Maria Camilla Bergonzi

Essential Oils Loaded in Nanosystems: A Developing Strategy for a Successful Therapeutic Approach

Essential oils are complex blends of a variety of volatile molecules such as terpenoids, phenol-derived aromatic components, and aliphatic components having a strong interest in pharmaceutical, sanitary, cosmetic, agricultural, and food industries. Since the middle ages, essential oils have been widely used for bactericidal, virucidal, fungicidal, antiparasitical, insecticidal, and other medicinal properties such as analgesic, sedative, anti-inflammatory, spasmolytic, and locally anaesthetic remedies. In this review their nanoencapsulation in drug delivery systems has been proposed for their capability of decreasing volatility, improving the stability, water solubility, and efficacy of essential oil-based formulations, by maintenance of therapeutic efficacy. Two categories of nanocarriers can be proposed: polymeric nanoparticulate formulations, extensively studied with significant improvement of the essential oil antimicrobial activity, and lipid carriers, including liposomes, solid lipid nanoparticles, nanostructured lipid particles, and nano- and microemulsions. Furthermore, molecular complexes such as cyclodextrin inclusion complexes also represent a valid strategy to increase water solubility and stability and bioavailability and decrease volatility of essential oils.

Evaluation of chemical stability of St. John's wort commercial extract and some preparations

Thermal and photostability of a commercial dried extract and capsules of St. Johns wort (Hypericum perforatum L.) were evaluated under the ICH test conditions. The extract was considered as drug substance and its preparations as drug products. In addition, capsules of different colours corresponding to different opaficient and pigment contents were also evaluated as primary package of drug product and the tests in the secondary pack were performed with amber containers, as well. A selective high-performance liquid chromatography (HPLC) for determination of stability of all the characteristic constituents, namely flavonols, hyperforins and hypericins, was carried out. Photostability testing showed all the constituents to be photosensitive in the tested conditions. However, different opaficients and pigments present in the capsules influenced the stability of the different classes of constituents. Amber containers suggested as secondary packages influenced only in part the photostability of the investigated constituents. Long-term thermal stability testing showed a very low (less than 4 months) hyperforins and hypericins t(90), even if ascorbic and citric acids were added to the formulation. From the results we have obtained it is clear that for St. Johns wort preparations, a mere translation of the ICH guidelines to the field of herbal products, as suggested by the WPHMP of the EMEA, cannot be accepted. A revision and adaptation of the storage conditions should be elaborated.

Artemisinin and artemisinin plus curcumin liposomal formulations: Enhanced antimalarial efficacy against Plasmodium berghei-infected mice

The therapeutic efficacies of novel liposomal delivery systems based on artemisinin or artemisinin-based combination therapy with curcumin have been investigated and reported in this study. The developed liposomal formulations had proper characteristics as drug carriers for parental administration in terms of particle size, polydispersity, encapsulation efficacy and ζ-potential. Their physical and chemical stabilities were also evaluated. Furthermore, the in vivo antimalarial activity of artemisinin-based liposomal formulations was tested in Plasmodium berghei NK-65 infected mice, a suitable model for studying malaria because the infection presents structural, physiological and life cycle analogies with the human disease. Artemisinin, alone or in combination with curcumin, was encapsulated in conventional and PEGylated liposomes and its in vivo performance was assessed by comparison with the free drug. Mice were treated with artemisinin at the dosage of 50 mg/kg/days alone or plus curcumin as partner drug, administered at the dosage of 100 mg/kg/days. Artemisinin alone began to decrease parasitaemia levels only 7 days after the start of the treatment and it appeared to have a fluctuant trend in blood concentration which is reflected in the antimalarial effectiveness. By contrast, treatments with artemisinin-loaded conventional liposomes (A-CL), artemisinin-curcumin-loaded conventional liposomes (AC-CL), artemisinin-loaded PEGylated liposomes (A-PL), artemisinin-curcumin-loaded PEGylated liposomes (AC-PL) appeared to have an immediate antimalarial effect. Both nanoencapsulated artemisinin and artemisinin plus curcumin formulations cured all malaria-infected mice within the same post-inoculation period of time. Additionally, all formulations showed less variability in artemisinin plasma concentrations which suggested that A-CL, AC-CL, A-PL and AC-PL give a modified release of drug(s) and, as a consequence, a constant antimalarial effect during time. In particular, A-PL seems to give the most pronounced and statistically significant therapeutic effect in this murine model of malaria. The enhanced permanency in blood of A-PL suggests the use of these nanosystems as suitable passive targeted carriers for parasitic infections; this strong effect of formulation is added up to the mechanism of action of artemisinin which acts in the erythrocyte cycle stage of human host as a blood schizonticide.

