Cultivable endophytic fungal community associated with the karst endemic plant Nervilia fordii and their antimicrobial activity.

Endophytic fungi from medicinal plants with specific pharmacological functions attract much attention to provide the possibility of discovering valuable natural drugs with novel structures and biological activities. Nervilia fordii is a rare and endangered karst endemic plant that is used as medicine and food homology in Guangxi, China. These plants have been reported to have antimicrobial, antitumor, antiviral, and anti-inflammatory activities. However, few studies have focused on the diversity and antibacterial activity of endophytic fungi from N. fordii. In the present study, 184 endophytic fungi were isolated from the healthy tissues of N. fordii, and their molecular diversity and antimicrobial activities were analyzed for the first time. These fungi were categorized into 85 different morphotypes based on the morphological characteristics and the similarity between the target sequence and the reference sequence in the GenBank database. With the exception of 18 unidentified fungi, the fungal isolates belonged to at least 2 phyla, 4 classes, 15 orders, 45 known genera, and 45 different species, which showed high abundance, rich diversity, and obvious tissue specificity. All isolates were employed to screen for their antimicrobial activities via the agar diffusion method against Escherichia coli, Staphylococcus aureus, and Candida tropicalis. Among these endophytes, eight strains (9.41%) displayed inhibitory activity against E. coli, 11 strains (12.94%) against S. aureus, and two strains (2.35%) against C. tropicalis, to some extent. In particular, our study showed for the first time that the fungal agar plugs of Penicillium macrosclerotiorum 1151# exhibited promising antibacterial activity against E. coli and S. aureus. Moreover, the ethyl acetate (EA) extract of P. macrosclerotiorum 1151# had antibacterial effects against E. coli and S. aureus with a minimum inhibitory concentration (MIC) of 0.5 mg ml-1. Further research also confirmed that one of the antimicrobial compounds of P. macrosclerotiorum 1151# was methyl chloroacetate and exhibited excellent antibacterial activity against E. coli and S. aureus up to 1.71-fold and 1.13-fold compared with tetracycline (TET) (5 mg ml-1), respectively. Taken together, the present data suggest that various endophytic fungi of N. fordii could be exploited as sources of novel natural antimicrobial agents.

Novel Substituted Thiophenes and Sulf-Polyacetylene Ester from Echinops ritro L.

Three new substituted bithiophenes (1⁻3), and one new sulf-polyacetylene ester, ritroyne A (16) were isolated from the whole plant of Echinops ritro together with twelve known substituted thiophenes. The structures were elucidated on the basis of extensive spectroscopic analysis including 1D and 2D NMR as well as MS. Furthermore, the absolute configuration of ritroyne A (16) was established by computational methods. In bioscreening experiments, four compounds (2, 4, 12, 14) showed similar antibacterial activity against Staphylococcus aureus ATCC 2592 with levofloxacin (8 µg/mL). Five compounds (2, 4, 9, 12, 14) exhibited antibacterial activities against Escherichia coli ATCC 25922, with minimum inhibitory concentration (MIC) values of 32⁻64 µg/mL. Three compounds (2, 4, 12) exhibited antifungal activities against Candida albicans ATCC 2002 with MIC values of 32⁻64 µg/mL. However, compound 16 did not exhibit antimicrobial activities against three microorganisms.

[Comparative study on the infrared fingerprint of Abrus cantoniensis based on the methods of sequential analysis of dual-indexes and cluster analysis].

The methods of sequential analysis of dual-indexes and cluster analysis were utilized to investigate the infrared fingerprints of A. cantoniensis planted in different years and different places in Guangxi, China. The results showed that 6 samples were able to be completely separated only through 13 point smoothing, when the dual-indexes analysis was applied in the present research, and the accurate relationship between these samples could be inspected and expressed by quantitative relationships under 6-dimensional spaces; however, the effect of cluster was bad only through 13 point smoothing of raw spectra, and it was very difficult to find out the regular sequences while the cluster analysis was applied. Furthermore, the 6 samples were able to be completely separated if raw spectra were dealed with 1st derivative after 13 point smoothing, and the clustering effects were more obvious and 6 samples of A. cantoniensis were completely separated. The above two methods could be used to evaluate the quality of Chinese medicinal materials easily when the sample was not excessive quantitatively, but the method of dual-indexes analysis was more difficult than the clustering analysis if the sample size was too large, since a mass of data such as common peak ratio and variation peak ratio of the IR fingerprint spectra were processed and analyzed statistically, while this method could accurately find out the closest relationship between any samples through comparing the quantitative relationships of common peak ratio and variation peak ratio of each sample under 6-dimensional space; the precision of cluster analysis was less than dual-indexes analysis, but it was more convenient than dual-indexes analysis when large sample data were analysed. Finally the above two methods all showed that the chemical composition of the A. cantoniensis was similar in the same cultivated area, but the difference in chemical composition of A. cantoniensis in different years was distinct even they were in the same place.

