Abstract
There has been a lively interest on macrocyclic polyamine alkaloids due to their remarkable pharmacological activities such as anti-tumor, anti-inflammatory, anti-Alzheimer's disease and anti-parasitic. Tripterygium wilfordii is a widely used traditional Chinese medicine, which is abundant in alkaloids including macrocyclic polyamine alkaloids. However, there are rarely studies on macrocyclic spermidine alkaloids of T. wilfordii so far. In this article, we use three known macrocyclic spermidine alkaloids celafurine, celabenzine and celacinnine, and successfully develop a simple and sensitive HPLC method for simultaneous quantification of macrocyclic spermidine alkaloids in root, stem and leaf of T. wilfordii.
Introduction
Spermidine alkaloids are polyamine heterocycles, biosynthetically derived from L-Orn or L-Arg via putrescine. The interests in the isolation and synthesis of these alkaloids result from the structural complexity and therapeutical properties attributed to these macrocycles [1]. A broad biological activity has been revealed by members of the polyamine family, such as anti-tumor [1], anti-inflammatory [2], anti-Alzheimer's disease [3] and anti-parasitic [4, 5]. However, although possessing remarkable activities, few macrocyclic polyamines have been isolated from natural products so far.
Tripterygium wilfordii is a widely used traditional Chinese medicine with a long history. Alkaloids, the main active components of T. wilfordii, possess many beneficial pharmacological activities, such as immunosuppression, anti-inflammation, analgesia, anti-tumor, anti-HIV, insecticidal, neuroprotection and so on [6]. The alkaloids of T. wilfordii include sesquiterpene alkaloids and macrocyclic polyamine alkaloids. In previous study, we found a new macrocyclic spermidine alkaloid of T. wilfordii, which possessed significant anti-inflammatory activity [2]. In addition, we also found other new unknown macrocyclic spermidine alkaloids existing in T. wilfordii [7]. Up to now, there are rarely studies on macrocyclic spermidine alkaloids of T. wilfordii and no researches on spermidine alkaloids analysis are reported. Therefore, it is meaningful to develop a simple and sensitive method for identify and determine macrocyclic spermidine alkaloids in T. wilfordii.
The macrocyclic polyamine alkaloids of T. wilfordii have 13 membered macrocyclic mother nucleus, with a unique structure of three nitrogen atoms. In this study, we use three known macrocyclic spermidine alkaloids celafurine, celabenzine and celacinnine (Fig. 1), which have been isolated and identified in root of T. wilfordii by our research group, to develop a simple and sensitive HPLC method for simultaneous quantification of macrocyclic spermidine alkaloids in root, stem and leaf of T. wilfordii.
Chemical structures of celafurine, celabenzine and celacinnine
Citation: Acta Chromatographica 35, 2; 10.1556/1326.2022.01014
Experimental
Materials
T. wilfordii was collected from Pingxiang City, Jiangxi Province in July 2020. The root, stem and leaf were identified by professor Jianqun Liu, key laboratory of modern preparation of traditional Chinese medicine, ministry of education, Jiangxi university of Chinese medicine. Celafurin, celabenzine and celacinnine were isolated from Tripterygium Wilfordii in our laboratory. The structures were identified by nuclear magnetic resonance and UPLC-Q-TOF-MS/MS, and which were consistent with the literature [8, 9]. The purities were more than 99% by HPLC.
Preparation of standard and sample solutions
Stand solutions
Celafurine, celabenzine and celacinnine were accurately weighed to 10 mL volumetric flask and dissolved in methanol to make a mixed standard solution 1 of 111.6, 130.9 and 129.8 μg mL−1, respectively. The solutions were stored at 4°C and warmed to room temperature before use. The stock solutions were diluted with methanol to get a series of mixed standard solutions. In addition, the solutions of each compound are prepared to determine the elution order.
Root, stem and leaf extract solutions
10 g powder of root, stem and leaf of T. wilfordii were accurately weighed to conical bottle with stopper, respectively. Ultrasonic extraction was carried out with 100 mL methanol twice. Filtered and collected the extracting solutions, then concentrated up to dryness, the residue was dissolved in 20 mL 2% H2SO4 solution. The acid water extracts were further refined to pH10.0 with 3 mL aqueous ammonia. Filtered and extracted with 20 mL chloroform solution three times, then concentrated up to dryness, the residue was dissolved in 2 mL methanol. Filtered with 0.22-μm microporous membrane, then diluted 1–10 mL with methanol. The solutions were stored at 4°C and warmed to room temperature before use. Besides, the effects of different aqueous ammonia concentrations on the contents of macrocyclic polyamine alkaloids were studied. The acid water extracts were further adjusted to pH 9.0 and 11.0 with 2 and 4 mL aqueous ammonia, respectively.
