The genus Juniperus (Cupressaceae), one of the most diverse of the conifers, comprises approximately 75 species of evergreen aromatic shrubs or trees native to northern hemisphere. Juniperus species have been widely used as herbal medicine from ancient time [1],[2]. The present study aimed to evaluate antioxidant and acetylcholinesterase (AChE) inhibitory activities as well as ex vivo neuroprotective effect of hydroalcoholic extracts of leaves and branches of three Juniperus species (J. communis L., J. oxycedrus L. and J. phoenicea L.) growing wild in Croatia. All tested Juniperus extracts were rich in polyphenols (3.0-3.9%). They possessed DPPH (IC50: 3-6 μg/mL) and nitric oxide (IC50: 115-186 μg/mL) free radical scavenging activities, ferric reducing properties (IC50: 14-22 μg/mL) and ability to inhibit lipid peroxidation (IC50: 51-60 μg/mL). The most potent inhibitory activity against AChE showed J. phoenicea and J. oxycedrus (IC50: 42 μg/mL and 72 μg/mL, respectively). Neuroprotection induced by extract treatment was evaluated in a model of cortical spreading depression described as a potential triggering mechanism in migraine. The extracts of J. communis and J. oxycedrus (800 µg/mL) significantly blunted K+ 60mM-induced decrease of serotonin turnover, evaluated as 5-HIAA/5-HT ratio in isolated rat cortex. The brine shrimp lethality assay showed that Juniperus extracts at the concentrations up to 800 μg/mL have no toxicity. Our results highlighted the potential for further in vivo research in order to confirm the protective effects of Juniperus species in neurological diseases characterized by increased burden of oxidative stress.

Skin aging can be related to the damage of the connective tissue and melanin over production. They may lead a loss of skin elasticity and pigmentation that are major signs of skin aging. Four traditional Chinese formulations were investigated for their anti-elastase and anti-tyrosinase properties. Methanolic extracts of the four traditional preparations Ermiao Wan (EW), Fang Feng Tong Sheng San (FFTSS), Si Miao San (SMS), and BuShenYiShou Capsules (BSYS) were evaluated along with standards by in-vitro enzymatic assays. The IC50 of the elastase inhibition was 0.004, 0.330, 0.028 and 0.830 µg/mL for EW, FFTSS, SMS and BSYS, respectively while the IC50 of the reference quercetin dihydrate was 0.480 µg/mL. On the other hand the IC50 of the tyrosinase inhibitory activity was 0.77, 0.27, 0.98 and 1.68 µg/mL for EW, FFTSS, SMS and BSYS, respectively compared to L(+)-ascorbic acid (IC50 38.89 µg/mL). The most potent formulation among the selected traditional preparations was Ermiao Wan with a 100 fold stronger inhibitory activity of elastase and over a 50 fold higher inhibitory potential of tyrosinase compared to references. This study confirms not only the mode of action for traditional Chinese herbal formulations as anti-aging medicines but also proves that the treatment with extracts can be more effective as a single compound treatment. It would potentially establish new alternative therapeutics for skin disorders based on traditional medicines.

Traditional Chinese Medicines include a large number of component herbs. Each of the herbal constituents contains many compounds that may even with low concentrations be relevant to the medicinal activity. Some plants are rich in volatile ingredients. Fang Feng Tong Sheng San (FFTSS) consists of 17 components [1]. This reputed TCM formulation is recommended for all wind/heat that is showing signs of internal heat so as not to let pathogens penetrate deeper. This study is carried out to determine the major volatile compounds and their possible role in the effectiveness of FFTSS. The hexane extract of the grinded powder of FFTSS was analyzed by GC-MS with a capillary column (30 m × 0.25 mm × 0.25 µm film thickness). In this study an analytical procedure was developed for optimization and rapid analysis of the major volatile constituents in FFTSS. Optimal analytical conditions: initial temperature 60 °C (held for 1 min), increased to 280 °C at 4 °C min-1, final temperature was held for 15 min.; carrier gas helium at a constant flow rate of 1.2 mL min-1.The temperatures of the interface, ion source and quadrupole were 280, 230 and 150 °C, respectively. Over 50 compounds have been identified ranging from alkanes, ketones to sterols. Seven compounds were considered as the most essential constituents in the FFTSS preparation (menthol, pulegone, butylidene phthalide, ligustilide, methyl linoleate, anthracenedione, isolongifolol). The proposed analytical method and the identified volatile compounds can contribute to the quality control of the FFTSS formula modernization and internationalization of this important preparation.

