Keywords: reversed-phase high-performance liquid chromatography; wood genus; flavonoids; coumarin abstract Objective: To conduct chemical taxonomic studies on two natural groups of herbs and woody plants in wood genus. Method: Reversed-phase high-performance liquid chromatography was used to determine the four chemical constituents in the woody plant [neoliguiritin (I), 5,8-dimethoxycoumarin (II), isoliquritoside (III), 8-［(2 ″, 3 ″)- prenyl] -4′-methoxy-flavone-7-O-β-D-glucopyranosyl- (2 → 1) -α-L-rhamnopyranoside (IV)], quantitative analysis of the four components in 11 plants of this genus . The stationary phase of the column is Supelco SIL-LC-18; the mobile phase is 40% methanol-methanol gradient elution, and the detection wavelength is changed according to the ultraviolet absorption wavelength of the four components; the flow rate is 1.0 ml.min-1; the four components The linear ranges are (0.5600 ～ 0.0175) μg, (0.3760 ～ 0.0118) μg, (0.2875 ～ 0.0090) μg and (0.3090 ～ 0.0097) μg, and the recoveries are 98.92%, 98.27%, 94.78% and 97.41%, respectively. Results: The content of coumarin in woody natural group was relatively high. Conclusion: Coumarin is one of the characteristic components of woody natural group different from herbal natural group.
DETERMINATION OF FLAVONES AND COUMARIN IN ARALIA BY RP-HPLC
Xu Xudong (Xu XD), Zhang Shuming (Zhang SM), Zhang Yumei (Zhang YM), Lin Geng (Lin G) and Yang Junshan (Yang JS)
(Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100094)
ABSTRACT AIM: To reveal the difference between the groups of herbal and woody in Aralia. METHODS: An accurate RP-HPLC method with variable wavelength for the determination of neoliguiritin (I), 5,8-dimethoxycoumarin (II), isoliquritoside (III) and 8-[(2 ″, 3 ″)-prenyl] -4′-methoxy-flavone-7-O-β-D-glucopyranosyl- (2 → 1) -α-L-rhamnopyranoside (IV) in 11 species of Aralia has been developed. The analytical column used was Supelco SIL-LC-18. The mobile phase was 40% MeOH to absolute MeOH. Gradient elution was employed. The flow rate was 1.0 ml.min-1. From 0.00 ～ 8.30 min the elute was detected at 276 nm, 8.30 ～ 14.00 min at 343 nm, 14.00 ～ 20.00 min at 361 nm and 20.00 ～ 30.00 min at 272 nm. During the detection, the wavelength was changed to obtain high sensitivity and precision. The linear ranges of I ～ IV were 0.0175 ～ 0.5600 μg, 0.0118 ～ 0.3760 μg, 0.0090 ～ 0.2875 μg and 0.0097 ～ 0.3090 μg, respectively. The recoveries of I ～ IV were between 94.78% and 98.92%. RESULTS: The c ontent of coumarin was higher in woody than in herbal. CONCLUSION: Coumarin is the specific constituent in the group of herbal, which is the difference from the woody.
KEY WORDS RP-HPLC; Aralia; flavonoids; coumarin
Aralia linn. Plants can be clearly divided into two natural groups, woody natural groups and herbaceous natural groups from the morphology and geographical distribution [1,2]. By analyzing the characteristic chemical composition of the genus plants, the difference between the main chemical components of the two natural groups can be found, and the two natural groups can be further divided into two independent subgenuses, namely the woody subgenus and the herbaceous subgenus. . Previous article [3] We have used reversed-phase high-performance liquid chromatography to determine the content of diterpene acids, triterpene acids and saponins in the genus Lignans. On this basis, we further established four chemical components in the genus Lignans [neoliguiritin (I), 5,8-dimethoxycoumarin (II), isoliquritoside (III), 8-[(2 ″, 3 ″)-prenyl] -4′-methoxy-flavone-7-O-β-D-glucopyranosyl- ( 2 → 1) -α-L-rhamnopyranoside (IV)] RP-HPLC method. According to the different ultraviolet absorption wavelengths of these four components, the detection wavelength is changed during the measurement process, so that the components are detected at the maximum absorption wavelength, and the interference of adjacent chromatographic peaks is avoided, and the sensitivity and accuracy of the analysis are improved. The four components are well separated within 25 minutes and can be used for sample analysis.