Chromatographic conditions
HPLC analysis was performed on Agilent 1260 liquid chromatography system (Agilent Technologies, Palo Alto, CA, USA), equipped with a thermostated-column device, a DAD detector and an auto-sample injector. Chromatographic conditions were as following, the analytical column Diamonsil®5 μm C18(250 × 4.6 mm) was purchased from Dikma Technologies Inc. (Beijing, China), equipped with a guard column. We used water and acetonitrile as the mobile phase, gradient elution condition (water: acetonitrile): 0–20 min, 70:30–60:40; 20–30 min, 60:40; 30–40 min, 60:40–30:70. Column temperature was set as 30°C. The spectrums were scanned by DAD detector, and finally 210 nm was chosen as the detection wavelength for all analytes. Injection volume was 20 μL.
Method validation
The method for the determination of celafurine, celabenzine and celacinnine in stem of T. wilfordii was validated in terms of specificity, linearity, limit of detection and quantification, precision, repeatability, stability and accuracy.
Results and discussion
Wavelength
The wavelengths of analytes were scanned from 210 to 400 nm by DAD detector (Fig. 2). Celafurine and celabenzine both have highest absorption at 210 nm. Celacinnine has maximum absorptions at 210 and 280 nm. When at 210 nm, the solvents did not interfere with the baseline. Therefore, we chose 210 nm as detection wavelength for the analytes.
The UV wavelengths of celafurin, celabenzine, celacinnine and an unknown compound
Citation: Acta Chromatographica 35, 2; 10.1556/1326.2022.01014
Method validation
Specificity
Specificity of the assays was determined by comparing the chromatograms of mixed standard solutions and samples solutions of root, stem and leaf of T. wilfordii. Celafurine, celabenzine and celacinnine were all base line separated with a resolution over 1.5, and the number of theoretical plates shall not be less than 4,000 according to the peak of celafurine. The chromatograms of mixed standard solutions and samples solutions of root, stem and leaf are shown in Fig. 3. Besides, we found an interesting phenomenon that one compound exists in stem and leaf, while not in root of T. wilfordii, at least lower than the limit of detection. The compound is separated between celabenzine and celacinnine (Figs 2–3), and suspected to be a new macrocyclic polyamine which had not been reported in T. wilfordii.
The baseline separated chromatograms of mixed standard solutions and samples solutions of root, stem and leaf of Tripterygium wilfordii (a-celafurine, b-celabenzine, c-celacinnine, d-unknown compound)
Citation: Acta Chromatographica 35, 2; 10.1556/1326.2022.01014
Linearity
Calibration curves were prepared by using methanol spiked with known concentrations of celafurine, celabenzine and celacinnine. The calibration curves of each compound were linear over the concentration by performing a regression linear analysis of the peak area (A) of each compound vs the concentrations (C). Celafurine, celabenzine and celacinnine were linear over concentrations 1.744–111.6 μg mL−1, 2.045–130.9 μg mL−1 and 2.027–129.8 μg mL−1, respectively. The regression equation of the calibration curves was A = 57.688C + 44.567, with regression coefficient (r2) of 1 for celafurine, A = 62.53C – 13.374 (r2 = 1) for celabenzine, and A = 55.865C – 13.286 (r2 = 1) for celacinnine, respectively.
Limit of detection and quantification
The limit of detection (LOD) and the lower limit of quantification (LOQ) were determined as 3 and 10 times the signal to noise ratios, respectively. LODs were 0.436 , 2.046 and 2.028 ng, LOQs were 0.872 , 4.090 and 4.054 ng for celafurine, celabenzine and celacinnine, respectively.
Precision
Precision of the methods were studied in terms of repeatability by determining mixed standard solution 1 in “Stand solutions” section, and which was assayed in six times. RSD values of the peak area was 0.15 for celafurine, 0.18 for celabenzine, and 0.17 for celacinnine, which indicated that the instrument had good precision.
Repeatability
Accurately weigh six 10 g stem powder, and prepare the sample solutions according to the method in “Root, stem and leaf extract solutions” section. The average contents of celafurine, celabenzine and celacinnine in stem were 0.070 mg g−1, 0.012 mg g−1 and 0.050 mg g−1, with RSD values of the peak area 4.08, 3.67 and 3.89, respectively.
Stability
One sample solution of “Repeatability” section was injected at 0, 2, 4, 8, 12, and 24 h respectively. RSD values of the peak area for celafurine, celabenzine and celacinnine were 0.82, 2.55 and 0.62, respectively. The results showed that the solution remained stable within 24 h.
Recovery
The recoveries were carried out by adding known amounts of celafurine, celabenzine and celacinnine to 5 g stem powder at three different levels, prepare the sample solutions according to the method in “Root, stem and leaf extract solutions” section. Each level was repeated three times and the amount of each compound was found by the assay method in “Chromatographic conditions” section. The recovery was calculated by dividing the amount of found by the added, then multiplied by 100%. The results presented in Table 1 indicated that the method was accurate.
