Rapid screening of 28 alkaloids in food poisoning samples by liquid chromatography-tandem mass spectrometry / 中国热带医学

@#Abstract: Objective　To investigate a poisoning incident caused by eating eight treasure congee, and establish liquid chromatography (LC)-mass spectrometry (MS)/MS screening method of 28 alkaloids to provide references for disposal of similar poisoning incidents. Methods　LC-MS/MS was used for screening 28 alkaloids in the urine, eight treasure congee and food raw material, and the detected alkaloids were quantified. Samples were extracted with 0.4% formic acid aqueous solution and separated by a Acquity UPLC BEH C18 column (1.7 μm, 100 × 2.1 mm). Acetonitrile-0.2% formic acid aqueous solution was used as the mobile phase and gradient elution was adopted. The ionization mode was electrospray positive ionization mode, and the detection method was multi-reaction monitoring (MRM). Analytes were quantified with the external standard method. Results　In the concentration range of 0-100 ng/mL, the linear correlation coefficient r were greater than 0.999 for 28 alkaloids. The recovery of 28 alkaloids in urine sample ranged from 63.0% to 105.0%, and the relative standard deviations (RSDs) were between 5.8% and 8.6%. The recovery of 28 alkaloids in eight treasure congee sample ranged from 72.0% to 109.0%, and the RSDs were between 6.3% and 9.7%. The recovery of 28 alkaloids in semen sesami nigrum sample ranged from 60.0% to 95.0%, and the RSDs were between 4.8% and 8.2%. Hyoscyamine (2 380.0 ng/mL), scopliamine (3.6 ng/mL) and rac-anisodamine (4.7 ng/mL) were detected in the patient's urine. Hyoscyamine (63.3 μg/g), scopliamine (5.7 μg/g) and rac-anisodamine (2.1 μg/g) were detected in eight treasure congee. Hyoscyamine (901.0 μg/g), scopliamine (80.0 μg/g) and rac-anisodamine (30.1 μg/g) were detected in the seed of Datura stramonium L. The ratio of scopliamine and hyoscyamine in the seed of D. stramonium was 1∶11, which complies with the characteristics of D. stramonium L. In urine sample, the proportion of scopliamine and rac-anisodamine was 0.15% and 0.20%, and hyoscyamine accounted for 99.65%. Conclusion　Seed morphology, the content range and proportion of three alkaloids are all in accord with the characteristics of D. stramonium. Combined with the clinical symptoms of atropine poisoning, it can be deduced that this incident is a family food poisoning caused by accidental consumption of seed of D. stramonium L. The method can provide technical support for the clinical diagnosis and treatment of alkaloid poisoning patients, and also provide a basis for emergency detection and disposal of alkaloid poisoning events.

Effect of the rolB gene on phenotypic development and tropane alkaloids biosynthesis in Atropa belladonna / 药学学报

The rol genes on pRiA4 plasmid of Agrobacterium rhizogenes are potent genes that promote secondary metabolism. Molecular breeding of Atropa belladonna can be conducted by introducing rol genes to increase tropane alkaloids (TAs) content in A. belladonna. In this study, the rolB gene was overexpressed in A. belladonna plants to study the effect of rolB gene on the biosynthesis of TAs. The phenotype, TAs content and expression levels of key enzyme genes in the pathway of TAs biosynthesis of transgenic A. belladonna were analyzed. The results showed that transgenic A. belladonna had developed root system, enlarged leaves, increased leaf fresh weight, deepened leaf color, enlarged flowers, changed flower shape, reduced pistil height and decreased pollen vitality. The content of TAs in the stems of transgenic A. belladonna was significantly higher than that of the control, and the contents of scopolamine, anisodamine, hyoscyamine can reach 2.11-2.91, 1.23-2.37 and 4.88-5.20 times of the control, respectively. Compared with the control group, the expressions of key enzymes putrescine N-methyltransferase (PMT), type III polyketide synthase (PYKS), tropinone reductase I (TRI), aromatic amino acid aminotransferase 4 (ArAT4), UDP-glycosyltransferase 1 (UGT1) and hyoscyamine 6-β-hydroxylase (H6H) in the TAs biosynthesis pathway were up-regulated, and the expression of tropinone reductase II (TRII) as a metabolic shunting gene was down-regulated. The results indicated that rolB gene enhanced TAs synthesis ability in roots and accumulation in stems of A. belladonna by enhancing metabolic flow of TAs synthesis pathway and weakening the metabolic shunt of competing pathway. This study laid a foundation for molecular breeding of A. belladonna with high-yield TAs content using rolB gene.