Experimental part
1 Instruments, pharmaceuticals and reagents HPLC, including Waters 510, Waters 490E detector, Waters U6K sampler, Waters Baseline 810 chromatographic workstation (all are products of Waters Liquid Chromatography Company); CX-250 ultrasonic Cleaner (Beijing Second Medical Equipment Factory).
Neoliguiritin (I), 5,8-dimethoxycoumarin (II), isoliquritoside (III), 8-[(2 ″, 3 ″)-prenyl] -4′-methoxy-flavone-7-O-β-D-glucopyranosyl- (2 → 1) -α-L-rhamnopyranoside (IV) was extracted and isolated from the genus by the author and identified by four spectrums, and the purity was above 95%.
Except for the edible soil Angelica sinensis, the head order wood was collected by Professor Tan Ceming, and the rest were provided and identified by Professor Zhu Zhaoyi of our institute.
Redistilled water, methanol (analytically pure, chromatographically pure), etc.
2 Chromatographic conditions Column: Supelco SIL-LC-18 (4.6 mm × 150 mm, 5 μm); mobile phase: A: 40% methanol, B: methanol, gradient elution, 0 ～ 10 min, A: 100%, 10 ～ 25 min, A: 10%, B: 90% linear change; column temperature: room temperature; sensitivity: 0.008AUFS; flow rate: 1.0 ml.min-1; detection wavelength: 0.00 ～ 8.30 min detection with 276 nm, 8.30 ～ 343 nm was used for 14.00 min, 361 nm was used for 14.00-20.00 min, and 272 nm was used for 20.00-30.00 min.
3 Selection of detection wavelength According to the ultraviolet absorption spectrum of 4 components, I has the maximum absorption at 214.8 nm and 276 nm, II has the maximum absorption at 228.9 nm and 343 nm, III has the maximum absorption at 361 nm, and IV at 271.5 There is maximum absorption at nm. Due to the large difference in the maximum UV absorption of the four components, if only a certain detection wavelength is used for detection, it will cause a decrease in sensitivity. Therefore, based on the retention time (tR) and ultraviolet spectrum data of the four components under the above chromatographic conditions, this method chooses 0.00 to 8.30 min to detect at 276 nm, 8.30 to 14.00 min at 343 nm, and 14.00 to 20.00 min at 361 In the detection of nm, 20.00 ～ 30.00 min is detected at 272 nm, which not only makes each component detected at the optimal absorption wavelength, but also avoids the interference of adjacent chromatographic peaks, and improves the sensitivity and accuracy of the analysis.
4 The standard curve accurately weighs I ～ IV standard products 2.24, 3.76, 1.15 and 2.06 mg respectively, put in 2, 2, 1 and 2 ml measuring flasks, dissolve with methanol and make volume to make standard stock solution; respectively, accurate Pipette 50, 20, 25 and 30 μl into a 1 ml volumetric flask and make up to volume with methanol to make a mixed standard stock solution. Dilute the above mixed standard stock solutions with methanol to 4, 8, 16, and 32 times to form 5 concentration gradient standard solutions. Aspirate 10 μl samples in turn and detect under the above chromatographic conditions, with the peak height (X) as the abscissa and the component content (Y) as the ordinate. The regression equation, correlation coefficient and linear range are shown in Table 1.
Tab 1 Calibration data of compounds I ～ IV (n = 5)
Compound tR / min Regression equation γ Linear range / μg
I 6.62 Y = 2.8751 × 10-7X-1.3622 × 10-2 0.9991 0.018 ～ 0.560
II 8.18 Y = 1.6031 × 10-7X-6.5835 × 10-3 0.9997 0.012 ～ 0.376
III 16.68 Y = 2.2618 × 10-7X-1.7228 × 10-3 0.9991 0.009 ～ 0.288
IV 22.14 Y = 1.4429 × 10-7X-4.5736 × 10-3 0.9994 0.010 ～ 0.309
I. Neoliguiritin; II. 5,8-Dimethoxycoumarin; III. Isoliquritoside; IV. 8-[(2 ″, 3 ″)-Prenyl] -4′-methoxy-flavone-7-O-β-D-glucopyranosyl- ( 2 → 1) -α-L-rhamnopyranoside.
