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J. Sep. Sci. 2015, 38, 9–17

Yali Chen1,2 Min Li1 Jianjun Liu3 Qian Yan1 Mei Zhong1 Junxi Liu1 Duolong Di1 Jinxia Liu4 1 Key

Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou, PR China 2 Institute of Medicinal Chemistry, School of Pharmacy, Lanzhou University, Lanzhou, PR China 3 University hospital of Gansu Traditional Chinese Medicine, Lanzhou, PR China 4 Institute of Biology, Gansu Academy of Sciences, Lanzhou, PR China Received August 19, 2014 Revised September 30, 2014 Accepted October 9, 2014

Research Article

Simultaneous determination of the content of isoquinoline alkaloids in Dicranostigma leptopodum (Maxim) Fedde and the effective fractionation of the alkaloids by high-performance liquid chromatography with diode array detection A simple and efficient method was developed for the simultaneous determination of eight isoquinoline alkaloids in methanol extracts of Dicranostigma leptopodum (Maxim) Fedde and the effective fractionation of the alkaloids of D. leptopodum by high-performance liquid chromatography with diode array detection. The chromatographic conditions were optimized on a SinoChrom ODS-BP column to obtain a good separation of the four types of alkaloid analytes, including two aporphines (isocorydine, corydine), two protopines (protopine and allocryptopine), a morphine (sinoacutine), and three quaternary protoberberine alkaloids (berberrubine, 5-hydroxycoptisine, and berberine). The separation of these alkaloids was significantly affected by the composition of the mobile phase, and particularly by its pH value. Acetonitrile (A) and 0.2% phosphoric acid solution adjusted to pH 6.32 with triethylamine (B) were selected as the mobile phase with a gradient elution. With this method, a new quaternary protoberberine alkaloid was isolated and the two structural isomers (isocorydine and corydine) were baseline separated. The appropriate harvest period for D. leptopodum was also recommended based on our analysis. The method for the effective fraction of the alkaloids of D. leptopodum was optimized under this method with regard to the varying significant pharmacological activities of the alkaloids. Keywords: Dicranostigma leptopodum (Maxim) Fedde / High-performance liquid chromatography / Isocorydine / Isoquinoline alkaloids DOI 10.1002/jssc.201400905



Additional supporting information may be found in the online version of this article at the publisher’s web-site

1 Introduction Alkaloids are important natural products that exhibit several pharmacological effects. Isoquinoline alkaloids are reported to contribute to the biological activity of this species and isoquinoline alkaloids can be divided into approximately 20 categories, including aporphines, protopines, morphine, and quaternary protoberberine alkaloids. The alkaloid family Correspondence: Professor Junxi Liu, Lanzhou tianshuizhonglu No18, Lanzhou, Gansu 730000, China E-mail: [email protected] Fax: +86-931-8277088

Abbreviations: AL, allocryptopine; BE, berberrubine; BR, berberine; CO, corydine; DAD, diode array detection; DLF, Dicranostigma leptopodum (Maxim) Fedde; EFA, effective fraction of the alkaloids; HY, 5-hydroxy-coptisine; IS, isocorydine; PO, protopine; SI, sinoacutine

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contains the most potential natural product candidates for the discovery of new drugs and therefore, the rapid and direct characterization of alkaloids in crude plant extracts is very important in pharmaceutical science [1–5]. Dicranostigma leptopodum (Maxim) Fedde (DLF) is an ornamental plant that grows mainly in the Qinling mountain area in the northwest of China. During phytochemical investigations on this plant, some isoquinoline alkaloids were isolated and identified, such as isocorydine (IS), berberrubine, berberine, dihydrosanguinaline, sinoacutine, corydine, isocorydione, N-methylhernovine, protopine, allocryptopine, and a new compound, 5-hydroxy-coptisine, which was isolated by our group. Previous studies have shown that the whole plant of DLF displays antipyretic, analgesic, detumescence, and, notably, antitumor activity. [6–11]. These isoquinoline alkaloids of DLF, particularly IS and the quaternary protoberberine alkaloids, all have significant pharmacological activities. In recent research,

