劉淑萍,王蕾(華北理工大學化學工程學院,河北唐山063009)

栗蓬抗菌成分提取工藝的優(yōu)化與聯(lián)合測定

劉淑萍*,王蕾
(華北理工大學化學工程學院,河北唐山063009)

摘要正交試驗優(yōu)化栗蓬抗菌成分的提取工藝,進一步對提取液進行分級醇沉及去除蛋白操作后,采用反相高效液相色譜(reversed-phase high-performance liquid chromatography,RP-HPLC)法對所提抗菌成分進行定性及聯(lián)合定量測定.結(jié)果表明,在最優(yōu)條件下,一次的提取率可達88.2%.采用Lichrospher C(18)色譜柱(150 mm×4.6 mm,5 μm),以甲醇0.1%的磷酸水溶液為流動相,梯度洗脫程序,檢測波長為280 nm,流速1 m L/min,柱溫30℃可較好地測定栗蓬中沒食子酸、原兒茶酸、槲皮素的含量.各成分質(zhì)量濃度與峰面積在測定范圍內(nèi)均呈良好的線性關(guān)系(r＞0.999),平均加樣回收率(n=9)分別為98.9%、96.0%和97.1%,相對標準偏差(relative standard deviation,RSD)分別為2.36%、0.77%和4.73%.

關(guān)鍵詞栗蓬;抗菌成分;提取;聯(lián)合測定

浙江大學學報(農(nóng)業(yè)與生命科學版) 42(1):23～29,2016

Journal of Zhejiang University(Agric.&Life Sci.)

http://www.journals.zju.edu.cn/agr

E-mail:zdxbnsb@zju.edu.cn

URL:http://www.cnki.net/kcms/detail/33.1247.S.20151215.1754.010.html

Optimization of the extraction process and combined determination of antibacterial ingredients of Li Peng.Journal of Zhejiang University(Agric.&Life Sci.),2016,42(1):23-29

LIU Shuping*,WANG Lei(College of Chemical Engineering,North China University of Science and Technology,Tangshan 063009,Hebei,China)

Summary Li Peng is the Chaetomium of chestnut(involucre of plant chestnut),also called the involucre of chestnut,thorn shell of chestnut,or Sarg.of chestnut etc.In the areas rich in Chinese chestnut,large amounts of waste Li Peng was produced,besides a small amount of which can be used for firewood,the rest will be discarded to rot or burned;however,it is not only a waste of resources,but also cause serious environmental pollution.Reports showed that Li Peng could be used as a raw material to cultivate chestnut mushrooms,but it was only an accessory ingredient,and the main ingredient was cotton seed shell of chestnut tree,so Li Peng has yet to be fully used.In some areas,the phenomenon of hyphae overgrowth occurred in chestnut mushrooms cultivated by Li Peng,the reason was due to its antibacterial effect.

The purpose of this paper was to establish a simple and low-cost technique for extraction of antibacterial components from Li Peng.At the same time,a high sensitive,good repeatable and stable,simple and rapid method was used to determine the antibacterial ingredients of Li Peng.

This paper used the single factor experiment and the orthogonal experiment optimization for the extraction process of antibacterial components in Li Peng.Furthermore,polysaccharide in the samples was precipitated byethanol and the qualitative and quantitative combined determination of antibacterial components were carried out using reversed-phase high-performance liquid chromatography(RP-HPLC)method after protein removal.

The results showed that the best way to extract the antibacterial composition of the 60-mesh fine slag in Li Peng was under 1∶40 material liquid ratio,70 W ultrasonic reflux 20 min.Using Lichrospher C18column (150 mm×4.6 mm,5μm),methanol-0.1%phosphoric acid solution as the mobile phase,the flow rate of 1 m L/min,the detection wavelength at 280 nm,column temperature of 30℃with the gradient elution program,the gallic acid,protocatechuic acid,and quercetin content in Li Peng could be better determined.The quality concentrations of each component with peak area showed a good linear relationship(r＞0.999)in the measurement range.The average sample recovery(n=9)rate were 98.9%,96.0%,97.1%.The relative standard deviation(RSD)were 2.36%,0.77%,4.73%.

