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Development of the HPLC-ELSD method for the determination of phytochelatins and glutathione in Perilla frutescens under cadmium stress conditions.

ABSTRACT: A rapid, accurate and simple method was developed for the simultaneous determination of glutathione (GSH) and phytochelatins (PCs) by high-performance liquid chromatography (HPLC) with an evaporative light-scattering detector. GSH, phytochelatin 2 (PC2), PC3, PC4, PC5 and PC6 can be separated with baseline separation within 9?min using a Venusil AA column (250?mm?×?4.6?mm i.d., 5?µm particle sizes). Acetonitrile and water containing 0.1% trifluoroacetic acid (0.1%) were employed as the mobile phase for the gradient elution. The drift tube temperature and flow rate of the carrier gas (N2) were 50°C and 1.5?l?min-1, respectively. Under optimum conditions, good linear regression equations of six analytes were obtained with the detection limits ranging from 0.2 to 0.5?µg?ml-1. The proposed method has been applied successfully for the quantification of GSH and PCs in Perilla frutescens (a cadmium hyperaccumulator) under cadmium stress. The recoveries were between 82.9% and 115.3%.

SUBMITTER: Zheng X

PROVIDER: S-EPMC5990822 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Development of the HPLC-ELSD method for the determination of phytochelatins and glutathione in Perilla frutescens under cadmium stress conditions.

Zheng Xinyu X Chen Shen S Zheng Meiqin M Peng Jun J He Xiaosan X Han Yongming Y Zhu Jingjing J Xiao Qingtie Q Lv Rixin R Lin Ruiyu R

Royal Society open science 20180509 5

A rapid, accurate and simple method was developed for the simultaneous determination of glutathione (GSH) and phytochelatins (PCs) by high-performance liquid chromatography (HPLC) with an evaporative light-scattering detector. GSH, phytochelatin 2 (PC2), PC3, PC4, PC5 and PC6 can be separated with baseline separation within 9 min using a Venusil AA column (250 mm × 4.6 mm i.d., 5 µm particle sizes). Acetonitrile and water containing 0.1% tri ...[more]

PMID: 29892363

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Project description:BACKGROUND:Polyunsaturated fatty acids such as linoleic acid (LA) and ?-linolenic acid (ALA) are abundant in vegetable oils and are important for human health. In the body, LA and ALA are respectively converted to the omega-6 fatty acid ?-linolenic acid (GLA) and the omega-3 fatty acid stearidonic acid (SDA) by ?6 desaturase (D6DES). Currently, dietary GLA and SDA are mainly obtained from marine organisms, but given their benefits to human health, many studies have aimed to enhance their accumulation in transgenic crops. Perilla frutescens (perilla) accumulates more ALA in its seed oil compared to other oilseed crops, making it a good candidate for the production of fatty acids via the fatty acid desaturase D6DES. RESULTS:In this study, we cloned the D6DES gene from Phytophthora citrophthora and confirmed its function in budding yeast. We then transformed the functional D6DES gene under the control of the seed-specific vicilin promoter into the perilla cultivar Yeobsil. The resulting transgenic perilla seeds accumulated significant levels of GLA and SDA, as well as putative C18:2?6,9 at minor levels. Developing seeds and leaves also accumulated GLA and SDA, although PcD6DES expression and GLA and SDA levels were much lower in leaves compared to developing seeds. GLA and SDA accumulated in both polar lipids and neutral lipids in mature perilla seeds expressing PcD6DES, especially in neutral lipids. Although the seed weight in PcD6DES perilla was 87-96% that of wild type, the total oil content per seed weight was similar between lines. The PcD6DES perilla plants contained very high content (over 45%) of both GLA and SDA in seed oil. CONCLUSIONS:Thus, PcD6DES perilla plants may represent a feasible alternative to traditional marine sources for the production of omega-3 oil capsules and to evening primrose seed oil for GLA as health food. In addition, these plants can be used to create other transgenic lines harboring additional genes to produce other desirable fish-oil like oils.

