Jianglin Zhao

Effects of biotic and abiotic elicitors on cell growth and tanshinone accumulation in Salvia miltiorrhiza cell cultures

This study examined the effects of biotic and abiotic elicitors on the production of diterpenoid tanshinones in Salvia miltiorrhiza cell culture. Four classes of elicitors were tested, heavy metal ions (Co2+, Ag+, Cd2+), polysaccharides (yeast extract and chitosan), plant response-signaling compounds (salicylic acid and methyl jasmonate), and hyperosmotic stress (with sorbitol). Of these, Ag (silver nitrate), Cd (cadmium chloride), and polysaccharide from yeast extract (YE) were most effective to stimulate the tanshinone production, increasing the total tanshinone content of cell by more than ten-fold (2.3xa0mgxa0g-1 versus 0.2xa0mgxa0g-1 in control). The stimulating effect was concentration-dependent, most significant at 25xa0μM of Ag and Cd and 100xa0mgxa0l-1 (carbohydrate content) of YE. Of the three tanshinones detected, cryptotanshinone was stimulated most dramatically by about 30-fold and tanshinones I and IIA by no more than 5-fold. Meanwhile, most of the elicitors suppressed cell growth, decreasing the biomass yield by about 50% (5.1–5.5xa0gxa0l-1 versus 8.9xa0gxa0l-1 in control). The elicitors also stimulated the phenylalanine ammonia lyase activity of cells and transient increases in the medium pH and conductivity. The results suggest that the elicitor-stimulated tanshinone accumulation was a stress response of the cells.

Flavonoids from Halostachys caspica and Their Antimicrobial and Antioxidant Activities

Seven flavonoids have been isolated from the aerial parts of Halostachys caspica C. A. Mey. (Chenopodiaceae) for the first time. By means of physicochemical and spectrometric analysis, they were identified as luteolin (1), chrysin (2), chrysin 7-O-β-D-glucopyranoside (3), quercetin (4), quercetin 3-O-β-D-glucopyranoside (5), isorhamentin-3-O-β-D-glucopyranoside (6), and isorhamentin-3-O-β-D-rutinoside (7). All flavonoids were evaluated to show a broad antimicrobial spectrum of activity on microorganisms including seven bacterial and one fungal species as well as pronounced antioxidant activity. Among them, the aglycones with relatively low polarity had stronger bioactivity than their glycosides. The results suggested that the isolated flavonoids could be used for future development of antimicrobial and antioxidant agents, and also provided additional data for supporting the use of H. caspica as forage.

Chemical analysis and biological activity of the essential oils of two valerianaceous species from China: Nardostachys chinensis and Valeriana officinalis.

In order to investigate essential oils with biological activity from local wild plants, two valerianaceous species, Nardostachys chinensis and Valeriana officinalis, were screened for their antimicrobial and antioxidant activity. The essential oils were obtained from the roots and rhizomes of the two plants by hydro-distillation, and were analyzed for their chemical composition by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Calarene (25.31%), aristolone (13.35%), α-selinene (7.32%) and β-maaliene (6.70%) were the major compounds of the 23 identified components which accounted for 92.76% of the total oil of N. chinensis. Patchoulol (16.75%), α-pinene (14.81%), and β-humulene (8.19%) were the major compounds among the 20 identified components, which accounted for 88.11% of the total oil of V. officinalis. Both oils were rich in sesquiterpene hydrocarbons as well as their oxygenated derivatives. Essential oils were shown to have broad spectrum antibacterial activity with MIC values that ranged from 62.5 μg/mL to 400 μg/mL, and IC50 values from 36.93 μg/mL to 374.72 μg/mL. The oils were also shown to have moderate antifungal activity to Candida albicans growth as well as inhibition of spore germination of Magnaporthe oryzae. Two essential oils were assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging, β-carotene bleaching and ferrozine-ferrous ions assays, respectively, to show moderate antioxidant activity. Results suggest that the isolated essential oils could be used for future development of antimicrobial and antioxidant agents.

