荷叶化学成分的研究
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摘要
荷叶为睡莲科植物莲（Nelumbo nucifera Gaertn.）的干燥叶，味苦、平，归肝、脾、胃经，具有清热解暑、升发清阳、凉血止血之功效，用于暑热烦渴、暑湿泄泻、便血便漏。
荷叶中含有多种化学成分，包括：生物碱、黄酮、有机酸、氨基酸以及挥发油等，其中主要的有效成分为生物碱和黄酮。
本实验将荷叶用乙醇加热回流提取，提取物经过多种柱层析分离和重结晶等方法，得到了7个单体化合物。利用质谱、红外、核磁共振等多种波谱学方法，结合其理化性质，鉴定了这7个化合物的结构，分别是槲皮素-3-O-β-D-吡喃木糖(1→2)-β-D-吡喃葡萄糖苷、异槲皮苷、胡萝卜苷、槲皮素-3-O-β-D-葡萄糖醛酸苷、β-谷甾醇、2-羟基-1-甲氧基阿朴啡和荷叶碱。
本实验还建立了荷叶中槲皮素-3-O-β-D-吡喃木糖(1→2)-β-D-吡喃葡萄糖苷和槲皮素-3-O-β-D-葡萄糖醛酸苷的HPLC测定方法，同时对其进行了方法学验证。结果表明本测定方法具有快速、准确、灵敏等特点。此外还对以上两个化合物清除自由基活性进行了研究。结果表明，槲皮素-3-O-β-D-吡喃木糖(1→2)-β-D-吡喃葡萄糖苷和槲皮素-3-O-β-D-葡萄糖醛酸苷对DPPH产生的自由基具有较好的清除作用。
本实验的研究结果为荷叶质量标准的完善以及荷叶的进一步开发利用提供了新的科学依据。
Folium nelumbinis is the dry leaves of Nelumbo nucifera Gaertn, which is highly valuedin traditional medicine field and can be widely used in many aspects. It is stated that Foliumnelumbinis can be introduced into the effective treatment of stroke, acute diarrhea,hematochezia and phalacrosis etc.
The objective of this research is to systematically separate and determine the chemicalcomposition of Folium nelumbinis. Seven compounds were extracted from Folium nelumbinis,the structures of which were identified by employing several spectral methods such as NMR,MS, UV and IR, combining with their physical and chemical properties. The seven isolatedcompounds are identified as quercetin-3-O-β-D-xylopyranosyl(1→2)-β-D-glucopyranoside,isoquercitrin,daucosterol,quercetin-3-O-β-D-glucuronide,sitosterol,2-hydroxy-1-methoxyaporphine and nuciferine.
In this paper, high pressure liquid chromatography(HPLC) was used to establish thequantification analysis method of the two flavonoids, quercetin-3-O-β-D-xylopyranosyl(1→2)-β-D-glucopyranoside and Quercetin-3-O-β-D-glucuronide, the contents of which werealso determined on three batches of samples of Folium nelumbinis.
1. Extraction, separation and structural identification
1.1Extraction
6Kg dried leaves of Nelumbo nucifera Gaertn were crushed and then extracted by refluxin80%(V/V) ethanol solution three times (1h,0.5h and0.5h, respectively). The mixed filtratewas evaporated under vacuum distillation to recycle ethanol and finally condensed tonon-ethanol.
1.2Separation and purification
Dilute ten folds of the above condensed solution and then rinsed on the macroporousadsorption resin with0.1%NaOH solution until to colorless. The macroporous adsorption resin was first rinsed by distilled water to colorless（pH=7.0） and then eluted with80%ethanol. The left elution was condensed to extract the540g part A. The alkali leftovers wasprocessed to pH=7.0by0.1%hydrochloric acid. The left elution was condensed to extract the205g part B.
1.2.1A1and A2were isolated from A by silica gel column chromatography with eluent I.
White powder precipitated in A1and then we rinsed it with ethyl acetate, finally,compound LL-3(58mg) was isolated.
A2′and A2″were isolated from A2by alkaline aluminum oxide columnchromatography with eluent II. The two extracts were evaporated to dryness and thenheated to dissolve in elutant II. When it was cooled to room temperature, there are someprecipitations and then it was filtered. Finally, LL-S1(62mg) and LL-S2(480mg) wereisolated after recrystallization in ethyl acetate.
1.2.2B1, B2, B3and B4were isolated from B by silica gel column chromatography whenusing III as mobile phase.
B1′was isolated from B1by polyamide column chromatography when usingdeveloping solvent VIII as mobile phase. B1〞was isolated from B1′by ODS columnchromatography when using developing solvent IX as mobile phase. The compoundLL-2(140mg) was separated from B1〞by gel filtration chromatography.
