多胺对白桦悬浮细胞生长和三萜积累的影响

目的 分析外源多胺对白桦悬浮细胞生长和三萜积累的影响。方法 在白桦悬浮细胞的生长末期添加0.1 mmol/L和1.0 mmol/L的腐胺（Put）、精胺（Spm）和亚精胺（Spd）,采用比色法和RT-PCR方法分析白桦三萜量及其合成关键酶羽扇豆醇合酶（LUS）基因的表达。结果 除1.0 mmol/L Spm处理使白桦悬浮细胞的活力和干质量积累降低外,其他处理均提高了白桦悬浮细胞的活力、干质量积累和三萜产量,且随着处理时间的延长,干质量积累和三萜产量呈增加趋势。其中1.0 mmol/L Put在处理的第2天对细胞干质量和三萜产量的促进效应最大,分别比对照增加了38.89%和116.35%。LUS基因的RT-PCR检测结果进一步证实了多胺对白桦三萜积累的促进作用。结论 Put可有效促进白桦悬浮细胞生长和三萜积累。     

白桦总三萜类成分高效液相色谱指纹图谱研究

目的建立白桦总三萜类成分的HPLC指纹图谱分析方法,研究不同采收期白桦药材的质量。方法以Kromasil C18柱（4.6mm×250mm,5μm）为色谱柱,流动相乙腈-水,采用梯度洗脱,流速为1.0ml/min,柱温25℃,检测波长280nm;进样量20μl;检测时间60min,对10批白桦总三萜类成分的HPLC指纹图谱进行研究,并运用相似度评价法对数据进行分析。结果实验标定9个共有峰,不同采收期的白桦指纹图谱相似度较好。结论该方法准确可靠,重复性好,用于白桦的质量评价切实可行;不同采收期白桦总三萜类化学组成相似,其相对比例较稳定。     

Quantification in mass units of Bet v 1, the main allergen of Betula verrucosa pollen, by a monoclonal antibody based-ELISA

Background Fagales pollens are considered among the main agents responsible for allergic diseases in many countries of the northern hemisphere and single major allergens have been shown to be responsible for these responses. Objective To develop a solid phase immunoassay for the quantification of Bet v I, the main allergen from Betula verrucosa (birch), and to assess its suitability for quantitating the equivalent major allergen in other Fagales species as well. Methods The assay is based on the use of two different anti-Bet v 1 monoclonal antibodies which were immobilized on the solid phase and. as a primary standard, affinity purified Bet v 1, the protein content of which was determined by amino acid analysis. Results The ELISA proved to measure less than 0.2 ng/mL of Bet v 1 with a practical range of 0.4–40 ng/mL and could be suitable lo quantify the equivalent major allergen in other Fagales species such as Corylus avellana (hazel), Carpinus betulus (horbeam) and Alnus glutinosa (alder). The method was compared witb quantitative electrophoresis and rocket immuno-electrophoresis for the determination of the allergen content in several Betula verrucosa extracts, and a very good quantitative correlation was found. Likewise, the Bet V 1 content exhibited a good correlation (r = 0.87; P < 0.005) with the allergenic potency values obtained by RAST inhibition. Conclusions The results indicate that the Bet v 1-assay could be useful for standardization purposes in Fagales pollen extracts intended for clinical use.     

白桦树皮挥发油成分的研究

采用气相色谱－质谱联用方法，对白桦树皮挥发油进行了研究，共鉴定出２９种化合物，主要成分为２－氧代丙酸（５３．５４％）。     

Preparation of chromosomes from plant leaf meristems for karyotype analysis and in situ hybridization

A reliable method for preparing metaphase chromosomes from plant leaf tissues is described. The chromosomes are suitable for karyotype analysis and gene mapping by fluorescence in situ hybridisation (FISH). The method is based on enzymatic digestion of young leaf tissues (shoot-tips) after which the resulting protoplasts are treated hypotonically before being dropped onto microscopic slides. Compared to root-tip chromosomes, leaf chromosomes tend to be longer, or less condensed, and hence more karyotypically differentiated. Metaphase index in young leaf tissues is also very high. Metaphase spread consists of evenly and well-distributed chromosomes and this allows accurate counting. The plant used to demonstrate this method is birch (Betula L.), a group of tree species that has extremely small chromosomes. Root-tip chromosomes of these plants are difficult to obtain, as cutting does not produce roots readily. Seedling chromosomes do not represent the same genomic constitution as their mother trees due to introgressive hybridisation. Furthermore, sample collection in the field is convenient and actively growing leaf buds are available throughout the growing season. FISH experiments with these leaf chromosomes also give good results comparable to those obtained with root-tip chromosomes or even better as mapping on long or extended chromosomes has high resolution in general. Mapping of the 16S–28S ribosomal genes on birch leaf chromosomes has been shown to differentiate between birch species and therefore can accurately confirm their interspecific hybrids.     

