王 棟,楊 歡,楊光明,樓成華,童 麗,蔡寶昌＊

1南京中醫(yī)藥大學(xué)藥學(xué)院,南京 210046;2江蘇省中藥炮制重點(diǎn)實(shí)驗(yàn)室,南京 210029; 3青海大學(xué)醫(yī)學(xué)院藏醫(yī)藥研究中心,西寧 810001

Introduction

GenusOxytropis,including 300 species[1],grows in the high latitude zones of the world,especially in Russia, America,China and Canada.In China,the entire plant ofOxytropis falcateBunge,which was called“E Da Xia”,has always been widely used as traditional Tibetan and Mongolian medicine[2].The aglycones of total flavonoids fromO.falcateBunge also have good effect on treatment for chronic bronchitis[3].In addition,the alkaloid ofO.falcateBunge can restrain growth of tumor cells.Swainsonine as an anticancer drug has entered Phase II clinical study.

In the past several years,this species had been widely investigated from a chemical point of view[4-13].In this study the volatile compositions fromO.falcateBunge were extracted by water distillation,supercritical CO2and headspace extraction techniques and analyzed by GC and GC/MS.As far as our knowledge,it is the first report about the analysis of the volatile constituents fromO.falcateBunge in three differentmethods.

Exper imental

Plantmaterial

The plants ofO.falcateBunge were gathered at the full flowering stage from plants wild growing at Bangor County in Tibet autonomous region(35°47′N,88°17’E,4700 m,China),in May 2007,and identified by Prof.Chen Jian-wei,College of Pharmacy,NanjingUniversity of Chinese Medicine.A voucherspecimen (OX:200705)was dried in the shade,chopped,then put into the glass dryer for preservation and experiment,Jiangsu KeyLaboratory of ChineseMedicine Processing.

Reagents and apparatus

All standards were purchased from National Institute. Commercial carbon dioxide(purity 99%,Tongguang Co.,Ltd.Nanjing,China).Mettler-Toledo AE240 E-lectron analytical balance(0.01 mg).HA221-50-06 Supercritical fluid extraction apparatus(Huaan SFE Co.,Ltd.Nantong,China).Agilent 4890D gas chromatograph(Agilent GmbH,USA)and Hewlett-Packard 6890D-MSD 5975B(Agilent GmbH,USA),software ChemStation(version D.03.00.611,Agilent GmbH, USA)and automatic sampling device of headspace extraction(G1888,USA).

Volatile chem ical compositions extraction

W ater distillation extraction

The oil from 240 g air-dried aerialpartsof plantswas isolated bywater distillation with cohobation for 5 h according to the method recommended in the Pharmacopoeia of China[14].The yellow oil was calculated on dryweight basis corresponding to 0.23% (v/w)and dried over anhydrous sodium sulphate,and it was sealed,frozen,stored at 4℃ in the dark for GC and GC/MS analysis.

Supercritical CO2extraction

Supercritical CO2extraction was perfor med in a laboratory apparatus,equipped with a 200 cm3 separator vessels connected in series.The driedO.falcateBunge powder(120 g,20 mesh)was extracted for 2 h under the supercritical conditions:Pressure,25 MPa,Temperature,313 K.The flow rate of CO2was 45 L/h.The separation of extractwas carried out at 4.4MPa and at 301 K.The oily extract was put into a mixed solution (distilled water and ether)three times,and then the ether extraction was dried at low temperature.The end product(0.637 g)was solid and dark yellow and stored at 4℃in the dark for GC and GC/MS analysis.

Headspace extraction

O.falcateBunge sample powder 6.0 g(40 mesh)was placed in a teflon vial to equilibrate for 30 min(100℃)before sampling.The needle of automatic sampling device was conditioned in a GC injector at 250℃for 0.5 h,prior to use in order to remove contaminants. The needle was pierced through the septum of 20 mL vial.Once extraction equilibrium reached for 30 min, the needle would immediately transfer the extraction to the GC injection port.The needle was exposed so that the volatile compounds were ther mally desorbed for 4 min in an injection port held at 250℃ and deposited onto the column on which subsequent chromatographic analysiswasperformed according to our GC conditions.

GC analysis

Analytical gas chromatography was carried out on an Agilent 4890D gas chromatograph fitted with a HP-5 capillary column(30 m ×0.53 mm),2.65μm fi lm thickness.Nitrogen was the carrier gas(1.0 mL/ min).Column temperature was initially kept at 70℃for 3 min,gradually increased to 230℃ at 4℃/min rate,then to 240℃at 1℃/min rate and finally raised to 290℃at 4℃/min rate(held for 5 min).Diluted samples oil(1/1000,v/v,in ether)of 1.0 mL extracted from water distillation,supercritical CO2extraction were manually injected at 250℃,and Flame Ionization Detection(F ID)was performed at 290℃.

GC/M S analysisl

GC/MS analysis was performed on a Hewlett-Packard 6890D apparatus coupled to a HP mass/selective detector(MSD 5975B HP),fitted with a HP-5MS capillary column(30 m ×0.25 mm),0.25μm film thickness;ionization voltage 70 eV,mass range m/z:30-550 amu;electron multiplier energy 2000 V.The sample(1 μL)was injected in the split mode(1∶15).Helium (99.999%,1.0 mL/min)was used as carrier gas.Gas chromatographic conditionswere perfor med on the basis of GC.

