There are few reports about the determination of alkaloids in Mahonia. The contents of ⅳ, ⅵ and ⅶ [12] in Cortex Phellodendri were determined by TLC scanning. Determination of ⅳ, ⅵ, ⅶ and magnolin [13] in stems of four Mahonia species by thin-layer optical density method; determination of ⅶ [14] in leaves of Mahonia species by HPLC method. Based on the determination of alkaloids in Berberis roots by variable wavelength high performance liquid chromatography [15], in order to develop and utilize Mahonia resources, a method for the determination of ⅰ ~ ⅶ in Mahonia plants by high performance liquid chromatography was established. These seven components can be detected 1 time at a fixed wavelength of 203 nm and twice at a variable wavelength of 203 ~ 346 nm. The latter can not only make seven components be detected at their respective maximum absorption wavelengths, but also avoid the interference of adjacent chromatographic peaks with different absorption wavelengths, thus improving the sensitivity and accuracy of analysis. In this paper, the roots, bark, wood, bark and wood of three species of Mahonia in China and the roots of 10 species of plants from different habitats were quantitatively analyzed by variable wavelength high performance liquid chromatography. Due to the small amount of reference materials, only I, III and V were qualitatively analyzed.
1 instrument and reagent testing
Waters high performance liquid chromatograph (5 10 pump, 490E detector, U6K sampler, baseline 8 10 chromatographic workstation).
Reference substances: berberine hydrochloride, palmatine hydrochloride and jatrorrhizine hydrochloride were purchased from China Institute for the Control of Pharmaceutical and Biological Products; Researcher Tu of China Institute for the Control of Pharmaceutical and Biological Products introduced Tetrahexylamine in Africa. Berbamine was purchased from Institute of Forestry and Soil, China Academy of Sciences and purified by recrystallization. Tetrandrine and tetrandrine were provided by Institute of Medicinal Plants, China Academy of Medical Sciences (the reference substance was separated into single spots by silica gel thin layer chromatography and two solvent systems; By HPLC, the peak height and peak area of the main components are above 97%. The plant specimens were identified by Ying Sheng Jun, a researcher at Institute of Botany, Chinese Academy of Sciences. Acetonitrile is chromatographically pure, methanol and potassium dihydrogen phosphate are analytically pure, and deionized water is used.
2 experimental methods and results
2. 1 chromatographic conditions [15]
Chromatographic column: μ-Bondapak phenyl (10μm, 3.9mm×300mm, Waters Corporation, USA); Mobile phase: 0.02 mol l-1potassium dihydrogen phosphate-acetonitrile (7 ∶ 3); Column temperature: room temperature; Flow:1.0 ml min-1; Sensitivity: 0.08AUFS；; Detection wavelength: fixed wavelength 203nm, variable wavelength 203nm after 0.0- 10.0min, and 346 nm after 10.0min. See figure 1 for the chromatogram of 7 alkaloids reference substances.
Figure/Kloc-Chromatograms of 0/7 alkaloids (detected at 203 nm)
I. echinone Ⅱ. Berberine ⅲ. Tetradecylamine ⅳ. jatrorrhizine
ⅴ. Tetrandrine ⅵ. Palmatine VII. berberine
2.2 Regression Equation and Linear Range
According to the reference [15], the regression equation was obtained by taking the sample volume (μg) as the abscissa (x) and the peak area (microvolt-second) as the ordinate (y). See table 1.
2.3 Preparation of sample solution
Accurately weigh 0.65438 0 g sample dry powder into a 50mL ground triangular bottle, add 20mL methanol, weigh, soak for 65438 02 h, ultrasonically extract for 60min, weigh, make up the lost methanol, shake well, and seal and stand; 5.0mL of supernatant was put in a centrifuge tube, sealed and centrifuged (10000 r min- 1,10 min); Take the supernatant (2.0mL) as the sample solution.
Table 1 Regression Equation and Linear Range of Quantitative Analysis of Four Alkaloids
raw material
regression equation
r
Linear range /μg
Ⅱ
y = 9.0 17x× 104+4.3 18× 105
0.9993(n=7)
0.0 19~0.296
Ⅳ
y = 4.239 x× 104-8.228× 104
0.9999(n=9)
0.0 19~0.996
Ⅵ
y = 4. 19 1x× 104+ 1.446× 105
0.9998(n=9)
0.020~ 1.008
Ⅶ
y = 4.209 x× 104+ 1.278× 105
0.9998(n=9)
0.020~ 1.0 10
2.4 accuracy test [15]
Accurately suck 10μL sample solution for injection, and repeat the determination of 10 times. The RSD(%) of the concentration (μ g mL-1) of each component in the sample was obtained: berbamine 0.28, jatrorrhizine 0.84, palmatine 0.75, berberine 1.2 1.
2.5 sample recovery test
Accurately weigh 0.05g5 parts of Mahonia angustifolia dry powder into a 50mL ground triangular flask, operate according to the document [15], and repeat the determination for 3 times for each part; The calculated recoveries of ⅱ, ⅳ, ⅵ and ⅶ are 97.2% ~ 102. 1%, 98.9% ~ 100.3% and 97.2% ~1respectively.
2.6 sample analysis [15]
Accurately suck 65438 00μ l sample solution for injection, detect it at the variable wavelength of 203 ~ 346 nm, quantitatively analyze Ⅱ, Ⅳ, ⅵ and ⅶ, and qualitatively analyze Ⅰ and Ⅲ. Then accurately inject 10μL, and detect at a fixed wavelength of 203nm to obtain the characteristic spectra of each sample, and qualitatively analyze the component ⅴ. The analysis results of seven alkaloids in roots, barks, barks, stems and trees of three Mahonia species and 10 species from different habitats are shown in Table 2 and Table 3.
