Lan-qing Ma

Characterization of glycosyltransferases responsible for salidroside biosynthesis in Rhodiola sachalinensis

Salidroside, the 8-O-β-D-glucoside of tyrosol, is a novel adaptogenic drug extracted from the medicinal plant Rhodiola sachalinensis A. Bor. Due to the scarcity of R. sachalinensis and its low yield of salidroside, there is great interest in enhancing production of salidroside by biotechnological manipulations. In this study, two putative UDP-glycosyltransferase (UGT) cDNAs, UGT72B14 and UGT74R1, were isolated from roots and cultured cells of methyl jasmonate (MeJA)-treated R. sachalinensis, respectively. The level of sequence identity between their deduced amino acid sequences was ca. 20%. RNA gel-blot analysis established that UGT72B14 transcripts were more abundant in roots, and UGT74R1 was highly expressed in the calli, but not in roots. Functional analysis indicated that recombinant UGT72B14 had the highest level of activity for salidroside production, and that the catalytic efficiency (Vmax/Km) of UGT72B14 was 620% higher than that of UGT74R1. The salidroside contents of the UGT72B14 and UGT74R1 transgenic hairy root lines of R. sachalinensis were also ∼420% and ∼50% higher than the controls, respectively. UGT72B14 transcripts were mainly detected in roots, and UGT72B14 had the highest level of activity for salidroside production in vitro and in vivo.

A tyrosine decarboxylase catalyzes the initial reaction of the salidroside biosynthesis pathway in Rhodiola sachalinensis.

Salidroside, the 8-O-β-d-glucoside of tyrosol, is the main bioactive component of Rhodiola species and is found mainly in the plant roots. It is well known that glucosylation of tyrosol is the final step in the biosynthesis of salidroside; however, the biosynthetic pathway of tyrosol and its regulation are less well understood. A summary of the results of related studies revealed that the precursor of tyrosol might be tyramine, which is synthesized from tyrosine. In this study, a cDNA clone encoding tyrosine decarboxylase (TyrDC) was isolated from Rhodiola sachalinensis A. Bor using rapid amplification of cDNA ends. The resulting cDNA was designated RsTyrDC. RNA gel–blot analysis revealed that the predominant sites of expression in plants are the roots and high levels of transcripts are also found in callus tissue culture. Functional analysis revealed that tyrosine was best substrate of recombinant RsTyrDC. The over-expression of the sense-RsTyrDC resulted in a marked increase of tyrosol and salidroside content, but the levels of tyrosol and salidroside were 274 and 412%, respectively, lower in the antisense-RsTyrDC transformed lines than those in the controls. The data presented here provide in vitro and in vivo evidence that the RsTyrDC can regulate the tyrosol and salidroside biosynthesis, and the RsTyrDC is most likely to have an important function in the initial reaction of the salidroside biosynthesis pathway in R. sachalinensis.

Componential Analysis and Acaricidal Activities of Stellera chamaejasme Extracts by Supercritical Fluid Extraction

Extracts of Stellera chamaejasme by supercritical fluid extraction (SFE) were shown to have acaricidal activities against Tetranychus cinnabarinus. In these experiments, we extracted the components of Stellera chamaejasme through optimizing the supercritical parameters of CO2 in SFE by uniform experimential design, and analyzed the extracts of Stellera chamaejasme, which were isolated under the optimal supercritical condition, by GC/MS analysis. Our results showed that the optimal condition of supercritical CO2 extraction was extracting pressure 49 MPa, extracting temperature 15 °C, resolution pressure 43 MPa, and resolution temperature 6.25°C. Under the optimal condition, the theoretical extract rate was 3.7731%, and actual measurement extract rate was 3.7534 %. Extracts of Stellera chamaejasme from the optimal SFE condition had contacting and systemic toxicity against Tetranychus cinnabarinus, which were more active than extracts by cold-soaked extraction method, with LC50 value of 2.407 mg/mL and 2.990 mg/mL, respectively. There were 13 compounds detected in the extracts from Stellera chamaejasme by supercritical fluid extraction, 99.99% of all components. The major compounds were Squalene, 9,12-Octadecadienoic acid (Z,Z)-, n- Hexadecanoic acid, Quinoline, and Campesterol. Squalene was the first time found from Stellera chamaejasme. Here, we also showed that Squalene had contacting and systemic toxicity against Tetranychus cinnabarinus, with LC50 value of 9.918 mg/mL and 12.918 mg/mL, respectively.