Project description:BackgroundPerilla frutescens (L.) Britt. is not only an edible plant but also a traditional medicinal plant commonly used for treating common cold. It is widely cultivated in southern China. The anatomical parts of P. frutescens that are recorded as medicines in the Chinese material medica are: Perillae Caulis, Perillae Folium and Perillae Fructus, which are the dried stems, the dried leaves and the dried ripe fruits, respectively. Rosmarinic acid is one of major polyphenolic ingredients found in all three Perillae Caulis, Perillae Folium and Perillae Fructus. The characteristic volatile oil of P. frutescens is believed to be another essential composition of the herb, giving its wide range of use.ResultsA simple, rapid and accurate HPLC-DAD method was set up, suitable for the assay of rosmarinic acid in Perillae Fructus, Perillae Folium and Perillae Caulis. 12 batches of Perillae Caulis, 12 batches of Perillae Folium and 13 batches of Perillae Fructus from 8 different regions of mainland China and Hong Kong herbal markets were collected for evaluating the quality of P. frutescens. Results showed that Perillae Folium typically had the highest content of rosmarinic acid. Certain macroscopic characteristics were related to the concentration of rosmarinic acid. The volatile components were identified and compared in Perillae Fructus, Perillae Folium and Perillae Caulis by gas chromatography-mass spectrometry (GC-MS). Extracts were prepared by steam distillation. Twelve, seventeen and nine compounds were identified and accounted for 69.71%, 50.54% and 81.73% of all identified peak areas in Perillae Caulis, Perillae Folium and Perillae Fructus respectively. The identified components were analyzed for comparison of Perillae Caulis, Perillae Folium and Perillae Fructus more effectively.ConclusionsThis work provides a means by which samples of various parts of P. frutescens can be evaluated in terms of their pharmacologically active components. It should be of value in the efficient exploitation of P. frutescens plant material in clinical applications and drug development.

Project description:BackgroundWhile severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection presents with mild or no symptoms in most cases, a significant number of patients become critically ill. Remdesivir has been approved for the treatment of coronavirus disease 2019 (COVID-19) in several countries, but its use as monotherapy has not substantially lowered mortality rates. Because agents from traditional Chinese medicine (TCM) have been successfully utilized to treat pandemic and endemic diseases, we designed the current study to identify novel anti-SARS-CoV-2 agents from TCM.MethodsWe initially used an antivirus-induced cell death assay to screen a panel of herbal extracts. The inhibition of the viral infection step was investigated through a time-of-drug-addition assay, whereas a plaque reduction assay was carried out to validate the antiviral activity. Direct interaction of the candidate TCM compound with viral particles was assessed using a viral inactivation assay. Finally, the potential synergistic efficacy of remdesivir and the TCM compound was examined with a combination assay.ResultsThe herbal medicine Perilla leaf extract (PLE, approval number 022427 issued by the Ministry of Health and Welfare, Taiwan) had EC50 of 0.12 ± 0.06 mg/mL against SARS-CoV-2 in Vero E6 cells - with a selectivity index of 40.65. Non-cytotoxic PLE concentrations were capable of blocking viral RNA and protein synthesis. In addition, they significantly decreased virus-induced cytokine release and viral protein/RNA levels in the human lung epithelial cell line Calu-3. PLE inhibited viral replication by inactivating the virion and showed additive-to-synergistic efficacy against SARS-CoV-2 when used in combination with remdesivir.ConclusionOur results demonstrate for the first time that PLE is capable of inhibiting SARS-CoV-2 replication by inactivating the virion. Our data may prompt additional investigation on the clinical usefulness of PLE for preventing or treating COVID-19.

Dataset Information

Development of the HPLC-ELSD method for the determination of phytochelatins and glutathione in Perilla frutescens under cadmium stress conditions.

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Development of the HPLC-ELSD method for the determination of phytochelatins and glutathione in <i>Perilla frutescens</i> under cadmium stress conditions.

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