Extracts from Leonurus sibiricus L. increase insulin secretion and proliferation of rat INS-1E insulinoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Mongolian medicine (TMM) uses preparations from herbs as one form of medication for the treatment of a diversity of diseases including diabetes mellitus (DM). We evaluated the effect of extracts from the plant Leonurus sibiricus L. (LS), used in TMM to treat typical symptoms of type 2 DM, on insulin secretion, electrophysiological properties, intracellular calcium concentration and cell proliferation of INS-1E insulinoma cells under standard cell culture conditions (SCC; 11.1mM glucose). MATERIALS AND METHODS: Insulin secretion was measured by ELISA, electrical properties were assessed by whole cell patch clamping, intracellular calcium concentration (Cai) by Fluo-4 time lapse imaging, insulin receptor expression was verified by RT-PCR and cell proliferation assessed by CellTiter-Glo® cell viability assay. RESULTS: Insulin released from INS-1E cells into the culture medium over 24h was significantly increased in presence of 500 mg/L aqueous LS extract (LS OWE) as well as methanolic LS extract (LS MeOH/H2O) but not in the presence of the butanol-soluble extract (LS MeOH/BuOH). Acute application of LS OWE resulted in a depolarization of the cell membrane potential paralleled by an initial increase and subsequent decline and silencing of action potential frequency, by KATP channel inhibition, persisting depolarization and an increase in Cai. The electrophysiological effects were comparable to those of 100 µM tolbutamide, which, however failed to elevate insulin secretion under SCC. Furthermore all LS extracts stimulated INS-1E cell proliferation. CONCLUSIONS: The finding that extracts from Leonurus sibiricus L. enhance insulin secretion and/or foster cell proliferation may provide possible explanations for the underlying therapeutic principles in the empirical use of LS-containing formulations in DM and DM-related disorders as applied in TMM.

Yarrow (Achillea millefolium L. s.l.): pharmaceutical quality of commercial samples.

Yarrow (Achillea millefolium L. s.l.) is traditionally used against inflammatory and spasmodic gastrointestinal complaints, hepato-biliary disorders, as an appetite enhancing drug, against skin inflammations and for wound healing due to its antiphlogistic, choleretic and spasmolytic properties. The main pharmacologically active principles were shown to be the essential oil (antimicrobial), proazulenes and other sesquiterpene lactones (antiphlogistic), dicaffeoylquinic acids (choleretic) and flavonoids (antispasmodic). In order to assess the pharmaceutical quality of the drug we evaluated the content of these bioactive compounds in 40 commercial drug samples. The essential oil and the proazulenes were analysed according to the European Pharmacopoeia, whereas the content of dicaffeoylquinic acids and flavonoids was determined by solid phase extraction (SPE)-HPLC. This comprehensive survey revealed that the quality of the drug material was very heterogenous, and only 50% of the samples met the standards of the European Pharmacopoeia. Moreover, this study gives information about the content of phenolic compounds in the drug and allowed to establish tentative reference values which may be used as additional parameters in the quality control of the drug.

TLC and HPLC characteristics of desacetylmatricarin, leucodin, achillin and their 8alpha-angeloxy-derivatives.

Five guaianolides, including two pairs of isomers, from a Hungarian taxon of the Achillea millefolium group were characterized analytically. Different chromatographic systems on TLC and HPLC were developed for the analysis of these compounds. TLC of leucodin, 8alpha-angeloxy-leucodin, achillin, 8alpha-angeloxy-achillin and desacetylmatricarin was performed on silica gel using dichloromethaneacetone and cyclohexane-ethylacetate mixtures as mobile phases. HPLC on stationary phases as LiChrospher RP2, LiChrospher RP8, LiChrospher RP18e, Hypersil BDS C18 and Aquasil C18 required isocratic and gradient systems with different methanol-water mixtures as mobile phases. The presented RF values and retention times allow the identification of the respective 2-oxo-guaianolides which are marker substances for certain non-proazulene containing species. Their TLC and HPLC fingerprints are compared to those of proazulene containing species and are relevant for quality control.

[Acid oligosaccharides as the active principle of aqueous carrot extracts for prevention and therapy of gastrointestinal infections]. / Saure Oligosaccharide als Wirkprinzip von wässrigen Zubereitungen aus der Karotte in der Prophylaxe und Therapie von gastrointestinalen Infektionen.

