investigated extracts, their antioxidative properties, but also to pH of the investigated gels and their mechanical characteristics. MATERIALS AND METHODS: Antioxidant activity of investigated extracts was estimated using both 2,2-diphenyl-1-picrylhydrazyl and ß-carotene/linoleic acid models. Chemical profile was achieved applying spectrophotometric and HPLC methods. In vitro scratch assay with L929 fibroblasts, and in vivo study of skin barrier repair potential of hydrogels with A. vulgaris extracts on human skin employing biophysical measurements, were performed in order to confirm the wound healing potential of A. vulgaris. Texture analysis of the gels was performed alongside the pH measurements. RESULTS: All tested extracts and gels accelerated the wound healing process while the effect of ethanolic extract on migration of fibroblasts was the most pronounced. The highest extent of wound closure was also observed for the ethanolic extract. The most favorable effect on in vitro wound healing was observed for gel with propyleneglycolic extract. Results of in vivo study were in line with in vitro findings. Healing potential may be attributed to phenolic compounds found in A. vulgaris extracts, low pH of the gels, and the satisfying antioxidant activity of the extracts. Parameters obtained by textural analysis indicated satisfying mechanical properties of the gels, relevant to topical application. CONCLUSION: Our study offers pharmacological evidence on the folkloric use of A. vulgaris in wound treatment, particularly after incorporation into hydrogel, and underlines an importance of a proper vehicle for incorporation of herbal extracts intended for topical treatment.

The estimation of the traditionally used yarrow (Achillea millefolium L. Asteraceae) oil extracts with anti-inflamatory potential in topical application.

ETHNOPHARMACOLOGICAL RELEVANCE: Olive or sunflower oil yarrow extracts have been extensively used against inflammatory disorders and skin wound healing in traditional medicine. AIM OF THE STUDY: To evaluate oil yarrow extracts traditional use in treatment of topical/dermatological skin impairments, sodium lauryl sulfate test was applied, and in vivo measuring of the biophysical parameters (erythema index, skin capacitance and the pH of the skin) in the artificially irritated skin was performed. As traditionally olive and sunflower oil have been used equally for extracts production, the experiment was carried out to investigate whether any of the oil extractants has the advantage over the other, and if the method of extraction might influence the desired activity. The observed activity has been connected to the chemical profile of the investigated extracts and their antioxidative properties. MATERIALS AND METHODS: In vivo measurements were performed using the appropriate probes for measuring skin capacitance, pH of the skin and erythema index (EI). The designed experiment enabled the evaluation of the anti-inflammatory effects of a seven-day application of oil yarrow extracts known in traditional medicine, on artificially irritated skin of volunteers. The chemical profile for the investigated samples was achieved applying the HPLC and UHPLC-MS methods. Also, ferric reducing antioxidant power (FRAP) test was performed to assess the antioxidant properties of the investigated samples. RESULTS: The application of tested oil extracts on artificially irritated skin in vivo demonstrated the ability to re-establish their optimal pH and hydration of skin to the values measured prior to the irritation. Considering the EI transition, the investigated samples succeeded in re-establishing the baseline values, with no significant difference after three- and seven-day application. CONCLUSIONS: The data obtained in the study showed that the oil yarrow extracts had an evident anti-inflammatory property. Namely, the investigated extracts demonstrated significant anti-inflammatory effect in an in vivo double blind randomized study, using a sodium lauryl sulfate test. The skin parameters assessed in the study (skin capacitance, pH and EI) were restored to the basal values after three- and seven-day treatment with the tested extracts. The shown effects were attributed to yarrow oil extracts composition. The yarrow oil extracts might be used as promising base in the phytopreparations designed for dermatological application as anti-inflammatory agents with a positive impact on the skin pH and its moisture content.

Evaluation of Anticancer and Antioxidant Activity of a Commercially Available CO2 Supercritical Extract of Old Man's Beard (Usnea barbata).