Improvement on Quality Standard of Belladonnae Herba / 中国实验方剂学杂志

ObjectiveTo improve the current standard of Belladonnae Herba in the 2020 edition of Chinese Pharmacopoeia. MethodTaking hyoscyamine sulfate， atropine sulfate and scopoletin as reference substances， and ethyl acetate-methanol-concentrated ammonia（17∶4∶2）as developing solvent， thin layer chromatography （TLC） was applied in the qualitative identification of Belladonnae Herba. The moisture， total ash and ethanol-soluble extract of Belladonnae Herba were determined based on the general principles in the 2020 edition of Chinese Pharmacopoeia （volume Ⅳ）. The contents of hyoscyamine sulfate and scopolamine hydrobromide were analyzed by high performance liquid chromatography （HPLC） with mobile phase of acetonitrile-54 mmol·L-1 phosphate buffer solution （14∶86）， flow rate of 1.0 mL·min-1 and detection wavelength at 210 nm. ResultThe spots in the TLC were clear with good separation and specificity. Hyoscyamine sulfate and scopolamine hydrobromide showed a good linearity with peak area in the range of 0.024 7-0.789 6 g·L-1 （r=0.999 9） and 0.003 9-0.124 0 g·L-1 （r=0.999 9）， the average recoveries of these two ingredients were 100.29% （RSD 1.6%） and 99.04% （RSD 1.4%）， respectively. The limits for moisture， total ash in Belladonnae Herba should be less than 13.0% and the limit for the ethanol-soluble extract should be more than 10.0%. Due to the low content and wide variation of scopolamine hydrobromide， the content of hyoscyamine sulfate should not be less than 0.098%. ConclusionThe established method is simple， specific and reproducible， which can be used to improve the quality control standard of Belladonnae Herba.

Effect of the rolC gene on biosynthesis of tropane alkaloids in Atropa belladonna / 药学学报

Hair roots induced by Agrobacterium rhizogene produce higher levels of secondary metabolites than non-induced plants, and the enhanced metabolic capacity is driven by the rol gene. We hypothesized that rol genes can be utilized to improve the biosynthesis of tropane alkaloids (TAs) in Atropa belladonna. In this study, the rolC gene from Agrobacterium rhizogene pRiA4 plasmid, driven by a CaMV35S promoter, was overexpressed in A. belladonna. The phenotypes, TAs content and transcriptional expression of key genes in TAs biosynthesis were analyzed in transgenic A. belladonna plants. Results show that transgenic A. belladonna exhibited a well-developed root system, male sterility, higher stamen column length than pistil, early flowering, internode shortening, smaller but more flowers, increased axillary buds and lateral buds, decreased apical dominance, and long and narrow leaves as compared to wild-type plants. Transgenic A. belladonna produced more TAs than wild-type plants, with the content of hyoscyamine, anisodamine and scopolamine reaching 2.58, 3.59 and 15.77-fold that of the control group, respectively. The gene expression of putrescine N-methyltransferase (PMT), tropinone reductase I (TRⅠ) and hyoscyamine 6-β-hydroxylase (H6H), key enzymes in TAs biosynthesis, were up-regulated compared with the control group. The above results indicate that the rolC gene enhances TAs biosynthesis in A. belladonna by up-regulating the expression of key enzymes in the TAs biosynthesis pathway, laying a foundation for genetic manipulation of A. belladonna to increase TAs content by increasing rolC gene expression.

Effects of nitrogen form on accumulation of alkaloids and expression of relative genes in Atropa belladonna / 中国中药杂志

Hyoscyamine and scopolamine are two main alkaloids in Atropa belladonna with great medicinal value. In this paper, the contents of hyoscyamine and scopolamine, the upstream products in alkaloid synthesis, and the expression levels of key enzyme genes PMT, TRⅠ and H6H in secondary metabolism of A. belladonna seedlings were measured to clarify the mechanism of nitrogen forms regulating alkaloids synthesis.The results showed that the 50/50 (NH⁺₄/NO⁻₃) treatment was more favorable for the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine. The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine, they both increased with the rise of ammonium ratio, reaching the highest at 75/25 (NH⁺₄/NO⁻₃). The detection of signaling molecule nitric oxide (NO) showed that the NO concentration decreased with the decrease of nitrate proportion. Further detection of gene expression levels of PMT, TRⅠ and H6H in TAs synthesis pathway showed that a certain amount of ammonium promoted the expression of PMT and H6H in roots. When the ratio of ammonium to nitrate was 50/50, PMT, TRⅠ and H6H in leaves and roots had higher expression levels. It can be speculated that the regulation of the formation of hyoscyamine to scopolamine by nitrogen forms mainly through affecting the expression of key enzyme genes. 50/50 (NH⁺₄/NO⁻₃) treatment increased the gene expression of TRⅠ in both leaves and roots as well as PMT and H6H in roots, promoting the synthesis of putrescine to hyoscyamine and the conversion of hyoscyamine to scopolamine.