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IS has been demonstrated to not only inhibit cell proliferation in hepatocellular carcinoma cell lines by induced G2/M cell cycle arrest and apoptosis, but also to target the drug-resistant cellular side population (or cancer stem cells) through PDCD4-related apoptosis [12–14]. Our group modified the structure of IS to obtain a series of derivatives. After screening research, 8-acetamino-isocorydine was selected as a candidate for new drug development owing to its good inhibitory effect on murine hepatoma H22 -induced tumors [15]. Berberine, as a quaternary protoberberine alkaloid, has been demonstrated to possess anticancer activity in different types of human cancer cells [16, 17]. Recent research also finds berberine can induce apoptosis and DNA damage in MG_63 human osteosarcoma cells [18]. Berberrubine is the metabolite of berberine in vivo, which is more lipophilic than berberine. Then, berberrubine has more efficient intestinal absorption, suggesting that berberrubine possesses more potential pharmacological activity than berberine [19]. As so many antitumor effects compounds in DLF, we predicted that the isoquinoline alkaloids are the material basis for the antitumor effects of DLF, although it possesses a limited content of this alkaloid [20–23]. To ensure the effectiveness and safety of traditional Chinese medicines in clinical applications, it is necessary to develop an analytical method for the quantitative determination of major alkaloids in DLF. Many effective analytical techniques [24–29], such as CE, CE–ESI-MS, and HPLC–MS have been applied for the determination of the content of isoquinoline alkaloids from natural sources. HPLC is the most extensively used technique, owing to its high resolution, sensitivity, great versatility, and simple sample pretreatment. To date, although there are some reports on the separation and determination of alkaloids in extracts of DLF by HPLC, the effectiveness of the method and the advancement of the technology have certain defects. In those literatures,

only one or two alkaloid standards were selected for quantitative analysis. At the same time, gradient elution has not be used widely because it can lead to poor separation of the chromatographic peak [30, 31]. In the present research, a convenient and sensitive method has been developed for the simultaneous quantitative determination of the eight isoquinoline alkaloids, including IS, corydine (CO), protopine (PR), allocryptopine (AL), sinoacutine (SI), berberrubine (BE), 5-hydroxy-coptisine (HY), and berberine (BR) (Fig. 1), from the methanol extracts of DLF using HPLC coupled with diode array detection (DAD). Through this method, two isomers (IS and CO) have been separated on the baseline in one run and a new quaternary protoberberine alkaloid (HY) was serendipitously discovered by the established chromatographic method [10]. The method was successfully applied to the established preparative method for the effective fraction of the alkaloids (EFA) of DLF.

2 Materials and methods 2.1 Chemicals, reagents, and samples The DLF plants were collected at Chongxin in the northwest of China (Supporting Information Fig. S1), and authenticated by Prof. Zhigang Ma of Lanzhou University, China. Voucher specimens (ZYC20120525) were deposited in the Key Laboratory of Chemistry of Northwestern Plant Resources, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences. The reference compounds of IS, CO, SI, PR, AL, BE, HY, and BR were isolated and purified from the whole DLF plant in our laboratory and the alkaloids obtained were found to be more than 99.8% pure through HPLC–DAD analysis, based on the peak area normalized method. The purified compounds were identified by various spectroscopic methods, including

Figure 1. Chemical structures of isoquinoline alkaloids isolated from DLF.

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intensive 2D-NMR techniques, and high-resolution (HR) ESIMS analysis. Acetonitrile, purchased from J&K Chemicals (USA), was of chromatographic grade. Methanol, phosphoric acid, and triethylamine were of analytical grade, and were purchased from Tianjin Chemical Reagent (Tianjin, China). Distilled and deionized water were obtained with a Spring-R10 water purification system (Research Scientific Instrument, Xiamen, China).