B2′was isolated from B2by silica gel column chromatography when usingdeveloping solvent VI as mobile phase. The compound LL-1(130mg) was finallyseparated from B2′by dry column chromatography repeatedly when using developingsolvent VII as mobile phase.
B3′was isolated from B3by silica gel column chromatography when usingdeveloping solvent IV as mobile phase. The compound LL-4(120mg) was finallyseparated from B3′by dry column chromatography repeatedly when using developingsolvent V as mobile phase.
White powder precipitated in B4and then it was filtered under vacuum. Thecompound LL-5(87mg) was separated after recrystallization in chloroform.
1.3Identification
On the basis of the physical and chemical properties with various spectral methods suchas UV, IR, MS, NMR, all the isolated compounds were identified asquercetin-3-O-β-D-xylopyranosyl(1→2)-β-D-glucopyranoside(LL-1),isoquercitrin(LL-2),daucosterol(LL-3), quercetin-3-O-β-D-glucuronide(LL-4), sitosterol(LL-5),2-hydroxy-1-methoxyaporphine(LL-S1) and nuciferine(LL-S2).
2. The Contents Determination of two kinds of Flavonoids Compounds in
Nelumbo nucifera Gaertn. by HPLC
2.1Equipments, Chromatographic conditions and control samples
Chromatograph: Agilent1200Series HPLC (UV detector);
Balance: Mettlertoledo AG135electronicbalance;
Chromatography column: ZORBAX Extend C18column（250mm×4.6mm，5um）;
Mobile phase: methanol-acetonitrile-acetic acid (4:1:15)
Flow rate:1.0mL/min;
Chromatography column temperature:35℃;
Controlsamples:quercetin-3-O-β-D-xylopyranosyl(1→2)-β-D-glucopyranoside(LL-1) andquercetin-3-O-β-D-glucuronide(LL-4). According to the HPLC normalized method, weconcluded that the purities of the two compounds are98.5%and99.6%, respectively.
2.2Contents determination and methodology
I. Theoretical plate number and Resolution
The results show that the theoretical plate of number of LL-1and LL-4are4803and6612, respectively. The resolution of LL-1and LL-4are2.03and1.51, respectively.
II. Intra-day precision test
The solution of compounds LL-1and LL-4with the same volume was injected into theHPLC for six times in one day. It was shown that the RSD(n=6) of peak area for thecompounds LL-1and LL-4were1.22%and1.34%, respectively, and the retention time were0.58%and0.14%, respectively.
III. Inter-day precision test
The same solution was injected into the HPLC once a day for six days and the resultsshowed that the RSD(n=6) of peak area for LL-1and LL-4were0.84%and0.50%, respectively, while the retention time were0.20%and0.14%, respectively.
IV. The standard curve
The linearity was in the concentration range of0.1μg-1μg for compounds LL-1andLL-4, and the correlation coefficients were0.9995and0.9999, respectively. The regressionequations of the two compounds were y=3417.6x-45.795(LL-1) andy=3761.2x-4.7947(LL-4).
V. Repeatability test
Six copies of the same compound were injected into HPLC and the RSD of peak area forLL-1and LL-4were0.14%and0.11%, respectively.
VI. Stability test
The same solution of samples was injected into the HPLC once every two hours in24h.The RSD of peak area for compounds LL-1and LL-4were0.58%and0.25%, respectively,while the retention time were0.43%and0.46%, respectively. The above results clearlydemonstrated the good stability of the two compounds in24h.
VII. LOD and LOQ
For both compounds LL-1and LL-4，based on the experiments conditions, theirdetection limit（LOD）was5ng, while quantification limit（LOQ）was also5ng.
VIII. Recoveries test
The average recovery for compound LL-1was100.4%and the RSD (n=6) was0.50%,while the average recovery for compound LL-4was100.1%and the RSD (n=6) was0.33%.
Ⅸ. Contents determination of three batches of samples
Three batches of samples of the leaves of Folium nelumbinis were determined, and itwas showed that the average content of compound LL-1was0.357%,0.328%and0.371%,and the average content of compound LL-4was0.994%,0.965%and1.029%, respectively.
All in all, this research provided valid and scientific theoretical evidence to furtherexplore and promote the technological upgrading in determination, extraction and purificationof chemical compositions in Folium nelumbinis.
3. The lotus two flavonoids study on the free radical scavenging effects
The experimental results show that, quercetin-3-O-β-D-xylopyranosyl-(1→2)-
β-D-glucopyranoside(LL-1) and quercetin-3-O-β-D-glucuronide have the ability ofscavenging capacity. quercetin-3-O-β-D-glucuronide is better than quercetin-3-O-β-D-xylopyranosyl(1→2)-β-D-glucopyranoside.
