Siniša Tomić

Contaminants of Medicinal Herbs and Herbal Products

Contaminants of Medicinal Herbs and Herbal Products Medicinal plants have a long history of use in therapy throughout the world and still make an important part of traditional medicine. Thus, medicinal plants and herbal products must be safe for the patient (consumer). This review addresses biological contaminants (microbes and other organisms) and chemical contaminants (mycotoxins, toxic elements such as heavy metals, and pesticide residues) as major common contaminants of medicinal herbs and herbal products. To prevent and screen for contamination and ensure safety and conformity to quality standards, medicinal herbs and herbal products should be included in appropriate regulatory framework. Onečišćenja u ljekovitom bilju i biljnim proizvodima Ljekovito bilje i biljni proizvodi već tisućljećima nalaze široku primjenu u različitim sustavima tradicionalnog liječenja. Stoga je njihova neškodljivost, ponajprije uvjetovana kakvoćom biljne sirovine, od izuzetne važnosti za javno zdravstvo. Od mogućih čimbenika koji utječu na kakvoću ljekovitog bilja i biljnih proizvoda ovaj pregledni rad osvrće se na najčešće prisutna biološka (mikroorganizmi) i kemijska onečišćenja (mikotoksini, toksični elementi poput teških metala te ostaci pesticida). S ciljem postizanja ujednačenih standarda kakvoće te osiguranja sigurnosti primjene biljnih proizvoda od vitalne su važnosti zakonski propisi koji moraju u odgovarajućim regulatornim okvirima obuhvatiti ovu skupinu proizvoda s naglaskom na sprječavanju i ispitivanju njihovih mogućih onečišćenja.

Evaluation of antiaggregatory activity of flavonoid aglycone series

BackgroundAmong natural compounds, present in every day diet, flavonoids have shown beneficial effect in prevention of cardiovascular diseases that can be attributed, at least partially to the described antiaggregatory activity i.e. antiplatelet effects of flavonoids. Due to the ever increasing pharmacological interest in antiplatelet agents a systematic experimental evaluation of large flavonoid series is needed.MethodsA set of thirty flavonoid aglycones has been selected for the evaluation. All measurements of aggregation were done under standardized and firmly controlled in vitro conditions. The whole blood samples, multiple platelet functional analyzer and adenosine diphosphate (ADP) as a weak agonist of aggregation were selected for this purpose.ResultsThe results were expressed as minimal concentration of flavonoid that can significantly lower the platelet aggregation compared to the corresponding untreated sample (minimal antiaggregatory concentration - MINaAC). All analyzed flavonoids exhibited antiaggregatory activity MINaAC ranging from 0.119 μM to 122 μM, while the most potent representatives were 3,6-dihydroxyflavone (0.119 μM) and syringetin (0.119 μM).ConclusionsMeasurable antiplatelet activity established at submicromolar flavonoid concentrations suggests that even a dietary consumption of some flavonoids can make an impact on in vivo aggregation of platelets. These findings also point out a therapeutical potential of some flavonoids.

Adverse drug reactions caused by drug-drug interactions reported to Croatian Agency for Medicinal Products and Medical Devices: a retrospective observational study

Aim To analyze potential and actual drug-drug interactions reported to the Spontaneous Reporting Database of the Croatian Agency for Medicinal Products and Medical Devices (HALMED) and determine their incidence. Methods In this retrospective observational study performed from March 2005 to December 2008, we detected potential and actual drug-drug interactions using interaction programs and analyzed them. Results HALMED received 1209 reports involving at least two drugs. There were 468 (38.7%) reports on potential drug-drug interactions, 94 of which (7.8% of total reports) were actual drug-drug interactions. Among actual drug-drug interaction reports, the proportion of serious adverse drug reactions (53 out of 94) and the number of drugs (n = 4) was significantly higher (P < 0.001) than among the remaining reports (580 out of 1982; n = 2, respectively). Actual drug-drug interactions most frequently involved nervous system agents (34.0%), and interactions caused by antiplatelet, anticoagulant, and non-steroidal anti-inflammatory drugs were in most cases serious. In only 12 out of 94 reports, actual drug-drug interactions were recognized by the reporter. Conclusion The study confirmed that the Spontaneous Reporting Database was a valuable resource for detecting actual drug-drug interactions. Also, it identified drugs leading to serious adverse drug reactions and deaths, thus indicating the areas which should be in the focus of health care education.

