Introduction
Chuanwu, the dry mother root of Aconitum carmichaelii Debx., which is mainly cultivated in Sichuan Province, Shaanxi Province, Yunnan Province, and some other places, has the efficacy of treating various rheumatic diseases and relieving pain as an anesthetic. In recent years, the output of the authentic production areas in Jiangyou City in Sichuan Province has decreased, while the output of other major producing areas has increased [1]. The mixed trading from different producing areas resulted in the unstable production quality of Chuanwu in the market. In order to standardize the various local markets, it is necessary to make a clear distinction among Chuanwu from different producing areas.
Alkaloids are the main active substances in Chuanwu, including diester alkaloids, monoester alkaloids, and water-soluble alkaloids [2]. Diester alkaloids, such as aconitine, hypaconitine, and mesaconitine (Fig. 1.), are the main toxic substances in Chuanwu, whose toxicity mainly affects the nervous system, muscle tissue, and heart [3]. Water-soluble alkaloids, such as neoline, fuziline, and zongorine (Fig. 1.), are also important active components of aconite, which have the effects of cardiotonic, antiarrhythmic, anti-inflammatory, and analgesic [4]. High performance liquid chromatography is an efficient and accurate method for the determination of alkaloids [5]. Solid phase microextraction combined with high performance liquid chromatography (SPE-HPLC) was used to determine the 6 alkaloids in Chuanwu preparation products [6]. The content of 6 alkaloids in processed aconite was determined by HPLC [7]. It was found that the content of monoester alkaloids increased significantly, and the content of diester alkaloids disappeared or decreased significantly. The UPLC/Q-TOF-MS/MS method was used by Ye [8] to analyze the chemical composition differences of raw and processed products of Chuanwu. It was found that different heating methods and accessories would affect the chemical composition of Chuanwu.
Due to the different ecological environments and cultivation conditions, the morphological habits of the original plants, the shape and structure, and the active components of traditional Chinese medicine may change. Ma [9] used HPLC to determine the contents of 6 alkaloids in 12 batches of commercially available Chuanwu. It was found that the contents of 6 alkaloids in 12 batches of samples were quite different. He speculated that this may be related to origin, harvest time, genetic basis, growth climate, and ecological environment. Luo [10] simultaneously determined the contents of five main alkaloids in the Fuzi water-soluble alkaloid extract and corresponding steamed aconite tablets by HPLC-ELSD. Subsequently, Luo [11] used the same method to determine the contents of three water-soluble alkaloids in Fuzi from different producing areas and found that the content of neoline can be used as an important evaluation index of Fuzi. Alkaloid content is one of the important indices in the quality evaluation of Chuanwu. In this study, HPLC-ELSD was applied to determine the contents of six representative alkaloids in Chuanwu from different producing areas, which were fuziline, neoline, zongorine, mesaconitine, hypaconitine, and aconitine. The characteristic indexes of different productions were explored. It will provide a theoretical basis for the quality evaluation of Chuanwu.
Experimental
Experimental Materials
From 2019 to 2020, 48 batches of Chuanwu samples were collected from Sichuan Province, Shaanxi Province, and Yunnan Province. (Table 1), they were identified as the dry mother root of Aconitum carmichaelii Debx. of Ranunculaceae by the researcher Yanli Xia of Chengdu University.
Instruments
The Agilent 1100 high performance liquid chromatograph, Agilent 1260 Infinity evaporative light scattering detector (Agilent Technology Co., Ltd., Chengdu, China), SB25-12YDTD ultrasonic cleaner (Xinzhi Biotechnology Co., Ltd., Ningbo, China), Tgl-1650 bench type refrigerated centrifuge (Shuke Instrument Co., Ltd., Sichuan, China), and WP-UFT-20 ultrapure water meter (Woter Water Treatment Equipment Co., Ltd., Sichuan, China) were provided by Chengdu University.
Table 1.
Chuanwu Sample Collection.