5 The extraction method and sample preparation method are the same as in Reference 3.
6 Method precision determination Take 5 samples (n = 5) of the same crude drug, and determine by the above method, the RSD (%) of I to IV were: 1.05, 1.26, 2.13 and 2.08. Prove that this method is feasible.
7 The sample recovery rate test accurately weighs 6 parts of 1 g of heading wood powder, of which 3 parts are used as a control, and the remaining 3 parts are mixed with a certain amount of mixed standard solution, and after natural drying, the sample solution is prepared according to the above method. Determine the recovery rate. The results are shown in Table 2. The average recoveries of the four components are from 94.78% to 98.92%.
Tab 2 Recoveries of compounds I ～ IV (n = 3)
Compound Added / μg Found / μg Recovery /% RSD /%
I 1.120 1.108 ± 0.060 98.92 ± 0.01 0.48
II 0.752 0.739 ± 0.100 98.27 ± 0.02 0.52
III 0.575 0.545 ± 0.180 94.78 ± 0.02 1.06
IV 0.618 0.602 ± 0.080 97.41 ± 0.03 0.98
I ～ IV cf Tab 1.
8 The sample analysis is carried out according to the preparation of the sample liquid. The quantitative determination of each sample and the calculation of the content are shown in Table 3, and the chromatogram is shown in Figure 1.
Tab 3 Content of compounds I ～ IV in different species
Classification Group Species Content /μg.g-1
I II III IV
Woody Sect. Aralia A.echinocaulis 0.047 0.016 0.022 0.117
A.decaisneana 0.019 1.744 0.182 0.051
A.chinensis var. Nuda-0.028 0.148 0.036
A.chinensis var.dasyphylloides 0.080 0.021 0.053 +
Sect.Digitipanicula Hoo A.elata 0.082 0.022 0.028 0.029
A.hupehensis 0.652 0.165 0.310 +
Sect. Capituli-gerae Harms A. dasyphylla 0.019 0.022 0.018 0.050
Herbal Sect. Genuinae Harms A. cordata 0.085 +-4.257
Sect.Anomalae Harms A.henryi 5.227 + 0.051 4.051
A.fargesii--+-
A.atropurpurea 0.037-3.059 0.039
I ～ IV cf Tab 1. +: The content is less than 0.015 μg.g-1;-: Not detected.
Fig 1 Chromatogram of standards (A), A.cordatad (B) and A.asphyphylla (C) Elute was detected at 276 nm (0.00 ～ 8.30 min), 343 nm (8.30 ～ 14.00 min), 361 nm (14.00 ～ 20.00 min) and 272 nm (20.00 ～ 30.00 min). I ～ IV cf Tab 1.
Discussion This study determined four chemical constituents of 11 species (including two variants) of the genus Wood, namely flavonoids, dihydroflavonoids, chalcone and coumarin. Since the plant of this genus is clearly divided into two natural groups in terms of morphology and geographical distribution, after comparing the relative contents of the above four chemical components in the two natural groups through this analysis method, it can be clearly found that, in the natural group of herbs, except longan and solitary , The content of flavonoids is high, and the difference between different species is large, but the content of coumarin ingredients is low or undetectable, and the coumarin ingredients are contained in the woody natural group, so it can be considered fragrant Bean extracts are characteristic components of woody natural groups that differ from herbal natural groups. Combining the results of the previous article [3], it can be found that both the natural herbal group and the woody natural group have their own characteristic components, that is, the content of diterpenes in the herbal natural group is higher, while the content of coumarin in the woody natural group Relatively high. This provides a basis for chemical taxonomy for the division of the two natural groups of herbs and woody species in the genus Wood.