The ancient Chinese formulation Fang Feng Tong Shen San (FFTSS) consists of 17 components. The purpose of this study was to investigate the anti-inflammatory, antioxidant, anti-diabetic, anti-tyrosinase, anti-neurodegenerative and antimicrobial effects of five commercial preparations of FFTSS and two laboratories constituted mixtures. In-vitro 2, 2-diphenyl-1picrylhydrazyl (DPPH), lipid peroxidation (LPO), α-glycosidase, tyrosinase, acetylcholinesterase, butyrylcholinesterase, elastase inhibitory and antimicrobial assays were carried out in order to determine the active potential of FFTSS. All commercial preparations have shown antioxidant properties after applying DPPH and LPO assays of an average IC50 of 117.68μg/ml and 2.86μg/ml, respectively. In addition, all commercial preparations exhibited a moderate inhibition against tyrosinase of an average IC50 of 0.353mg/ml while the activity were higher in the formula without salts and excipients reaching an IC50 of 0.077mg/ml. The anti-neurodegenerative property of FFTSS was weak while the inhibitory activity of elastase was stronger. The formula was able to inhibit αglucosidase in an average of 60.22%. FFTSS showed stronger effect against Staphylococcus aureus but less antimicrobial activity against Gram-negative bacteria. In summary FFTSS has shown efficient scavenging effects of superoxide and peroxide radicals as well as a strong anti-inflammatory and antidiabetic potential. Therefore the formulation can be considered as a valuable source of a natural antioxidant and anti-inflammatory, anti-diabetic and antiaging agent but also as a candidate in the search for modern pharmaceuticals. INTRODUCTION: Traditional Chinese Medicine (TCM) is not only an ancient medicine but continues to be in use today. Indeed, it is still a major part of healthcare provision in China as it is provided in many state hospitals alongside standard

In Asia, medicinal plants are applied for the treatment of many diseases including diabetes mellitus [1]. In this study Bauhinia malabarica (Roxb.) leaves, Caesalpinia pulcherrima (Linn.) Swartz flowers and leaves and C. coriaria (Jacq.) Willd leaves [Fabaceae] were investigated for their antidiabetic activity. The dried and powdered plant parts were extracted by percolation with solvents of different polarities: n-hexane, ethyl acetate, ethanol and water. The activity of all of the gained extracts at 8 mg/mL was compared to acarbose using an in vitro α-glucosidase inhibitory bioassay. The sample solution and α-glucosidase were mixed with phosphate buffer solution that contained bovine serum albumin and sodium azide. Acarbose 8 mg/ml was used as the positive control, while 5% DMSO was the negative control. The yellow product, p-nitrophenol (pNP) was monitored at 405nm every 30 seconds by micro plate reader. The velocity was determined by the linear relationship equation between absorbance and time [2]. The most significant inhibitory activity was detected from all ethanolic extracts ranging from 89% to 96%, while acarbose resulted in 84% inhibition. The other extracts inhibited the α-glucosidase to a lower extent. This is due to different polarities of the extracted compounds. Further separation of the highly active ethanolic extracts was performed using different chromatographic methods. Isoquercitrin, quercitrin, myricitrin, and luteolin were isolated. The isolated compounds (8 mg/ml) showed α-glucosidase inhibition. Isoquercitrin isolated from B. malabarica and quercitrin from C. pulcherrima leaves showed inhibition of 57% and 71%, respectively. This implies that other ingredients may contribute to the α-glucosidase inhibitory activity. This study confirmed the importance of these investigated medicinal plants for their potential antidiabetic activity.