There is a worldwide ongoing investigation for novel natural constituents with cytotoxic and antioxidant properties. The aim of this study was to investigate chemical profile and stated biological activities of the supercritical CO2 extract (SCE) of old man's beard compared to the extracts obtained using the conventional techniques (Soxhlet extracts and macerate). The most abundant compound identified was usnic acid, which content was inversely proportional to the polarity of the solvent used and was the highest in the SCE, which was the sample revealing the highest cytotoxic activity in tested tumor cell lines (B16 mouse melanoma and C6 rat glioma), with lower IC50 values compared to pure usnic acid. Further investigations suggested both SCE and usnic acid to induce apoptosis and/or autophagy in B16 and C6, indicating higher cytotoxicity of SCE to be related to the higher degree of ROS production. A good correlation of usnic acid content in the extracts and their antioxidant capacity was established, extricating SCE as the most active one. Presented results support further investigations of SCE of old man's beard as a prospective therapeutic agent with potential relevance in the treatment of cancer and/or in oxidative stress-mediated conditions.

[Manufacturing process of high quality phytopreparation on example of herbal sedative].

INTRODUCTION: Rational phytotherapy is a modern concept of using plant-originated drugs which has emerged from the need to improve phytotherapy in order to make the use of herbal remedies more efficient and safer. The aim of this study was to give the health-care workers more information on the manufacturing process of high quality phytopreparation following principles of Good Manufacturing Practice and Good Laboratory Practice on the example of herbal sedative, Odoval S capsules. MATERIAL AND METHODS: This study was designed to reflect the production process of a high-quality and safe herbal remedy, starting from defining the formulation and the production procedure to the quality control of raw materials, characterization of the final product, and testing stability of active ingredients in the capsules. RESULTS: Formulation of the phytopreparation, validation of the production process, quality control and stability testing, all together have resulted in the production of capsules with defined valeric acid content (1 mg valeric acid per capsule). DISCUSSION: The preparation is recommended to relieve the symptoms caused by chronic stress (anxiety, irritability, fatigue, lack of concentration, heart palpitations) and for mild insomnia. CONCLUSION: This paper presents the complete cycle of the production of a phytopreparation on the example of a new herbal sedative--Odoval S capsules.

Anti-inflammatory, gastroprotective, free-radical-scavenging, and antimicrobial activities of hawthorn berries ethanol extract.

Hawthorn [Crataegus monogyna Jacq. and Crataegus oxyacantha L.; sin. Crataegus laevigata (Poiret) DC., Rosaceae] leaves, flowers, and berries are used in traditional medicine in the treatment of chronic heart failure, high blood pressure, arrhythmia, and various digestive ailments, as well as geriatric and antiarteriosclerosis remedies. According to European Pharmacopoeia 6.0, hawthorn berries consist of the dried false fruits of these two species or their mixture. The present study was carried out to test free-radical-scavenging, anti-inflammatory, gastroprotective, and antimicrobial activities of hawthorn berries ethanol extract. Phenolic compounds represented 3.54%, expressed as gallic acid equivalents. Determination of total flavonoid aglycones content yielded 0.18%. The percentage of hyperoside, as the main flavonol component, was 0.14%. With respect to procyanidins content, the obtained value was 0.44%. DPPH radical-scavenging capacity of the extract was concentration-dependent, with EC50 value of 52.04 microg/mL (calculation based on the total phenolic compounds content in the extract). Oral administration of investigated extract caused dose-dependent anti-inflammatory effect in a model of carrageenan-induced rat paw edema. The obtained anti-inflammatory effect was 20.8, 23.0, and 36.3% for the extract doses of 50, 100, and 200 mg/kg, respectively. In comparison to indomethacin, given in a dose producing 50% reduction of rat paw edema, the extract given in the highest tested dose (200 mg/kg) showed 72.4% of its activity. Gastroprotective activity of the extract was investigated using an ethanol-induced acute stress ulcer in rats with ranitidine as a reference drug. Hawthorn extract produced dose-dependent gastroprotective activity (3.8 +/- 2.1, 1.9 +/- 1.7, and 0.7 +/- 0.5 for doses of 50, 100, and 200 mg/kg, respectively), with the efficacy comparable to that of the reference drug. Antimicrobial testing of the extract revealed its moderate bactericidal activity, especially against gram-positive bacteria Micrococcus flavus, Bacillus subtilis, and Lysteria monocytogenes, with no effect on Candida albicans. All active components identified in the extract might be responsible for activities observed.