[Evaluation on germplasm resources of main production area of Artemisia annua in China].

OBJECTIVE: To evaluate and select excellent germplasm resources of Artemisia annua L., providing basic data for data base and breeding of A. annua. METHOD: Seventy-two germplasms of A. annua, which were collected from main production areas, were planted in germplasm resources garden in Jingxi and Nanning under the same conditions. The samples were gathered in the squaring stage. Artemisinin was extracted by supersonic wave and determined by ultraviolet spectrophotometry. RESULT: The leaf's yield and the content of artemisinin in the samples of different germplasms were varied significantly. Effect of environment on artemisinin content was more than that of hereditary factors. The content of artemisinin was different when the same material of A. annua grew in different place. The content of artemisinin in next year's plant was decreasing. CONCLUSIONS: Seven germplasms from south of China have been selected, their content of artemisinin was above 0.9% and calculated yield was more than 2250 kg x hm(-2). The content of artemisinin has been affected by hereditary factors and variation of the growth environment.

[Determination of total flavonoids in Abrus cantoniensis and its dynamic changes].

OBJECTIVE: To develop a method for ultrasonic extraction and determination of total flavonoids in Abrus cantoniensis, and to analyze its dynamic changes. METHOD: The optimized condition of extraction of total flavonoids was studied with orthogonal design. The contents of total flavonoids in different organs and of different growth stages were determined by UV-visible spectrophotometer. RESULT: The ethanol volume and extraction times were the main factors impacting the effects of ultrasonic extraction. The content of total flavonoids in stems were higher than in roots and the lowest in leaves. The dynamic changes of total flavonoids contents in roots and stems of A. cantoniensis were in similar trends. Its total flavonoids content in the two parts of plant increased gradually with the growth and reached the maximum in October, and the content decreased significantly in Feburay of next year. The content of total flavonoids in leaves reached also to the highest value before leaves fell off. CONCLUSION: The optimized extraction method of total flavonoids in Abrus contoniensis was obtained with three times with 80% ethanol at 20 times of volume for 30 min. The results implied that the best yield and quality may be obtained before leaves fall.

[Isolation and biological characteristics of rhizobia strains from Abrus cantoniensis].

OBJECTIVE: The research aimed at studying the biological characteristics of rhizobia isolated from Abrus cantoniensis. METHOD: The rhizobia strains, isolated from different environments in Guangxi, were studied for their growing characters and the generation time. They were also compared for survival capabilities under stresses caused by NaCl, pH, temperature, and different kinds and concentration of antibiotics. RESULT: The strains obtained from A. cantoniensis in subtropical zone produced alkali in YMA medium, the average generation time was 14.8 hours, and thus they belong to slow-growing rhizobia. Rhizobia strains differed greatly in respect to tolerance of high temperature, adaptability of acidic environment and sensitivity to four antibiotics, but they had the same abilities of using different carbon and nitrogen sources. After 70 days from inoculated strains, the seedling formed nodules on the root (85.0%), and the dry matter of vine was increased by 51.1%. CONCLUSION: The rhizobia strains isolated from different ecological environments are good germplasm resources of tolerances to high temperature and acidic environment. The research will greatly help utilize the rhizobia resources and enhance the quality of crude drugs of medicinal leguminosae.

[Effects of different nitrogenous compounds on growth and nodulation of Abrus cantoniensis].

OBJECTIVE: The research aimed at the effects of different nitrogenous compounds on growth and nodulation of Abrus cantoniensis. METHOD: After the seedlings of the herb were inoculated with rhizobia in potted culture, they were supplied with nutrition solutions which contained the three nitrogenous compounds, KNO3, NH4NO3, (NH4)2SO4 of different nitrogen concentration. The growth and nodulation of seedlings was determined after 70 days. RESULT: Different nitrogenous compounds were able to enhance the vegetable growth of seedlings variously. The effect of (NH4)2SO4 and NH4NO3 on growth was better than that of KNO3. Seedlings nodulation was obviously inhibited by these nitrogenous compounds. Their inhibitory effects ranked NH4NO3 > (NH4)2SO4 > KNO3. The treatments of KNO3 and the lower concentration treatments of NH4NO3 and (NH4)2 SO4 didn't inhibit the nodulation of seedlings, but the higher concentration treatment of NH4NO3 and (NH4)2SO4 severely inhibited nodulation or even made a no formation of nodule. CONCLUSION: The results showed that ammonium nitrogen the higher inhibitory ability to the nodulation of seedlings of A. cantoniensis than nitrate nitrogen. Therefore, the application of ammonium nitrogen fertilizer should be controlled in culture of the herb, which is in favor of increasing the function of biological nitrogen fixation and the quality of the medicinal materials of A. cantoniensis.