Qualitative analysis

Partial constituents were identified under the same chromatographic conditions by GC by comparison of their GC retention timeswith those of the literatures or with those of standards available in our laboratories. Further identification was made by comparison of their mass spectra collected with N IST.05 or previous reports[7-10].Component relative amountswere calculated based on GC peak areas normalization method without using correction factors.

Table 1 Volatile chem ical compositions fromO.falcateBunge

Note:Content(%):the ratio of the peak area of a compound to total peak area;Compounds less than 0.1%are not reported;-:not detected.All constituentswere identified byN IST.05;(1)identified by literatures or standards;(2)identified by our previous reports.

Results and D iscussion

Although no apparent toxic substance was found in our study,the genusOxytropisplantswere reported to have strong stimulating effecton the human eyes and respiratory system,and cause eyes congestion,headache and throat discomfort.It is necessary to study on their volatile components[15].The main a im of this study is to examine the volatile compositions fromO.falcateBunge in China.

Overall,58 compounds were identified from the aerial partsofO.falcateBunge bywater distillation,supercrit-ical CO2and headspace extraction methods.The percentage of compounds is given in Table 1,where the compounds are listed in order of elution on the HP-5MS column.Our results agreed that Paraffin hydrocarbon compounds amounted 30.1%of the oil extracted bywater distillation.The majority of the volatile products extracted by supercritical CO2extraction were flavonoids,which accounted for 68.5%.Headspace extraction analytic results showed that aldehyde compounds 53.8%and organic acid 18.6%were the principal volatile compositions.In addition,micromolecular terpenoids,esters,aldehydes and nitrogenous compounds were also identified in the analyzed volatile products.

According to our results,different extraction methods led to special selectivity for extracting volatile compositions of each sample.In our study,the oil from water distillation was characterized by a high content of heneicosane(22.2%),6,10,14-tr imethyl-2-pentadecanone(5.4%),2-methylbenzyl cyanide(5.1%),3,7-dimethyl-1,6-octadien-3-ol(3.7%) (Fig.1).The comparison with the water distillation oil revealed that the significative difference of supercritical CO2extraction was the content of flavonoids,among which,5-hydroxy-2-phenyl-4-chromanone (21.3%),2,4-dihydroxyphenyl-2-phenylethyl ketone(18.9%),2′,4′-dihydroxy-chalcone(17.4%),and 2,3-dihydro-5,7-dihydroxy-2-phenyl-4H-1-benzopyran-4-one(10.9%),

Fig.3 Volatile Compositions from O.falcateBunge (headspace extraction)

were the major compounds(Fig.2).Meanwhile,HSGC/MS analysis revealed the presence of the aldehyde compounds(53.8%),e.g.2-methyl-propanal(24. 4%),2-methyl-butanal(22.6%),benzaldehyde(6. 8%)(Fig.3).

In spite of quantitative differences,we found that retention times of the volatiles extracted using three methods were consecutive which could provide a comprehensive volatile composition ofO.falcateBunge.Additionally, we concluded that headspace extraction was the most effective and time-saving technique of the three extraction methods and suitable for screening micromolecular compounds,such as organic acids and aldehyde compounds.However,in our study we found the main disadvantage of headspace extraction methodswas low extraction yield.In order to optimize the extraction yield, we chose 20 and 40 mesh dried sample powders,equilibrated for 30 min(100℃)before sampling and completely ther mally desorbed for 4 min(250℃).

The conditionsof supercriticalCO2extractionwere chosen based on previous report on similar material[16].In order to extract the volatile compositions completely,we perfor med on a condition(Pressure,25 MPa;Temperature,313 K)and chose appropriate organic reagents to re-extract the supercritical CO2extraction products to remove non-volatile substances.After comparing with three organic reagents(petroleum ether,ether and chloroform),we finally decided to use ether to remove the non-volatile impurities.This workable method was speedy,convenient and labor-saving.

Zheng Shang-zhen previously reported 2-pheny1-5,7-dihydroxy flavanone as principal compound in essential oil ofO.falcateBunge extracted by petroleum ether[13].In this study,we also found flavonoids were the major compounds in volatile product of supercritical CO2extraction.In addition,our recent research found that flavonoid compounds had prominently effective biological activities[17],such as anti-inflammatory,analgesia and so on.2′,4′-Dihydroxy-chalcone detected fromO.falcateBunge could also effectively restrain growth of tumor cells.These resultswere similar to the clinical usage ofO.falcateBunge for the treatment of fever and cold as a medicinal herb.So we suggest that flavonoid compounds ofO.falcateBunge should be studied in detail in the future.

Conclusion

Comparison of the volatiles extracted by three methods showed that the compositions fromO.falcateBunge were complex and were only a portion using a single method.So water distillation,supercritical CO2and headspace extraction techniques coupled to GC and GC/MS are perfor med on identification of volatile compositions.Combining utilization of the above three methods improves the reliability of analytic results and could be used for examining the volatile compositionsof plantmedicine.

Acknowledgment The GC and GC/MS spectra were perfor med at Nanjing University of Chinese Medicine, China.The assistance of the staff is gratefully appreciated.Thanks are also due to Dr Yu Sheng and Dr Deng Hai-shan for their guidance,advises and useful suggestions.

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