Stability of the constituents of Calendula, Milk-thistle and Passionflower tinctures by LC-DAD and LC-MS

As a part of our investigations on the stability of tinctures, we evaluated 40 and 60% v/v tinctures of Calendula flower, Milk-thistle fruit and Passionflower. These preparations are widely employed in phytotherapy, thus Calendula is used externally for anti-inflammatory properties, Milk-thistle and Passionflower are employed for hepatic injuries and in tenseness with difficulty in falling asleep, respectively. Aim of this work was to assess the chemical stability of their active or marker constituents from accelerated and long-term testing by using HPLC. For Calendula flower and Passionflower active constituents are not known, however, flavonoids seem to have a crucial importance for the activity, and thus are considered the markers of Calendula and of Passionflower. Active constituents of Milk-thistle are represented by silymarin that is a phytocomplex mainly constituted by three flavolignans: silybin, silychristin and silydianin. Our investigation showed a very low thermal stability of the constituents from accelerated and long-term testing and determined by HPLC-DAD and -MS analyses and was related both to the class of flavonoids and water content of the investigated tinctures. Thus, shelf-lives at 25 degrees C of the most stable tincture (Passionflower 60% v/v) was about 6 months and only about 3 months the stability of Milk-thistle tinctures.

Variability in the content of the constituents of Hypericum perforatum L. and some commercial extracts

Seven samples of Hypericum perforatum L. (St. Johns wort) were collected throughout Tuscany; the dried extracts were assayed to determine the concentration of the constituents. Total flavonol content ranged from 4.58% to 15.90%; hypericins ranged from 0.05% to 0.11%; and hyperforins ranged from 1.37% to 20.80%. In addition, four commercially dried extracts were analyzed using the same high-performance liquid chromatographic (HPLC) method; their flavonol contents varied from 10.64% to 15.01%, hypericins varied from 0.03% to 0.20%, and hyperforins varied from 1.18% to 6.54%. The aim of this investigation was to evaluate the contents of the different constituents depending on environmental factors and drying and storage conditions of the wild samples. In addition, the contents of the constituents of the products available to the consumer that were related to quality and the relation of this to safety and efficacy were also evaluated.

Oral Metabolism and Efficacy of Kalanchoe pinnata Flavonoids in a Murine Model of Cutaneous Leishmaniasis

Leishmaniasis is a parasitic disease that threatens 350 million people worldwide. In a search for new antileishmanial drugs, the in vitro activity of flavonoids from Kalanchoe pinnata (Crassulaceae) was previously demonstrated in infected cells. In order to demonstrate the safety and oral activity of K. pinnata, flavonoids were evaluated in vivo in a murine model of cutaneous leishmaniasis. Daily oral doses of quercetin 3-O-alpha-L-arabinopyranosyl (1-->2)-alpha-L-rhamnopyranoside, quercetin 3-O-alpha-L-rhamnopyranoside, and free quercetin (16 mg/kg body weight) all were able to control the lesion growth caused by Leishmania amazonensis and to significantly reduce parasite load. These flavonoids were as effective as the crude K. pinnata aqueous extract given at 320 mg/kg body weight. HPLC-DAD-MS analysis of the plasma of extract-treated mice suggested that quercetin and quercetin glucuronides are the main metabolites of K. pinnata quercetin glycosides. Our results indicate that K. pinnata quercetin glycosides are important active components of the aqueous extract and that they possess potent oral efficacy against cutaneous leishmaniasis.

Conventional and long-circulating liposomes of artemisinin: preparation, characterization, and pharmacokinetic profile in mice

Artemisinin (qinghaosu), a unique endoperoxide sesquiterpene lactone isolated from Artemisia annua L., is a very active antimalarial drug, including severe and cerebral malaria. However, its therapeutical efficacy is limited due to its scarce bioavailability. In this article, artemisinin-loaded conventional and polyethylene glycol (PEGylated) liposomes were proposed as carriers to increase biopharmaceutical properties of the drug. Encapsulation efficacy was determined by high-performance liquid chromatography/diode array detection/electrospray ionization–mass spectrometry, dimensional analysis was performed by dynamic light scattering, and morphology was performed by trasmission electron microscopy. After dialysis, both liposomal formulations showed an encapsulation efficacy of more than 70%; mean diameter of all the artemisinin-loaded vesicles was approximately 130–140 nm. The polydispersity index of the formulations ranged from 0.2 to 0.3 and resulted as suitable for intraperitoneal (i.p.) administration. Pharmacokinetic profile and the main pharmacokinetic parameters of the carriers were evaluated in healthy mice i.p. Free artemisinin was rapidly cleared from plasma and hardly detected 1 hour after administration. Conversely, both liposomal formulations showed much longer blood-circulation time than free artemisinin; artemisinin was still detectable after 3 and 24 hours of administration, respectively, for conventional and PEGylated liposomes. AUC0–24h values were increased by approximately 6 times in both of the liposomal formulations, in comparison with free artemisinin. A strong effect of formulation on the half-life of artemisinin was enhanced by more than 5-fold by the incorporation of PEG into liposomes. Liposomes loaded with artemisinin, especially the long-circulating vesicles, could really represent a new strategy for developing smart, well-tolerated, and efficacious therapeutic nanocarriers to treat tumors, but could also be very useful to treat parasitic disease.