Reagents
Mesaconitine (MUST-17041010), aconitine (MUST- 17022206), hypaconitine (MUST-17041011), neoline (MUST-17102710), zongorine (DST180116-073), and fuziline (DST171130-013) were purchased from Chengdu Dester Biotechnology Co., Ltd. (Chengdu, China). Chromatographic grade acetonitrile and methanol were purchased from Fisher Scientific International Inc. (Shanghai, China). Chromatographic grade triethylamine was purchased from the Kelong Chemical Reagent Factory (Chengdu, China).
Experimental Methods
Investigation of the Main Producing Areas of Chuanwu
Researchers conducted field investigations in Sichuan Province, Shaanxi Province, and Yunnan Province, collected samples, and consulted literature. The climate, altitude, soil, harvest time, and cultivation management methods were investigated and recorded.
Investigation of the Appearance Properties of Chuanwu
The appearance characteristics of Chuanwu such as shape, surface color, texture, section color, hollowness, and shrinkage, were investigated and identified.
Determination of Alkaloids in Chuanwu
1. Determination of Aconitine, Mesaconitine, and Hypaconitine
(1) Preparation of reference substance solution: The appropriate amount of each reference substance was weighed accurately. A 50% methanol solution was added to prepare a mixed reference stock solution containing 20 mg aconitine, 20 mg mesaconitine, and 20 mg hypaconitine per 1 mL.
(2) Preparation of the test solution: About 3.0 g of Chuanwu medicinal powder was weighed and dissolved in a conical flask with a stopper containing 75 mL 0.05 mol/L hydrochloric acid-70% acidic methanol solution. The mixture was ultrasonically treated for 30 min at a power of 250 W and a frequency of 40 kHz, and then centrifuged at 8000 r·min-1 for 6 min after cooling. 50 mL supernatant was recovered to about 1~2 mL under reduced pressure at 60ºC. The recovery solution was then fixed to the scale in a 5 mL measuring bottle and filtered by a 0.45 μm filter membrane.
(3) Chromatographic conditions: Chromatographic column: Agilent Extend C18 column (250 mm×4.6 mm, 5μm). Mobile phase: (A) acetonitrile/tetrahydrofuran (v/v 25:15), (B) 0.1 mol/ L ammonium acetate solution (0.5 mL glacial acetic acid per 1000 mL). Gradient elution: 0~48 min, 15%→26% A. 48~49 min, 26%→35% A. 49~58 min, 35% A. 58~65 min, 35%→15% A. Detection wavelength: 235 nm. Flow rate:1.0 mL·min-1. Column temperature: 35ºC. Detector: Diode array detector. Injection volume: 10 μL.
2. Determination of Fuziline, Neoline, and Zongorine
(1) Preparation of reference substance solution: The appropriate amount of each reference substance was weighed accurately. A 50% methanol solution was added to prepare a mixed reference stock solution containing 0.25 mg of neoline, 0.15 mg of fuziline, and 0.15 mg of zongorine per 1 mL.
(2) Preparation of test solution: The same as that of 1.
(3) Chromatographic conditions: Chromatographic column: Agilent Extend C18 column (250 mm×4.6 mm, 5 μm). Mobile phase: (A) acetonitrile, (B) 0.1% triethylamine. Gradient elution: 0~8 min,10%→20% A. 8~15 min, 20%→25% A. 15~25 min, 25%→25% A. 25~30 min, 25%→28% A. 30~35 min, 28%→45% A. 3~42 min, 45%→50% A. 42~48 min, 50%→65% A. 48~60 min, 65%→85% A. 60~70 min, 85%→85% A. Flow rate: 1.0 mL·min-1. Column temperature: 35ºC. Detector: Evaporative light scattering detector. Carrier gas: Air. An evaporation temperature: 50ºC. Carrier gas flow rate: 1.3 L·min-1. Gain: 1. Injection volume: 20 μL.
Results and Discussion
Investigations in Main Producing Areas
Environmental Factors of Different Producing Areas
It can be seen from Table 2 that, compared with the other two provinces, Jiangyou City in Sichuan Province, Chengdu County, and Nan Zheng County in Shaanxi Province have a lower altitude, less sunshine, and a longer frost-free period. Located in Yunnan Guizhou Plateau, Chuanwu producing areas in Butuo County in Sichuan Province, Yun long County, and Binchuan County in Yunnan Province have the characteristics of a higher altitude, stronger light, and a shorter frost-free period.