The content of essential and toxic elements in Croatian propolis samples and their tinctures

Summary There is a general lack of scientific literature dealing with the mineral composition of propolis, despite the fact that the presence of minerals in plant tissues can significantly affect the pharmacotherapeutic properties of derived products. Raw propolis samples collected from different Croatian regions and their respective tinctures were therefore analysed for mineral composition by atomic emission/absorption spectrometry. Contents of macro- and trace elements (Ca, K, Mg, Na, Al, B, Ba, Cr, Cu, Fe, Mn, Ni, Sr and Zn) were determined. Levels of toxic elements (As, Cd, Hg, and Pb) were also analysed, since they are possible environmental contaminants that could be transferred into propolis products for human consumption. Following the European Pharmacopoeia requirements for quality control of herbal drugs, crude propolis samples were also analysed for total ash content. Significant correlation between the total mineral content and total ash of raw propolis (r = 0.9273) showed that total ash might serve as the first information that justifies the need for detailed mineral analysis of tested samples. Among analysed minerals, Ca, Mg, K, Al, Fe and Zn were found to be the most abundant in raw propolis, ranging from 12.06 (Mg) to 932.6 mg/100g (Zn). Mineral contents of all investigated tinctures were significantly lower compared to the respective raw propolis samples. Extractability was variable due to influence of the sample matrix and correlated with the mineral content of raw propolis only in the case of Fe, Mg, Sr and Zn. All samples met the World Health Organization (WHO) standards regarding the maximum permitted amounts of Hg, Pb, As and Cd except for two raw propolis samples with increased lead content indicating pollution of the collection areas.

Hydroxytyrosol expresses antifungal activity in vitro

Hydroxytyrosol (HT) is a potent antioxidant found in olive oil and leaves. Using several in vitro approaches, we tested antifungal activity of HT. HT showed broad spectrum of antifungal activity against medically important yeasts and dermatophyte strains with MIC values ranging between 97.6 µgml⁻¹ and 6.25 mgml⁻¹. The antimicrobial activity of HT was also tested using the time-kill methodology. Below the MIC value, HT showed potent damage of cell wall of Candida albicans ATCC 10231 using fluorescent dye-exclusion method. At the subinhibitory concentration, HT also influenced dimorphic transition of Candida indicating that HT is inhibitor of germ-tube formation as one of the most important virulence factor of C. albicans. Furthermore, HT showed disturbances in cell surface hydrophobicity (CSH) of C. albicans. The in vitro results indicate that HT caused a significant cell wall damage and changes in CSH as well as inhibition of germ-tube formation as virulence factor of C. albicans. The study indicates that HT has a considerable in vitro antifungal activity against medically important yeasts.

Olive leaf extract activity against Candida albicans and C. dubliniensis – the in vitro viability study

Abstract Olive leaf extract is characterized by a high content of polyphenols (oleuropein, hydroxytyrosol and their derivatives), which is associated with its therapeutic properties. The objective of the present research was to evaluate the antifungal activity of olive leaf extract against Candida albicans ATCC 10231 and C. dubliniensis CBS 7987 strains. Minimum inhibitory concentrations (MIC) of the extract were determined by several in vitro assays. The extract showed a concentration depended effect on the viability of C. albicans with MIC value of 46.875 mg mL-1 and C. dubliniensis with MIC value 62.5 mg mL-1. Most sensitive methods for testing the antifungal effect of the extracts were the trypan blue exclusion method and fluorescent dye exclusion method while MIC could not be determined by the method according to the EUCAST recommendation suggesting that herbal preparations contain compounds that may interfere with this susceptibility testing. The fluorescent dye exclusion method was also used for the assessment of morphological changes in the nuclei of treated cells. According to the obtained results, olive leaf extract is less effective against the tested strains than hydroxytyrosol, an olive plant constituent tested in our previous study.

