Development of extraction technique and GC/FID method for the analysis of cannabinoids in Cannabis sativa L. spp. santicha (hemp).

INTRODUCTION: Nowadays, the interest in industrial Cannabis sativa L. herb has been increasing in the world. As a result, it is becoming one of the most studied plants due to its multifunctional benefits. OBJECTIVES: To the best of our knowledge, no study has been conducted so far to determine the impact of extraction methods and conditions on the extraction yields of CBD and CBG from the Cannabis sativa L. ssp. Santhica. Therefore, we aimed to investigate a simple and sensitive GC-FID method to determine CBD and CBG in hemp extract. METHODS: As regards sample preparation, three extraction techniques were compared, including maceration (ME), ultrasound-assisted extraction (UAE) and reflux-heat extraction (RHE), in order to obtain a high recovery of the CBD of interest from the plant material. The GC-FID method developed in this study represents a powerful tool for the extraction and analysis of non-psychoactive cannabinoids from hemp varieties to be used for the preparations of extracts with a high content of bioactive compounds for both pharmaceutical and nutraceutical applications. RESULTS: A simple extraction procedure for CBD and CBG from hemp was also optimized in this work, by using ultrasound assisted extraction method with 96% ethanol, material/solvent ratio 1:10 and extraction time 10 min at room temperature. CONCLUSION: The overall analytical method was fully validated in agreement with international guidelines. Therefore, proving a powerful and reliable tool for both the selection of hemp varieties with a high content of bioactive compounds and the quality control of its derivatives.

Liposomal Incorporation to Improve Dissolution and Stability of Rosmarinic Acid and Carvacrol Extracted from Oregano (O. onites L.).

The potential antimicrobial benefit of high levels of rosmarinic acid (RA) and carvacrol (CA) in oregano (O. onites L.) extract has been limited until now by poor bioavailability arising from the low aqueous-phase solubility and slow dissolution behaviour of the lyophilized extract (E). To address this issue, various ratios of phospholipon 90H (P90H) and 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol), sodium salt (DMPG) were sonicated, yielding four empty liposomes (L1, L2, L3, and L90). After an initial selection process, Turkish oregano extract was internalized into the more promising candidates. Each empty liposome, extract-loaded liposome (LE1, LE2, and LE3), and freeze-dried control (E) was assessed in terms of structure, composition, RA and CA dissolution profile, storage stability, and, when relevant, zeta potential. Empty liposome L1, which was prepared using P90H and DMPG in a 1:1 ratio, displayed the most convenient encapsulation traits among the four unloaded types. Loaded liposome LE1, obtained by combining oregano extract and L1 in a 1:1 ratio, proved superior as a vehicle to deliver RA & CA when compared against control freeze-dried E and test liposomes LE2 and LE3. Dissolution profiles of the active compounds RA and CA in loaded liposomes were determined using a semi-automated dissolution tester. The basket method was applied using artificial gastric juice without pepsin (AGJ, 50rpm, 500mL). The pH value was maintained at 1.5 (37 ± 0.5°C). Aliquots (5ml) were manually extracted from parallel dissolution vessels at 1, 3, 5, 7, 10, 15, 20, 25, 30, 45, and 60-minute time points. Dissolution tests, run to completion on LE1, showed that approximately 99% of loaded CA and 88% of RA had been released. Shorter dissolution times were also noted in using LE1. In particular, the release profile of CA and RA had levelled off after only 25 minutes, respectively, depicting an impressive 3.0-3.3 and 2.3-2.6 rate increase compared to the freeze-dried control extract. The improved dispersibility of RA and CA in the form of LE1 was supported by particle size and zeta potential measurements of the liposome, yielding 234.3nm and -30.9mV, respectively. The polydispersity index value was 0.35, indicating a reasonable particle size distribution. To study storage stability, liposomes were stored (4°C, 6 months) in amber coloured glass containers (4 oz.). Each container held 30 capsules, which were stored according to the ICH guidelines prescribed for long-term storage (25°C ± 2°C; 60% ± 5% RH). Triplicate samples were withdrawn after 0, 3, 6, 9, and 12 months for analysis. Lastly, LE1 displayed good storage stability. The results imply that RA and CA can be conveniently and routinely delivered via oral and mucosal routes by first internalizing oregano extracts into appropriately engineered liposomes.

The Influence of Different Oregano Species on the Antioxidant Activity Determined Using HPLC Postcolumn DPPH Method and Anticancer Activity of Carvacrol and Rosmarinic Acid.