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was placed into a 50 mL flask and extracted twice with 20 mL of methanol (0.5 h for each time) in an ultrasonic bath at room temperature. The combined extracts were mixed and diluted to 50 mL with methanol. The solution was filtered through a 0.45 ␮m membrane and 20 ␮L of the filtered solution was injected into the HPLC system. The chromatographic peaks of the analytes were confirmed by comparing their retention time and UV spectra with those of the reference standards. Quantitative analysis was carried out by the standard curve method.

2.2 Instrumentation An Agilent 1200 series LC system, equipped with a G1312A binary pump, G1315B diode array detector, and G1328B manual injector, with a wavelength range of 190–950 nm, and Agilent ChemStation software (version A.10.02) were used for the HPLC–DAD method. The chromatographic separation of analytes was performed on a SinoChrom ODS-BP (250 mm × 4.6 mm, 5 ␮m) column, which was purchased from Dalian Elite Analytical Instruments, Dalian, China. The solvent system used for the chromatographic separation was a gradient elution of acetonitrile (A) and 0.2% phosphoric acid in water adjusted by triethylamine solution to pH 6.32 (B). The gradient elution of the mobile phase was 20–26% (A) at 0–20 min, 26–50% (A) at 20–35 min, 50% (A) at 35–37 min, 50–20% (A) at 37–40 min, and 20% (A) at 40–45 min. The flow rate was maintained at 1.0 mL/min, while the column temperature was maintained at 30⬚C. The injection volume was 20 ␮L for each run. The wavelength of the DAD detector ranged from 190 to 400 nm and the detected wavelength was set at 270 and 360 nm. A Mettler Instrumente CH-8606 (Greifensee-Zurich, Switzerland) was employed to weigh the standard compounds. A Sartorius PB-10 Standard pH meter (Germany) was employed to adjust the pH value of the mobile phase.

2.3 Preparation of reference solutions Eight reference analytes of IS (1.544 mg), CO (1.056 mg), SI (0.726 mg), PR (1.132 mg), AL (1.248 mg), BE (1.257 mg), HY (1.095 mg), and BR (1.558 mg) were accurately weighed and diluted to 1 mL with methanol to produce the standard stock solutions. The stock solutions were stored at 4⬚C and brought to room temperature before use. A standard mixture solution was prepared by mixing 0.1 mL each of the eight standard stock solutions and then diluted to 1 mL with methanol. Then, a multiple proportion dilution of the standard mixture solution was employed to prepare the required concentration for the standard curves.

2.5 Preparation of the EFA of DLF The dried DLF from Chongxin, Gansu, China (1.0 kg) was pulverized and soaked in 15 L of a 0.5% aqueous hydrochloric acid (HCl) solution for 2 h. The mixture was then refluxed for 1 h three times. After neutralization with 10% aqueous sodium hydroxide solution, the solution containing the extracts was concentrated to 0.5 L under reduced pressure on a rotary evaporator. Ninety five percent ethanol was added to the concentrated residue with vigorous stirring until the concentration of ethanol reached 70%, and the mixture was then allowed to stand for 4 h. The mixture was filtered by vacuum suction filtration and the filtrate was concentrated to 1 L at reduced pressure on a rotary evaporator. The pH was adjusted to 10, and the mixture was extracted in chloroform (3 × 400 mL). The solvent of chloroform part was removed under reduced pressure and 100 mL methanol was added to the residue to recrystallize IS. Part of the water solution was added to a glass column packed with pretreated LX28 (500 g), which is a kind of macroporous adsorption resin that was chosen for its good selective adsorption of isoquinoline alkaloids found in DLF in our other work. After the adsorption process, the column bed was washed with 2 L of water. Finally, 95% ethanol (2000 mL) was applied to wash the macroporous adsorption resin bed for desorption of the alkaloids. The desorbed fraction and the mother liquor from the recrystallization were combined and concentrated to dryness under reduced pressure in a rotary evaporator to afford the EFA.

2.6 Preparation of a solution of the EFA of DLF for chromatographic analysis A powder of the EFA of DLF (10 mg) was accurately weighed and dissolved in a volumetric flask with methanol to a volume of 100 mL, to afford the sample solution for chromatographic analysis.