Planting Mode, Field Management, and Harvest Time in Different Producing Areas
It can be seen from Table 3 that Jiangyou City in Sichuan Province, Chengdu County, and Nan Zheng County in Shaanxi Province are generally intercropped with corn and rotated with rice. The field management of removing redundant lateral roots and terminal buds was carried out. The harvest time is earlier, from late June to early August. Buckwheat is widely planted in rotation in Butuo County in Sichuan Province, Yun long County, and Binchuan County in Yunnan Province, with rough field management and a later harvest time from mid-September to early November. It is not difficult to find that with the increase in altitude, the harvest time of Chuanwu in different producing areas is delayed. The harvesting time of Chuanwu in Jiangyou City in Sichuan Province, Chengdu County, and Nan Zheng County in Shaanxi Province meets the provisions of Chinese Pharmacopoeia 2020.
Analysis of the Appearance and Properties of Chuanwu
See Fig. 2, Fig. 3, Fig. 4, Fig. 5, and Table 4 for the investigation results of the appearance and shape of Chuanwu. The samples of Jiangyou have shrunk slightly, with a brown surface, a white section, and a firm and full texture. The samples of Shaanxi have serious shrinkage, a grayish brown to tan surface, a yellow section, a medium texture, and a slightly full texture. The samples of Butuo and Yunnan have serious shrinkage, a light weight, and obvious hollowness.
Analysis of the Content Determination Results of Three Diester Alkaloids
The chromatograms of three diester alkaloids are shown in Fig. 6. The determination results are shown in Table 5.
It can be found that the content distributions of the three alkaloids vary from region to region.
The average content of three diester alkaloids in Chuanwu from Jiangyou is as high as 0.1015%, among which the content of aconitine is as high as 0.0520%, accounting for 50.73% of the three diester alkaloids. The proportions of aconitine, mesaconitine, and hypaconitine were 50.73%, 36.04%, and 13.23%, respectively.
Table 2.
Comparison of meteorological factors in different producing areas.
Table 3.
Comparison of planting mode, field management, and harvest time of Chuanwu in different producing areas.
Table 4.
Appearance and shape of Chuanwu from different producing areas.
Table 5.
Determination results of three diester alkaloids in Chuanwu from different producing areas.
[i] *AC is the content ratio of Aconitine/total alkaloids. MAC is the content ratio of Mesaconitine/total alkaloids. HAC is the content ratio of Hypaconitine/total alkaloids. DA is the content ratio of three diester alkaloids/total alkaloids. AC/DA is the content ratio of Aconitine/three diester alkaloids. MAC/DA is the content ratio of Mesaconitine/total alkaloids. HAC/DA is the content ratio of Hypaconitine/three diester alkaloids. **p<0.05
The average content of the three diester alkaloids in Chuanwu from Shaanxi is as high as 0.0942%, among which the content of aconitine is as high as 0.0472%, accounting for 49.53% of the content of the three diester alkaloids. The proportions of aconitine, mesaconitine, and hypaconitine were 49.53%, 32.93%, and 17.54%, respectively. The distribution of alkaloids in Shaanxi is similar to that in Jiangyou City.
The sum of the three diester alkaloids in Butuo is the lowest, with an average value of only 0.0294%. The content of aconitine and mesaconitine was the lowest among the four producing areas. However, the ratio of the content of hypaconitine to the three diester alkaloids was as high as 31.44%, which was the highest among the four producing areas. The proportions of aconitine, mesaconitine, and hypaconitine were 13.94%, 54.62%, and 31.44%, respectively.
