Chemical composition and cardiotropic activity of Ziziphora clinopodioides subsp. bungeana (Juz.) Rech.f.

ETHNOPHARMACOLOGICAL RELEVANCE: Ziziphora clinopodioides subsp. bungeana (Juz.) Rech.f. is a subshrub that is widely distributed in China, Kazakhstan, Kyrgyzstan, Mongolia, Russia, Tajikistan, Turkmenistan, and Uzbekistan. The species is used in traditional medicine for the relief of symptoms connected to cardiovascular diseases like coronary heart disease or hypertension. AIM OF THE STUDY: was to validate traditional use of Z. clinopodioides subsp. bungeana for the treatment of coronary hearth diseases using in vivo models and to find active compounds responsible for the activity. MATERIALS AND METHODS: Multiple extracts were obtained from the aerial parts of Z. clinopodioides subsp. bungeana using maceration, liquid-liquid extraction, CO2 extraction and ultrasound-assisted extraction. Preliminary screening studies for the evaluation of the efficacy of Z. clinopodioides subsp. bungeana extracts on the model of hemic hypoxia were performed. The most effective samples were selected and included in the main study. Stage 2 of the study evaluated the cardiotropic activity of the selected extracts on a model of chronic heart failure. Preparations were administered to animals intragastrically once a day for 28 days. For the isolation of individual compounds plant material was extracted with 96% ethanol. The obtained crude extract was sequentially extracted with n-hexane and dichloromethane and separated by chromatography on a Diaion HP-20 column. The obtained fractions were further subjected to Sephadex LH-20 column chromatography and eluted isocratically with 96% ethanol (EtOH) to yield subfractions, which were further separated by preparative HPLC to obtain 13 individual compounds. RESULTS: Extracts obtained from Ziziphora clinopodioides subsp. bungeana (Juz.) Rech.f. herb were subjected to pharmacological screening for the evaluation of their efficacy on hemic hypoxia. Based on the obtained results, out of the sixteen tested extracts two (AR and US 60%) were selected for further evaluation of their cardiotropic activity. Modeling of chronic heart failure was carried out in accordance with the following stages: 1) anesthesia with chloral hydrate at a dose of 450 mg/kg, intraperitoneally, 2) artificial ventilation of the lungs, 3) thoracotomy, 4) modeling of permanent ischemic or ischemic-reperfusion damage. Both extracts effected the indicators of contraction and output, comparable to the reference drug - Monopril. Based on the extraction methods used to obtain RAF and US60 and data from the literature, it can be assumed that they contain compounds with medium polarity, including polyphenols and terpenoids. At the next stage three previously undescribed monoterpenoid derivatives - Ziziphoric acid (1), Ziziphoroside D (2) and 6'-malonylziziphoroside A (3), along with two previously described megastigmane glucosides - blumenol C glucoside (4), blumenol C 9-O-(6'-O-malonyl-beta-D-glucopyranoside (5) and two previously described monoterpenoids 7a-hydroxymintlactone (6), 7-hydroxypiperitone (7) together with six polyphenols - pinocembrine-7-O-rutinoside (8), chrysine-7-O-rutinoside (9), acacetin-7-O-rutinoside (10), luteolin-7-O-rutinoside (11), rutin (12) and rosmarinic acid (13) were isolated from Z. clinopodioides subsp. bungeana extracts. CONCLUSION: Our results support the traditional use of Z. clinopodioides subsp. bungeana for the treatment of coronary diseases. As a result of Z. clinopodioides subsp. bungeana extracts screening in vivo, two extracts were selected as potential cardiotropic agents. Phytochemical analysis of the plant material led to the isolation of five terpenoid derivatives, two megastigmane glycosides, five flavonoids and one cinnamic acid derivative, which could be responsible for the reported biological activity. Future experiments are required to understand the mechanisms of action for the isolated compounds.

[Cardioprotective effect of new functional food containing salmon oil with motherwort oil extract].