We optimized the extraction of antibacterial ingredients in Li Peng.By simple operation,low energy consumption and high extract efficiency method increased the feasibility of this technology,and provides theoretical basis of mass industrial production for the extraction of antibacterial ingredients in Li Peng.Under the optimum conditions,we can extract 88.2%antibacterial components from chestnut at one time.The antibacterial component of gallic acid,protocatechuic acid,quercetin content in Li Peng was 0.176 0 mg/g(RSD=0.23%),0.053 7 mg/g (RSD=0.16%),0.019 6 mg/g(RSD=0.21%),respectively.The RP-HPLC is a high sensitive,good repeatable and stable method,which is also simple,convenient and accurate.This paper enriched the reference basis for the combined determination of the antibacterial components of Li Peng.

Key words Li Peng;antibacterial constituents;extraction;combined determination

栗蓬為板栗毛殼(植物栗的總苞),又稱板栗總苞等.據(jù)焦啟揚等[1]報道栗蓬含有大量抗菌成分,具有很高的藥用價值.現(xiàn)代藥理研究表明:栗蓬的水煎液、醇溶物、乙酸乙酯提取物等均有不同程度的抗菌活性[2-4].其中沒食子酸(gallic acid)、原兒茶酸(protocatechuic acid)作為有機酸中的主要有效成分,以及黃酮類化合物槲皮素(quercetin),都具有抗真菌、抗感染、抗病毒、抗腫瘤等藥理活性及藥用價值[1,5-6].因此,對其提取測定具有實際意義并可代表抗菌成分的提取效果.對以上3種物質(zhì)現(xiàn)有測定的方法有流動注射化學發(fā)光法、高效液相色譜法、紫外分光光度法等[7 12];然而對于在栗蓬中這3種物質(zhì)的同時測定方法還未見報道.因此,本試驗采用反相高效液相色譜(reversed-phase high-performance liquid chromatography,RP-HPLC)法,以梯度洗脫程序?qū)跖钪袥]食子酸、原兒茶酸、槲皮素的含量進行測定.RP-HPLC具有分析速度快、分辨率和靈敏度高、分離效果好等特點,而且樣品經(jīng)過色譜柱后不被破壞,可以為栗蓬的進一步研究提供基礎(chǔ)數(shù)據(jù).

在盛產(chǎn)板栗的地區(qū),會產(chǎn)生大量的栗蓬廢棄物,除少量用作燒柴外,其余則被丟棄腐爛,這樣不僅浪費資源,還造成嚴重的環(huán)境污染.據(jù)報道未處理的栗蓬可作為原料培育栗蘑,但其僅為配料,主料仍是栗樹枝棉籽殼等[13],栗蓬仍未得到充分利用.因此,研究對栗蓬中抗菌成分提取的優(yōu)化工藝和測定是必要的.根據(jù)現(xiàn)有的板栗總苞化學成分的提取方法[14-19],本文采用正交試驗法對栗蓬中抗菌成分的提取進行了優(yōu)化.

1　材料與方法

1.1試驗材料與試劑

試驗材料:栗蓬采集于2014年唐山市遷西縣,板栗收獲采集后,風干栗蓬,破碎過60目篩.

試劑:甲醇(色譜純,天津市四有精細化學品有限公司),乙醇(河北建寧醫(yī)藥化工廠),原兒茶酸對照品(上海如吉生物科技發(fā)展有限公司,純度≥98%),沒食子酸(分析純,天津市科密歐化學試劑有限公司),槲皮素對照品(純度98%,上海如吉生物科技發(fā)展有限公司),磷酸(分析純),超純水.

1.2主要儀器設(shè)備

JY99-2D超聲波細胞粉碎機(寧波新芝生物科技股份有限公司);日立L-2000型高效液相色譜儀(包括L-2130高通量泵,L-2200自動進樣器,L-2300柱溫箱,L-2455二極管陣列檢測器).

1.3栗蓬抗菌成分的提取與優(yōu)化

取60目的板栗總苞,至圓底燒瓶內(nèi),加入一定體積的甲醇溶液,在一定的超聲波功率下提取一定時間,過濾殘渣,定容于容量瓶中備用.按此步驟,研究各條件對栗蓬中抗菌成分提取率的影響.

通過4因素3水平[L9(34)]正交試驗,以提取率為考核指標,進行優(yōu)化提取.

