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    Dynamic analysis of 10 components of the Chinese herbal compound Wuzhuyu-tang absorbed into rat plasma**☆

    2011-07-27 01:06:46MuxinGongYaxunWangJianSunQiweiZhangZhiminWang

    Muxin Gong, Yaxun Wang, Jian Sun, Qiwei Zhang, Zhimin Wang

    1School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China

    2Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing 100700, China

    3Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China

    INTRODUCTION

    Wuzhuyu-tangis an effective formula for treating migraines in the clinic[1-2].Even though there have been some studies on the chemical constituents ofWuzhuyu-tang[3],the active components in it are unclear because there are a number of ingredients in the complex formula.It is difficult to study and clarify the active fractions of Chinese herbal compounds.In line with Traditional Chinese Medicine the ingredient is usually extracted with boiling water, and a popular study method combines its pharmacochemistry with a pharmacodynamic study.Systematic isolation of a chemical presents numerous difficulties, and this method has three important drawbacks: (1) it ignores the relation between the proportions of various components and efficacy; (2) it ignores the bioavailability of the drug; and (3) there is a lack of systematic pharmacodynamic investigation[4].With the aim of addressing these problems, the Serum Pharmacochemistry of Chinese Medicine was introduced by some scholars.Through understanding the relation between changes in the components in serum and efficacy, the active ingredients could be clarified[5].However, the influence of endogenous substances, and the detection limit as a result of drug metabolism make it difficult not only to determine the active ingredients, but also to obtain sufficient components for identification, pharmacological evaluation and screening[4].

    How can this complex issue be made more simple? For this, a hypothesis was proposed to include that “pharmacological ingredients should exist in the absorption ingredients”and that “the ingredients in the complex prescription exceeded that of the absorption ingredients which exceeded that of the pharmacological ingredients”.

    Based on a method of a former researcher in the field, blood samples were taken from the portal vein of rat liver at different times after oral administration for determination of the absorbed components ofWuzhuyu-tangextract in plasma and their dynamic characteristics.According to the pathogenesis of migraines and the pharmacology of theWuzhuyu-tangextracts,we preliminarily discuss the effective components which are absorbed.

    RESULTS

    Quantitative analysis of experimental animals

    A total of 60 male adult Wistar rats were randomly divided into three groups: a normal control group, a moderate dosage group and a low dosage group.Blood samples were collected from the portal vein and analyzed with liquid chromatography-mass spectrometry at 15, 30, 60, 120,and 180 minutes after administration.A total of 60 rats were included in the final analysis.

    Determination of 10 representative ingredients in samples using a combination of high performance liquid chromatography-mass spectrometry, selection of optimal conditions and specificity

    Total ion chromatography (TIC) is shown in Figures 1 and 2 with positive and negative ion scan modes, respectively.The results showed that the detection point of most ingredients was higher in positive scan mode.

    Furthermore, the various components were confirmed by fragmentation regularities of the mass spectrum in positive and negative ion scan modes to determine m/z.The extracted ion chromatogram (EIC) of the mixture in positive mode is shown in Figure 3.

    Figure 1 Positive ion mode scanning total ion chromatography of 10 mixed reference substances.The Y-axis represents ion signal intensity.Irs: Isorhamnetin-3-O-β-D-glucosyl(6″→1″′)-α-L-rhamnoside; Lcs: limocitrin-3-O-β-D-glucoside; Rg1: ginsenoside-Rg1; Rb1:ginsenoside-Rb1; Re: ginsenoside- Re; Li: limonin; Ev:evodiamine; 6-Gi: gingerol; Dhe: dehydroevodiamine; Ru:rutaecarpine.

    Figure 2 Negative ion mode scanning total ion chromatography of 10 mixed reference substances.The Y-axis represents ion signal intensity.Irs: Isorhamnetin-3-O-β-D-glucosyl(6″→1″′)-α-L-rhamnoside; Rg1:ginsenoside-Rg1; Dhe: dehydroevodiamine; Re:ginsenoside-Re; Rb1: ginsenoside-Rb1; Li: limonin; Lcs:limocitrin-3-O-β-D-glucoside; Ru: rutaecarpine.