Traditional Chinese Medicine (TCM) has been playing an important role in health protection and disease control for thousands of years in China. TCM is gaining more and more attention all over the world due to the unique theory and long historical practice. In this study an analytical procedure was developed for optimum and rapid analysis of the major chemical constituents in the Chinese formula Fang Feng Tong Sheng (FFTS). FFTS has been already used in Jin dynasty in the 13th century. This well-known formulation of Chinese Medicine is recommended for all wind/heat that is showing signs of internal heat so as not to let pathogens penetrate deeper. It is also applied for skin disorders, intestinal wind, obesity, and influenza. Recently the formulation is recommended for the treatment of gout. Gout is one of the most painful forms of arthritis. Fang Feng Tong Sheng consists of 17 ingredients; however, the complexity presented is mainly due to the broad range of chemical compounds with various properties. The rational approach to the authentication and quality assessment is essential. Fingerprint techniques are a powerful tool for quality control. The chromatographic separation of the components of FFTS was performed using HPLC-DAD on an Intersil ODS-3 C18 column (4.6 mm × 250 mm, 5 µm) as stationary phase, whilst water with 0.05% acetic acid and acetonitrile were used as mobile phase for a gradient elution. On the basis of the characteristic UV absorption profile, the information of molecular weight, and the structure provided by ESI-MSn, a number of constituents deriving from the aqueous extract was detected and 20 of them were identified in this study. In addition preparations of five different suppliers (capsules, pills) were analysed in comparison. The proposed analytical method contributes substantially to the quality control of Fang Feng Tong Sheng formula.

Nowadays, there is an increasing interest in the use of natural botanical herbal extracts as active ingredients of functional cosmetic and pharmaceutical products. Lavandula x intermedia Emeric ex Loisel. ‘Budrovka’ is an indigenous cultivar of lavandin and, together with the common lavender (L. angustifolia), it has been widely cultivated in Croatia. The present study was undertaken to evaluate and compare the possible inhibitory effects of Lavandula ethanolic extracts on elastase and tyrosinase which are important therapeutic targets. In terms of anti-ageing, finding inhibitors of elastase enzyme can be useful to prevent loss of skin elasticity and wrinkle formation, but also an anti-elastase agent may be useful in the treatment of inflammation. Inhibition of elastase activity by the leaf, flower and inflorescence stalk extracts of Lavandula x intermedia ‘Budrovka’ at 1mg/mL was found to be 100%, 77.7% and 18.6%, respectively, while the inhibitory percentages for L. angustifolia extracts were 100% (leaf), 65.8% (flower) and 15.1% (inflorescence stalk). Investigation of tyrosinase inhibitors may lead to development of novel agents for the treatment of hyperpigmentation as well as skin-whitening agents. In the tyrosinase inhibition assay, flower extracts (1mg/mL) of L. x intermedia ‘Budrovka’ and L. angustifolia were the most effective, exhibiting similar inhibitory effects of 70.4% and 72.9%, respectively, while the leaf and inflorescence stalk extracts exerted lower activity. According to the results of the present in vitro enzyme inhibition studies, flowers and leaves of both Lavandula taxa may be promising sources of active ingredients in dermatological preparations.

Similarities of the morphological and physiological changes to mammalian cells make yeast a simple model system for cellular and perhaps organismic aging [1]. During the study of the chronological aging experiments differences between cells of Saccharomyces cerevisiae wildtype BY4741 treated with the TCM formulation Huang-Lian-Jie-Du-Tang (HLJDT) and its isolated pure main compounds (baicalein, baicalin, berberine, geniposide, jatrorrhizine, wogonin) in comparison to untreated yeast cells were investigated. HLJDT consists of Gardeniae fructus (zhi zi; 1.5 parts), Scutellariae radix (huáng qín; 1 part), Phellodendri cortex (huáng bai; 1 part) and Coptidis rhizoma (huáng lián; 1.5 parts). In preliminary investigations the most effective concentrations of the lyophilized decoction and its main ingredients were figured out. Different liquid media seem to influence the results. A significant life-span extension detected as colony forming units (CFU) could be observed with the decoction over a period of 15 and 18 days, respectively. The pure compounds showed only weak effect on the prolongation of the life-span of the yeast cells. These results might underline the use of the TCM formulation HLJDT to prevent age-related diseases.