Combination of fish oil with herbal oil extract can extend functional properties of the products. A new functional food of salmon oil with oil extract of motherwort (8:2) was developed and studied. Qualitative profiling was evidenced about presence of flavonoids and iridoids. Quantification of vitamins A (210±3 IU/g), Е (2.8±0.1 mg/g), D3 (12.2±0.4 IU/g) and PUFA (43.3±0.4%, incl. 9.4±0.1% of omega-3 and 33.9±0.1% of omega-6) was done by modern methods. The developed product was found nontoxic (LD50>15 000 mg/kg b.w., intragastriсally, LD50>3000 mg/kg b.w., intraperitoneal). The 14 days intragastric administration of product to the rats (initial b.w. 200-250 g) at the doses of 2340 and 1170 mg/kg b.w. significantly increased the left ventricular pressure after ischemia comparing with control, and normalized the contraction and relaxation of the left ventricle. Biochemical data confirmed the decrease of aspartate amino transferase and creatine kinase activity in rats treated at all experimental doses. Application of fish oil combination with motherwort oil extract is promising for development of new functional foods with cardio protective properties.

[EXPERIMENTAL STUDY OF PHARMACOKINETICS OF KETOPROFEN NANOSYSTEM FOR INHALATION ADMINISTRATION.]

Pharmacokinetics of ketoprofen nanosystem was studied in outbred rats after single endotracheal administration. Ketoprofen was analyzed in blood serum and tissues by HPLC with UV detection. After endotracheal administration of the nanosystem, ketoprofen rapidly appears in systemic blood flow and its concentration in blood serum reaches maximum after 15 min. The maximum drug concentration in tissues was observed near the site of introduction, namely, in lungs Ketoprofen showed moderate penetration in tissues with high vascularization, and weak penetration in tissues with moderate vascularization. After endotracheal administration of the nanosystem, only small amount of ketoprofen overcomes the blood brain barrier.

DEVELOPMENT OF THE METHOD FOR IDENTIFICATION AND GENOTYPING OF THE BACTERIA PASTEURELLA MULTOCIDA AND MANNHEIMIA HAEMOLYTICA ON THE BASIS OF THE PCR AND PHYLOGENETIC ANALYSIS OF BACTERIAL CULTURES ISOLATED FROM CATTLE.

The results of development of a method for detection and genotyping of the bacteria Pasteurella multocida capsular five groups and Mannheimia haemolytica Al based on the multiplex polymerase chain reaction (PCR) with electrophoretic detection are submitted. Diagnostic sensitivity of the developed method was 103 CFU/ml in the study of the pure cultures and 105 CFU/g in the study of biological material. A study of 260 samples of biological material from infected animals revealed Pasteurella multocida in 50.0%, and Mannheimia haemolytica in 11.2% of the investigated samples. Circulation among the tested livestock of capsular groups B and E of Pasteurella multocida was not revealed. The majority of the tested samples contained group A, in some cases, group D, and, in one case, group F. On the basis of the phylogenetic analysis circulation of two different genetic types of Pasteurella multocida of the capsular group A was revealed.

Comparative analysis of requirements for quality of fish oil for food and medical applications.

Fish oil is reach natural source of vitamins А, Е, D and polyunsaturated fatty acids (PUFA) which are useful for supplementation, prophylaxis and therapy in food industry, medicine and veterinary. The analysis of regulatory documents for the quality of fish oil (based on concentration of main nutrients) leads to conclusion that content of vitamins A and D, and PUFA is controlled in medicinal preparations. With regard to fish oil for food application there is no unified approach. In particularly, content of vitamin D in food supplements with fish oil available on Russian market is not indicated by manufacturer or its concentration varies in 3-10 folds. Because of important biological functions and pharmacological activity, it is very important to have uniform standardization for vitamins and PUFA in fish oil for application in food industry and medicine. Development and unification of analytical methods became special attention for these reasons.

[A prototype of oligonucleotide microarray for detection of pathogens relating to arena- and Filoviridae families].