The total content of the three diester alkaloids in Yunnan was also low, with an average value of only 0.0694%, which was significantly lower than that in Jiangyou and Shaanxi. However, the content of mesaconitine was as high as 0.0496%, accounting for 73.31% of the three diester alkaloids, which was significantly highest among the four producing areas. The content of hypaconitine was only 0.0029%, accounting for 4.07% of the three diester alkaloids, which was significantly lower than that of other producing areas. The proportions of aconitine, mesaconitine, and hypaconitine were 22.62%, 73.31%, and 4.07%, respectively.
Analysis of Content Determination Results of Three Water-Soluble Alkaloids
The chromatograms of three water-soluble alkaloids are shown in Fig. 7. The determination results are shown in Table 6.
It can be seen from Table 6 that the distribution content of the three water-soluble alkaloids in Chuanwu from different producing areas is obviously different. The content of the three water-soluble alkaloids in Chuanwu from Jiangyou City in Sichuan Province was as high as 0.1023%, which was the highest among the four producing areas. The content of fuziline was as high as 0.0145%, which was significantly higher than that of Butuo and Yunnan, accounting for 14.00% of the three water-soluble alkaloids. The content of neoline was as high as 0.0467%, which was the highest among the four producing areas, accounting for 45.50% of the three water-soluble alkaloids. The content of zongorine was as high as 0.0411%, accounting for 40.50% of the three water-soluble alkaloids. The ratio of the total content of the three water-soluble alkaloids to the total content of the three diester alkaloids is about 1.2.
Fig. 6.
a) Reference Standards, b) Jiangyou City, Sichuan Province, c) Shaanxi Province, d) unnan Province, e) Butuo Country, Sichuan Province.
As for Chuanwu from Shaanxi Province, the sum of the three water-soluble alkaloids was as high as 0.0739%, which was significantly higher than that in Butuo and Yunnan. The content of fuziline was as high as 0.0146%, which was obviously higher than that in Butuo and Yunnan, accounting for 15.44% of the three water-soluble alkaloids. The content of neoline was as high as 0.0283%, which was significantly higher than that of Butuo and Yunnan, accounting for 37.40% of the three water-soluble alkaloids. The content of zongorine was 0.0309%, accounting for 47.17% of the three water-soluble alkaloids. The ratio of the total content of the three water-soluble alkaloids to the total content of the three diester alkaloids is about 0.83.
Table 6.
Determination results of three water-soluble alkaloids in Chuanwu from different producing areas.
[i] *FL is the content ratio of fuziline/total alkaloids. NL is the content ratio of neoline/total alkaloids. ZR is the content ratio of zongorine/total alkaloids. WA is the content ratio of three water-soluble alkaloids/total alkaloids. FL/WA is the content ratio of fuziline/three water-soluble alkaloids. NL/WA is the content ratio of neoline/three water-soluble alkaloids. ZR/WA is the content ratio of zongorine/three water-soluble alkaloids. WA/DA is the content ratio of three water-soluble alkaloids/three diester alkaloids.
However, the sum of the three water-soluble alkaloids in Chuanwu from Butuo Country in Sichuan Province was only 0.0492%, which is significantly lower than that in Jiangyou and Shaanxi. The content of fuziline was only 0.0054%, which was significantly lower than that of Jiangyou and Shaanxi, accounting for 10.88% of the three water-soluble alkaloids. The content of neoline was only 0.0039%, which was significantly lower than that of Jiangyou and Shaanxi, accounting for only 7.70% of the three water-soluble alkaloids. The content of zongorine was 0.0399%, accounting for 81.43% of the three water-soluble alkaloids. The ratio of the total content of the three water-soluble alkaloids to the total content of the three diester alkaloids was about 2.07.
As for Chuanwu from Yunnan Province, the sum of the three water-soluble alkaloids was the lowest, which was only 0.0463% and significantly lower than that in Jiangyou and Shaanxi. The content of fuziline was only 0.0070%, accounting for 14.95% of the three water-soluble alkaloids. The content of neoline was only 0.0063%, which was significantly lower than that of Jiangyou and Shaanxi, accounting for only 12.87% of the three water-soluble alkaloids. The content of zongorine was 0.0331%, accounting for 72.18% of the three water-soluble alkaloids. The ratio of the total content of the three water-soluble alkaloids to the total content of the three diester alkaloids is about 0.71.
