Heme peroxidases are responsible for the dehydrogenation and oxidation metabolism of harmaline into harmine / 中国天然药物

Harmaline and harmine are β-carboline alkaloids with effective pharmacological effects. Harmaline can be transformed into harmine after oral administration. However, enzymes involved in the metabolic pathway remain unclear. In this study, harmaline was incubated with rat liver microsomes (RLM), rat brain microsomes (RBM), blood, plasma, broken blood cells, and heme peroxidases including horseradish peroxidase (HRP), lactoperoxidase (LPO), and myeloperoxidase (MPO). The production of harmine was determined by a validated UPLC-ESI-MS/MS method. Results showed that heme peroxidases catalyzed the oxidative dehydrogenation of harmaline. All the reactions were in accordance with the Hill equation. The reaction was inhibited by ascorbic acid and excess H2O2. The transformation of harmaline to harmine was confirmed after incubation with blood, plasma, and broken blood cells, rather than RLM and RBM. Harmaline was incubated with blood, plasma, and broken cells liquid for 3 h, and the formation of harmine became stable. Results indicated an integrated metabolic pathway of harmaline, which will lay foundation for the oxidation reaction of dihydro-β-carboline. Moreover, the metabolic stability of harmaline in blood should not be ignored when the pharmacokinetics study of harmaline is carried out.

Functional Antagonism of Sphingosine-1- Phosphate Receptor 1 Prevents Harmaline- Induced Ultrastructural Alterations and Caspase-3 Mediated Apoptosis

@#Background: There is a meaningful necessity for a targeted therapy of essential tremor (ET), as medications have not been developed specifically for ET. For nearly a century, many drugs have been applied in the treatment of tremor but the drug treatment of ET remains still unknown. Some potential therapeutic factors such fingolimod (FTY720) can be effectively used to treat ET in animals. In the present research, the effect of FTY720, the immunomodulatory sphingosine 1-phosphate (S1P) analog, on degeneration of cerebellar and olivary neurons induced by harmaline in male rats was investigated. Methods: The animals were allotted into control dimethyl sulfoxide (DMSO), saline + harmaline [30 mg/kg, intraperitoneally, (i.p.)], harmaline + FTY720 (1 mg/kg, i.p, 1 h and 24 h before harmaline injection) groups (n = 10). The cerebellum and inferior olive nucleus (ION) were studied for neuronal degeneration using immunohistochemistry (IHC) and ultrastructural study by transmission electron microscopy (TEM) techniques. Results: Harmaline caused neuronal cell loss, caspase-3 mediated apoptosis, astrocytosis and ultrastructural changes in cerebellar Purkinje cells and inferior olive neurons. FTY720 exhibited neuroprotective effects on cerebellar Purkinje cells and inferior olivary neurons. Conclusion: These results suggest that FTY720 has potential efficacy for prevention of ET neurodegeneration and astrocytosis induced by harmaline in male rats.

Study on Quality Standard for Kazakhstan Medicine Peganum harmala / 中国药房

OBJECTIVE： To establish the quality standard for Kazakhstan medicine Peganum harmala. METHODS： Ten batches of P. harmala collected in Xinjiang Kazakh region were selected as research objects to investigate their characteristics. Qualitative identification of harmaline and harmine was conducted by TLC. The contents of water， total ash， acid-insoluble ash and ethanol extract were tested according to Chinese Pharmacopoeia（2015 edition）. The contents of harmaline and harmine were determined by HPLC. The determination was performed on X-bridge C18 column（250 mm×4.6 mm，5 μm） with mobile phase consisted of acetonitrile-ammonium acetate buffer （adjusted to 6 with glacial acetic acid， gradient elution） at the flow rate of 1 mL/min. The detection wavelength was set at 267 nm， and column temperature was 25 ℃. The sample size was 5 μL. RESULTS： TLC identification results showed that 10 batches of medicinal material showed clear spots at the same position as harmaline and harmine reference substances. Water， total ash， acid-insoluble ash should not be more than 12％，22％，2％， respectively； ethanol extract must not be less than 16％. HPLC results showed that the linear ranges of harmaline and harmine were 15.22-301.40，15.09-301.80 μg/mL； RSDs of precision， reproducibility and stability tests were all lower than 4％； average recoveries were 100.22％ and 100.94％（all RSD＜2％）. The determination results showed that the content of total alkaloids （harmaline and harmine） should not be less than 6.5 mg/g. CONCLUSIONS： Based on the original standard， test items are added in this study. TLC method is established to identify harmaline and harmine. HPLC method is established to determine their contents. Established quality standard can be used for comprehensive quality control of P. harmala from Xinjiang Kazakh region.