references
1 Chinese Academy of Sciences Editorial Board of Chinese Academy of Sciences. Flora of China. Volume 54. Beijing: Science Press, 1978.150
2 Xu Xudong, Yang Junshan, Zhu Zhaoyi. Research progress of triterpenoid saponins in wood genus. Journal of Pharmaceutical Sciences, 1997, 32: 711
3 Xu Xudong, Zhang Shuming, Yang Junshan, et al. Determination of diterpenic acid, triterpenic acid and triterpene saponins in the wood genus by reversed-phase high performance liquid chromatography. Journal of Pharmaceutical Sciences, 1998, 33: 933
DETERMINATION OF FLAVONES AND COUMARIN IN ARALIA BY RP-HPLC
Xu Xudong (Xu XD), Zhang Shuming (Zhang SM), Zhang Yumei (Zhang YM), Lin Geng (Lin G) and Yang Junshan (Yang JS)
(Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100094)
ABSTRACT AIM: To reveal the difference between the groups of herbal and woody in Aralia. METHODS: An accurate RP-HPLC method with variable wavelength for the determination of neoliguiritin (I), 5,8-dimethoxycoumarin (II), isoliquritoside (III) and 8-[(2 ″, 3 ″)-prenyl] -4′-methoxy-flavone-7-O-β-D-glucopyranosyl- (2 → 1) -α-L-rhamnopyranoside (IV) in 11 species of Aralia has been developed. The analytical column used was Supelco SIL-LC-18. The mobile phase was 40% MeOH to absolute MeOH. Gradient elution was employed. The flow rate was 1.0 ml.min-1. From 0.00 ～ 8.30 min the elute was detected at 276 nm, 8.30 ～ 14.00 min at 343 nm, 14.00 ～ 20.00 min at 361 nm and 20.00 ～ 30.00 min at 272 nm. During the detection, the wavelength was changed to obtain high sensitivity and precision. The linear ranges of I ～ IV were 0.0175 ～ 0.5600 μg, 0.0118 ～ 0.3760 μg, 0.0090 ～ 0.2875 μg and 0.0097 ～ 0.3090 μg, respectively. The recoveries of I ～ IV were between 94.78% and 98.92%. RESULTS: The c ontent of coumarin was higher in woody than in herbal. CONCLUSION: Coumarin is the specific constituent in the group of herbal, which is the difference from the woody.
KEY WORDS RP-HPLC; Aralia; flavonoids; coumarin
Aralia linn. Plants can be clearly divided into two natural groups, woody natural groups and herbaceous natural groups from the morphology and geographical distribution [1,2]. By analyzing the characteristic chemical composition of the genus plants, the difference between the main chemical components of the two natural groups can be found, and the two natural groups can be further divided into two independent subgenuses, namely the woody subgenus and the herbaceous subgenus. . Previous article [3] We have used reversed-phase high-performance liquid chromatography to determine the content of diterpene acids, triterpene acids and saponins in the genus Lignans. On this basis, we further established four chemical components in the genus Lignans [neoliguiritin (I), 5,8-dimethoxycoumarin (II), isoliquritoside (III), 8-[(2 ″, 3 ″)-prenyl] -4′-methoxy-flavone-7-O-β-D-glucopyranosyl- ( 2 → 1) -α-L-rhamnopyranoside (IV)] RP-HPLC method. According to the different ultraviolet absorption wavelengths of these four components, the detection wavelength is changed during the measurement process, so that the components are detected at the maximum absorption wavelength, and the interference of adjacent chromatographic peaks is avoided, and the sensitivity and accuracy of the analysis are improved. The four components are well separated within 25 minutes and can be used for sample analysis.
Experimental part
1 Instruments, pharmaceuticals and reagents HPLC, including Waters 510, Waters 490E detector, Waters U6K sampler, Waters Baseline 810 chromatographic workstation (all are products of Waters Liquid Chromatography Company); CX-250 ultrasonic Cleaner (Beijing Second Medical Equipment Factory).