1.4提取液中多糖和蛋白質(zhì)的去除

提取液濃縮后,以90%乙醇對其醇沉12 h.取多次醇沉后的樣液按體積4∶1加入Sevage試劑,劇烈振蕩后離心,收集上清液,重復多次,直到?jīng)]有乳白色變性蛋白質(zhì)析出.

1.53種抗菌成分的定性及定量技術(shù)

1.5.1色譜條件的確定

在1.3節(jié)中所確定的最優(yōu)提取條件下,并經(jīng)過1.4節(jié)步驟去除多糖和蛋白質(zhì)后,得到待測提取液.

C18填料與硅膠基質(zhì)相比,具有更強的疏水性,反相色譜的流動相需選擇極性較強的溶液,因此本試驗選用Lichrospher C18色譜柱,以甲醇0.1%的磷酸水溶液為流動相對沒食子酸、原兒茶酸、槲皮素的標準品進行波長掃描,結(jié)果顯示均在280 nm附近有最大吸收,故選擇280 nm為3個成分的檢測波長.

比較了3種流速,結(jié)果顯示流速為0.5 m L/min時,各峰之間可以完全分離,但耗時太長;流速為1.5 m L/min及以上時,沒食子酸、原兒茶酸、槲皮素分離度較低;流速為1 m L/min時,12 min以內(nèi)3種成分可得到分離,且分離度較好.

柱溫較低時,由于采集時間長,后面洗脫出來的峰漂移嚴重;柱溫太高,長時間操作容易對柱子造成損傷.所以選擇柱溫為30℃,既解決了漂移問題,又延長了柱子的壽命.

3種成分由于結(jié)構(gòu)上的差異,選擇多種單一體系的流動相試驗,均不能將這3種成分同時分開,且干擾嚴重,分離度不能滿足測定需求.根據(jù)沒食子酸的極性最強,原兒茶酸次之,槲皮素極性最弱的性質(zhì),選擇梯度洗脫程序,先用甲醇0.1%的磷酸水溶液(15∶85),再將流動相比例線性改變至55∶45,將栗蓬中極性較小的物質(zhì)全部洗脫出來,使3種成分得到滿意的分離,兼顧了分離度和較好的出峰時間.

以此確定色譜條件為Lichrospher C18色譜柱(150 mm×4.6 mm,5μm);甲醇0.1%的磷酸水溶液為流動相;梯度洗脫程序(表1);檢測波長為280 nm;流速1 m L/min;柱溫30℃;進樣量10μL對提取液中沒食子酸、原兒茶酸、槲皮素進行測定.

1.5.2標準曲線的繪制

在1.5.1節(jié)色譜條件下,分別精密量取系列質(zhì)量濃度的混合對照品溶液10μL,注入液相色譜儀,記錄色譜圖.以峰面積為縱坐標(y),對照品質(zhì)量為橫坐標(x),進行線性回歸.

1.5.3精密度、穩(wěn)定性、重復性、加樣回收率試驗

1)精密度:精密量取供試樣品溶液,測定沒食子酸、原兒茶酸和槲皮素的峰面積,分別計算3種成分的相對標準偏差(relative standard deviation,RSD) (n=6).

2)穩(wěn)定性:精密量取供試品溶液,在常溫下放置24 h,在0、3、6、9、12、24 h分別進樣10μL,測定沒食子酸、原兒茶酸和槲皮素的峰面積,分別計算3種成分的RSD(n=6).

3)重復性:精密稱取栗蓬樣品6份,制備供試樣品溶液,測定沒食子酸、原兒茶酸和槲皮素的峰面積,分別計算3種成分的RSD.

4)加樣回收率:取栗蓬樣品9份,每份1 g,精密稱量,每3份1組,按高、中、低3個水平分別精密加入對照品溶液,制備供試樣品溶液,按1.4節(jié)去除多糖和蛋白質(zhì)后,分別測定沒食子酸、原兒茶酸和槲皮素的峰面積,計算平均回收率.

1.5.4樣品測定

取栗蓬樣品3份,按1.3節(jié)所確定的最優(yōu)提取條件制備供試樣品溶液,進樣測定,每份平行測定9次,以外標法計算沒食子酸、原兒茶酸、槲皮素的含量.