Study on the Quality Standard for Peganum Harmala Alkaloids Cream / 中国药师

Objective:To establish the quality standard for peganum harmala alkaloids cream ( CAPH) . Methods: The general quality of CAPH was inspected according to the general notices described in Chinese Pharmacopoeia volumeⅠ2010 edition. The qual-itative identification was carried out by TLC with harmine and harmaline as the index ingredients. The content determination was carried out by HPLC methods with harmine, harmaline and vasicine as the index ingredients. Results:The inspection items were all met the requirements. The experimental samples and the reference substances in TLC showed the identical spots with the same color and shape at the same position. The calibration curve of harmaline, harmine and vasicine was linear within the concentration range of 3. 440-110. 000 μg·ml-1 , 3. 340-107. 000 μg·ml-1 and 1. 380-22. 000 μg·ml-1 , respectively. The recovery was 98. 1%, 99. 8% and 99. 3% with RSD of 1. 75%, 1. 78% and 1. 95%, respectively (n=6). Conclusion: The established quality control methods meet the requirements of methodology, and the results lay foundation for the quality standard for CAPH.

Research on quality specification of the seeds of Peganum harmala L. of a uygur traditional medicine / 中国药学杂志

OBJECTIVE: To develop the quality specification of seeds of Peganum harmala. METHODS: According to the Chinese Pharmacopoeia (2010 Version, Volume 1) and its appendix method, the water, total ash, acid insoluble ash, 50% ethanol extractives, and heavy metal were analyzed for seeds of P. harmala. TLC method was used to separate harmaline (HAL), harmine (HAR) and vasicine (VAS) in seed samples using mixture of ethyl acetate-methanol-ammonia water (10:1.5:0.5) as a developing solvent on high performance silica G pre-coated plate with 254 nm fluorescent (GF254) and to identify them inspected under UV 366 nm, 254 nm, visualized by spraying with both Dragendorff reagent and by bioautographic assay. In the HPLC method, HAL and HAR were separated on a C18 column with acetonitrile-ammonium acetate water (19:81) as the mobile phase and detected at 330 nm. The HPLC fingerprints were performed on the same C18 column and eluted by using a linear gradient of acetonitrile (A) and 0.1 mmol · L-1 ammonium acetate buffer under the flow rate at 0.7 mL · min-1 and detected at 280 nm. RESULTS: In the TLC procedures, 254 and 366 nm fluorescent, Dragendorff reagent, and bioautographic assay for the detection of acetylcholinesterase inhibitor can be used for qualitative identification of the active ingredients. For the HPLC quantitative method, the calibration curve of HAR displayed ideal linearity over the range of 1.97-198.68 μg · mL-1 with average recovery of 99.69% (RSD of 1.89%). HAL displayed ideal linearity over the range of 1.70-345.30 μg · mL-1 with average recovery of 100.66% (RSD of 1.78%). The contents of HAL and HAR in 11 batches of seeds of P. harmala were 3.234% and 3.755%. In the characteristic fingerprints of seeds of P. harmala, four common peaks were identified. CONCLUSION: The results indicated that the water, total ash, acid insoluble ash, and 50% ethanol extractives were not more than 9.0%, 8.0%, 1.0%, and 22.0%, respectively. The heavy metal of plumbum, cadmium, arsenic, mercury, and copper were not more than 5 × 10-6, 3 × 10-6, 2 × 10-6, 2 × 10-6 and 20 × 10-6, respectively. The content limit of the sum of HAL and HAR was not lower than 5.5%. With the peak of HAL as reference peak, the variance of relative retention time of the four common peaks, in the characteristic fingerprints of seeds off. harmala, should be fluctuated in the range of 5% of the specified value. The qualitative and quantitative method established was suitable for the quality evaluation and assessment of seeds of P. harmala.

Degenerative Change of Cerebellar Purkinje Cells by Harmaline Treatment / 체질인류학회지

The indole alkaloid harmaline has been to cause tremor and ataxia, and produce cerebellar neurotoxicity in rat. Degeneration of Purkinje cell alligned in narrow parasagittal bands result from excitation of inferior olivary nucleus in harmaline-treated rats. The objective of this study was to investigate the hypothesis that excitation of climbing fiberinduced by harmaline mediates Purkinje cell injury or degeneration. For this purpose, the inferior olive of rats was chemically ablated by using 3-acetyl pyridine, a neurotoxic chemical, and cerebellar damage followed by administration of harmaline was analyzed using immunohistochemical markers for neurons, glial cells. The results demonstrated that a subset of Purkinje cell in the vermis and paravermis degenerated after harmaline treatment, but harmaline produced little or no Purkinje cell degeneration after inferior olivary ablation. These results suggested that harmalineinduced activation of inferior olivary neurons may lead to release of glutamate from climbing fiber synaptic terminal distributed over the Purkinje cells, and may lead to cytotoxic degeneration of Purkinje cells.