Results of recovery
| Compound | Amount of sample (g) | Content of the sample (µg) | Added (µg) | Measured (µg) | Recovery (%) | Average recovery (%) | RSD (%) |
| celafurine | 5.0158 | 291.07 | 103.6 | 419.26 | 106.2 | 107.4 | 1.6 |
| 5.0618 | 293.74 | 103.6 | 434.91 | 109.4 | |||
| 5.0077 | 290.60 | 103.6 | 420.33 | 106.6 | |||
| 5.0577 | 293.50 | 207.2 | 531.88 | 106.2 | 110.6 | 4.0 | |
| 5.0540 | 293.28 | 207.2 | 575.48 | 115.0 | |||
| 5.0028 | 290.31 | 207.2 | 550.62 | 110.7 | |||
| 5.0785 | 294.70 | 414.2 | 789.50 | 111.4 | 106.3 | 4.9 | |
| 5.0314 | 291.97 | 414.2 | 712.78 | 100.9 | |||
| 5.0104 | 290.75 | 414.2 | 750.84 | 106.5 | |||
| celabenzine | 5.0618 | 67.85 | 57.96 | 137.11 | 109.0 | 110.5 | 3.6 |
| 5.0158 | 67.23 | 57.96 | 144.04 | 115.0 | |||
| 5.0077 | 67.12 | 57.96 | 134.59 | 107.6 | |||
| 5.0577 | 67.79 | 115.92 | 190.51 | 103.7 | 105.3 | 5.2 | |
| 5.0540 | 67.74 | 115.92 | 204.71 | 111.5 | |||
| 5.0028 | 67.06 | 115.92 | 184.51 | 100.8 | |||
| 5.0785 | 68.07 | 231.84 | 305.47 | 101.8 | 98.4 | 3.5 | |
| 5.0314 | 67.44 | 231.84 | 284.15 | 94.9 | |||
| 5.0104 | 67.16 | 231.84 | 294.83 | 98.6 | |||
| celacinnine | 5.0618 | 65.84 | 60.3 | 136.69 | 108.4 | 104.5 | 4.5 |
| 5.0158 | 65.24 | 60.3 | 132.95 | 105.9 | |||
| 5.0077 | 65.14 | 60.3 | 124.59 | 99.3 | |||
| 5.0577 | 65.79 | 120.6 | 180.62 | 96.9 | 94.9 | 2.2 | |
| 5.0540 | 65.74 | 120.6 | 176.83 | 94.9 | |||
| 5.0028 | 65.08 | 120.6 | 172.25 | 92.8 | |||
| 5.0785 | 66.06 | 241.2 | 302.39 | 98.4 | 96.9 | 1.5 | |
| 5.0314 | 65.45 | 241.2 | 292.60 | 95.4 | |||
| 5.0104 | 65.18 | 241.2 | 296.91 | 96.9 |
The effect of different ammonia concentrations
In the experiment, we adjusted the acid water extracts to pH 9.0, 10.0, and 11.0 with 2, 3, and 4 mL aqueous ammonia, respectively. According to the chemical structures, the macrocyclic spermidine alkaloids celafurine, celabenzine and celacinnine all have three nitrogen atoms, possessing the structure with two amide bonds and secondary amine, leading to weak alkalinity. The role of aqueous ammonia was to free the alkaloids, and then the alkaloids could be extracted by chloroform easily. There were no significant differences of the contents of macrocyclic polyamines at pH 10.0 and 11.0. However, when at pH 9.0, the contents of macrocyclic polyamines were significantly lower (10%–20%) than pH 10.0 and 11.0. In addition, when at pH 11.0, there was interference at the peak position of celabenzine, which affected the specificity. Thus, finally we adjusted the acid water extracts to pH 10.0.
Macrocyclic spermidine alkaloids in root, stem and leaf of T. wilfordii
The established method was used to determine macrocyclic spermidine alkaloids in root and leaf of T. wilfordii as well. The contents of celafurine, celabenzine and celacinnine are 0.138 mg g−1, 0.019 mg g−1 and 0.010 mg g−1 in dry root powder, and 0.095 mg g−1, 0.009 mg g−1 and 0.012 mg g−1 in dry leaf powder, individually. Thus it can be seen, macrocyclic spermidine alkaloids are also distributed in stem and leaf, not only in root. T. wilfordii has a long growth cycle, with alkaloids grows year by year [10]. Moreover, root is non-renewable, while stem and leaf are renewable. Consequently, stem and leaf could be better alternatives for root considering macrocyclic spermidine alkaloids.
Conclusion
In this article, we established a simple and sensitive HPLC method for simultaneous quantification of macrocyclic spermidine alkaloids celafurine, celabenzine and celacinnine in root, stem and leaf of T. wilfordii. The macrocyclic spermidine alkaloids all have conjugate systems, leading to high UV responses. However, the contents of these macrocyclic spermidine alkaloids are very low, which limit further researches. We also find that stem and leaf contain comparable macrocyclic spermidine alkaloids with root. Besides, other new macrocyclic spermidine alkaloids may exist in stem and leaf of T. wilfordii, while not in root. These findings might have guiding significances for the phytochemical study of T. wilfordii.
Acknowledgments
This research was financially supported by the Project for Academic and Technical Leaders of Major Disciplines in Jiangxi (No. 20182BCB22004) and the National Natural Science Foundation of China (No. 81860686).
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