Antifungal Activity of Extracts by Supercritical Carbon Dioxide Extraction from Roots of Stellera chamaejasme L. and Analysis of Their Constituents Using GC-MS

In this work the supercritical fluid extraction with carbon dioxide was applied to obtain the extracts from roots of Stellera chamaejasme L. The extracts were evaluated through the antifungal activity against Monilinia fructicola. Furthermore, the active compounds from the extracts were separated by macro porous resin and analyzed using gas chromatography-mass spectrometry (GC-MS). The results showing that the extracts, obtained using supercritical carbon dioxide (SC-CO2) extraction technique, had strong antifungal activity against M. fructicola with inhibition ratio of 88.71% at 2000 μg/mL, minimum inhibitory concentration (MIC) of 250 μg/mL, and minimum fungicidal concentration (MFC) of 2000 μg/mL. After separation with macro porous resin chromatography, fourteen fractions were collected from the SC-CO2 extracts. At concentration of 2000 μg/mL, fraction 4, 7, and 12 presented highly antifungal activity against M. fructicola, with inhibition ratio of 83.97 %, 80.23 %, and 75.69 %, respectively. Followed by the GC-MS analysis, the main active compounds from fraction 4, 7, and 12 included hexanedioic acid, bis (2-ethylhexyl) ester, πsitosterol, 7-methyl-Z-tetradecen-1-ol acetate9, 9-hexadecenoic acid-hexadecyl ester (Z), 1, 2-benzenedicarboxylic acid-diisooctyl ester, (3π24Z) stigmasta-5, 24(28)-dien-3-ol, stigmastan-3, 5-diene, and squalene.

Inhibitory Effect and Antifunal Mechanism of Umbelliferone on Plant Pathogenic Fungi

Low concentrations of 7-hydroxycoumarin (umbelliferone) were found of antifungal activity towards plant pathogenic fungi using growth rate method. Umbelliferone inhibited mycelia growth in Monilia fructigena, Fusarium moniliforme, Botrytis cinerea and Colletotrichum capsici with the minimal inhibitory concentration (MIC) values of 250, 500, 1000, and 2000 μg mL-1, respectively. The results also showed its minimal fungicide concentration (MFC) values of M. fructigena and F moniliforme were both 1000 μg mL-1. But no MFC values towards B. cinerea and C. capsici were obtained in this study. It is noteworthy that umbelliferone was very active against the spore germination of the four fungi. 100% inhibition of spore germination of M. fructigen and F. moniliforme were observed, when the umbelliferone concentrations were up to 1000 μg mL-1 and 2000 μg mL-1, respectively. The antifungal mechanism of umbelliferone was further investigated using scanning electron microscope (SEM) and transmission electron microscope (TEM). After umbelliferone treatment, the fungal mycelia appeared to deform or adhere. The cell structure of fungal mycelium was destroyed, and the cell contents were spilled. Antifungal mechanism of umbelliferone is probably based on its destruction activity towards pathogenic fungal cellular organs. According to our investigation, umbelliferone has the potential to be useful as a phytogenous pesticide.

Acaricidal Activity of Ethyl Palmitate against Tetranychus cinnabarinus

Isolation and identification of bioactive compounds from medicinal plant of Inula flower is a potential approach for the development of new pesticides against the mite of Tetranychus cinnabarinus. Dry Inula Flower was used as the starting material to obtain active substances with acaricidal activity by organic solvent extraction and bioactivity assay verification. The main component of the active substances in fraction 33 was further identified by GC/MS and proven to be ethyl palmitate. In order to clarify the roles of ethyl palmitate playing against mites of Tetranychus cinnabarinus, the enzymatic activities were assayed regarding to four essential enzymes in the process of either nerve conduction or anti-oxidation reaction of the mites, including assays for Ca2+-ATPase, superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). In addition, the substructures of the ethyl palmitate treated adult female mite of T. cinnabarinus were observed by transmission electron microscopy. In the bioassay, ethyl palmitae showed strong activities against the mites with the corrected mortality rates of more than 90% for samples at 2mg/ml concentration. The activity increases of antioxidant enzymes indicate a certain degree of stimulation of the anti-oxidant mechanisms to response for the ethyl palmitate treatment in vivo. The Ca2+-ATPase plays essential role in the maintenance of nerve cell excitability and conduction. Its enzymatic activity was subject to a certain degree of declination, which might cause the death of the mites due to suppression in nerve conduction. The toxicity effect of the mites by ethyl palmitate was further confirmed by TEM observation. The significant destruction of the skin structures and cellular substructures can directly affect the normal function of the cells, and ultimately lead to the death of the mites. Based on the strong acaricidal activity of ethyl palmitate has against the carmine spider mite, it is valuable to be further investigated for a new type of botanical pesticides development.