By comparing the four producing areas, it can be found that the contents of the three water-soluble alkaloids, fuziline, and neoline, from Jiangyou and Shaanxi are significantly higher than those from Butuo and Yunnan. Particularly, the content of neoline in Jiangyou was significantly higher than that in the other three producing areas, with an average of 0.0467% accounting for about 45% of the three water-soluble alkaloids. It was about 1.65 times the average value of Shaanxi, 11 times that of Butuo, and 7 times that of Yunnan. Therefore, the content of neoline can be used as a characteristic difference component to distinguish Jiangyou from other producing areas.
Analysis of Characteristic Indexes and Establishment of Quality Standards for Chuanwu from Four Producing Areas
Combined with the analysis of the content determination results of three diester alkaloids and three water-soluble alkaloids, the contents of three diester alkaloids and three water-soluble alkaloids in the four main producing areas were comprehensively analyzed, and the different components in each producing area were screened out. Based on the average value of each production area, the key characteristic indexes of each production area were selected, respectively, and then combined with the original sample data. Based on the data range that each sample can reach, the quality standards of the characteristic indexes of Chuanwu in four producing areas were established. The results are shown in Table 7.
Results and Discussion
Screaming of Characteristic Indexes and Cause Analysis of Chuanwu in Jiangyou City, Sichuan Province
Due to its low altitude, Jiangyou City in Sichuan Province has an annual frost-free period of nearly 280 days and abundant rainfall throughout the year. Due to such climatic conditions and man-made management, the surface of the local Chuanwu is brown, the cross section is white, and the texture is solid and full, which is obviously different from other producing areas. Through comprehensive evaluation, the characteristic index of Jiangyou Chuanwu was preliminarily established. The content of neoline is greater than 0.02%. The sum of the three water-soluble alkaloids is greater than 0.05%. The content of neoline accounted for more than 35% of the sum of three water-soluble alkaloids. The content of zongorine accounted for less than 50% of the sum of the three water-soluble alkaloids. The ratio of the sum of three water-soluble alkaloids to the sum of three diester alkaloids was greater than 0.6. Chuanwu which meets all the above conditions can be identified as being produced in Jiangyou City, Sichuan Province.
Screaming of Characteristic Indexes and Cause Analysis of Chuanwu in Shaanxi Province
Hanzhong City in Shaanxi Province has a low altitude and an annual frost-free period of nearly 240 days. The field management there is relatively rough. Due to such climatic conditions and man-made management, the surface of the local Chuanwu is grayish brown to tan, the cross section is yellow, and the texture is medium and slightly plump. Through comprehensive quality evaluation, the Chuanwu from Hanzhong can be identified and distinguished by the *AC/DC is the content ratio of aconitine/three diester alkaloids. MAC/DA is the content ratio of mesaconitine/three diester alkaloids. HAC/DA is the content ratio of hypaconitine/three diester alkaloids. FL is the content ratio of fuziline/total alkaloids. NL is the content ratio of neoline/total alkaloids. WA is the content ratio of three water-soluble alkaloids/total alkaloids. NL/WA is the content ratio of neoline/three water-soluble alkaloids. ZR/WA is the content ratio of zongorine/three water-soluble alkaloids. WA/DA is the content ratio of three water-soluble alkaloids/three diester alkaloids. following indicators: The content of neoline was greater than 0.01%, accounting for more than 20% of the total content of the three water-soluble alkaloids. However, the content of zongorine accounted for less than 70% of the sum of the three water-soluble alkaloids. The Chuanwu that met all the above indicators could be identified as those that were produced in Hanzhong City, Shaanxi Province.
Table 7.
Summary of Characteristic Differences in Each Producing Area.