The aim of this study was to evaluate concentration-dependent antioxidant and anticancer activities of CA and RA in ethanol extracts of three different Oregano species (Origanum onites L., Origanum vulgare L., and Origanum vulgare ssp. hirtum). The study revealed the highest RA antioxidant activity in O. vulgare ssp. hirtum (9550 ± 95 mmol/g) and the lowest in O. vulgare L. (2605 ± 52 mmol/g) (p < 0.05). The highest CA amount was present in O. onites L., which was 1.8 and 4.7 times higher (p < 0.05) than in O. vulgare ssp. hirtum and O. vulgare L., respectively. The anticancer activity was evaluated on human glioblastoma (U87) and triple-negative breast cancer (MDA-MB231) cell lines in vitro. RA anticancer activity was negligible. CA and the extracts were about 1.5-2 times more active against MDA-MB231 cell line (p < 0.05) compared to U87 cell line. The anticancer activities of three tested extracts were similar against U87 cell line (p > 0.05) but they had different activities against MDA-MB231 cell line.

Formulation and characterization of Turkish oregano microcapsules prepared by spray-drying technology.

The aim of this study was optimization of spray-drying process conditions for microencapsulation of Turkish oregano extract. Different concentrations of maltodextrin and gum arabic as encapsulating agents (wall material) as well as influence of selected processing variables were evaluated. The optimal conditions were maintained on the basis of the load of main bioactive compounds - ursolic, rosmarinic acids and carvacrol - in prepared microparticles after comparison of all significant response variables using desirability function. Physicomechanical properties of powders such as flowability, wettability, solubility, moisture content as well as product yield, encapsulation efficiency (EE), density, morphology and size distribution of prepared microparticles have been determined. The results demonstrated that the optimal conditions for spray-drying mixture consisted of two parts of wall material solution and one part of ethanolic oregano extract when the feed flow rate was 40 mL/min and air inlet temperature -170 °C. Optimal concentration of wall materials in solution was 20% while the ratio of maltodextrin and gum arabic was 8.74:1.26.

Somatic complaints and refrain from buying prescribed medications. Results from a cross-sectional study on people 60 years and older living in Kaunas (Lithuania).

BACKGROUND: The use of medicines by elderly people is a growing area of concern in social pharmacy. A significant proportion of older people do not follow the recommendations from physicians and refrain from buying prescribed medications. The aim of this study is to evaluate associations between self-rated health, somatic complaints and refraining from buying prescribed medications by elderly people. FINDINGS: Data was collected in a cross-sectional study in 2009. We received 624 completed questionnaires (response rate - 48.9%) from persons aged 60-84 years living in Kaunas (Lithuania). Somatic complaints were measured with the 24 item version of the Giessen Complaint List (GBB-24). Logistic regression (Enter model) was used for evaluation of the associations between refraining from buying medications and somatic complaints. These associations were measured using odds ratio (OR) and calculating the 95% confidence interval (CI).The mean scores in total for the GBB scale and sub-scales (exhaustion, gastrointestinal and cardiovascular) were lowest among respondents who did not refrain from buying prescribed medications (means for GBB-24 scale: 21.04 vs. 24.82; p=0.001). Logistic regression suggests that somatic complaints were associated with a increased risk of refraining from buying prescribed medications (OR=1.35, 95% CI=1.15-1.60). CONCLUSIONS: Somatic complaints were significantly associated with the decision to refrain from buying prescribed medications.

Causes of refraining from buying prescribed medications among the elderly in Kaunas, Lithuania.

BACKGROUND AND OBJECTIVE: Accessibility to medications among the elderly is a source of concern in Lithuania and beyond. However, there are no studies carried out on this topic in Lithuania. Therefore, the aim of this study was to evaluate the causes of refraining from buying prescribed medications among the elderly in Kaunas, Lithuania. MATERIAL AND METHODS: The data were collected in a cross-sectional ABUEL study in 2009. A total of 624 filled-in questionnaires (response rate, 48.9%) from the elderly aged 60-84 years living in Kaunas (Lithuania) were received. For evaluation of the impact of explanatory variables on the analyzed event (binary dependent variable), an Enter model of logistic regression was used. RESULTS: The study showed that 32.7% of the respondents refrained from buying prescribed medications. The most common reasons (respondents could select several options) for this decision were financial problems (48.0%), disappearance of problems (40.7%), and fear of side effects (22.5%). Refraining from buying prescribed medications was positively associated with age (OR, 0.85; 95% CI, 0.74 to 0.99). Higher education was associated with a reduced risk of refraining from buying prescribed medications due to financial problems (OR, 0.49; 95% CI, 0.31 to 0.78) and an increased risk of refraining from buying medications due to the disappearance of health problems (OR, 1.75; 95% CI, 1.15 to 2.68). An opposite association with worries about daily expenses was observed. CONCLUSIONS: Study has revealed that one-third of the elderly refrained from buying prescribed medications, and the main reasons for this were financial problems and disappearance of health problems.