Antifungal activity of the essential oil of Illicium verum fruit and its main component trans-anethole.

In order to identify natural products for plant disease control, the essential oil of star anise (Illicium verum Hook. f.) fruit was investigated for its antifungal activity on plant pathogenic fungi. The fruit essential oil obtained by hydro-distillation was analyzed for its chemical composition by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). trans-Anethole (89.5%), 2-(1-cyclopentenyl)-furan (0.9%) and cis-anethole (0.7%) were found to be the main components among 22 identified compounds, which accounted for 94.6% of the total oil. The antifungal activity of the oil and its main component trans-anethole against plant pathogenic fungi were determined. Both the essential oil and trans-anethole exhibited strong inhibitory effect against all test fungi indicating that most of the observed antifungal properties was due to the presence of trans-anethole in the oil, which could be developed as natural fungicides for plant disease control in fruit and vegetable preservation.

Antimicrobial and antioxidant activities of the root bark essential oil of Periploca sepium and its main component 2-hydroxy-4-methoxybenzaldehyde.

The root bark essential oil of Periploca sepium Bunge (Asclepiadaceae/ Apocynaceae) obtained by hydrodistillation was investigated by GC and GC-MS. 2-Hydroxy-4-methoxybenzaldehyde was found to be the main component (78.8% of the total) among 17 identified compounds. 2-Hydroxy-4-methoxybenzaldehyde was separated and purified by preparative thin layer chromatography, and was further identified by means of physicochemical and spectrometric analysis. Both the essential oil and 2-hydroxy-4-methoxybenzaldehyde exhibited antimicrobial activities on all test bacteria and fungi, with MBC/MFC values ranging from 125 μg/mL to 300 μg/mL, MIC values from 80 μg/mL to 300 μg/mL, and IC50 values from 63.29 μg/mL to 167.30 μg/mL. They also showed moderate antioxidant activity in the DPPH free radical scavenging, β-carotene bleaching and ferrozine-ferrous ions assays. The results indicated that the major portion of these antimicrobial and antioxidant activities was due to the presence of 2-hydroxy-4-methoxy-benzaldehyde in the oil, which could potentially be developed as an antimicrobial and antioxidant agent in the future.

Diterpenoid Tanshinones and Phenolic Acids from Cultured Hairy Roots of Salvia miltiorrhiza Bunge and Their Antimicrobial Activities

Four diterpenoid tanshinones and three phenolic acids were isolated from the crude ethanol extract of the cultured hairy roots of Salvia miltiorrhiza Bunge by bioassay-guided fractionation. By means of physicochemical and spectrometric analysis, they were identified as tanshinone ΙΙA (1), tanshinone Ι (2), cryptotanshinone (3), dihydrotanshinone Ι (4), rosmarinic acid (5), caffeic acid (6), and danshensu (7). These compounds were evaluated to show a broad antimicrobial spectrum of activity on test microorganisms including eight bacterial and one fungal species. Among the four tanshinones, cryptotanshinone (3) and dihydrotanshinone Ι (4) exhibited stronger antimicrobial activity than tanshinone ΙΙA (1) and tanshinone Ι (2). The results indicated that the major portion of the antimicrobial activity was due to the presence of tanshinones and phenolic acids in S. miltiorrhiza hairy roots, which could be used as the materials for producing antimicrobial agents for use in agricultural practice in the future.

Preparative Separation of Phenolic Compounds from Halimodendron halodendron by High-Speed Counter-Current Chromatography