3 discussion
3. 1 The method established in this paper can be used for 25 minutes under the same chromatographic conditions.
Table 2 Analysis results of 7 alkaloids in different parts of 3 Mahonia plants (%), n=2.
Plant name
location
Ⅰ
Ⅱ
Ⅲ
Ⅳ
Ⅴ
Ⅵ
Ⅶ
Mahonia latibracteata subspecies. Ganping Wenzhong
(Sichuan Traditional Chinese Medicine School)
root
-
+
0.68
+
0.05
0.78
Root bark
+
0.0 1
-
1.66
+
0. 14
3.76
Genmu
+
+
0.69
+
0.06
0.84
Stem bark
-
-
0.34
+
0.05
0.47
Trunk wood
+
-
0.53
+
0.08
0.2 1
Kumquat ) Fedde: Ten Contributions of Narrow Leaves
(Chongqing Nanchuan Institute of Drug Planting)
root
+
0.34
+
1. 17
+
0.06
1.27
Root bark
+
0.98
+
3.02
+
0.20
3.54
Genmu
+
0. 18
+
0.54
+
0.70
Stem bark
+
0.29
+
1.47
+
0. 19
0.4 1
Trunk wood
+
0.28
+
0.98
+
0.05
1.36
Ten contributions of Adiantum longibracteatum and Longbract
(Ebian, Sichuan)
root
+
+
0.40
+
0.62
1.73
Root bark
+
+
1. 10
+
1.30
6.94
Genmu
-
+
0.33
+
0.80
0.98
Stem bark
-
+
0.74
-
0.60
1.24
Trunk wood
-
-
+
0.2 1
-
0.28
0.45
+:component detected-:component not detected+:content < 0.0 1%.
Table 3 10 Analysis Results of Seven Alkaloids in the Roots of Mahonia (%), n=2
Plant name
Place of production
Ⅰ
Ⅱ
Ⅲ
Ⅳ
Ⅴ
Ⅵ
Ⅶ
Ten contributions. ) Carl.
Ebian in Sichuan
+
+
0.44
+
0.78
1.44
Chongqing Fengjie
+
0.02
+
0.50
+
0.64
2.07
Rat stab score 10, M.bodinieri Gagnep.
Chongqing Jinfo Mountain
+
0. 10
+
0.8 1
+
0.09
0.77
Chongqing medicine planting suo
+
0.08
+
0.79
+
0.06
1. 1 1
Chongqing Institute of Traditional Chinese Medicine
+
+
0.4 1
+
0.05
0.78
Hunan Xinhua *
+
+
0. 16
+
0.53
1.44
Ten contributions of Sichuan and Hubei. patchouly
Emeishan, Sichuan
-
+
0.45
+
0. 14
1. 10
Ebian in Sichuan
-
+
0.82
+
0. 12
1.22
Chongqing Jinyun Mountain
+
+
0.35
+
0.02
0.73
Ten contributions of gan ping. Ganping Wenzhong
Chongqing Nanchuan
+
0.0 1
+
1.0 1
+
0.04
1.58
Sichuan province traditional Chinese medicine school
-
+
0.68
+
0.05
0.78
Kumquat ) Fedde: Ten Contributions of Narrow Leaves
Chongqing medicine planting suo
+
0.34
+
1. 17
+
0.06
1.27
Emeishan, Sichuan
+
+
0.82
+
0.06
1.42
Hunan Xinhua *
+
0. 19
+
0.88
+
0.07
0.59
M.gracilipes(Oliv)Fedde, ten contributions of slender stems.
Emeishan, Sichuan
-
+
0.67
+
0. 15
0.99
Ten contributions of Adiantum longibracteatum and Longbract
Ebian in Sichuan
+
+
0.40
+
0.62
1.73
Taenia saginata in purple and yellow rocks
Emeishan, Sichuan
-
0.02
+
1.05
+
0. 17
0.55
M.polyodota Fedde, Top Ten Contributors of Multi-Teeth
Emeishan, Sichuan
-
0.02
+
0.67
+
0. 10
0.52
Chongqing Jinfo Mountain
-
0.0 1
+
0.22
+
0. 13
0.30
M.sp (unknown species)
Chongqing Jinfo Mountain
-
0.0 1
+
0.28
+
1.38
1.30
+:component detection-:component not detected+:content < 0.0 1% *: the determination site is stem.
I, II and V dibenzyl isoquinoline and III, IV, VI and VII protoberberine alkaloids in Mahonia plants from different parts and different habitats were determined. This method has good reproducibility, high precision and wide application range.
3.2 Mahonia and Berberis are similar in genetic relationship and chemical composition. The results show that the same alkaloid components in these two genera can be determined under the same or similar HPLC chromatographic conditions.
3.3 In 10 plant, the peak value of III is very low. According to the molecular structure and peak area of ⅳ, ⅵ and ⅶ, the content of ⅲ is less than 0.0 1%. Except Mahonia angustifolia, I and II have low contents and weak peaks in other plants. The peak of ⅴ is very strong in most plants, and its content is high (in this experiment, it was not quantified because of the small amount of reference substance).
3.4 The distribution and content of these seven components in different parts of the genus are different, and the degree of difference is also different for different plant species. The content of root bark is basically the highest, followed by stem bark, followed by root wood and stem wood. In the past, the stem was used as medicine [1], but from the analysis results, its root is also a resource that can not be ignored.
Author: Lu Chengdu Guanghua School of Pharmacy, West China University of Medical Sciences 6 10044.
Wang Li is the director of the Institute of Medicinal Plants of China Academy of Medical Sciences of Peking Union Medical College.
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