A prototype of oligonucleotide microarray for detection of Lassa, Junin, Machupo, Guanarito viruses (Arenaviridae family), Ebola and Marburg viruses (Filoviridae family) was presented. An original approach founded on virus proteins (nucleocapsid protein for Junin, Guanarito, Machupo viruses and RNA-dependent RNA-polymerase for Lassa, Ebola and Marburg viruses) amino acid sequences analysis with subsequent transform of revealed unique peptides into due sets of oligonucleotides was used to design probes for hybridization and primers.

Imported cases of dengue fever in Russia during 2010-2013.

OBJECTIVE: To confirm dengue infection among Russian tourists returned from Southeast and Mexico in 2010-2013 with clinical signs of infection. METHODS: Blood and serum samples from patients were collected. NS1 antigen and human IgM/IgG antibodies to dengue virus were identified using commercial tests manufactured by "Standard Diagnostics, INC.", Korea. ELISA test was used for the quantitative analyses of human IgM/IgG antibodies to dengue virus ("Orgenics Ltd.", Israel). Viral RNA was detected using commercial real-time PCR tests manufactured by "Genome Diagnostics Pvt. Ltd.", India and "Vector", Russia. Genotypes of revealed dengue viruses were determined employing nucleotide sequencing and phylogenetic analysis of 5'-UTR of the viral genome. RESULTS: A total of 98 collected blood samples were analyzed. Fifty samples were positive for at least one of four markers of dengue infection. IgM to dengue virus were revealed in 38 samples, in 25 samples IgM were combined with IgG. NS1 antigen was detected in 43 samples. 22 serum samples were positive for dengue virus RNA. The majority of samples (12 patients) from tourists returned from Thailand were positive for genotype 1 of dengue virus, 2nd and 4th genotype were identified each in 1 patient. CONCLUSIONS: Due to laboratory confirmed cases of imported dengue fever in Russia, the differential diagnosis of dengue is strictly recommended for tourists returning from endemic areas.

[Effect of a complex preparation of sea urchin shells on blood glucose level and oxidative stress parameters in type II diabetes model].

The experimental preclinical study on mice showed that a complex of polyhydroxylated naphthoquinone pigments and minerals from shells of sea urchins decreases the concentration of glucose, stimulates the synthesis of phospholipids in liver, and has antioxidant properties. On the model of streptozotocin-nicotinamide-induced type II diabetes, this complex of polyhydroxylated naphthoquinone pigments and minerals from sea urchin shells exhibited activity after administration for 10 days in a dose of 1.8 mg/kg.

Development and verification of real-time PCR assay for identification of viral agents causing acute respiratory infections in human beings.

A multiplex polymerase chain reaction (PCR) for identification of four viruses causing acute respiratory diseases in human beings was developed. The analytical sensitivity of developed RT-PCR for identification of adenovirus, respiratory-syncytial virus, flu viruses types A and B, and actual subtypes of type A flu virus (seasonal and pandemic variants H1N1, seasonal H3N2, and viruses of bird flu that are pathogenic to human beings H5 and H7) was 1 × 103 genome equivalents per milliliter. Diagnostic sensitivity for flu virus type A and B, and also subtypes H1 (seasonal H1N1, pandemic variant of H1N1 of year 2009), H3, H5 was 1 × 103-104 viral particles per milliliter. The method developed has high specificity and does not have positive signal in experiments with DNA/cDNA of human beings and viral DNA. We have studied 50 samples using the developed set. Etiology was defined in 33 samples.

Infection of chickens caused by avian influenza virus A/H5N1 delivered by aerosol and other routes.