Identification and quantification of constituents of Gardenia jasminoides Ellis (Zhizi) by HPLC-DAD–ESI–MS

A simple, rapid and specific HPLC method was carried out for the analysis of characteristic constituents in Gardenia jasminoides Ellis (Zhizi), namely iridoids, caffeoyl quinic acid derivatives and crocins. The separation was successfully obtained using a C(18) column by gradient elution with mixtures of methanol and water as mobile phases; detection wavelength was set at 240 nm for iridoid glycosides, 315 nm for quinic acid derivatives and 438 nm for crocins. The analytical method was validated and the quantification of active compounds, namely iridoids, was performed. Linearity, precision, repeatability, stability, accuracy, limit of detection (LOD) and limit of quantification (LOQ) were also reported. This assay was successfully applied for qualitative and quantitative analysis of five commercial samples of G. jasminoides Ellis.

Influence of cultivation conditions, season of collection and extraction method on the content of antileishmanial flavonoids from Kalanchoe pinnata.

ETHNOPHARMACOLOGICAL RELEVANCE Leaves from Kalanchoe pinnata (Lamarck) Persoon (Crassulaceae) are popularly used for healing wounds. Its antileishmanial properties are established in experimental animals, and its active flavonoid components have been identified. AIM OF THE STUDY In this study, we attempted to standardize the extract from K. pinnata leaves by evaluating the influence of season of harvest, sunlight exposure and method of extraction on antileishmanial flavonoids content. MATERIALS AND METHODS HPLC-DAD-MS was used to identify and quantify the active antileishmanial flavonoids in different extracts. ANOVA test for analyses of variance followed by the Tukey test of multiple comparisons were used in the statistical analysis. The antileishmanial potential was assessed by the activation of nitric oxide production by murine macrophage using the Griess method. RESULTS We demonstrated that active flavonoids were significantly more abundant when the leaves were collected in the summer, and that aqueous extraction at 50°C allowed the highest flavonoid extraction. The benefit of sunlight exposure was confirmed in plants cultivated under direct sunlight when compared with those that grown under shade. Under sunny conditions the yield of the most active antileishmanial favonoid quercitrin was increased by 7-fold. All aqueous extracts tested were capable to enhance the macrophage nitric oxide production. However, hot aqueous extract from leaves collected in summer exhibited the higher activity, in agreement with HPLC-DAD-MS analysis tendency. In addition, with the aim of reducing the individual chemical variations of the plant constituents and optimizing the production of the active extract, it was obtained in vitro monoclonal KP specimens that were easily adapted to field conditions and were able to produce antileishmanial flavonoids. CONCLUSION Our study reports the better conditions of cultivation, harvest and extraction protocol for obtaining a K. pinnata extract exhibiting the highest antileishmanial activity. Additionally, we propose the flavonoids quercetin 3-O-α-L-arabinopyranosyl (1→2)-α-L-rhamnopyranoside and quercitrin, as satisfactory chemical markers for standardization purposes.

A Prolonged Protein Kinase C-Mediated, Opioid-Related Antinociceptive Effect of St John's Wort in Mice

UNLABELLED The antinociceptive profile of St. Johns Wort (SJW) was investigated in mice in a condition of acute thermal and chemical pain, together with the mechanism that might underlie this effect. A dried extract of SJW induced a prolonged antinociception that persisted for 120 minutes after administration. The thermal antinociception was prevented by naloxone and by the protein kinase C (PKC) activator PMA, whereas the chemical antinociception was prevented by PMA, remaining naloxone insensitive. A chloroform (CHL) and a methanol (MET) fraction, obtained to investigate the involvement of the SJW main components, hyperforin and hypericin/flavonoid, respectively, increased pain threshold with a time course comparable to the dried extract. The CHL antinociception was prevented by naloxone, whereas the MET antinociception was antagonized by PMA. Purified hyperforin and hypericin showed an antinociceptive efficacy comparable to CHL and MET, respectively. Conversely, flavonoids were devoid of any effect. The administration of yohimbine and atropine did not modify SJW, CHL and MET antinociception. These results indicate that both CHL and MET fractions mediate the SJW-induced antinociception. In particular, the presence of hypericin was fundamental to induce both thermal and chemical antinociception through the inhibition of the PKC activity, whereas hyperforin selectively produced a thermal opioid antinociception. PERSPECTIVE This article presents evidence of a persistent thermal and chemical antinociception of SJW that is mainly mediated by PKC-inhibiting mechanisms. These findings identify important targets for a longer-acting activation of endogenous pain systems and should potentially help clinicians who seek safe, tolerable, and prolonged treatments for pain relief.