Comparison of the chemical constituents of raw Fructus Aurantii and Fructus Aurantii stir-baked with bran, and the biological effects of auraptene.

ETHNOPHARMACOLOGICAL RELEVANCE: Fructus Aurantii (FA) is a Chinese herbal medicine commonly used in clinical practice to improve gastrointestinal motility, treat dyspepsia, and relieve constipation. More than 20 processing methods of FA have been recorded, among which FA stir-baked with bran is the earliest, most time consuming, and the most popular one. Raw FA has a strong ability to promote qi-moving and has middle-energizer-soothing effects; therefore, it is often used to relieve hypochondrium distension and pain, and to relax the stagnation of the liver Qi. FA stir-baked with bran is more effective in nourishing the stomach and curing indigestion. AIM OF THE STUDY: In this study, the chemical composition and differences between raw FA and FA stir-baked with bran were systematically compared. The chemical components that increased after stir-baking FA and bran were separated and their pharmacodynamic characteristics were determined. Lastly, the processing mechanism of FA was further explained. MATERIALS AND METHODS: Twelve main chemicals in raw FA and FA stir-baked with bran were compared using high-performance liquid chromatography (HPLC). The main differential components were identified, separated, purified, and then analyzed using pharmacodynamic tests. The intestine-pushing test, in vitro smooth muscle test, and in vitro acetylcholinesterase (AchE) activity test in mice were performed to explain the mechanism of auraptene in improving gastrointestinal motility. RESULTS: Using HPLC, the primary chemical that differed between raw FA and FA stir-baked with bran was identified as auraptene. The processed FA was extracted, separated, and purified to obtain pure auraptene. The intestine-pushing test in mice showed that low (0.6 mg·kg-1) and medium doses (1.2 mg·kg-1) of auraptene could promote peristalsis of the small intestine, whereas a high dose (2.4 mg·kg-1) inhibited peristalsis. In vitro studies on the smooth muscle of mice showed that a low dose of auraptene (0.2 mmol·L-1, 10-800 µL) could promote contraction, whereas a high dose (0.2 mmol·L-1, >1000 µL) had the opposite effect. Auraptene has a mechanism of action similar to that of the acetylcholinesterase inhibitor, neostigmine. Additionally, auraptene could inhibit AchE activity in vitro. CONCLUSIONS: Auraptene is the main chemical constituent that differs between raw FA and FA stir-baked with bran. Pharmacodynamic tests showed that auraptene has a cholinergic effect, by virtue of its role as an acetylcholinesterase inhibitor. Moreover, auraptene could dually regulate the gastrointestinal smooth muscle. Auraptene was present in low levels and its content varied in FA stir-baked with bran, depending on the origin and source of FA, and the treatment procedures it was subjected to. In the Chinese Pharmacopoeia, the recommended dose of FA stir-baked with bran is a low dose of 3-10 g, which effectively promotes small-intestinal peristalsis. The mechanism of action is attributed to an increase in the relative content of acetylcholine by the inhibition of AchE activity to promote gastrointestinal motility. The increased levels of auraptene in FA stir-baked with bran are the main reason and the primary purpose for the change in its medicinal properties. This technique, therefore, has potential to be used as one of the main processing mechanisms of raw FA.

Acute and subacute toxicity of the extract of Aristolochiae fructus and honey-fried Aristolochiae fructus in rodents.

Aristolochiae Fructus (AF) and honey-fried Aristolochiae Fructus (HAF) have been used in China for thousands of years as an anti-tussive and expectorant drug. Few clinical cases were reported associated with the toxicity of AF and HAF, although relatively high contents of aristolochic acids (AAs) were found in them. This work was designed to compare the acute and subacute toxicity of AF and HAF in order to provide references for safe clinical use and to evaluate the possibility of reducing toxicity of AF by honey-processing. The extracts of the herb were fed to mice or rats via gastric tube. Various toxic signs and symptoms, body weights, serum biochemical assay, organ weights and histopathology were used to evaluate the toxic effects. The median lethal dose (LD50) of AF and HAF are 34.1±7.2 g/kg/d and 62.6±8.0 g/kg/d with a 95% average trustable probability (p=0.95), respectively. The subacute results showed a dose-dependant relationship of the toxicity of AF and HAF. Even in the high dose groups, only moderate toxicity was observed. Honey-frying and decoction with water can decrease the contents of AAs, and attenuate the toxic effects of AF. But sufficient attention should be still paid to the safety of AF and HAF due to the existence of AAs.

[Effect of honey-toasting on the constituents and contents of aristolochic acid analogues in aristolochiae fructus].

OBJECTIVE: To evaluate the degree of de-toxification of Aristolochiae Fructus by honey-toasting technology from chemical viewpoint. METHODS: The contents of aristolochic acid analogues (AAs) in Aristolochiae Fructus and its honey-toasted product were determined by HPLC, and the degree of de-toxification was evaluated comprehensively. RESULTS: After honey-toasted, the contents of AAs decreased to varying degrees, and some new compounds were found. CONCLUSION: The constituents and contents of Aristolochiae Fructus change after honey-toasted, which indicate honey-toasting can reduce the toxicity of Aristolochiae Fructus.

[Determination of the active constituents in aurantii fructus from Jiangxi province at different harvest time by HPLC].