In this paper, new index of content determination was offered to improve qualitystandard of the leaves of Nelumbo nucifera Gaertn. The results of this paper provided atheoretical support for further study and a scientific basis for development and utilization ofOroxylum indicum.
荷叶中含有多种化学成分，包括：生物碱、黄酮、有机酸、氨基酸以及挥发油等，其中主要的有效成分为生物碱和黄酮。
本实验将荷叶用乙醇加热回流提取，提取物经过多种柱层析分离和重结晶等方法，得到了7个单体化合物。利用质谱、红外、核磁共振等多种波谱学方法，结合其理化性质，鉴定了这7个化合物的结构，分别是槲皮素-3-O-β-D-吡喃木糖(1→2)-β-D-吡喃葡萄糖苷、异槲皮苷、胡萝卜苷、槲皮素-3-O-β-D-葡萄糖醛酸苷、β-谷甾醇、2-羟基-1-甲氧基阿朴啡和荷叶碱。
本实验还建立了荷叶中槲皮素-3-O-β-D-吡喃木糖(1→2)-β-D-吡喃葡萄糖苷和槲皮素-3-O-β-D-葡萄糖醛酸苷的HPLC测定方法，同时对其进行了方法学验证。结果表明本测定方法具有快速、准确、灵敏等特点。此外还对以上两个化合物清除自由基活性进行了研究。结果表明，槲皮素-3-O-β-D-吡喃木糖(1→2)-β-D-吡喃葡萄糖苷和槲皮素-3-O-β-D-葡萄糖醛酸苷对DPPH产生的自由基具有较好的清除作用。
本实验的研究结果为荷叶质量标准的完善以及荷叶的进一步开发利用提供了新的科学依据。
Folium nelumbinis is the dry leaves of Nelumbo nucifera Gaertn, which is highly valuedin traditional medicine field and can be widely used in many aspects. It is stated that Foliumnelumbinis can be introduced into the effective treatment of stroke, acute diarrhea,hematochezia and phalacrosis etc.
The objective of this research is to systematically separate and determine the chemicalcomposition of Folium nelumbinis. Seven compounds were extracted from Folium nelumbinis,the structures of which were identified by employing several spectral methods such as NMR,MS, UV and IR, combining with their physical and chemical properties. The seven isolatedcompounds are identified as quercetin-3-O-β-D-xylopyranosyl(1→2)-β-D-glucopyranoside,isoquercitrin,daucosterol,quercetin-3-O-β-D-glucuronide,sitosterol,2-hydroxy-1-methoxyaporphine and nuciferine.
In this paper, high pressure liquid chromatography(HPLC) was used to establish thequantification analysis method of the two flavonoids, quercetin-3-O-β-D-xylopyranosyl(1→2)-β-D-glucopyranoside and Quercetin-3-O-β-D-glucuronide, the contents of which werealso determined on three batches of samples of Folium nelumbinis.
1. Extraction, separation and structural identification
1.1Extraction
6Kg dried leaves of Nelumbo nucifera Gaertn were crushed and then extracted by refluxin80%(V/V) ethanol solution three times (1h,0.5h and0.5h, respectively). The mixed filtratewas evaporated under vacuum distillation to recycle ethanol and finally condensed tonon-ethanol.
1.2Separation and purification
Dilute ten folds of the above condensed solution and then rinsed on the macroporousadsorption resin with0.1%NaOH solution until to colorless. The macroporous adsorption resin was first rinsed by distilled water to colorless（pH=7.0） and then eluted with80%ethanol. The left elution was condensed to extract the540g part A. The alkali leftovers wasprocessed to pH=7.0by0.1%hydrochloric acid. The left elution was condensed to extract the205g part B.
1.2.1A1and A2were isolated from A by silica gel column chromatography with eluent I.
White powder precipitated in A1and then we rinsed it with ethyl acetate, finally,compound LL-3(58mg) was isolated.
A2′and A2″were isolated from A2by alkaline aluminum oxide columnchromatography with eluent II. The two extracts were evaporated to dryness and thenheated to dissolve in elutant II. When it was cooled to room temperature, there are someprecipitations and then it was filtered. Finally, LL-S1(62mg) and LL-S2(480mg) wereisolated after recrystallization in ethyl acetate.
1.2.2B1, B2, B3and B4were isolated from B by silica gel column chromatography whenusing III as mobile phase.
B1′was isolated from B1by polyamide column chromatography when usingdeveloping solvent VIII as mobile phase. B1〞was isolated from B1′by ODS columnchromatography when using developing solvent IX as mobile phase. The compoundLL-2(140mg) was separated from B1〞by gel filtration chromatography.
B2′was isolated from B2by silica gel column chromatography when usingdeveloping solvent VI as mobile phase. The compound LL-1(130mg) was finallyseparated from B2′by dry column chromatography repeatedly when using developingsolvent VII as mobile phase.