    A specificity test showed the EIC of blank plasma where no impurities interfered in the peaks of isorhamnetin-3-O-β-D-glucosyl(6″→1″′)-α-L-rhamnoside (Irs), limocitrin-3-O-β-D-glucoside (Lcs), dehydroevodiamine (Dhe),ginsenoside-Rg1 (Rg1), ginsenoside-Re (Re),ginsenoside-Rb1 (Rb1), limonin (Li), gingerol (6-Gi),evodiamine (Ev), rutaecarpine (Ru) (supplementary Figures 1-2 online).

    Drug constituents in portal vein plasma after oral administration of different doses of Wuzhuyu-tang extract

    The TIC of the plasma samples after oral administration of different doses ofWuzhuyu-tangextract at different times is shown in Figure 4.The EIC of components in plasma at different times after oral administration is shown in supplementary Figure 3 online.The dynamic changes in the absorption ingredients at all time points are shown in Figure 5.Lcs, Dhe and Ru were not detected in either dose sample.

    Figure 3 Extracted ion chromatogram of 10 mixed reference substances.The Y-axis represents ion signal intensity.Irs: Isorhamnetin-3-O-β-D-glucosyl (6″→1″′)-α-L-rhamnoside; Lcs: limocitrin-3-O-β-D-glucoside; Dhe:dehydroevodiamine; Rg1: ginsenoside-Rg1; Re:ginsenoside-Re; Rb1: ginsenoside-Rb1; Li: limonin; 6-Gi:gingerol; Ev: evodiamine; Ru: rutaecarpine.

    Irs was detected in all samples of moderate- and lowdose groups after 30 minutes, and the concentration first increased then decreased, with a peak at 60 minutes.Rg1 was detected at all time points after moderate-dose administration, and the concentrations first increased and then decreased, while Rg1 was detected in the low-dose group after 30 and 60 minutes; the peak was at 30 minutes.Re was only detected in samples of the moderate-dose group after 30 minutes, and not in the low-dose group.Rb1 was detected in samples of the moderate-dose group after 30, 60 and 120 minutes, with a peak at 60 minutes,but was detected in the low-dose group only after 30 minutes.In the two samples after 180 minutes, there was no peak at m/z=1 132, but peaks at m/z=1 109 occurred at the same location; the results need further research on whether Rb1 was transformed in the body.

    Li was detected in samples of both dosage groups after 15 minutes, and the concentrations first increased then decreased, with a peak at 60 minutes.

    Figure 4 Total ion chromatography of plasma at different time points after administration of different doses.The Y-axis represents ion signal intensity.(A) 16.3 g/kg dose;(B) 8.15 g/kg dose.(1-5) 15, 30, 60, 120 and 180 minutes after administration.

    Figure 5 Dynamic changes of seven absorption components at 15-180 minutes (min) after administration.Results were obtained from a mixture of equal volume samples from 4 rats of each group.(1) 16.3 g/kg dose; (2)8.15 g/kg dose.Irs: Isorhamnetin-3-O-β-D-glucosyl(6″→1″′)- α-L-rhamnoside; Rg1: ginsenoside- Rg1; Re:ginsenoside-Re; Rb1: ginsenoside-Rb1; Li: limonin; 6-Gi:gingerol; Ev: evodiamine.

    6-Gi was only detected in the samples of both dosage groups after 15 and 30 minutes, and had a high value at 15 minutes.

    Ev was detected in all samples of both groups after 15 minutes, and the concentrations first increased then decreased, with peak points at 30 and 60 minutes, in the moderate- and low-dose groups, respectively.The time the majority of constituents absorbed into the blood were seen was 30 minutes to 60 minutes after administration.Re, 6-Gi and Rb1 were metabolized faster.