2　結(jié)果與分析

2.1栗蓬抗菌成分的提取技術(shù)

2.1.1栗蓬粒度對抗菌成分提取效果的影響

按1.3節(jié)試驗方法與步驟,改變栗蓬的目數(shù),考察其對抗菌成分(均以原兒茶酸為例)提取效率的影響.試驗結(jié)果(圖1)表明,當粒度大于50目時,抗菌成分提取率達到最大值(61.2%),這是由于隨著顆粒度的減小,比表面積增大,與提取劑接觸面增大,有利于抗菌成分溶脹浸出.選擇栗蓬最佳粒度值為60目.

2.1.2提取劑的選擇對抗菌成分提取效果的影響

按1.3節(jié)的試驗方法與步驟,改變栗蓬的提取劑種類,考察其對抗菌成分提取效率的影響.試驗結(jié)果見表2.

由表2數(shù)據(jù)可得,不同提取劑的提取率有較大差別.以水提取時雜質(zhì)較多,出峰復雜,分離較繁瑣,不利于定量分析,根據(jù)提取率的大小優(yōu)選甲醇.

2.1.3助提方法效果對比

按改變栗蓬的助提方法,考察其對抗菌成分提取效率的影響.試驗結(jié)果(表3)表明,在相同的試驗時間內(nèi)熱回流提取的抗菌成分較冷浸、溫浸高,而超聲波助提法在短時間內(nèi)能達到與熱回流相當?shù)牧?提取時間卻減少了72 min,效率明顯提高.這是由于超聲波的機械效應(yīng)、空化效應(yīng)與熱效應(yīng)等,使細胞壁上的有效成分更快地溶解于溶劑之中.以上所述,確定最佳提取方法為超聲波助提.

2.1.4提取工藝的正交試驗優(yōu)化

由表4的試驗數(shù)據(jù)表明,4因素均對抗菌成分提取有不同程度的影響,各因素影響的主次順序為D＞A＞C＞B.由正交試驗結(jié)果RD最大可知,時間的改變對試驗結(jié)果的影響最大,而甲醇濃度對提取率的影響最小;根據(jù)K值確定出最佳提取方案為A2B2C2D3.該條件可作為栗蓬中抗菌成分超聲波提取的最佳工藝條件.

在優(yōu)化條件下以提取4次的總和為抗菌成分含量,一次提取的提取率可達88.2%.

2.23種抗菌成分的定性及定量結(jié)果

2.2.1標準曲線的制備

按1.5.2節(jié)的試驗步驟和方法,繪制峰面積與質(zhì)量的工作曲線,得3種成分的回歸方程(表5).

2.2.2精密度、穩(wěn)定性、重復性、加樣回收率試驗結(jié)果

按1.5.3節(jié)試驗步驟測得結(jié)果(表6)表明,該工藝精密度與重復性良好,平均加樣回收率在96.0%～98.9%之間,供試品溶液在24 h內(nèi)相對穩(wěn)定.此方法準確可靠,可作為栗蓬中抗菌成分測定的參考依據(jù).

2.2.3抗菌成分測定結(jié)果

沒食子酸、原兒茶酸、槲皮素的混合對照品與栗蓬提取液的色譜圖(圖2)顯示,沒食子酸、原兒茶酸、槲皮素標準品的出峰時間分別為5.86、9.70和11.51 min,而樣液的出峰時間5.88、9.79和11.53 min,與標準品一致,據(jù)此可判斷各成分含量,試驗結(jié)果見表7.

3　結(jié)論

1)栗蓬中抗菌成分在最優(yōu)提取條件下,以提取4次的總和為抗菌成分含量,在最優(yōu)條件下提取一次,能將88.2%的抗菌成分提取出來.

2)RP-HPLC方法,梯度洗脫程序,同時聯(lián)合測定栗蓬中抗菌成分(沒食子酸、原兒茶酸、槲皮素成分)的含量.抗菌成分沒食子酸、原兒茶酸、槲皮素含量分別為0.176 0 mg/g(RSD=0.23%)、0.053 7 mg/g (RSD=0.16%)、0.019 6 mg/g(RSD=0.21%),此方法靈敏度高,重復性和穩(wěn)定性良好,測定簡便、快速,結(jié)果準確,豐富了栗蓬抗菌成分聯(lián)合測定的參考依據(jù).