This study presents results of the study of infectivity of avian influenza virus (AIV) A subtype H5N1 strains isolated from agricultural birds across the territory of the Russian Federation and CIS countries. The results of the susceptibility of chickens to the AIV isolates delivered by the aerosol route and the dissemination of the virus in the organs of infected birds are presented. As was observed, the sensitivity of birds to AIV by the aerosol route of infection is 30 times higher than by intranasal route, 500 times higher than by the oral route and 10000 times higher than by the intragastric route of infection, which is indicative of higher permissivity of respiratory organs to AIV. The highest titres of AIV A subtype H5N1(A/Chicken/Kurgan/05/2005 strain) in aerosol-infected chickens were found in nasal cavity mucosa, lungs, cloaca, serum and kidney, where viable virus accumulation was detected by 18h post-infection (p.i.). The highest virus titres were observed 54h p.i. in lungs, serum and kidney, reaching the value of 8.16 lg EID50 /g(ml) in the lungs. The results showed that birds infected by the aerosol route developed higher titres of virus than those infected by other routes.

[Development and validation of the real-time PCR assay for the identification of viral agents causing acute respiratory infections in humans].

The real time PCR assay targeting influenza A and B virus, 5 subtypes of influenza A virus (seasonal H1N1, pandemic H1N1 (2009), seasonal H3N2, pathogenic for human subtypes of avian influenza H5 and H7), respiratory syncytial virus, and adenovirus was developed. The analytical sensitivity of the developed assay was 1 x 10(3) genome equivalents per ml. The diagnostic sensitivity of the method was 1 x l0(3)-10(4) viral particles per ml. Experiments with human DNA/cDNA and viral cDNA showed a markedly high diagnostic specificity of the developed PCR assay. In the assay of the developed PCR test, 50 nasopharyngeal swab specimens were tested. The etiology was identified in 33 samples.

[Studies of properties of pandemic influenza virus A/H1N1 circulated in Russian Federation in 2009 - 2010].

AIM: Studies of cultural, virologic, antigenic properties of 89 samples of pandemic influenza A(H1N1) 2009 virus isolated in Russian Federation from May 2009 to March 2010. MATERIALS AND METHODS: Properties of isolated samples were compared with those of the reference strain A/ California/04/2009 (H1N1). RESULTS: Studies of biological properties and analysis of genome nucleotide sequences of the isolated samples showed that those strains are closely related to the reference strain. CONCLUSION: Monitoring of genetic, virologic and antigenic properties of pandemic influenza A(H1N1) 2009 virus isolates carried out from May 2009 to March 2010 did not reveal significant changes in the abovementioned properties of the virus or emergence of mutations that can lead to such changes.

[Genetic variability of isolates of pandemic influenza A virus H1N1 isolated in Russia in 2009].

Complete nucleotide sequence of the genome segments encoding the surface glycoproteins, hemagglutinin, and neuraminidase of influenza A virus H1N1 derived from the patients with influenza in the context of pandemic (H1N1) 2009 was determined out of 14 isolates of pandemic influenza. The philogenetic analysis of these sequences demonstrated their genetic similarity to the corresponding genes of the pandemic influenza virus A (H1N1) 2009 isolates obtained in other countries; each gene homology was greater than 99%. Neuraminidase mutations causing virus resistance to oseltamivir and other neuraminidase inhibitors, known from the literature, were not detected. The hemagglutinin gene mutation D222G was found in 4 isolates from autopsy material. In the hemagglutinin of pandemic A/Salekhard/01/2009(H1N1) isolate a mutation G155E leading to the increase in viral replication in developing chick embryos was detected. The nature and frequency of nucleotides substitutions within HA and NA genes were determined in the current research.

[Pharmacokinetics of hopantenic acid upon peroral administration].

A simple, specific and sensitive RP-HPLC method with UV detection for the determination of hopantenic acid in human blood plasma has been developed. The pharmacokinetics of drug pantocalcin upon single peroral administration was investigated on 18 healthy volunteers. The peak of hopantenic acid in blood plasma was achieved at 1.56 h and the elimination half life was 6.68 h. No hopantenic acid in blood plasma is found in 48 h.

Anti-inflammatory activity of a HPLC-fingerprinted aqueous infusion of aerial part of Bidens tripartita L.