Neoliguiritin (I), 5,8-dimethoxycoumarin (II), isoliquritoside (III), 8-[(2 ″, 3 ″)-prenyl] -4′-methoxy-flavone-7-O-β-D-glucopyranosyl- (2 → 1) -α-L-rhamnopyranoside (IV) was extracted and isolated from the genus by the author and identified by four spectrums, and the purity was above 95%.
Except for the edible soil Angelica sinensis, the head order wood was collected by Professor Tan Ceming, and the rest were provided and identified by Professor Zhu Zhaoyi of our institute.
Redistilled water, methanol (analytically pure, chromatographically pure), etc.
2 Chromatographic conditions Column: Supelco SIL-LC-18 (4.6 mm × 150 mm, 5 μm); mobile phase: A: 40% methanol, B: methanol, gradient elution, 0 ～ 10 min, A: 100%, 10 ～ 25 min, A: 10%, B: 90% linear change; column temperature: room temperature; sensitivity: 0.008AUFS; flow rate: 1.0 ml.min-1; detection wavelength: 0.00 ～ 8.30 min detection with 276 nm, 8.30 ～ 343 nm was used for 14.00 min, 361 nm was used for 14.00-20.00 min, and 272 nm was used for 20.00-30.00 min.
3 Selection of detection wavelength According to the ultraviolet absorption spectrum of 4 components, I has the maximum absorption at 214.8 nm and 276 nm, II has the maximum absorption at 228.9 nm and 343 nm, III has the maximum absorption at 361 nm, and IV at 271.5 There is maximum absorption at nm. Due to the large difference in the maximum UV absorption of the four components, if only a certain detection wavelength is used for detection, it will cause a decrease in sensitivity. Therefore, based on the retention time (tR) and ultraviolet spectrum data of the four components under the above chromatographic conditions, this method chooses 0.00 to 8.30 min to detect at 276 nm, 8.30 to 14.00 min at 343 nm, and 14.00 to 20.00 min at 361 In the detection of nm, 20.00 ～ 30.00 min is detected at 272 nm, which not only makes each component detected at the optimal absorption wavelength, but also avoids the interference of adjacent chromatographic peaks, and improves the sensitivity and accuracy of the analysis.
4 The standard curve accurately weighs I ～ IV standard products 2.24, 3.76, 1.15 and 2.06 mg respectively, put in 2, 2, 1 and 2 ml measuring flasks, dissolve with methanol and make volume to make standard stock solution; respectively, accurate Pipette 50, 20, 25 and 30 μl into a 1 ml volumetric flask and make up to volume with methanol to make a mixed standard stock solution. Dilute the above mixed standard stock solutions with methanol to 4, 8, 16, and 32 times to form 5 concentration gradient standard solutions. Aspirate 10 μl samples in turn and detect under the above chromatographic conditions, with the peak height (X) as the abscissa and the component content (Y) as the ordinate. The regression equation, correlation coefficient and linear range are shown in Table 1.
Tab 1 Calibration data of compounds I ～ IV (n = 5)
Compound tR / min Regression equation γ Linear range / μg
I 6.62 Y = 2.8751 × 10-7X-1.3622 × 10-2 0.9991 0.018 ～ 0.560
II 8.18 Y = 1.6031 × 10-7X-6.5835 × 10-3 0.9997 0.012 ～ 0.376
III 16.68 Y = 2.2618 × 10-7X-1.7228 × 10-3 0.9991 0.009 ～ 0.288
IV 22.14 Y = 1.4429 × 10-7X-4.5736 × 10-3 0.9994 0.010 ～ 0.309
I. Neoliguiritin; II. 5,8-Dimethoxycoumarin; III. Isoliquritoside; IV. 8-[(2 ″, 3 ″)-Prenyl] -4′-methoxy-flavone-7-O-β-D-glucopyranosyl- ( 2 → 1) -α-L-rhamnopyranoside.
5 The extraction method and sample preparation method are the same as in Reference 3.
6 Method precision determination Take 5 samples (n = 5) of the same crude drug, and determine by the above method, the RSD (%) of I to IV were: 1.05, 1.26, 2.13 and 2.08. Prove that this method is feasible.