    DISCUSSION

    In this experiment, the representative ingredients (Ev, Ru,Li, Irs, Rg1, Re, Rb1, 6-Gi ) could be detected inEvodiae Fructus, Rhizoma Zingiberis Recens and Ginseng Radix et Rhizoma andWuzhuyu-tangextract by high performance liquid chromatography, Lcs, Dhe which can be isolated from water-soluble parts of Evodiae Fructus could not be detected in the extract.Using these as the reference substances, liquid chromatography-mass spectrometry for simultaneous determination of components was established.Using three kinds of mobile phase (acetonitrile-water-methanol) and with control of the column temperature, the samples separated well without addition of acid, alkali or buffer.The peak shape was improved through increasing the alcohol content after 35 minutes.However, the result for the composition of ginseng saponins in these conditions was not satisfactory.A higher response value of Rg1, Re[M + formic acid]for determination was obtained by adding 0.1% formic acid in the aqueous phase.The results proved that each component achieved complete separation in the selected liquid chromatography-mass spectrometry conditions, and there was no interference from the blank plasma or other components in the chromatographic peaks.The conditions were stable and reproducible, thus suitable for biological sample analysis.Methanol, acetonitrile and acetone were added in the samples as plasma protein precipitation agents and the ratio of sample:methanol of 1: 5 was chosen.The methanol was added in portions, and then ultrasonic extraction was used to ensure maximum dissolution of the components.As the components Li, Ev, Ru were easily modified, the samples were rapidly centrifuged after extraction from the blood to prevent metabolism by plasma components in storage.The plasma was mixed with methanol immediately to denature enzymes, then stored at low temperature and analyzed as quickly as possible.

    Usually, ion trap mass spectrometry is used to identify the elements in a complex mixture, but ongoing changes decreased the amount of the absorption ingredients,while there was a difference between different doses in component quantities.The results showed that 7 types of component absorbed into the blood from theWuzhuyu-tangextract could be recognized in this experimental condition.The absorption rate of Li, Ev,6-Gi, and Rg1 exceeded that of Irs, Rb1 and Re, with appearance of the components in the blood at 30 minutes to 60 minutes after extract administration.In addition, analysis of the dynamic processes of the absorption ingredients showed that Re, 6-Gi and Rb1 were metabolized faster, which is consistent with the results of previous studies[6-8].Due to time and other reasons, the production of metabolites was not examined and should be studied further.

    The major components absorbed fromWuzhuyu-tangextract have been identified; the next step is to look for the efficacious component of the absorbed components.According to previous studies, the common symptoms of migraine patients may be associated with changes in 5-HT[9].In vivo, 90% of 5-HT exists in the total digestive tract, and 5-HT2is also distributed in the eyes.Loss of 5-HT can not only lead to headaches, but also can cause nausea, vomiting or diarrhea, and visual disturbances and anomalies.In addition, migraine headache was also associated with abnormal vascular contraction and expansion[10].Most patients experienced small artery contraction, and aorta expansion.Small artery contraction can induce microcirculatory disturbance which causes hand, foot and cold chills.Artery expansion may stimulate a headache-sensitive area of the middle meningeal artery, causing a severe headache.Based on high performance liquid chromatography analysis ofWuzhuyu-tangextract, the main ingredients from Evodiae Fructus include three categories: indole alkaloids-Ev, Ru as its representatives; Ev has analgesic(with structure similar to sumatriptan succinate, a chemical drug for migraine), anti-inflammatory,anti-allergic and mild sedative effects[11].Ev can also improve serotonin transporter (5-HTT) expression[12].In addition, Ev can relax blood vessels, reduce hypoxia,increase then decrease body temperature, and inhibit gastric emptying and gastric transit in male rats;Lactones-Li as its representative, can regulate the rhythm of vascular endothelial cells, improve microcirculation and has anti-platelet aggregation,anti-inflammatory, and anti-nociceptive properties[13];Flavonoids-Lcs, Irs as its representatives, can suppress platelet aggregation, reduce capillary permeability, and is anti-inflammatory[14].The above-described components have potential values in various aspects of migraine pathology.Apart from Ru and Lcs, they are confirmed to be active ingredients[15-17].