參考文獻(References):

[1]焦啟揚,吳立軍,黃建,等.板栗總苞化學成分的分離與鑒定.沈陽藥科大學學報,2009,26(1):23-26.JIAO Q Y,WU L J,HUANG J,et al.Chemical constituents from the involucre of Castanea mollissima Blume.Journal of Shenyang Pharmaceutical University,2009,26(1):23-26.(in Chinese with English abstract)

[2]張琳,高慧媛,馬場正樹,等.板栗總苞中的抗糖尿病活性成分.沈陽藥科大學學報,2010,27(7):530-532.ZHANG L,GAO H Y,MASAKI B,et al.Constituents with anti-diabetic activity from the involucre of Castanea mollissima Blume.Journal of Shenyang Pharmaceutical University,2010,27(7):530-532.(in Chinese with English abstract)

[3]劉淑萍,陳立棉.HPLC法同時測定板栗總苞中3種黃酮苷元含量.天然產(chǎn)物研究與開發(fā),2013,25(11):1541-1544.LIU S P,CHEN L M.Simultaneous determination of three flavone glycosides in chestnut involucre by HPLC.Natural Product Research and Development,2013,25(11):1541-1544.(in Chinese with English abstract)

[4]石恩慧,李紅,谷明燦,等.板栗總苞多酚純化工藝優(yōu)化及其對細胞活力的影響.中國食品學報,2013,13(6):134-140.SHI E H,LI H,GU M C,et al.Purification technology of polyphenols from Castanea mollissima Blume with macroporous absorbent resin.Journal of Chinese Institute of Food Science and Technology,2013,13(6):134-140.(in Chinese with English abstract)

[5]周家駒,謝桂榮,嚴建新.中藥原植物化學成分手冊.北京:高等教育出版社,2004:9.ZHOU J J,XIE G R,YAN J X.Traditional Chinese Medicines Molecular Structures,Natural Sources and Applications.Beijing:Higher Education Press,2004:9.(in Chinese)

[6]張連茹,陳喀林,林路,等.鋅(Ⅱ)沒食子酸的配位行為及其與DNA的相互作用.武漢大學學報(理學版),2004,50(2):234-238.ZHANG L R,CHEN K L,LIN L,et al.Study on the complexes of gallic acid-Zn(Ⅱ)and the interaction with DNA.Journal of Wuhan University(Natural Science Edition),2004,50(2):234-238.(in Chinese with English abstract)

[7]PHAKTHONG W,LIAWRUANGRATH B,LIAWRUANGRATH S.Determination of gallic acid with rhodanine by reverse flow injection analysis using simplex optimization.Talanta,2014,130(1):577-584.

[8]DENDERZ N,LEHOTAY J.Using of molecularly imprinted polymers for determination of gallic and protocatechuic acids in red wines by high performance liquid chromatography.Journal of Chromatography A,2014,1372:72-80.

[9]YAO Y Y,ZHANG L,WANG Z F,et al.Electrochemical determination of quercetin by self-assembled platinum nanoparticles poly(hydroxy-methylated-3,4-ethylenedioxythiophene) nanocomposite modified glassy carbon electrode.Chinese Chemical Letters,2014,25(4):505-510.

[10]郭增軍,孫啟時,龍麗輝,等.九牛造中總鞣質(zhì)提取方法和沒食子酸含量測定研究.中藥材,2007,30(11):1398-1401.GUO Z J,SUN Q S,LONG L H,et al.Studies on the methods of extract and determination of total tannins and gallic acids of the root of Euphorbia hylonoma.Journal of Chinese Medicinal Materials,2007,30(11):1398-1401.(in Chinese with English abstract)

[11]顏流水,溫振東,黃智敏,等.毛細管電泳法測定金銀花中綠原酸、蘆丁和槲皮素.藥物分析雜志,2007,27(3):367-370.YAN L S,WEN Z D,HUANG Z M,et al.CE determination of chlorogenic acid,rutin and quercetin in Flos lonicerae.Chinese Journal of Pharmaceutical Analysis,2007,27(3):367-370.(in Chinese with English abstract)

[12]張淑蓉,裴香萍,裴妙榮,等.不同采收期紫丁香葉中原兒茶酸、原兒茶醛和酪醇的含量測定.中國現(xiàn)代應(yīng)用藥學雜志,2009,26(6):457-460.ZHANG S R,PEI X P,PEI M R,et al.Determination of protocatechuic acid,protocatechuic aldehyde and tyrosol in different seasons collections of syringa oblate leaves.Chinese Journal of Modern Applied Pharmacy,2009,26(6):457-460.(in Chinese with English abstract)