In recent years the incidence of Alzheimer's is increasing especially in Western countries caused by the fact that people get older than in former times. Therefore it is important to find new compounds which have a significant effect on this special form of dementia. Therefore in this study 50 plants used in Traditional Mongolian Medicine were tested for their acetylcholinesterase inhibitory effect. MeOH extracts of different plant parts (leaves, flowers, aerial parts, roots) were tested by the modified photometric method of Ellman [1]in vitro. The methanolic extracts of six different plants showed an activity over 25% at a concentration of 1,000µg/ml: Bergenia crassifolia Fritsch. (Saxifragaceae) 44.4%, Carduus crispus L. (Asteraceae) 60.5%, Juniperus sibirica Burgsd. (Juniperaceae) 28.4%, the two Lespedeza species L. dahurica (Laxm.) Schindl. 37.1% and L. hedysaroides (Pall.) Kitag. (Fabaceae) 36.0% and Potentilla viscosa G. Don. (Rosaceae) 26.0%. Further fractionation resulted in higher activities. Purification of the Carduus crispus extract led to an activity up to 90.9% at 1.000µg/ml. The natural resources, especially plants combined with the knowledge of traditional medicine give a pool of potential new drugs.

As people are becoming older different forms of dementia are a major public health concern. After a screening of 50 plants used in the Traditional Mongolian Medicine on their acetylcholinesterase inhibitory effect the MeOH extract of Carduus crispus L. (Welted Thistle; Asteraceae) showed a pronounced activity of 60.5% at a conc of 1.000µg/ml. The biological test was performed by the method of Ellman [1, modif.]. Different plant parts (leaves, flowers, stem, roots) were extracted with solvents of different polarities. The highest activity was found in the MeOH mazerate of the leaves (60.9% at 1.000µg/ml) whereas the traditionally used hot water extracts were not as active (32.4% at 1.000µg/ml). The alcoholic extracts showed stronger effects (MeOHcold> MeOHhot> EtOHhot>> H2Ohot). The differences between leaf, flower and stem extracts have not been significant. Separation of MeOH extracts by polarity differences showed a pronounced activity of the n-butanol fraction (stem 59.2%, flower 66.2%, leaf 81.6% at 1.000µg/ml). Fractions with an activity up to 90.9% at 1.000µg/ml were obtained by the use of SPE. The compounds from these fractions isolated by SPE and RP-HPLC could be identified by spectroscopic methods as chlorogenic acid; neochlorogenic acid; p-coumarylquinic acid and apigenin derivatives. These results may substantiate the traditional use of the investigated plant for the improvement of cognition.

Apoptosis is very important for the development and homeostasis of organisms. Various stimuli e.g. low doses of H2O2 can induce apoptosis in Saccharomyces cerevisiae [1]. S. cerevisiae wildtype BY4741 were used as test organisms. Apoptosis under stress conditions induced by H2O2 or acetate was investigated using the TCM formulation Huang-Lian-Jie-Du-Tang (HLJDT; it consists of Gardeniae fructus, zhi zi; 1.5 parts, Scutellariae radix, huáng qín; 1 part, Phellodendri cortex, huáng bai; 1 part, Coptidis rhizoma, huáng lián; 1.5 parts.) and its main compounds (baicalein, baicalin, berberine, geniposide, jatrorrhizine, wogonin) as substrates. In preliminary investigations the influence of the incubation time as well as the influence of different concentrations of the substrates on the model organism was determined. HLJDT did not show any anti apoptotic effect. After H2O2 treatment baicalein reduced the cell death significantly. Berberine and geniposide prevented the yeast cells from dying under acetate stress conditions. These pure compounds which reduced apoptosis under stress conditions may play an important role in future in the therapy of neurodegenerative diseases.