Adherence of microorganisms to the intestinal mucosa is an important and initial step in the pathogenesis of gastrointestinal infections and mediated by carbohydrate structures on the cell surface. Adherence can be blocked by carbohydrate receptor analogues. Aqueous extracts from carrots (carrot soup) contain acidic oligosaccharides, which are able to block adherence of various enteropathogenic microorganisms to HEp-2 cells and human intestinal mucosa in vitro. Dependent on the grade of polymerisation the most potent blocking ability was seen for trigalacturonic acid. Clinical studies revealed, that aqueous carrot extracts are significantly superior to the basic glucose-electrolyt-solution for oral rehydration in acute gastrointestional infections of children.

Highly hydroxylated guaianolides of Achillea asiatica and Middle European Achillea species.

From flower heads of Achillea asiatica (L.) Serg., three new guaianolides were isolated by repeated column chromatography and HPLC. The constitution and the stereochemistry of these new, labile compounds were determined by MS, one ((1)H, (13)C, selective (1)H-TOCSY and (1)H-NOESY) and two-dimensional NMR experiments ((1)H, (1)H-COSY, (1)H, (13)C-HSQC, (1)H, (13)C-HMBC). The substances were identified as 8 alpha-angeloxy-2 alpha, 4 alpha,10 beta-trihydroxy-6 beta H,7 alpha H, 11 beta H-1(5)-guaien-12,6 alpha-olide (1), 8 alpha-angeloxy-1 beta,2 beta:4 beta,5 beta-diepoxy-10 beta-hydroxy-6 beta H, 7 alpha H, 11 beta H-12,6 alpha-guaianolide (2) and 8 alpha-angeloxy-4 alpha,10 beta-dihydroxy-2-oxo-6 beta H,7 alpha H, 11 beta H-1(5)-guaien-12,6 alpha-olide (3). They were also detected in Middle European species (Achillea collina, Achillea ceretanica (2x and 4x), Achillea roseoalba, Achillea asplenifolia) by HPLC, TLC and off line MS and have not been described before. The possibility that these compounds might be products of an oxidation process is discussed.

Topical anti-inflammatory activity of a new germacrane derivative from Achillea pannonica.

The topical anti-inflammatory activity of a germacrane derivative [1,4-dihydroxy-germacra-5E-10(14)-diene; DHGD] isolated from Achillea pannonica Scheele (Asteraceae) was investigated employing the Croton oil-induced dermatitis in the mouse ear. Its effects on the oedematous response and on leukocytes infiltration are described. The germacrane derivative significantly inhibited ear oedema in a dose-dependent manner, with an ID(50) of 0.40 micromol/cm(2). DHGD (0.75 micromol/cm(2)) provoked a global inhibition of the oedematous response (61 %) higher than that induced by an equimolar dose of indomethacin (43 %) within 24 hours; the reduction induced by hydrocortisone (0.10 micromol/cm(2)) was 68 %. The effect of DHGD (61 % inhibition) was higher than that of the equimolar dose of indomethacin (51 % inhibition) also on granulocytes recruitment at the site of inflammation. Hydrocortisone (0.10 micromol/cm(2)) reduced the cellular infiltrate by 44 %.

Hereditary non-polyposis colorectal cancer (HNPCC): new germline mutation (190-191 del AA) in the human MLH1 gene and review of clinical guidelines for surveillance of affected families.

Hereditary non-polyposis colorectal cancer (HNPCC) is one of the most common genetic diseases comprising at least 5-6% of all colorectal cancers. It is characterized by early onset and mostly right-sided tumors (proximal to the splenic flexure). Molecular analyses are useful methods for diagnosis in index patients and for the detection of risk persons in affected families. A 37-year-old female patient whose family history fulfilled the criteria for hereditary non-polyposis colorectal cancer (HNPCC) was studied using PCR and DNA sequencing for the detection of mutations in the mismatch repair genes hMSH2 and hMLH1. Additionally, literature was reviewed (MEDLINE research until 2000) concerning clinical guidelines for surveillance in HNPCC families. A new deletion of two adenosine nucleotides (190-191 del AA) at codon 64 in exon 2 of the hMLH1 gene was found. The frameshift led to a stop codon at amino acid position 75. This mutation is considered to be disease causing in the development of the colorectal cancer of this family. Six publications with detailed recommendations for the surveillance of risk persons were found in the literature. Following their guidelines, colonoscopy is recommended from 20-30 years on for members of a family who fulfills either the Amsterdam criteria or the Bethesda criteria in combination with a detection of microsatellite instability. Female risk persons should be investigated gynecologically, including a transvaginal ultrasound examination, from 25-35 years on for the early detection of endometrial or ovarian cancer. Recommendations for gastroscopy, abdominal ultrasound examination and urine analysis are not given in all publications. Genetic counseling is recommended from 18 years on for all members of affected families.