一氧化碳对白桦悬浮细胞生长和三萜累积的影响

目的分析外源一氧化碳(CO)对白桦悬浮细胞生长和三萜累积的影响。方法在白桦悬浮细胞培养的第8天添加5、10、20、30μmol/L的CO供体高铁血红素处理0.25～8 d,采用高效液相色谱法、比色法和实时荧光定量PCR方法分析白桦三萜含量及其合成关键酶基因的表达。结果除20和30μmol/L CO处理2 d后显著降低白桦悬浮细胞干质量外,CO处理未对白桦悬浮细胞干质量产生显著影响。除30μmol/L CO处理提高pH值和丙二醛含量外,CO处理未对pH值和丙二醛含量产生显著影响。CO处理不同程度地增加了三萜、白桦酯醇和齐墩果酸含量,其中,30μmol/L CO处理1 d时三萜积累量达最大值,为同期对照的1.3倍;10μmol/L CO处理4 d时白桦酯醇含量达最高值,为对照的1.5倍;10μmol/L CO处理2 d时齐墩果酸含量达最高值,为对照的4.2倍。三萜合成关键酶FPPS、LUS、CAS1、CAS2和β-AS基因的实时荧光定量PCR检测结果进一步证实了CO对白桦三萜累积的促进作用。结论 CO处理促进了白桦悬浮细胞中三萜的合成。     

Anti-inflammatory and anti-nociceptive effect of Betula platyphylla var. japonica in human interleukin-1β-stimulated fibroblast-like synoviocytes and in experimental animal models

Ethnopharmacological relevance

Traditional medicine has widely been used Betula platyphylla var. japonica to treat various inflammatory diseases including arthritis.

Aim of the study

To determine the anti-inflammatory, anti-nociceptive, and anti-arthritic effects of Betula platyphylla in interleukin-1β (IL-1β)-stimulated fibroblast-like synoviocytes from human rheumatoid arthritis and in nociceptive and inflammatory animal model.

Materials and methods

The inflammatory mediators such as IL-6, tumor necrosis factor (TNF)-α matrix metalloproteinase (MMP)-1, MMP-13, inducible nitric oxide synthesis (iNOS), nitrites, prostaglandin E₂ (PGE₂) and cyclo-oxygenase 2 (COX-2) activity of Betula platyphylla were tested in IL-1β-stimulated fibroblast-like synoviocytes. Tail withdrawal in response to thermal stimulation in tail flick test or paw flinching and shaking in response to sc hind paw formalin injection was measured 1 h after oral administration of Betula platyphylla. The former was evaluated with a paw pressure test, and the latter was measured using the squeaking score, and paw volume in inflammatory arthritis tests.

Results

Betula platyphylla significantly inhibited proliferation of IL-1β-induced synoviocytes. Betula platyphylla reduced the levels of inflammatory mediators, such as IL-6, TNF-α, MMP-1, MMP13, and PGE₂. In particular, Betula platyphylla significantly inhibited the releases of nitrites and iNOS, as well as release of NFκB, into the nucleus of IL-1β-treated synoviocytes, even at concentrations as low as 1 μg/ml. Oral administrant of Betula platyphylla at 400 mg/kg significantly decreased about 27.8% of tail flick withdrawal and inhibited about the number of paw flinches in both phases 1 and 2 of the formalin test. In the carrageenan-induced acute pain and arthritis model, Betula platyphylla dose dependently reduced the nociceptive threshold and the arthritic symptoms at day 8, respectively, and Betula platyphylla at 400 mg/kg markedly reduced the inflammatory area about 48% in the ankle joints. This capacity of Betula platyphylla at 400 mg/kg was similar to that of the celecoxib-2 inhibitor in carrageenan-induced nociceptive and inflammatory arthritis model.

Conclusions

These results suggest that Betula platyphylla has anti-nociceptive and anti-inflammatory effects in IL-1β-stimulated RA FLS and in an animal model of arthritis. Thus, the use of Betula platyphylla as a pharmaceutical candidate for the treatment of arthritis should be further studied.     

The bark of Betula platyphylla var. japonica inhibits the development of atopic dermatitis-like skin lesions in NC/Nga mice

The bark of Betula platyphylla Sukat. var. japonica Hara (Asian White Birch, AWB) is one of the herbs used in Eastern countries for the treatment of various inflammatory diseases including atopic dermatitis (AD). The present study was performed to examine if and how the bark of AWB inhibits the development of AD-like skin lesions in NC/Nga mice induced by repeated application of picryl chloride (PC). With this aim, we examined the skin symptom severity, itching behavior, serum immunoglobulin (Ig) E level and mRNA expression of cytokines at iliac and cervical lymph nodes in the mice. Oral administration of AWB extracts (25, 100 and 250 mg/kg) to the PC-treated mice inhibited the development of AD-like skin lesions as exemplified by a significant decrease in the total skin severity scores, itching behavior and a decrease in hypertrophy and infiltration of inflammatory cells into dermis. The serum IgE level was also significantly reduced by AWB extract. In the RT-PCR results, the expression of interleukin-4 mRNA was reduced by AWB extract, whereas the expression of interferon-gamma mRNA was not changed. These results suggest that AWB inhibits the development of AD-like skin lesions in NC/Nga mice through the suppression of the T-helper 2 cell response.     

Xanthine oxidase inhibitory properties of Czech medicinal plants

Aim of the study

To investigate in vitro xanthine oxidase inhibitory properties of plants traditionally used in Czech Republic and Central-East Europe region for gout, arthritis or rheumatism treatment.

Materials and methods

Methylene chloride-methanolic and two ethanolic extracts of 27 plant species were screened for in vitro xanthine oxidase inhibitory activity using a spectrophotometric method.

Results

Around 50% of the species exhibited some degree of xanthine oxidase inhibitory properties at 200 μg/mL, showing a moderate correlation (r = 0.59) with total phenol content. The most active were methylene chloride-methanolic extracts of Populus nigra and Betula pendula, with IC₅₀ of 8.3 and 25.9 μg/mL, respectively, followed by 80% ethanolic extract of Caryophyllus aromaticus and Hypericum perforatum, both under 50 μg/mL.

Conclusions

Populus nigra and Betula pendula were identified as species with the highest xanthine oxidase inhibitory potential in our study. This correlates with the ethnobotanical data on their use in Central European folklore and provides the basis for further investigation on these plants.