In Croatia, two indigenous species of the Berberis L. genus, common barberry (B. vulgaris L.) and Croatian barberry (B. croatica Horvat), occur. While common barberry is a well known species growing in many parts of Europe, Croatian barberry is endemic to the illyric-balcanic region (Croatia, Bosnia and Herzegovina, Montenegro, and FYROM) [1]. As a contribution to chemotaxonomy, differences of RP-HPLC chromatograms of B. croatica and B. vulgaris were investigated. Herbal samples were collected at six different locations (three locations per species) in Croatia. Methanolic extracts of roots, twigs, leaves and fruit were analysed with regard to their berberine content and chromatographic fingerprints. The HPLC conditions were: isocratic (TEA-adjusted 0.02 mol L-1H3PO4 (pH 4.82)–acetonitrile (75:25)) and gradient elution (H2O/HCOOH, 9:1 followed by acetonitrile/HCOOH). Signals were detected at 205.4, 254.2 and 330.2nm. Cluster analysis (UPGMA) was employed for evaluation of fingerprints. The average quantity of berberine in extracts of B. vulgaris was higher than in extracts of B. croatica. In extracts of roots it ranged from 7.85 to 12.19% and from 2.44 to 7.29%, for B. vulgaris and B. croatica, respectively. The quantity of berberine in extracts of twigs varied from 0.82 to 1.53% and from 0.48 to 0.63%, for B. vulgaris and B. croatica, respectively. Even though differences in the individual chromatograms of two species could be noted and UPGMA tended to group samples of the same species together, cluster analyses of fingerprints did not reveal important differences between the two species. This finding indicates that common and Croatian barberry are probably very closely related.

Microbiological assays referring to antibiotics were first mentioned in 1955 in the US Pharmacopedia XV and in the Pharmacopedia of India I. In the pharmacopedias two general methods are employed: The first group of methods is grounded on diffusion (disc assay, cylinder-plate and hole-plate assay), the second one is based on the determination of optical density (turbidimetric method). Both methods involve certain problems, particularly with respect to the testing of low-active natural substances. In the course of our investigations those test methods were evaluated and the advantages and disadvantages discussed. The methods were compared to test systems not being described in the pharmacopedias (microdilution test, bioautographic TLC assay). In addition to that we examined spectrophotometrically (OD580nm) the influence of an antimicrobial substance and of a solutizer on the bacterial growth. Tetracycline hydrochloride was used as reference and naringenin as test substance. The results were analyzed with statistical methods cited in the pharmacopedias, e.g. straight-line method and compared to other common methods, e.g. analysis of variance.

Microbiological assays referring to antibiotics were first mentioned in 1955 in the US Pharmacopeia XV and in the Pharmacopeia of India I. In the pharmacopeias two general methods are employed: The first group of methods is grounded on diffusion (disc assay, cylinder-plate and hole-plate assay), the second one is based on the determination of optical density (turbidimetric method). Both methods involve certain problems, particularly with respect to the testing of low-active natural substances. In the course of our investigations those test methods were evaluated and the advantages and disadvantages discussed. The methods were compared to test systems not being described in the pharmacopeias (microdilution test, bioautographic TLC assay). In addition to that we examined spectrophotometrically (OD580nm) the influence of an antimicrobial substance and of a solutizer on the bacterial growth. Tetracycline hydrochloride was used as reference and naringenin as test substance. The results were analyzed with statistical methods cited in the pharmacopeias, e.g. straight-line method and compared to other common methods, e.g. analysis of variance.

The need to examine germ counts of raw materials, of natural or synthetic drugs during manufacture, of final products and of crude herbal drugs brings along an increasing demand for total viable counts in the pharmacopeias. In the present publication a comparison of the viable cell count limits is made between the European Pharmacopeia, the United States Pharmacopeia and pharmacopeias of the following European countries [in alphabetical order: Austria, Croatia (= Slovenian Republic(3))), Czech Republic (= Republic of Slovakia(4))), Germany, Hungary, Italy, Switzerland and USA].