The anti-inflammatory potential of three doses of an aqueous infusion of aerial parts Bidens tripartita L. against carrageenan-induced acute paw edema in rats was investigated. A phytochemical study and qualitative-quantitative analyses revealed the presence of flavonoids, tannins, polysaccharides, phenols, amino acids, ascorbic acid, organic acids and polyacetylenes. Infusion doses of 20ml/kgbody wt. exhibited significant anti-inflammatory activity in rats, as compared with indomethacin. In addition, the infusion showed analgesic properties in a hot-plate test and antipyretic properties in carrageenan-induced local hyperthermia, both in rats. The effects were dose-dependent. Our results provide evidence for the potential usefulness of B. tripartita infusion in the treatment of inflammatory disorders.

[Phytochemical analysis of Hippophae rhamnoides L. fruits from Russian varieties cultivated in Finland and food products containing them].

Results of phytochemical analysis of the berries from six Russian Hippophae rhamnnoides L. varieties (Augustinka, Botanitseskaja, Ljubitelskaja, Vorobjvskaja, Pertsik, Trofimovskaja), cultivated in Finland, are presented. The contents of ascorbic acid (vitamin C), tocopherols (vitamin E), total flavonoid (vitamin P), total carotenoid (vitamin A) and total organic acids were analyzed in unfreezed berries. The same parameters were defined in food products (jelly, sweet sauce and compote) containing fruits of three Hippophae rhamnoides L. varieties (Botanitseskaja, Ljubitelskaja and Trofimovskaja). According to the results, Russian Hippophae rhamnoides L. varieties can be cultivated in South-Finland and different kinds of food products with high quality and concentration of biologically active compounds may be prepared from these berries.

[Investigation of therapeutic and protective effect of Reaferon-ES Lipint on mice infected with avian influenza virus (subtype H5N1)].

The efficacy of the therapeutic, prophylactic and urgent prophylactic schemes for the use of Reaferon-ES lipint, a liposomal human recombinant alpha-interferon for oral use, was studied on mice infected with the avian influenza virus. Strain A/Chicken/Kurgan /05/2005 (subtype H5N1) of the avian influenza virus showed high virulence with respect to mice ICR. Theoretically-based calculations allowed to design an optimal therapeutic and prophylactic dose of the drug for the mice (1000 units/animal). It was observed that only after prophylactic use of Reaferon-ES lipint it was effective in protection of the mice infected with 10 LD50 of the avian influenza virus (A/Chicken/Kurgan/05/2005, H5N1). The protection coefficient was 0.35. Under such conditions the drug efficacy was comparable with that of Tamiflu. Therefore, Reaferon-ES lipint could be recommended for prophylaxis of the infection due not only to the season strains of the influenza virus, but also to the strains of the avian influenza virus.

[Light-absorbing and antiradical properties of a product with lutein and zeaxanthin in vitro and kinetics of carotinoids at single oral administration on rats].

Light-absorbing and antiradical properties of the new product on a basis of lutein and zeaxanthin for correction of eye diseases in model system of initiated oxidation of isopropylbenzene were investigated. It is shown, that the product is the effective light-absorbing agent and inhibitor of free-radical oxidation in vitro. In experiments on animals (rat) the pharmacokinetics of the product was investigated at single oral administration. A simple, specific and sensitive RP-HPLC method for the determination of lutein in rat plasma was developed, which was applied to pharmacokinetic investigation in rats after oral administration of lutein at dose 20 mg/kg. It was established, that the peak plasma levels was achieved to 2 hour and the mean elimination half life was 2,4 hours.

[Experimental and clinical study of preparation Iridol action on the central nevros system].

In experiment on adult rats male the anxiolitic action of new plant origin drug Iridol, enriched with compounds iridoids, is shown. This effect of Iridol was approximately the same as by diazepam. In patients with arterial hypertension I - II degree, accompanied by psychoemotional disturbances, the use of Iridol in complex treatment increases efficacy of therapy.