Evaluation of phenolic acids and phenylpropanoids in the crude drugs.

Phenolic acids and phenylpropanoids have an important biological activity and are therapeutic agents of crude drugs. Development of validated analysis techniques of these phytotherapeutic agents (fingerprinting and assay procedures) is an important practice for efficacy, safety, and quality control of herbal drug preparations. The aim of the present work was to study analytical capabilities of the evaluation of selected phenolic acids and phenylpropanoids: caffeic acid, chlorogenic acid, cinnamic acid, coumaric acid, ferulic acid, gallic acid, protocatechuic (3,4-dihydroxybenzoic) acid, rosmarinic acid, vanillic acid, and vanillin. Optimization and validation procedures of rapid and simple method of reversed-phase high-performance liquid chromatography were carried out. The mobile phase of the optimized chromatographic method consisted of methanol and 0.5% acetic acid solvent in water. For the application of method, two kinds of raw materials were chosen: propolis and the Herba Origani. Coumaric acid is the dominating phenolic acid of propolis (2785 microg/g). Results of analysis of Herba Origani demonstrated high quantities (6376 microg/g) of rosmarinic and protocatechuic (1485 microg/g) acids in the samples.

[Antimicrobial activity of soft and purified propolis extracts]. / Tirstojo ir isgryninto propolio ekstrakto antimikrobinis aktyvumas.

OBJECTIVE: To evaluate the antimicrobial activity of soft and purified propolis extracts. STUDY OBJECT AND METHODS: Antimicrobial activity of soft and purified propolis extracts was determined with reference cultures of Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 33499, Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis ATCC 12459, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 8035, and fungus Candida albicans ATCC 60193. Microbiological tests were performed under aseptic conditions. Minimum inhibitory concentration (MIC)--the highest dilution of preparation (the lowest concentration of preparation) that suppresses growth of reference microorganisms--was determined. RESULTS: Concentration of phenolic compounds in soft propolis extract that possesses antimicrobial activity against gram-positive (Staphylococcus aureus, Enterococcus faecalis) and gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis) is 0.587+/-0.054 mg and 0.587+/-0.054-0.394+/-0.022 mg (P>0.05) and in purified propolis extract--0.427+/-0.044 mg and 0.256+/-0.02 mg (P>0.05). Klebsiella pneumoniae is most resistant to soft propolis extract when the concentration of phenolic compounds is 1.119+/- 0.152 mg and to purified propolis extract when the concentration of phenolic compounds is 1.013+/-0.189 mg (P>0.05). Spore-forming Bacillus subtilis bacteria are more sensitive to soft and purified propolis extracts when the concentration of phenolic compounds is 0.134+/-0.002 mg and 0.075+/-0.025 mg, respectively, and Bacillus cereus--when the concentration is 0.394+/-0.022 mg and 0.256+/-0.02 mg (P>0.05). Sensitivity of fungus Candida albicans to soft and purified propolis extracts is the same as Bacillus subtilis. Encapsulated bacterium Klebsiella pneumoniae is most resistant to antimicrobial action of soft and purified propolis extracts as compared with gram-positive Staphylococcus aureus and Enterococcus faecalis bacteria (P<0.05), gram-negative Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis (P<0.05), spore-forming Bacillus subtilis and Bacillus cereus bacteria (P<0.05), and fungus Candida albicans (P<0.05). There is no statistically significant difference between antimicrobial effect of soft propolis extract and purified propolis extract on gram-positive bacteria, gram-negative bacteria, spore-forming bacteria, encapsulated bacteria, and Candida fungus. CONCLUSIONS: Soft and purified propolis extracts possess antimicrobial activity. They could be recommended as natural preservatives in the manufacture of pharmaceutical products.

[Technology of extract improving the immune system]. / Imunine sistema gerinancio ekstrakto technologijos ypatybes.

The article deals with the production of the extract of Echinacea purpurea herbs when selecting an extractant concentration, the size of particles in a raw material and the method of extraction. Using 60% (v/v) ethanol for production of the immune system enhancing extract, the extract becomes green due to pigmental substances; therefore, 60% (v/v), 70% (v/v), and 96% (v/v) ethanol was not used for extraction. It is recommended to use 50% (v/v) ethanol for extraction. Using 40% (v/v) ethanol not all active substances are extracted and resistance of the extract to microbial contamination is weaker. It was determined that the method of repercolation should be used to yield the largest amount of active substances. The largest amount of active substances in the immune system enhancing extract was obtained when the flow speed of extract was 0.2 ml/min. The quality of extract was tested within the period of 26 months by the monitoring of following parameters: appearance of the extract, refraction index, relative density, ethanol concentration, the amounts of dry residue and chicoric acid. The analysis was carried out every three months. The samples of the extract remained stable during storage: no alterations of color, smell or taste were observed. Some of the samples had deposits, which is quite natural in the production of drugs from herbal material, as they have no influence on the quality of the drug (the amounts of active substances were found to be within acceptable limits). On the basis of the obtained results, the period of expiry of the immune system enhancing tincture was confirmed to be 2 years.