Combination of chromatographic and spectroscopic methods for the isolation and characterization of polar guaianolides from Achillea asiatica.

Four polar guaianolides, 8alpha-angeloxy-2alpha,4alpha, 10beta-trihydroxy-6betaH,7alphaH, 11betaH-1(5)-guaien- 12,6alpha-olide; 8alpha-angeloxy-1beta,2beta:4beta,5beta-diepoxy- 10beta-hydroxy-6betaH,7alphaH,11betaH-12,6alpha-guaianolide; 8alpha-angeloxy-4alpha, 10beta-dihydroxy-2-oxo-6betaH, 7alphaH, 11betaH- 1(5)-guaien- 12,6alpha-olide and 8-desacetyl-matricarin, were isolated from Achillea asiatica and characterized by TLC, MS, IR, HPLC and diode array detection. Purified extracts were separated by means of flash chromatography. HPLC separations were achieved using different methanol-water gradients as mobile phase and LiChrospher 100-RP8 5 microm or Zorbax SB-C8 3.5 microm as stationary phases. The chromatographical data are compared to those of the proazulene 8alpha-tigloxy-artabsin which shows antiinflammatory effects. By means of these characteristics the identification of the guaianolides with potential antiphlogistic properties is also possible from other sources.

Novel germline mutation (300-305delAGTTGA) in the human MSH2 gene in hereditary non-polyposis colorectal cancer (HNPCC).

Hereditary non-polyposis colorectal cancer (HNPCC) is a common hereditary syndrome characterized by the high incidence and early onset of colorectal cancer. The majority of the HNPCC families carry germline mutations in either the MSH2 or the MLH1 mismatch repair gene. A 46 year-old female patient whose family history fulfilled the Amsterdam criteria for HNPCC was diagnosed with undifferentiated adenocarcinoma of the transverse colon. Recognizing the Lynch 2 syndrome (the existance of multiple HNPCC related cancers in a pedigree), we used polymerase chain reaction followed by direct sequencing to screen the coding regions of both the MSH2 and the MLH1 genes for germline mutations in DNA from the patient. We detected a novel germline mutation (300-305delAGTTGA) in exon 2 of human MSH2. We noted microsatellite instability in four microsatellite loci. Immunohistochemistry showed a lack of expression of the MSH2 gene product in the tumor, suggesting that the mutation is a disease-causing mutation.

Qualitative and quantitative determination of sesquiterpenoids in Achillea species by reversed-phase high-performance liquid chromatography, mass-spectrometry and thin-layer chromatography.

A reversed-phase high-performance liquid chromatographic method was developed as a universal analysis system in order to determine and quantify antiphlogistic sesquiterpenoids in different Achillea species. Identification was performed by HPLC and diode array detection as well as by monitoring the HPLC fractions by TLC and MS. Using santonin as internal standard, HPLC separations were achieved with a methanol-water gradient system using RP 8 LiChrospher 100 (5 microm) as stationary phase. For validation, sample analyses were performed, using the two tetraploid species A. collina and A. pratensis. The method allows the identification and quantification of the main compounds achillicin, 8alpha-tigloxy-artabsin, 8alpha-angeloxy-artabsin, arglanin and santamarin with variation coefficients between 3.4 and 4.7% (total content) using santonin as internal standard. For the different compounds recovery was found between 81 and 107% performing multiple analyses of A. collina and A. pratensis.

Guaianolide-endoperoxide and monoterpene-hydroperoxides from Achillea nobilis.

From flower heads of Achillea nobilis two chrysanthemol derivatives (1, 2), tanaparthin-beta-peroxide (3), and 5-hydroxy-3,6,7,4'-tetramethoxyflavone (4) were isolated by repeated column chromatography and HPLC. Structure elucidation has been performed by means of MS, 1H-NMR, 13C-NMR, and 2D-NMR experiments.