[13]隗永青,曹均,曹慶昌.北京郊區(qū)板栗林下栗蘑栽培技術(shù)及效益分析.中國食用菌,2010,29(2):21-23,53.WEI Y Q,CAO J,CAO Q C.Analysis of chestnut mushroom cultivation technique and benefit of Beijing suburb of chestnut under forest.Edible Fungi of China,2010,29 (2):21-23,53.(in Chinese)

[14]賈陸,席芳,王娜,等.板栗殼化學成分研究.中國醫(yī)藥工業(yè)雜志,2010,41(2):98-102.JIA L,XI F,WANG N,et al.Chemical constituents of Castanea mollissima Blume shell.Chinese Journal of Pharmaceuticals,2010,41(2):98-102.(in Chinese with English abstract)

[15]席芳.板栗殼化學成分的研究.河南:鄭州大學,2007:1-60.XI F.Study on chemical constituents of Castanea mollissima Blume shell.Henan:Zhengzhou University,2007:1-60.(in Chinese with English abstract)

[16]石恩慧,李紅,谷明燦,等.響應(yīng)面法優(yōu)化超聲提取板栗總苞多酚工藝條件.中國食品學報,2013,5(13):69-76.SHI E H,LI H,GU M C,et al.Optimization of technology for polyphenols extraction of Castanea mollissima Blume using response surface analysis.Journal of Chinese Institute of Food Science and Technology,2013,5(13):69-76.(in Chinese with English abstract)

[17]金秀梅,吳迪,黃健,等.板栗總苞化學成分的分離與鑒定(Ⅱ).沈陽藥科大學學報,2009,1(26):23-26.JIN X M,WU D,HUANG J,et al.Isolation and identification of chemical constituents of Castanea mollissima Blume(Ⅱ).Journal of Shenyang Pharmaceutical University,2009,1(26):23-26.(in Chinese with English abstract)

[18]石恩慧,郭凱軍,李紅,等.板栗總苞多酚提取工藝優(yōu)化及其抗氧化性研究.動物營養(yǎng)學報,2013,25(2):406-414 SHI E H,GUO K J,LI H,et al.Castanea mollissima Blume polyphenols:extraction technology optimization and its antioxidant activity.Chinese Journal of Animal Nutrition,2013,25(2):406-414.(in Chinese with English abstract)

[19]葉君,孫博航,黃健,等.板栗總苞中總黃酮提取工藝及含量測定.現(xiàn)代中醫(yī)藥,2010,6(30):102-104.YE J,SUN B H,HUANG J,et al.The extraction and determination of total flavonoids in Chestnut involucre.Modern Traditional Chinese Medicine,2010,6(30):102-104.(in Chinese)

書 名:《白塔湖植物》(“浙江植物志”系列叢書)

作 者:吳建人 金孝鋒

.新書架.

浙江大學出版社出版

內(nèi)容簡介:本書是作者對浙江省諸暨市白塔湖濕地公園數(shù)次調(diào)查的基礎(chǔ)上,對該區(qū)內(nèi)維管植物和植被較為詳盡的記錄.共篩選出維管植物358種(含種下等級和栽培品種,其中少數(shù)作為附種)介紹,包括栽培植物200種、野生植物158種,每個種均有中文名、拉丁學名、所屬科名,以及簡單的鑒別特征、分布介紹(野生植物還有在本區(qū)內(nèi)分布狀況,如常見、偶見、少見等),并附有彩色照片以便野外鑒定時參考.還簡單介紹了區(qū)內(nèi)的主要植被類型.

本書圖文并茂,內(nèi)容詳實,實用性強,可作為生物、林業(yè)、園林、環(huán)保、旅游等專業(yè)的實踐教學用書,也可供苗木種植、園林設(shè)計、戶外愛好者參考.

收稿日期(Received):2015-03-21;接受日期(Accepted):2015-06-25;網(wǎng)絡(luò)出版日期(Published online):2015-12-15

*通信作者(

Corresponding author):劉淑萍(http://orcid.org/0000-0002-1972-9710),E-mail:wzgdlq@126.com

基金項目:河北省科技廳科技支撐項目(13227308).

DOI:10.3785/j.issn.1008-9209.2015.03.211

中圖分類號Q 5-3;R 978.1;O 652.1

文獻標志碼A