[Estimation of maximum acceptable concentration of lead and cadmium in plants and their medicinal preparations]. / Svino ir kadmio leistinu koncentraciju ivertinimas augalinese zaliavose ir is ju pagamintuose vaistuose.

UNLABELLED: Heavy metals (lead, cadmium) are possible dashes which quantity is defined by the limiting acceptable contents. Different drugs preparations: infusions, decoctions, tinctures, extracts, etc. are produced using medicinal plants. The objective of this research was to study the impurities of heavy metals (lead, cadmium) in medicinal plants and some drug preparations. MATERIALS AND METHODS: We investigated liquid extracts of fruits Crataegus monogyna Jacq. and herbs of Echinacea purpurea Moench., tinctures--of herbs Leonurus cardiaca L. The raw materials were imported from Poland. Investigations were carried out in cooperation with the Laboratory of Antropogenic Factors of the Institute for Biomedical Research. Amounts of lead and cadmium were established after "dry" mineralisation using "Perkin-Elmer Zeeman/3030" model electrothermic atomic absorption spectrophotometer (ETG AAS/Zeeman). It was established that lead is absorbed most efficiently after estimation of absorption capacity of cellular fibers. About 10.73% of lead crosses tinctures and extracts, better cadmium--49.63%. Herbs of Leonurus cardiaca L. are the best in holding back lead and cadmium. About 14.5% of lead and cadmium crosses the tincture of herbs Leonurus cardiaca L. We estimated the factors of heavy metals (lead, cadmium) in the liquid extracts of Crataegus monogyna Jacq. and Echinacea purpurea Moench., tincture of Leonurus cardiaca L. after investigations of heavy metals (lead, cadmium) in drugs and preparations of it. The amounts of heavy metals (lead, cadmium) don't exceed the allowable norms in fruits of Crataegus monogyna Jacq., herbs of Leonurus cardiaca L. and Echinacea purpurea Moench. after estimation of lead and cadmium extraction factors, the maximum of acceptable daily intake and the quantity of drugs consumption in day.

[Analysis of possibilities of pharmaceutical care elements implementation in Lithuanian community pharmacies]. / Farmacines rupybos elementu igyvendinimo Lietuvos visuomenes vaistinese galimybiu analize.

OBJECTIVE: Objective of this survey--possibilities of implementation of pharmaceutical care elements in daily work of Lithuanian community pharmacies. All pharmacists have to ensure the best quality of pharmaceutical services which they render to their patients. Pharmacists also must improve the quality of pharmaceutical services, according to recommendations of pharmaceutical care. For a start they should implement at least separate elements of pharmaceutical care if they are going to provide pharmaceutical care services in their community pharmacies in the future. That's why this survey was designed to clarify the possibilities of implementation of separate pharmaceutical care elements in to daily work of community pharmacies. MATERIAL AND METHODS: Material of this survey--pharmaceutical services rendered in Lithuanian community pharmacies. This survey has an analytical character and was accomplished with questionnaire. Chiefs of community pharmacies were asked to complete of questionnaire. We gave 150 questionnaires and received answers from 128 pharmacists. RESULTS: After gathered data analysis we found out that only 2% of pharmacies are almost prepared and well equipped for provision of pharmaceutical care services; however, they still have to rearrange their facilities. CONCLUSIONS: Lithuanian community pharmacies are not ready to work according to recommendations of pharmaceutical care today. Some elements of pharmaceutical care (such as blood pressure measurement, provision of written information for patient, etc.) are already implemented by almost all Lithuanian community pharmacies. If pharmacists want to provide pharmaceutical care services they should create a database of patient medication records in pharmacy. Unfortunately Lithuanian pharmacists do not have such databases today. Only with such databases there is a possibility to render pharmaceutical services of the best quality.

[Development of intranasal lactocin (oxytocin) drops technology]. / Laktocino (oksitocino) intranazaliniu lasu technologijos sukurimas.

Pure oxytocin substance was obtained from posterior part of cattle pituitary gland by high pressure liquid chromatography. Biological activity of the substance--450-500 IU/mg. Chromatographically pure Oxytocin substance was used in developing two different compositions of Lactocin intranasal drops (40 IU/ml). Stability evaluation was performed for 2 year period. The technical documentation was prepared on the basis of the research results. Lactocin is active preparation helping lactation and is indicated for lactostasis treatment and its prophylaxis after delivery.