OBJECTIVE: To determine the contents of the main active constituents in Aurantii Fructus from Jiangxi at different harvest time and make sure the best harvest time. METHODS: RP-HPLC was used to assay the active constituents (including naringin, neohesperidin, synephrine, nobiletin, tangeretin, meranzin hydrate, meranzin, marmin and auraptene) contents in the Aurantii Fructus at different harvest periods from Xingan and Zhangshu countries. RESULTS: The trend of the contents of those active constituents was basically decreased as the day trailing. CONCLUSION: The best harvest time of Aurantii Fructus from Jiangxi is about the Great Heat.

[Effect of ferulic acid on learning and memory impairments of vascular dementia rats and its mechanism of action].

This study is to investigate the effect of ferulic acid on learning and memory impairments of vascular dementia (VD) rats and its mechanism of action. VD rats model was replicated by permanent bilateral common carotid artery occlusion (2VO). The learning and memory capability of VD rats was evaluated by Morris water maze. The activity of acetylcholinesterase (AChE) and superoxide dismutase (SOD) and the content of glutamic acid (Glu) and malondialdehyde (MDA) in hippocampus of VD rats' brain were determined, separately. The results showed that ferulic acid could alleviate learning and memory deficits of VD rats significantly. Ferulic acid was found to inhibit the activity of AChE and increased the activity of SOD in rat hippocampus. In addition, ferulic acid could also decrease the content of Glu and MDA in rat hippocampus. These results suggested that ferulic acid could alleviate VD rats' learning and memory deficits, which might be due to antioxidation, the improvement of cholinergic system in brain, or the inhibitory of nerve injury by excitatory amino acids.

Simultaneous quantification of polymethoxylated flavones and coumarins in Fructus aurantii and Fructus aurantii immaturus using HPLC-ESI-MS/MS.

The major lipid-soluble constituents in Fructus aurantii (zhiqiao) and Fructus aurantii immaturus (zhishi) are polymethoxylated flavones (PMFs) and coumarins. In the present study, a high-performance liquid chromatography with electrospray ionization tandem mass spectrometry method was developed to quantify PMFs (nobiletin, tangeretin, 5-hydroxy-6,7,8,4'-tetramethoxyflavone, and natsudaidai) and coumarins (marmin, meranzin hydrate, and auraptene) simultaneously. PMFs and coumarins were detected by electrospray ionization tandem mass spectrometry in positive ion mode and quantified with multiple reaction monitor. Samples were separated on a Diamonsil C18 (150 mm × 4.6 mm, 5 µm) column using acetonitrile and formic acid-water solution as a mobile phase in gradient mode with a flow rate at 0.5 mL/min. All calibration curves showed good linearity (r² > 0.9977) within the test ranges. Variations of the intraday and interday precisions were less than 4.07%. The recoveries of the components were within the range of 95.79%-105.04% and the relative standard deviations were less than 3.82%. The method developed was validated with acceptable accuracy, precision, and extraction recoveries and can be applied for the identification and quantification of four PMFs and three coumarins in citrus herbs.

Identification, synthesis and quantification of process-related impurities in auraptene.

Impurities in chemically synthesized auraptene, an active pharmaceutical ingredient (API), were detected by a gradient reverse-phase high-performance liquid chromatography (RP-HPLC) method. Molecular weights and major product ions of these chemical compounds were determined by liquid chromatography/Triple Quadrupole (LC-MS/MS) analysis. Structural assignments were presumed as umbelliferone (Imp-I), (E)-6,7-dihydroxy-3,7-dimethyl-2-octene-umbelliferone (Imp-II), (E)-6,7-epoxy-3,7-dimethyl-2-octene-umbelliferone (Imp-III) and 4-methylauraptene (Imp-IV). The impurities were authentically synthesized, confirmed by nuclear magnetic resonance spectroscopy (NMR) and infrared spectroscopy (IR), and subsequently used as reference samples in routing HPLC system suitability testing for method specificity and detectability. Method specificity was further verified by forced degradation studies. The developed method was validated for characterization of impurities in synthesized auraptene according to the guidelines of the International Conference on Harmonization (ICH) in our laboratory.

[Studies on the chemical constituents from Daphne tangutica].

OBJECTIVE: To research the chemical constituents from Daphne tangutica. METHODS: Column chromatography with silica gel, ODS-C18, Sephadex LH-20 and re-crystallization were employed to isolate and purify the constituents. According to physical and chemical properties and spectral data to identify the structure of compounds. RESULTS: Eight compounds were isolated and identified as palmitic acid (1), laurostearic acid(2), beta-sitosterol(3), 7-methoxy-8-hydroxycumarin(4), daphnetin(5), genkwanin(6), hydroxygenkwanin(7), p-hydroxybenzoic acid(8). CONCLUSION: Compounds 1,2,7 and 8 are isolated from Daphne tangutica for the first time.

[HPLC determination of strychnine and brucine in Semen Strychni and its processed products].

OBJECTIVE: This paper reports a HPLC method for determinition of strychnine and brucine in Semen Strychni and its processed products of Jiangxi method and innovated methed. METHOD: SiO2 was used as the stationary phase, n-hexane-dichloromethane-methanol-ammonia(47.5:47.5:5:0.35) as the mobile phase, with detection wavelength of 254 nm. RESULT: The contents of strychnine and brucine in the processed products of Jiangxi are lower. CONCLUSION: This method is accurate, simple and reliable.