    The main components derived from Ginseng Radix et Rhizoma: Saponins-Rg1, Re, Rb1 as its representatives,play an important role in blood rheology, are anti-inflammatory and can protect brain cells[18-19].They also have sedative[20]and anti-stress effects[21], enhance immunity[22], and repair the damage caused by migraine headaches.The main active component from Zingiberis Rhizoma Recens, gingerol, inhibited vomiting-inducing cyclophosphamide completely by oral administration; it can antagonize 5-HT and may exert its antiemetic by regulating the 5-HT3receptor[23].It also has analgesic,antipyretic, antioxidative effectsin vitroandin vivoand may be of value in treating the major symptoms accompanying migraine[24].

    Thus, seven different compounds of the absorption ingredients, namely Irs, Ev, Li, Rg1, Re, Rb1 and 6-Gi,are likely to have a pharmacodynamic effect on all aspects of migraine.

    The liquid chromatography-mass spectrometry measurement of the 10 components was established to to enable the fast measurement and dynamic detection of microconstituents in plasma.Among the absorbed compounds, Irs and Li had not been reported in previous studies, either in theWuzhuyu-tangformula or in each of its elements.Thus, it is possible and easy to determine the active ingredients based on analysis of the absorbed components.

    MATERIALS AND METHODS

    Design

    Pharmacokinetics of Chinese herbal compound components.

    Time and setting

    The experiment was performed at the School of Traditional Chinese Medicine, Capital Medical University,China, from July 2009 to July 2010.

    Materials

    Animals

    A total of 60 male Wistar rats, aged 4 months, weighing 270-310 g, were obtained from the Vital River Laboratory Animal Technology Co.Ltd., China (Certification No.SCXK (Jing) 11-00-0006).Protocols were conducted in accordance with theGuidance Suggestions for the Care and Use of Laboratory Animals, formulated by Ministry of Science and Technology of the People’s Republic of China[25].

    Medicinal components

    Evodiae Fructus was purchased from Hunan B&A Scientific Chinese Herbal Medicine Co., Ltd., lot number Guangxi050803; it was identified as the fruit ofEvodia rutaecarpa(Juss.) Benth., and the control sample was preserved in School of Traditional Chinese Medicine,Capital Medical University, with serial number WZY-0709.Ginseng Radix et Rhizoma was purchased from JI’AN City Xinkaihe Co., Ltd., Jujubae Fructus was purchased from Xinzhen city, Henan province, and the control sample was preserved in our laboratory, the serial number is RS-0803 and DZ-0709 respectively.Zingiberis Rhizoma Recens was purchased from Beijing.All of medicinal components were identified to conform with the stipulations under each type inPharmacopoeia of the People’s Republic of China, issued by the China Pharmacopoeia Committee (2005)[26].

    Preparation of Wuzhuyu-tang extract

    Aliquots (1 400 g) equivalent to 10 times the prescribed human daily dose were added to water (1: 10) and soaked for 30 minutes at room temperature, then decocted twice for 40 and 30 minutes.Precipitation was performed in 70% alcohol, and then stored for 24 hours at 0-4°C.After leaching, ethanol was retrieved under 60°C, followed by vacuum drying.The assay results of representative constituents in the extract are shown in Table 1.

    Table 1 Content of representative components in Evodiae Fructus, Ginseng Radix et Rhizoma, Zingiberis Rhizoma Recens and Wuzhuyu-tang extract

    Methods

    Animal treatment

    Rats were allocated to one of three groups then fed for 7 days for adaptation, and then fasted for 12 hours, with free access to water before the experiment.Two concentrations of suspension ofWuzhuyu-tangextract were prepared for administration.Rats were given normal saline, or 3 mL ofWuzhuyu-tangextract equivalent to 16.3 g/kg or 8.15 g/kg of raw material.