3 Results and discussion 2.4 Preparation of sample solutions for chromatographic analysis

3.1 Selection of chromatographic conditions

Dried DLF was pulverized and the powder was passed through 30 mesh sieves. A total of 0.5 g of the sample powder

The quantitative determination of such alkaloids by chromatographic methods was always a challenge in analytical

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chemistry. To address this challenge, we test two newgeneration columns, including the Waters XTerra MS C18 (250 mm × 4.6 mm, 5 ␮m) and the SinoChrom ODS-BP C18 (250 mm × 4.6 mm, 5 ␮m) columns. Both these two columns can be fully endcapped, decrease the number of surface silanols and ensure low silanol activity, which could improve their ability to separate alkaloids. However, the former column was more suitable for the separation of the quaternary protoberberine alkaloids not for all four types of isoquinoline alkaloids. And it was also better for separating alkaloids at lower pH values of the mobile phase, not suitable for this experiment. Therefore, the SinoChrom ODS-BP C18 column was selected for the subsequent experiments [32, 33]. With regard to the representative content of chemical constituents and the significant pharmacological activities in this traditional Chinese medicine, the eight isoquinoline alkaloids, IS, CO, PR, AL, SI, BE, HY, and BR (Fig. 1 and Supporting Information Fig. S1), were selected as the reference analytes for the development of the quantitative analytical method for DLF. To achieve good separation and an ideal distribution, various system conditions were all investigated, including the mobile phase, use of buffers of various strengths, pH of the mobile phase, gradient, and the wavelength. Several HPLC methods report the use of phosphate buffers, ion-pairing, ionic liquid additive, and triethylamine solutions in the mobile phase to overcome peak broadening and tailing issues [32–37]. During the selection of the mobile phase, various systems, such as methanol/phosphoric

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acid, methanol/triethylamine, acetonitrile/phosphoric acid or phosphate buffers, acetonitrile/triethylamine solutions, ionic liquid (1-butyl-3-methylimidazole tetrafluoroborate, BMIM BF4 ) and SDS were tested to identify the optimum conditions. Finally, acetonitrile was selected as the organic phase and a phosphoric acid/triethylamine solution was selected as the aqueous phase because this system gave better resolution, peak shape, and a more stable baseline when compared with other buffers systems or additive. To improve the resolution and peak symmetry, various concentrations of phosphoric acid and triethylamine, as buffers of different strengths, were tested. It was found that the presence of the phosphate buffer (0.2% phosphoric acid adjusted to pH 6.32 with triethylamine) in the mobile phase resulted in a significant improvement in the retention behavior of the eight alkaloids. The pH of the mobile phase was the most important factor in the RP-HPLC separation of basic alkaloid compounds, because it could greatly affect the elution patterns and the retention behavior of the alkaloids. Thus, the use of an acidic mobile phase is generally preferable for HPLC analysis of alkaloids [35]. To select an optimal pH for the mobile phase, eight pH values (pH 3.89, 4.31, 4.76, 5.31, 5.65, 6.32, 6.57, and 6.92) were examined for their effect on the retention behavior of the standard analytes. The results are shown in Fig. 2. As the pH increased from 3.89 to 6.92, the retention times of tertiary amine alkaloids (protopine, aporphine, and morphine alkaloids) were increased due to their decreased polarity and hydrophilicity. In contrast, the retention times of the quaternary

Figure 2. Chromatogram of standard solution at different pH values (pH from 3.89 to 6.92). Conditions: 270 nm; Mobile phase: acetonitrile (A) and 0.2% phosphoric acid in water, adjusted by triethylamine solution to pH 6.32 (B); Gradient: 20–26% (A) at 0–20 min, 26–50% (A) at 20–35 min, 50% (A) at 35– 37 min, 50–20% (A) at 37–40 min, and 20% (A) at 40–45 min; Injection volume: 20 ␮L; Flow rate: 1.0 mL/min.