Preliminary investigation of metal and metalloid contamination of homeopathic products marketed in Croatia

BACKGROUND Due to their popularity as a complementary therapy in many diseases, homeopathic products of animal, vegetable, mineral and chemical origin should be tested for the presence of contaminants to prevent eventual toxic effects. OBJECTIVE Thirty samples of homeopathic products were analyzed to estimate possible contamination with potentially toxic elements: Pb, Cd, As, Hg, Cr, Ni and Zn, and to assess human exposure to these metals/metalloid as a consequence of their consumption. METHODS Atomic absorption spectrometry was used to determine metal and metalloid concentrations. RESULTS Most tested products had very low metal/metalloid levels (below the limit of quantification of the method), but the metal/metalloid levels in the remaining products were in the following ranges (in microg g(-1)): Pb 0.33-1.29 (6 samples), Cd 2.78 (1 sample), As 0.22 (1 sample), Hg 0.02-0.12 (24 samples), Cr 0.40-10.27 (10 samples), Ni 0.43-55.00 (19 samples), and Zn 2.20-27.80 (11 samples). In the absence of regulatory standards for homeopathic products, the obtained results were compared to maximum allowable levels (MALs) as proposed by USP Ad Hoc Advisory Panel. Some analyzed preparations had metal levels above MALs (Pb: 2 samples; Cd: 1 sample; Ni: 2 samples). However, estimated cumulative daily intakes from tested homeopathic products were in all cases lower than permitted daily exposures for all dosage forms. CONCLUSION The risk of bioaccumulation of metals/metalloid from the homeopathic medicines seems to be rather low, due to small quantities of those products prescribed to be applied per day, as well as insignificant metal contamination of the majority of tested products. However, the fact that particular formulations were contaminated by metals above MALs indicates potential risk and points to the necessity of regular monitoring of homeopathic products for metal contamination, due to their frequent and mostly unsupervised use.

Antifungal Activity of Oleuropein against Candida albicans—The In Vitro Study

In the present study we investigated activity of oleuropein, a complex phenol present in large quantities in olive tree products, against opportunistic fungal pathogen Candida albicans. Oleuropein was found to have in vitro antifungal activity with a minimal inhibitory concentration (MIC) value of 12.5 mg·mL−1. Morphological changes in the nuclei after staining with fluorescent DNA-binding dyes revealed that apoptosis was a primary mode of cell death in the analyzed samples treated with subinhibitory concentrations of oleuropein. Our results suggest that this antifungal agent targets virulence factors essential for establishment of the fungal infection. We noticed that oleuropein modulates morphogenetic conversion and inhibits filamentation of C. albicans. The hydrophobicity assay showed that oleuropein in sub-MIC values has significantly decreased, in both aerobic and anaerobic conditions, the cellular surface hydrophobicity (CSH) of C. albicans, a factor associated with adhesion to epithelial cells. It was also demonstrated that the tested compound inhibits the activity of SAPs, cellular enzymes secreted by C. albicans, which are reported to be related to the pathogenicity of the fungi. Additionally, we detected that oleuropein causes a reduction in total sterol content in the membrane of C. albicans cells, which might be involved in the mechanism of its antifungal activity.

Granting marketing authorisation for medicines in South East European countries: The point of view of the authority

European legislation for medicines places the emphasis on an assessment of quality, safety and efficacy during the procedure for the granting of marketing authorisations for medicines, in order to protect patient health. The integrated European regulatory system involves the participation of a network of experts from the agencies of the member states that takes part in the European procedures for the authorisation of medicines. On the way to full membership in the EU, candidate countries and potential candidates have to transpose and implement the European directives for medicinal products; they must also strengthen their scientific and administrative capacities. Croatia acquired good experience in implementing the simplified marketing authorisation procedure for medicines authorised in the EU pursuant to the New Collaboration Agreement between Drug Regulatory Authorities in Central and East European Countries (nCADREAC), which helps it to exchange information and prepare for the implementation of European procedures. However, there are still some provisions to transpose before actual full membership, and also dossier upgrading, in which the marketing authorisation holder has to harmonise its documentation about a medicinal product with the requirements of the directives, if a product already on the market was not previously approved in line with current European legislation. Collaboration with the European Medicines Agency (EMA) through an Instrument for Pre-Accession (IPA) provides candidate countries and potential candidates the opportunity for education and training in some regulatory activities as well as the participation of their representatives as observers in some EMA committees and working groups. Some characteristics of the national regulatory frameworks of the countries of South East Europe in their efforts to achieve harmonisation with EU legislation are presented in this paper.