Effects of Mentha Piperita Extracts on Activities of Several Enzymes of Tetranychus Cinnabarinus

The specific acaricidal mechanisms of Mentha piperita extracts were studied. The acaricidal and relative enzyme activity in Tetranychus cinnabarinus after treatment with Mentha piperita extracts at the concentration of 2mg·ml- 1 was studied by using the slide dip technique and colorimetric method, respectively. The effects of Mentha piperita extracts on the poisonous symptom and ultrastructure of the treated mites were also observed. Our results showed that fraction V from Mentha piperita extracts had stronger toxicity than others. The mites had experienced four phases namely quiescence, excitement, spasm and death after treatment, indicating that the Mentha piperita extracts were able to cause the poisonous symptom of the treated mites. Enzyme activity assay showed that the activities of glutathione-S-transferase (GSTs) and monoamine oxidase (MAO) were strongly induced whereas the activities of acetylcholine esterase (AChE) and protease were restrained after treatment. The changes of these enzymes led to the death of mites due to the destruction in the nervous transmission and digestive system. The transmission electron microscopic observations revealed that the treated mites suffered from the rupture of ultrastructure such as endoplasmic reticulum, mitochondria and nuclear membrane. These data provided useful information for the use of Mentha piperita as a novel resource of botanical acaricide.

Acaricidal Activity of Stigmasterol from Inula Britannica against Tetranychus cinnabarinus

The activity of fraction 12 identified as stigmasterol by GC/MS from the petroleum ether extract of Inula britannica was evaluated against Tetranychus cinnabarinus in laboratory. The bioactivities and mechanism of stigmasterol against Tetranychus cinnabarinus was further investigated. Superoxide dismutase, peroxidase and catalase in T. cinnabarinus treated with stigmasterol were examined through biochemical methods. The ultrastructure of the treated mites was observed by using transmission electron microscope (TEM). The stigmasterol showed lethal effects on the mite adults, and corrected mortality was over 50% with the concentration of 2 mg/ml. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in treated mites were enhanced in activity. The results showed that stigmasterol can destroy the balance between oxidation and anti-oxidation. Glutathione-S-transferase (GSTs) activity was strongly induced after treatment. The results indicated that stigmasterol was perhaps attributing to over excitation and dead of treated mites. TEM observations revealed that the treated mites suffered from the ruptures of cuticle, muscular filament, nuclear membrane, mitochondria and endoplasmic reticulum. Thus, stigmasterol appears a strong acaricidal activity against Tetranychus cinnabarinus, can be exploited as new resources for developing potential botanical acaricides.

Acaricidal Activities of the Polygonum Aviculare Extracts against Tetranychus cinnabarinus and Its Affection to the Enzyme Activities

This paper showed a study on acaricidal activities of the plant extracts from Polygonum aviculare against Tetranychus cinnabarinus and its affection to the activity among several relative enzymes. The plant extracts were prepared using different solvents such as chloroform, petroleum ether and methanol. It was found that the corrected mortality of female adults could reach 89.95 % (2 mg·mL-1) and the main lethal concentrations (LC50) was 0.7153 ± 0.1253 mg·mL-1 when using chloroform as the solvent, which exhibited best acaricidal activities among three solvents (P < 0.05). Through further extraction and column chromatography, we obtained 19 components from the chloroform extracts, and the number 2, 7, 8, 15, 16, 17 and 18 components had acaricidal activities higher than 70%, among them, number 16 had highest motality (87.12 %). Furthermore, colorimetry assay showed that the component number 16 could affect the activity of acetylcholinesterase (AChE), Na+, K+-ATPase, GSH-S-transferase (GSTs) of T. cinnabarinus.

Isolation and Identification of the Acaricidal Components Extracted from Stellera chamaejasme

The research of Botanical acaricide is an important aspect of integrated agricultural mite management. Eight kinds of different organic solvents were used for cold-soak extraction from Stellera chamaejasme. Combined with tracking activity, the fraction E7-4 was isolated from ethyl acetate extract using medium-pressure liquid chromatography (MPLC) and preparative HPLC. Using nuclear magnetic resonance, it was determined that fraction E7-4 was Scopoletin. The results showed that the scopoletin in Stellera chamaejasme was the important acaricidal activity ingredient which exhibited contact and systemic activity against Tetranychus cinnabarinus. The Lethal concentrations (LC50) to Tetranychus cinnabarinus were 1.267 mg/mL and 7.388 mg/mL respectively.