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protoberberine alkaloids did not change significantly with the pH value. These phenomena can be justified, as quaternary protoberberine alkaloids are charged and do not change their chemical structures under acidic conditions, but tertiary amine type alkaloids are converted into their protonated form in acidic media, resulting in a decreased retention time on the C18 column. In the literature [32–35], a 0.1% v/v concentration of formic acid was selected as the mobile phase for the separation of quaternary protoberberine alkaloids. However, in our experiment, with the low pH of the mobile phase (pH 3.89), BE, CO, and IS were eluted at almost the same time (at about 10 min), and the chromatographic peaks overlapped substantially. Therefore, a strongly acidic mobile phase was not suitable for the separation of the four types of alkaloids in our case. Finally, a mobile phase with a pH of 6.32 was selected for the separation of the eight alkaloids, and under these conditions, baseline separation of the two protopine alkaloids, PR and AL, and the two aporphine alkaloids, CO and IS, was achieved. In summary, the optimum resolution was achieved when acetonitrile (A) and 0.2% phosphoric acid, adjusted by triethylamine (B) to pH 6.32, were selected as the mobile phase. Under the optimal conditions, good resolution and satisfactory peak shape were achieved and the eight target compounds could be eluted with baseline separation within 45 min (Fig. 2, Supporting Information Figs. S1 and S2). A typical chromatogram for the mixed standard solution is shown in Supporting Information Fig. S1. It can be seen that the eight standard analytes were well separated, though there is some tailing for the quaternary protoberberine alkaloids (BE, HY, and BR) in the chromatogram of the standard mixed solution (Supporting Information Fig. S1). This is a common problem in isolating quaternary alkaloids on silica gel. Undoubtedly, the tailing peak will have an impact on the validation of the content determination, as can be deduced from the linear correlation coefficient. Due to the diverse physicochemical properties, with hydrophilic and lipophilic components co-existing in the DLF extract, the gradient elution was used in the present experiment. The content of the aqueous phase (B) was slowly decreased from 80 to 74% in 20 min to elute the majority of the hydrophilic impurities. Then, the content of acetonitrile was increased from 26 to 50% in the next 15 min, while maintaining the proportion of the mobile phase for 2 min which allowed the resolution of similar chemical structures, including CO and IS, and PR and AL. In particular, the two isomers, CO and IS, had a good separation. To reduce the analysis time and maintain stable baselines, the content of acetonitrile (A) was reduced from 50 to 20% for 3 min and this was maintained for 5 min. The optimum gradient of the mobile phase using acetonitrile (A) and an aqueous buffer solution (containing 0.2% phosphoric acid, adjusted with triethylamine to pH 6.32) (B) was found to be: 20–26% (A) at 0–20 min, 26–50% (A) at 20–35 min, 50% (A) at 35–37 min, 50–20% (A) at 37–40 min, and 20% (A) at 40–45 min. Under

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this condition, the two categories of components were first separated chromatographically. In the present study, 270 nm was selected as the detection wavelength for all but the quaternary protoberberine alkaloids, which were detected at 360 nm. The details are shown in the Supporting Information.

3.2 Optimization of sample preparation conditions For optimal extraction efficiency, reflux and ultrasonic extraction were selected for the sample preparation conditions (Table 1). Methanol and a 0.5% HCl aqueous solution were selected as the extraction solvents as methanol possesses higher solubility and extraction capability than other solvent systems, such as water/methanol, ethanol, or ethanol solution. In addition, 0.5% HCl aqueous extracted DLF was more useful for the industrial production and those target compounds were all alkaloids and were, therefore, suitable for extraction in acidic solution. Temperature, solvent, amount, and extract times were all examined for extraction efficiency. The results indicated that the eight target compounds could be extracted efficiently in methanol using ultrasonic extraction at room temperature and reflex extraction in 0.5% HCl aqueous. Ultrasonic extraction is a simple, efficient, and classical extraction method for natural products but it is not suitable for the industrial production. Therefore, we chose ultrasonic extraction with methanol for the small amount of DLF, and considering industrial production the reflex extraction with 0.5% HCl aqueous was selected for the large amount of DLF.