Three phenolic compounds, p-hydroxybenzoic acid (1), isorhamnetin-3-O-β-D-rutinoside (2), and 3,3′-di-O-methylquercetin (5), along with a phenolic mixture were successfully separated from the ethyl acetate crude extract of Halimodendron halodendron by high-speed counter-current chromatography (HSCCC) with chloroform-methanol-water-acetic acid (4:3:2:0.05, v/v) as the two-phase solvent system. The phenolic mixture from HSCCC was further separated by preparative HPLC and purified by Sephadex LH-20 to afford quercetin (3) and 3-O-methylquercetin (4). Seven hundred mg of ethyl acetate crude extract was separated by HSCCC to obtain six fractions which were then analyzed by high performance liquid chromatography (HPLC). The HSCCC separation obtained total of 80 mg of the mixture of quercetin (3) and 3-O-methylquercetin (4) (26.43% and 71.89%, respectively) in fraction 2, 14 mg of 3,3′-di-O-methylquercetin (5) at 95.14% of purity in fraction 3, 15 mg of p-hydroxybenzoic acid (1) at 92.83% of purity in fraction 5, 12 mg of isorhamnetin-3-O-β-D-rutinoside (2) at 97.99% of purity in fraction 6. This is the first time these phenolic compounds have been obtained from H. halodendron, and their chemical structures identified by means of physicochemical and spectrometric analysis.

Enhancement of Diepoxin ζ Production by Yeast Extract and Its Fractions in Liquid Culture of Berkleasmium-Like Endophytic Fungus Dzf12 from Dioscorea zingiberensis

This study was to examine the effects of yeast extract (YE) and its fractions (YE1 and YE2) on the growth and diepoxin ζ (a spirobisnaphthalene with a diversity of bioactivities) production in liquid culture of Berkleasmium-like endophytic fungus Dzf12 from Dioscorea zingiberensis. When YE was applied to the liquid medium at 10 g/L on day 3 of culture, the diepoxin ζ production was most effectively enhanced 3.2-fold (378.70 mg/L versus 120.09 mg/L in control) after another 10 days culture. Feeding with 15 g/L of YE on day 9, the mycelia biomass reached 16.44 g/L, about 2.3-fold in comparison with the control (7.15 g/L). The polysaccharide fraction (YE1) was mainly responsible for stimulating diepoxin ζ accumulation, and the non-polysaccharide fraction (YE2) was mainly responsible for promoting mycelia growth. The results showed that the diepoxin ζ production in liquid culture of endophyte Dzf12 could be effectively enhanced by YE and its fractions.

Enhancement of diepoxin ζ production in liquid culture of endophytic fungus Berkleasmium sp. Dzf12 by polysaccharides from its host plant Dioscorea zingiberensis

This study is the first report of the enhancement of diepoxin ζ production in liquid culture of the endophytic fungus Berkleasmium sp. Dzf12 by the polysaccharides from its host plant Dioscorea zingiberensis which serve as elicitors. Three polysaccharides, namely water-extracted polysaccharide (WEP), sodium hydroxide-extracted polysaccharide and acid-extracted polysaccharide were sequentially prepared from the rhizomes of D. zingiberensis. Among them, WEP was found to be the most effective elicitor to enhance diepoxin ζ production. When WEP was added to the medium at 400xa0mgxa0l−1 on day 3 of culture, the maximal diepoxin ζ yield (intracellular diepoxin ζ in mycelia plus extracellular diepoxin ζ in medium) of 350.76xa0mgxa0l−1 on day 15 was achieved, which was about 2.69-fold in comparison with that (130.43xa0mgxa0l−1) of the control.

Preparative separation of helvolic acid from the endophytic fungus Pichia guilliermondii Ppf9 by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied for preparative separation of helvolic acid from the crude extract of the endophytic fungus Pichia guilliermondii Ppf9, associated with the medicinal plant Paris polyphylla var. yunnanensis for the first time. The two-phase solvent system consisted of n-hexane–ethyl acetate–methanol–water (4.5:4.5:5.0:5.0, v/v) appending with phosphoric acid (0.2%, v/v) was employed. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature of the apparatus were 800xa0rpm, 3xa0mlxa0min−1 and 25°C, respectively. About 6.8xa0mg of helvolic acid was successfully obtained from 450xa0mg of the crude extract by HSCCC within 4xa0h separation procedure, and its purity reached to 93.2% according to the HPLC analysis. The product was further characterized by MS, 1H-NMR and 13C-NMR spectra.