Effects of methyl jasmonate on metabolism of topical alkaloids and expression of relate genes in Atropa belladonna / 中国中药杂志

Hyoscyamine and scopolamine are important secondary metabolites of tropane alkaloid in Atropa belladonna with pharmacological values in many aspects.In this study, the seedlings of A.belladonna were planted by soil culture and treated with different concentrations of methyl jasmonate (MeJA). The contents of hyoscyamine and scopolamine,the upstream products in alkaloid synthesis,and the expression levels of key enzyme genes PMT, TR Ⅰ and H6H in secondary metabolites of A. belladonna seedlings were measured to clarify the mechanism of MeJA regulating alkaloids synthesis.The results showed that MeJA(200 μmol·L⁻¹) treatment was more favorable for the accumulation of alkaloids.The content of putrescine was almost consistent with the change of key enzymes activities in the synthesis of putrescine,the both increased first and then decreased with the increased MeJA concentration and the content of putrescine reached the highest at 200 μmol·L⁻¹ MeJA.Further detection of gene expression of PMT, TR Ⅰ and H6H in TAs synthesis pathway showed that no significant trend in PMT gene expression levels.The expression levels of TR Ⅰ and H6H in leaves and roots under 200 μmol·L⁻¹ MeJA were the highest.It can be speculated that the regulation of the formation of hyoscyamine and scopolamine by MeJA mainly through affecting the expression of key enzyme genes.Appropriate concentration of MeJA increased the gene expression of TR Ⅰ in both leaves and roots as well as H6H in roots,promoting the accumulation of alkaloids and the conversion of hyoscyamine to scopolamine.

Improvement in Quality Control Methods for Belladonna Oral Solution / 中国药师

Objective:To improve the identification and determination methods in the original quality standard for belladonna oral solution.Methods:Belladonna oral solution was identified by a specific chromatogram of HPLC,and scopoletin and hyoscyamine sulfate in belladonna oral solution were detected by dual wavelength spectrophotometry.The detection of fingerprints was performed on a Waters SunFire C18 (250 mm×4.6mm,5 μm) column.The mobile phase was methanol-0.05% phosphoric acid solution at a flow rate of 1.0 ml·min-1.The detection wavelength was 344 nm and the column temperature was 30℃.The detection of scopoletin and hyoscyamine sulfate was performed on the same C18 column.The mobile phase was 10 mmol·L-1 heptanesulfonate sodiumat (pH value was 3.3 adjusted by glacial acetic acid)-absolute ethanol-acetonitrile (68.75∶6.25∶25) at a flow rate of 1.0 ml·min-1.The detection wavelengths were 344 nm and 210 nm,and the column temperature was 30℃.Results:The specific chromatogram of belladonna oral solution was accordance with that of belladonna tincture raw material.The retention time and relative peak area of each characteristic peak and reference peak all met requirements of Chinese Pharmacopoeia.Scopoletin and hyoscyamine sulfate were completely separated from the other compositions under the above mentioned conditions.The calibration curves were linear within the range of 5.168-103.360 μg·ml-1 (r=1.000 0) for scopoletin and 50.560-758.400 μg·ml-1 (r=0.999 9) for hyoscyamine sulfate.The average recovery was 101.79% (RSD=1.05%,n=6) and 100.92% (RSD=0.97%,n=6),respectively.Conclusion:After the quality control method improvement,the identification shows high specificity and the quality of belladonna oral solution can be better controlled by the two selected index components.The method is easy and accurate,which can provide a reliable way to control the quality.