3.3 Validation of the HPLC assay The validation of the HPLC–DAD method was evaluated through validation parameters that included linearity, work range, sensitivity, precision, and recovery. A gradient elution program was developed for the separation and quantification of eight analytes by a single HPLC run within 45 min (Supporting Information Fig. S2). BE, PR, AL, HY, SI, BR, CO, Table 1. Influence of different pretreatments on the eight alkaloid constituents of DLF

Extract method

Reflex by methanol Reflex by 0.5% HCl aqueous Ultrasonic by methanol Ultrasonic by 0.5% HCl aqueous

Contents (mg/g) BE

PR

AL

HY

SI

BR

CO

IS

0.12

2.40

0.56

0.05

0.22

0.08

0.18

3.00

0.17

3.73

0.43

0.06

0.07

0.12

0.23

4.10

0.28

3.42

0.66

0.09

0.11

0.20

0.26

5.04

0.16

3.35

0.64

0.08

0.03

0.12

0.25

3.70

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Table 2. Regression equations, correlation coefficients of linear calibration graphs, and detection limits for the eight analytes

Analytes

Linear range (␮g/mL)

Regression equationa)

Correlation coefficient

LOD (ng/mL)

LOQ (ng/mL)

BE PR AL HY SI BR CO IS

0.20–126 1.56–100 1.88–120 0.21–110 0.56–72 0.30–156 0.21–106 0.15–154

y = 48.86 x − 9.610 y = 0.142 x + 0.050 y = 5.853 x + 0.089 y = 51.77 x − 33.66 y = 19.55 x + 7.499 y = 63.00 x − 3.662 y = 31.4 x + 30.69 y = 36.66 x +13.46

0.9994 0.9995 0.9998 0.9995 0.9998 0.9995 0.9980 0.9995

25 100 115 55 70 80 50 20

65 250 265 110 140 160 120 50

a) y represents the peak area, x represents the concentration (␮g/mL).

and IS were well-resolved and eluted at 10.9, 20.9, 21.5, 22.9, 26.4, 29.6, 34.2, and 35.1 min, respectively. These eight analytes of DLF were identified by comparing their retention time and UV spectra with their reference compounds. The peak purity was confirmed by comparing the DAD data with the peaks of the respective analytes. Impurities or overlapping peaks were not found in every targeted chromatographic peak. Integrated chromatographic peak areas were plotted against the corresponding concentration of the injected standard solutions to obtain the calibration curves. The injection concentration, which could be detected at the S/N of 3, was considered to be the LOD. The LOQ was the injection concentration corresponding to the peak heights with S/N of 10. The regression equations were established using seven concentration levels on six consecutive days. The detailed descriptions of the regression curves are presented in Table 2. The correlation coefficients of all the calibration curves were higher than 0.9995 (except CO for 0.9980). The precision was expressed as the RSD, six replicates of each analyte were injected on three consecutive days. The analytical precision assessed through the statistical results of the intraday and interday determination of eight analytes were 1.10–2.24 and 1.80–4.16, respectively, which implied Table 3. Intraday and interday precisions and recoveries of analytes spiked in the sample (Chongxin, Gansu, China, May) (n = 6)

Analytes

BE PR AL HY SI BR CO IS

Precision (RSD%)

Intradaya)

Inerdayb)

2.24 1.18 1.81 1.22 1.32 1.10 1.66 1.88

2.45 2.52 4.16 2.21 1.80 1.92 2.92 2.56

Reccovery mean (%)

RSD (%)

92.4 95.8 101.5 100.1 99.2 99.5 105.2 101.9

2.57 2.03 2.03 1.40 1.22 1.54 1.53 1.64

a) Determination in a day. b) Determination in three successive days.

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these analytes were all stabilized under the optimum condition (Table 3). A certain amount of the authentic standards was added into the known real sample, The mixtures were extracted as described in Section 2.5 and analyzed using the developed HPLC method mentioned above. Then, the quantity of each component was subsequently achieved from the corresponding calibration curves. As shown in Table 3, the average recoveries of the investigated components were 92.4–105.2%. Therefore, the results of our experiments demonstrated that the HPLC–DAD method definitely possessed the advantage of high precision, high resolution, and relatively short analysis time for the analysis of herbal DLF extracts.