    Sampling

    After administration, rats were intraperitoneally anesthetized with pentobarbital sodium.Samples were taken from portal vein blood and collected in heparin infiltration-off centrifuge tubes after 15, 30, 60, 120, and 180 minutes.Samples were immediately centrifuged at 3 000 r/min for 10 minutes, and separated from plasma.An equal amount of methanol was added and the sample vortexed for 1 minute to inactivate enzymes, followed by storage at -20°C for analysis within one week.Equimolar amounts of plasma of four rats were mixed and methanol was added, then subjected to ultrasound, and centrifuged for 10 minutes at 10 000 r/min at 4°C.Samples were dried with nitrogen for about 30-40 minutes.The residue was dissolved using ultrasound at a constant volume of 2 mL with methanol, filtered through a 0.22 μm membrane, and 3 μL of extract was used for injection.

    Analysis of blood samples from portal vein of rats

    Chromatographic conditions: ZORBAX Extend C18 column (Agilent Inc., Santa Clara, CA, USA, 4.6 mm×250 mm, 5 μm); gradient elution using a mobile phase consisting of methanol-acetonitrile-0.1% formic acid solution at a flow rate of 1 mL/min at a column temperature of 30°C, and injection volume of 3 μL, split ratio 7: 3.The gradient is shown in Table 2.

    Table 2 Mobile phase composition in high performance liquid chromatography-mass spectrometry determination of absorbed components in the blood

    Mass spectrometry conditions: Mass Range Mode: Ultra Scan; Ion Polarity: Positive; Ion Source Type: ESI; Dry Temp (Set): 350°C; Nebulizer (Set): 50 psi; Dry Gas (Set):N2,10 L/min; Scan range: 100-1200 m/z; capillary voltage: 3 500 V.

    Methodological investigation

    Detection mode selection: The mixed reference substances (Irs, Lcs, Dhe, Rg1, Re, Rb1, Li, 6-Gi, Ev,Ru), their source and purity testing are as follows:

    ubstance Source Ev, 110802-200505,Ru, 110801-200504,Li, 110800-200404,Rb1, 110704-200318,Rg1, 110703-200322,Re, 110754-200217 National Institute for the Control of the Pharmaceutical and Biological products,China Lcs, purity ≥ 95%,Irs, purity ≥ 98%,6-Gi, purity ≥ 98%,Dhe, purity ≥ 98%Newly synthesized, purity inspection was assessed under two different high performance liquid chromatography conditions with DAD, and then calculated with statistical analysis.

    TIC was obtained with positive and negative ion scan modes.

    Detection of select m/z: The various components were confirmed by the fragmentation regularity of three-grade mass spectrum in positive and negative ion scan mode,the results are shown in supplementary Figures 1-4 online.The detection response was strong for the majority of constituents.EIC of the mixture in positive ion mode was obtained.

    Specificity: The blank plasma was analyzed according to the m/z of the reference sample of the component under test.The impurity in the peaks of Irs, Lcs, Dhe, Rg1, Re,Rb1, Li, 6-Gi, Ev, and Ru was determined.

    Author contributions: Muxin Gong and Jian Sun had full access to all data and participated in data integrity and data analysis.Muxin Gong and Yaxun Wang participated in data collection.Qiwei Zhang and Zhimin Wang participated in study design, study supervision, and manuscript development.

    Conflicts of interest: None declared.

    Funding: The present study was financially supported by the Scientific Research Program of Beijing Municipal Education Commission-Capital Key Research Program of Chinese Medicine and Nursing, No.10ZYH01; the Supporting Program of the “Eleventh Five-year Plan” for Science and Technology Research of China, No.2006BAI08B03.

    Ethical approval: All animal experiments were approved by the Animal Ethics Committee of Capital Medical University in China.

    Supplementary information: Supplementary data associated with this article can be found in the online version, by visiting www.nrronline.org, and entering Vol.6, No.33, 2011 after selecting the “NRR Current Issue” button on the page.

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