Biofilms are communities of microorganisms that can be harmful and/or beneficial, depending on location and cell content. Since in most cases (such as the formation of biofilms in laboratory/medicinal equipment, water pipes, high humidity-placed structures, and the food packaging machinery) these bacterial and fungal communities are troublesome, researchers in various fields are trying to find a promising strategy to destroy or slow down their formation. In general, anti-biofilm strategies are divided into the plant-based and non-plant categories, with the latter including nanoparticles, bacteriophages, enzymes, surfactants, active peptides and free fatty acids. In most cases, using a single strategy will not be sufficient to eliminate biofilm, and consequently, two or more strategies will inevitably be used to deal with this unwanted phenomenon. According to the analysis of potential biofilm inhibition strategies, the best option for the food industry would be the use of hydrolase enzymes and peptides extracted from natural sources. This article represents a systematic review of the previous efforts made in these directions.

Several QSPR models for predicting the properties of flavonoid glycosides isolated from Paliurus spina-christi Mill. and of some related flavonoids were described and evaluated. Log P values for all of them were calculated according to the method of Rekker. All investigated flavonoids showed expressive hydrophobicity. Significant correlation between the partition coefficient, log P, and van der Waals volume, V-w (calculated according to the method described by Moriguchi(34) et al.) was obtained. Topological indices used for correlation analysis include the Wiener index, W(G), valence connectivity index, chi(V)(G), Balaban index, J(G), and information-theoretic index, I(G). The best models were obtained with the valence connectivity index for all the flavonoids studied.

Here we report on investigations into the effects of microwave irradiation on secondary plant constituents in vitro and in plant materials. In the in vitro tests from 12 dissolved substances only vincamine was decomposed by microwave energy. Investigations on a greater number of plant organs containg various glycosidic compounds or alkaloids made evident that in most cases microwaves drying does not have a negative influence on the content of pharmacologically active compounds. In some cases microwave dried drugs showed higher contents of the determined constituents than air dried drugs

Plant material is to a different extent contaminated by microorganisms. As DAB 10 demands limiting values for aerobic bacteria, for yeasts and moulds, a reduction of microorganisms of the crude herbal drug is necessary. An effective method is the application of microwave irradiation.The microorganisms isolated and identified on different herbal drugs (leaves. flowers, herbs) were mainly spore-forming aerobic bacteria, Micrococcus luteus, and moulds of the genera Aspergillus and Penicillium. Various parameters such as the use of different microwave powers and the influence of humidity on the total count of microorganisms were tested according to the method described in DAB 10. It can be concluded from the results that microwave treatment allows careful drying with a germ-reducing effect, provided testing takes place under optimized and accurately defined conditions.

Ida, Y., Satoh, Y., Ohtsuka, M., Nagasao, M., Shoji, J. (1994) Phytochemistry 35, 209—215. Roberts, M. F. (1991) in: Biotechnology in Agriculture and Forestry, Vol. 15, Medicinal and Aromatic Plants, Ill, (Bajaj, V. S. P., ed.), Springer-Verlag, Heidelberg, pp. 39—57. 6 Obmoto, T., Tanaka, H., Nikaido, T. (1976) Chem. Pharm. Bull. 24, 1532. Ohmoto, T., Koike, K. (1989) in: The Alkaloids, Academic Press Inc., NewYork, Vol. 36, pp. 135—170. 6 Sheridan, H., Bhandari, P. (1992) Planta Med. 58, 299. wounds (2). Previous reports state that the herb contains flavonoids, mainly quercetin and kaempferol aglycones (3, 4), without giving any further details. Since flavonoids show a diuretic effect too, we investigated the flavonoid pattern. We isolated and identified four flavonol O-glycosides: quercetin 3-O-rhamnoglucoside (rutin), quercetin 3-O-glucoside (isoquercitrin), quercetin 3-O-rhamnoside (quercitrin), and kaempferol 3-O-glucoside (astraglin). Another substance which was obtained in very small amounts could be tentatively identified by hydrolytic, cochromatographic, and standard spectroscopic techniquos. It is supposed to be a quercetin 5-O-glucoside. We could not find any C-glycosyl flavonoids, which confirms the results obtained by Delorme et al. (4).