Screaming of Characteristic Indexes and Cause Analysis of Chuanwu in Butuo Country, Sichuan Province
Butuo Country in Sichuan Province has high altitude as well as strong sunshine, and the frost-free period is 200 days throughout the year. The field management there is rather rough. Due to such climatic conditions and man-made management, the local Chuanwu shrinks seriously, with a grayish brown to tan surface, a yellow section, a light texture, and an obvious hollow. The local ebony shrank seriously, with a grayish brown to yellowish brown surface, a yellow section, a light texture, and an obvious hollowness. Through comprehensive quality evaluation, Chuanwu from Butuo can be distinguished by the following indicators: The content of aconitine accounted for less than 30% of the total of the three diester alkaloids. The contents of fuziline and neoline were both less than 0.01%. The contents of neoline and zongorine accounted for less than 20% and 70% of the sum of the three water-soluble alkaloids, respectively. The content ratio of the three water-soluble alkaloids to the three diester alkaloids was greater than 0.8. The Chuanwu that met all the above indicators can be identified as Chuanwu produced in Butuo Country, Sichuan Province.
Screaming of Characteristic Indexes and Cause Analysis of Chuanwu in Lijiang City, Yunnan Province
The climatic conditions in Lijiang City, Yunnan Province, are similar to those in Butuo County, Sichuan Province, with an annual frost-free period of about 100-200 days. On-site management is also relatively rough. Due to such climatic conditions and man-made management, Chuanwu from this produce area shrank seriously, with a grayish brown to tan surface, a yellow section, a light texture, and an obvious hollow. The following indicators can be used to distinguish Chuanwu from Yunnan: The content of mesaconitine accounted for more than 45% of the sum of the three diester alkaloids. The content of hypaconitine was less than 10% of the sum of the three diester alkaloids. The content of fuziline was less than 0.01%. The content of neoline was less than 30% of the sum of the three water-soluble alkaloids. The content of zongorine accounted for more than 50% of the total content of three water-soluble alkaloids. The Chuanwu produced in accordance with all the above indexes could be identified as Chuanwu produced in Yunnan Province.
Reason Analysis of Quality Formation of Chuanwu from Different Producing Areas
There were few studies on the quality of Chuanwu from different producing areas, but there were some studies on the quality differences of Fuzi from different producing areas. Luo [12] collected 42 batches of Fuzi samples from four producing areas. It was reported that the geographical origin might be the main factor affecting the geographical authenticity of Fuzi. Neoline might be the key component to identifying the geographical authenticity of Fuzi. Yue Conghui [13] analyzed and evaluated Fuzi in different cultivation areas and compared the cultivation system, yield, alkaloid content, polysaccharide, and protein content of Fuzi cultivated in Butuo County and Jiangyou City in Sichuan Province and Mianxian County and Chengdu County in Shaanxi Province. It was reported that the Jiangyou cultivation area was the best, Butuo and Mianxian were the second, and the Chengdu cultivation area was the worst. Jiang [14] determined the content of fuziline and zongorine in Fuzi, which was cultivated in Butuo County in Sichuan Province, Mianxian County, and Chengdu County in Shanxi Province, by HPLC-MS. It was found that the sum of the two alkaloids in Jiangyou was the highest, while that in Chengdu was the lowest. The contents of aconite alkaloids in different cultivation areas were compared by Shu [15]. It was found that there were great differences in the contents of aconite extract, total alkaloids, and diester alkaloids, which were speculated to be related to the growth environment and cultivation mode of Fuzi.
Conclusions
In conclusion, it can be speculated that the growth environment, cultivation management modes, and harvesting time of producing Chuanwu might jointly contribute to the difference in product quality. Jiangyou City in Sichuan Province and Hanzhong City in Shaanxi Province both have low altitudes, weak sunshine, and early harvesting times, showing a similar law that the content of neoline is relatively high and the content of zongorine is relatively low. Butuo Country in Sichuan Province and Lijiang City in Yunnan Province both have high altitudes, strong sunshine, and later harvesting times, showing a similar law that the content of neoline is relatively low and the content of zongorine is relatively high. Therefore, the proportion of neoline content and zongorine content can be used as a characteristic identification index for the quality difference of Chuanwu in different producing areas, which provides a reference for establishing scientific quality evaluation methods and quality control standards for Chuanwu.