Identification of Counterfeit Medicines for Erectile Dysfunction from an Illegal Supply Chain

Identification of Counterfeit Medicines for Erectile Dysfunction from an Illegal Supply Chain The appearance of counterfeit medicines in supply chains is a global public health problem that may seriously affect patients. Counterfeit drugs do not meet quality standards and do not declare their real composition and/or source for the purposes of fraud. They may be generic or innovative, they may contain genuine constituents in a fake packaging, or wrong ingredients, or inactive ingredients, or an incorrect quantity of the active substance. In Croatia, no cases of counterfeit medicines have been detected so far, but the Agency for Medicinal Products and Medical Devices has received 34 samples of medicines and other products for testing from Zagreb City Police. The samples included medicines for erectile dysfunction: sildenafil, tadalafil, and vardenafil. Twenty-three samples of tablets without marketing authorisation in Croatia were tested with high-performance liquid chromatography (HPLC) for the declared sildenafil and tadalafil content. Samples labelled 1 (batch T/33), 3 (batch T/33), 5 (batch 4), 6 (batch M0016J), 10 (batch T-070235), 12 (batch T-070544), 15 (batch 314833201), 16 (batch 832718474), and 17 (batch 504830028) containing sildenafil and samples labelled 20 (batch 070356), 21 (batch 05668), and 22 (batch T 378 5) containing tadalafil did not contain the active substance within the acceptable 95 % to 105 % margin of deviation from the declared content. While most samples cannot be described as fake with a reasonable amount of certainty, there is still a suspicion of counterfeit. A correct conclusion can be drawn only with the assistance of the manufacturers and by conducting additional laboratory tests. Identifikacija Krivotvorenih Lijekova za Erektilnu Disfunkciju iz Ilegalnog Lanca Opskrbe Pojava krivotvorenih lijekova u lancima opskrbe globalni je javnozdravstveni problem koji može imati ozbiljnih posljedica za pacijenta. Krivotvoreni lijekovi ne zadovoljavaju propisane standarde kakvoće te su u svrhu prijevare drugačije deklarirani u odnosu na njihov stvarni sastav i/ili podrijetlo. Mogu biti generički, ili inovativni, mogu sadržavati ispravne sastojke, ali pogrešno pakiranje, pogrešne sastojke, ne sadržavati djelatnu tvar ili ne sadržavati dovoljnu količinu djelatne tvari. U Hrvatskoj do sada nisu zabilježeni slučajevi krivotvorenih lijekova, ali je Agencija za lijekove i medicinske proizvode od policijske uprave Zagrebačke zaprimila 34 uzorka lijeka i ostalih proizvoda u svrhu ispitivanja. Od lijekova, radilo se o lijekovima za erektilnu disfunkciju sildenafilu, tadalafilu i vardenafilu. 23 uzorka tableta koji nemaju odobrenje za stavljanje lijeka u promet u Hrvatskoj ispitana su metodom tekućinske kromatografije visokog učinka (HPLC) na deklarirani sadržaj sildenafila i tadalafila. Ustanovljeno je da uzorci pod oznakama 1 (serija T/33), 3 (serijaT/33), 5 (serija 4), 6 (serija M0016J), 10 (serija T-070235), 12 (serija T-070544), 15 (serija 314833201), 16 (serija 832718474), 17 (serija 504830028) koji su sadržavali sildenafil te uzorci 20 (serija 070356), 21 (serija 05668), 22 (serija T 378 5) i 23 (702099) koji su sadržavali tadalafil, ne sadržavaju djelatnu tvar unutar prihvatljivih granica odstupanja od 95 % do 105 % od deklarirane vrijednosti. Iako se ne može sa sigurnosti za većinu uzoraka reći da se radi o krivotvorini, ipak se može govoriti o sumnji na krivotvorinu, a cjeloviti zaključak može se izvesti jedino uz suradnju i mišljenje proizvođača te provedbom dodatnih laboratorijskih ispitivanja.