3.4 Method application The method was applied for the determination of the eight analytes in DLF and the EFA of DLF. Twenty samples of DLF were collected from different locations at different times, and the contents of BE, PR, AL, HY, SI, BR, CO, and IS were simultaneously determined by HPLC–DAD method. The data are listed in Table 4. The results indicated that their chromatographic patterns were generally the same, although the peak intensities were different, indicating that the contents of the targeted chemical constituents varies greatly in different samples. The reason for this may be the variation in habitat, climate, circumstances or soil conditions. Different growing environments of DLF in different locations results in the variation of the flowering stage and maturing stage, which could impact the content of chemical components in DLF. On the whole, of the eight alkaloids tested, the content of IS was consistently higher than that of the other alkaloids. It can be seen that the content of IS in samples from Longnan was lower than that in other locations and the highest content of PR was determined in the samples from Wudu. Meanwhile, as shown in Table 4, the content of alkaloids in DLF is different in samples from the same locations because there harvest times are different. Dynamic analysis and evaluation results showed that the appropriate harvest period of DLF was during the full-bloom and initial fruiting stages, which are from the end of May to the middle of June in the www.jss-journal.com

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Table 4. Contents (mg/g) of the eight alkaloids in the extracts of DLF collected from different locations or at different times in 2012

Cultivar

Chongxin Chongxin Heshui Heshui Heshui Zhengning Zhengning Lingtai Lingtai Jingchuan Pingliang Guanzizhen Ningxian Gangu Qinchengqu Longnan Kangxian Wuduqu Hanwangzhen Dielytra

Date

5.26 6.15 5.26 6.15 7.15 5.26 6.15 5.26 6.15 5.29 5.29 5.29 5.29 5.29 5.29 5.29 5.29 5.29 5.29 5.29

Contents of each alkaloids (mg/g ± SD of dry sample, n = 3)a) BE

PR

AL

HY

SI

BR

CO

IS

0.24 ± 0.03 0.28 ± 0.07 0.24 ± 0.02 0.65 ± 0.10 0.13 ± 0.04 0.31 ± 0.00 0.47 ± 0.01 0.30 ± 0.02 0.21 ± 0.04 0.20 ± 0.03 0.47 ± 0.12 0.19 ± 0.00 0.24 ± 0.05 0.23 ± 0.01 0.44 ± 0.36 0.01 ± 0.003 0.03 ± 0.01 Un0.02 ± 0.01 0.04 ± 0.02

0.11 ± 0.01 0.15 ± 0.02 0.14 ± 0.02 0.92 ± 0.02 0.09 ± 0.01 2.48 ± 0.07 1.28 ± 0.07 1.30 ± 0.01 1.32 ± 0.06 0.26 ± 0.09 0.12 ± 0.05 0.27 ± 0.10 0.11 ± 0.03 3.01 ± 0.05 0.28 ± 0.02 5.25 ± 0.16 3.95 ± 0.04 10.44 ± 0.18 3.59 ± 0.04 1.25 ± 0.06

0.18 ± 0.01 0.15 ± 0.01 0.13 ± 0.00 0.10 ± 0.01 0.08 ± 0.00 0.11 ± 0.014 0.14 ± 0.10 0.05 ± 0.00 0.17 ± 0.01 0.13 ± 0.05 0.13 ± 0.01 0.19 ± 0.04 0.04 ± 0.00 0.57 ± 0.05 0.08 ± 0.01 3.10 ± 0.09 0.07 ± 0.004 0.13 ± 0.01 2.65 ± 0.05 0.03 ± 0.00

0.07 ± 0.01 0.05 ± 0.01 0.04 ± 0.00 Un

Simultaneous determination of the content of isoquinoline alkaloids in Dicranostigma leptopodum (Maxim) Fedde and the effective fractionation of the alkaloids by high-performance liquid chromatography with diode array detection.

A simple and efficient method was developed for the simultaneous determination of eight isoquinoline alkaloids in methanol extracts of Dicranostigma l...
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