Overexpression of NtPMT and HnH6H changed hyoscyamine-rich Atropa belladonna to scopolamine-rich varieties / 药学学报

Atropa belladonna L. is the commercial plant material for production of tropane alkaloids, including hyoscyamine and scopolamine. The wild-type Atropa belladonna is characterized by the hyoscyaminerich chemotype, in which the hyoscyamine content is much higher than the scopolamine content. It is the common goal for the pharmaceutical industry to increase the content of scopolamine in A. belladonna. Based on the T0 progeny of transgenic A. belladonna with NtPMT and HnH6H overexpression, T1 progeny of transgenic A. belladonna were obtained through self-pollination and used in a field trial. The 461 bp fragment of NtPMT and the 1 077 bp HnH6H were simultaneously expressed from T1 progeny of transgenic A. belladonna, but were not obtained from the wild-type A. belladonna. At the transcription level, the expression of NtPMT and HnH6H were detected in T1 progeny of transgenic A. belladonna, but were not detected in the wild-type plants. Further, the alkaloids were analyzed by HPLC. In the stems and leaves of T1 progeny of transgenic A. belladonna, hyoscyamine was not detected and scopolamine was detected at very high levels; in the stems and leaves of wild-type A. belladonna, hyoscyamine was detected at much higher levels. In the leaves of T1 progeny of transgenic A. belladonna, the content of scopolamine was 15-36 folds higher than that of wildtype leaves; in the stems of T1 progeny of transgenic A. belladonna, the scopolamine content was 37-108 folds higher than that of wild-type stems. In conclusion, overexpression of NtPMT and HnH6H greatly enhanced conversion of hyoscyamine into high-value scopolamine and improved the commercial value of A. belladonna.

Chemical constituents from roots and rhizomes of Physochlaina infundibularis / 中草药

Objective: To investigate the chemical constituents from the dried roots and rhizomes of Physochlaina infundibularis. Methods: Repeated chromatography with silica gel, macroporous resin, MCI, and Sephadex LH-20 columns was used to isolate the chemical constituents and their structures were elucidated based on physicochemical properties and spectral data. Results: Seventeen compounds were isolated and identified as hyoscyamine (1), scopolamine (2), anisodamine (3), scopoletin (4), scopolin (5), umbelliferone (6), 6, 7-dimethoxycoumarin (7), 3-methoxyquercetin (8), isoquercitin (9), kaempferol-7-O-β-D-glucoside (10), syringarenol (11), protocatechuic acid (12), methyl 4-hydroxybenzoate (13), 2-hydroxybenzoic acid (14), tropic acid (15), 4- hydroxybenzoic acid (16), and palmitic acid (17). Conclusion: Compounds 7-17 are firstly obtained from the plants in Physochlaina G. Don, and compound 5 is firstly obtained from P. infundibularis.

Improvement of mental retardation plus epilepsy in children following management / 中国组织工程研究

Objective To investigate the effective management approaches for mental retardation plus epilepsy in children.Method Sodium valproat[15～ 60 mg/（ kg· day)] or nitrazepam[0.5～ 1 mg/（ kg· day） ] and guanmaishu containing hyoscyamine(0.0045～ 0.045 mg/（ kg· day)] as adjuvant were administered for 1.5～2 consecutive years.EEG,three dimensional Doppler ultrasonic examination of cerebral vessels were performed.Result Favorable therapeutic effect was obtained in 25 cases(48.1％ ),good effect in 12 cases(23.1％ ).Conclusion Favorable therapeutic effect of hyoscyamine in epilepsy is correlated with improved cerebral microcirculation,cortical choline receptor blocking ,reduced conduction between synapses.These factors all inhibit onset of epilepsy.

Study on quality standard for Zangfukang Suppository / 中成药

AIM: To establish the method for quality control of Zangfukang Supposit ory (Radix Przewalskii tanguticae, Pericarpium Zanthoxyli, Herba Oxytropis falca tae, etc.). METHODS: Radix Przewalskii tanguticae, Pericarpium Zanthoxyli, Her ba Oxytropis falcatae were identified by TLC, and the content of hyoscyamine in Radix Przewalskii tanguticae was determined by HPLC. RESULTS: The ethyl acetate-methanol-ammonia(17∶2∶1) was applied as the developer for the identification of Radix Przewalskii tanguticae on silica gel G coating plate. The cyclohexane-ethyl acetate-formic acid (30∶15∶1) was applied as the developer for the identification of Pericarpium Zanthoxyli on s ilic a gel G coating plate. The cyclohexane-ethyl acetate-formic acid (50∶10∶1) w as taken as the developer for the identification of Herba Oxytropis falcatae als o on silica gel G coating plate. Hyoscyamine showed a good linear relationship a t a range of 2.5～25?g,r=0.9999. The average recovery was 99.59%, and RSD was 1.34%. CONCLUSION: The methods are accurate and reliable, and can control the quality of this preparation effectively.