B3′was isolated from B3by silica gel column chromatography when usingdeveloping solvent IV as mobile phase. The compound LL-4(120mg) was finallyseparated from B3′by dry column chromatography repeatedly when using developingsolvent V as mobile phase.
White powder precipitated in B4and then it was filtered under vacuum. Thecompound LL-5(87mg) was separated after recrystallization in chloroform.
1.3Identification
On the basis of the physical and chemical properties with various spectral methods suchas UV, IR, MS, NMR, all the isolated compounds were identified asquercetin-3-O-β-D-xylopyranosyl(1→2)-β-D-glucopyranoside(LL-1),isoquercitrin(LL-2),daucosterol(LL-3), quercetin-3-O-β-D-glucuronide(LL-4), sitosterol(LL-5),2-hydroxy-1-methoxyaporphine(LL-S1) and nuciferine(LL-S2).
2. The Contents Determination of two kinds of Flavonoids Compounds in
Nelumbo nucifera Gaertn. by HPLC
2.1Equipments, Chromatographic conditions and control samples
Chromatograph: Agilent1200Series HPLC (UV detector);
Balance: Mettlertoledo AG135electronicbalance;
Chromatography column: ZORBAX Extend C18column（250mm×4.6mm，5um）;
Mobile phase: methanol-acetonitrile-acetic acid (4:1:15)
Flow rate:1.0mL/min;
Chromatography column temperature:35℃;
Controlsamples:quercetin-3-O-β-D-xylopyranosyl(1→2)-β-D-glucopyranoside(LL-1) andquercetin-3-O-β-D-glucuronide(LL-4). According to the HPLC normalized method, weconcluded that the purities of the two compounds are98.5%and99.6%, respectively.
2.2Contents determination and methodology
I. Theoretical plate number and Resolution
The results show that the theoretical plate of number of LL-1and LL-4are4803and6612, respectively. The resolution of LL-1and LL-4are2.03and1.51, respectively.
II. Intra-day precision test
The solution of compounds LL-1and LL-4with the same volume was injected into theHPLC for six times in one day. It was shown that the RSD(n=6) of peak area for thecompounds LL-1and LL-4were1.22%and1.34%, respectively, and the retention time were0.58%and0.14%, respectively.
III. Inter-day precision test
The same solution was injected into the HPLC once a day for six days and the resultsshowed that the RSD(n=6) of peak area for LL-1and LL-4were0.84%and0.50%, respectively, while the retention time were0.20%and0.14%, respectively.
IV. The standard curve
The linearity was in the concentration range of0.1μg-1μg for compounds LL-1andLL-4, and the correlation coefficients were0.9995and0.9999, respectively. The regressionequations of the two compounds were y=3417.6x-45.795(LL-1) andy=3761.2x-4.7947(LL-4).
V. Repeatability test
Six copies of the same compound were injected into HPLC and the RSD of peak area forLL-1and LL-4were0.14%and0.11%, respectively.
VI. Stability test
The same solution of samples was injected into the HPLC once every two hours in24h.The RSD of peak area for compounds LL-1and LL-4were0.58%and0.25%, respectively,while the retention time were0.43%and0.46%, respectively. The above results clearlydemonstrated the good stability of the two compounds in24h.
VII. LOD and LOQ
For both compounds LL-1and LL-4，based on the experiments conditions, theirdetection limit（LOD）was5ng, while quantification limit（LOQ）was also5ng.
VIII. Recoveries test
The average recovery for compound LL-1was100.4%and the RSD (n=6) was0.50%,while the average recovery for compound LL-4was100.1%and the RSD (n=6) was0.33%.
Ⅸ. Contents determination of three batches of samples
Three batches of samples of the leaves of Folium nelumbinis were determined, and itwas showed that the average content of compound LL-1was0.357%,0.328%and0.371%,and the average content of compound LL-4was0.994%,0.965%and1.029%, respectively.
All in all, this research provided valid and scientific theoretical evidence to furtherexplore and promote the technological upgrading in determination, extraction and purificationof chemical compositions in Folium nelumbinis.
3. The lotus two flavonoids study on the free radical scavenging effects
The experimental results show that, quercetin-3-O-β-D-xylopyranosyl-(1→2)-
β-D-glucopyranoside(LL-1) and quercetin-3-O-β-D-glucuronide have the ability ofscavenging capacity. quercetin-3-O-β-D-glucuronide is better than quercetin-3-O-β-D-xylopyranosyl(1→2)-β-D-glucopyranoside.
In this paper, new index of content determination was offered to improve qualitystandard of the leaves of Nelumbo nucifera Gaertn. The results of this paper provided atheoretical support for further study and a scientific basis for development and utilization ofOroxylum indicum.
引文
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