7 The sample recovery rate test accurately weighs 6 parts of 1 g of heading wood powder, of which 3 parts are used as a control, and the remaining 3 parts are mixed with a certain amount of mixed standard solution, and after natural drying, the sample solution is prepared according to the above method. Determine the recovery rate. The results are shown in Table 2. The average recoveries of the four components are from 94.78% to 98.92%.
Tab 2 Recoveries of compounds I ～ IV (n = 3)
Compound Added / μg Found / μg Recovery /% RSD /%
I 1.120 1.108 ± 0.060 98.92 ± 0.01 0.48
II 0.752 0.739 ± 0.100 98.27 ± 0.02 0.52
III 0.575 0.545 ± 0.180 94.78 ± 0.02 1.06
IV 0.618 0.602 ± 0.080 97.41 ± 0.03 0.98
I ～ IV cf Tab 1.
8 The sample analysis is carried out according to the preparation of the sample liquid. The quantitative determination of each sample and the calculation of the content are shown in Table 3, and the chromatogram is shown in Figure 1.
Tab 3 Content of compounds I ～ IV in different species
Classification Group Species Content /μg.g-1
I II III IV
Woody Sect. Aralia A.echinocaulis 0.047 0.016 0.022 0.117
A.decaisneana 0.019 1.744 0.182 0.051
A.chinensis var. Nuda-0.028 0.148 0.036
A.chinensis var.dasyphylloides 0.080 0.021 0.053 +
Sect.Digitipanicula Hoo A.elata 0.082 0.022 0.028 0.029
A.hupehensis 0.652 0.165 0.310 +
Sect. Capituli-gerae Harms A. dasyphylla 0.019 0.022 0.018 0.050
Herbal Sect. Genuinae Harms A. cordata 0.085 +-4.257
Sect.Anomalae Harms A.henryi 5.227 + 0.051 4.051
A.fargesii--+-
A.atropurpurea 0.037-3.059 0.039
I ～ IV cf Tab 1. +: The content is less than 0.015 μg.g-1;-: Not detected.
Fig 1 Chromatogram of standards (A), A.cordatad (B) and A.asphyphylla (C) Elute was detected at 276 nm (0.00 ～ 8.30 min), 343 nm (8.30 ～ 14.00 min), 361 nm (14.00 ～ 20.00 min) and 272 nm (20.00 ～ 30.00 min). I ～ IV cf Tab 1.
Discussion This study determined four chemical constituents of 11 species (including two variants) of the genus Wood, namely flavonoids, dihydroflavonoids, chalcone and coumarin. Since the plant of this genus is clearly divided into two natural groups in terms of morphology and geographical distribution, after comparing the relative contents of the above four chemical components in the two natural groups through this analysis method, it can be clearly found that, in the natural group of herbs, except longan and solitary , The content of flavonoids is high, and the difference between different species is large, but the content of coumarin ingredients is low or undetectable, and the coumarin ingredients are contained in the woody natural group, so it can be considered fragrant Bean extracts are characteristic components of woody natural groups that differ from herbal natural groups. Combining the results of the previous article [3], it can be found that both the natural herbal group and the woody natural group have their own characteristic components, that is, the content of diterpenes in the herbal natural group is higher, while the content of coumarin in the woody natural group Relatively high. This provides a basis for chemical taxonomy for the division of the two natural groups of herbs and woody species in the genus Wood.
references
1 Chinese Academy of Sciences Editorial Board of Chinese Academy of Sciences. Flora of China. Volume 54. Beijing: Science Press, 1978.150
2 Xu Xudong, Yang Junshan, Zhu Zhaoyi. Research progress of triterpenoid saponins in wood genus. Journal of Pharmaceutical Sciences, 1997, 32: 711
3 Xu Xudong, Zhang Shuming, Yang Junshan, et al. Determination of diterpenic acid, triterpenic acid and triterpene saponins in the wood genus by reversed-phase high performance liquid chromatography. Journal of Pharmaceutical Sciences, 1998, 33: 933
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