
==== Front
Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)13436-6
10.1016/j.heliyon.2024.e37405
e37405
Research Article
Quality control of traditional Chinese medicine Chelidonii herba based on 2D-Q-NMR
Zhang Huanbang a1
Chen Mengjie a1
Xu Yang a
Wang Kun b
Li Hua 2022041@fjtcm.edu.cn
ac⁎⁎
Chen Lixia syzyclx@163.com
a⁎⁎⁎
Huang Chao huangchao426@126.com
a⁎
a Wuya College of Innovation, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, China
b Ningxia Institute for Drug Control, Ningxia, 750004, China
c Institute of Structural Pharmacology & TCM Chemical Biology, College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, 350122, China
⁎ Corresponding author. huangchao426@126.com
⁎⁎ Corresponding author. Wuya College of Innovation, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, China. 2022041@fjtcm.edu.cn
⁎⁎⁎ Corresponding author. syzyclx@163.com
1 These authors contributed equally to this work.

04 9 2024
30 9 2024
04 9 2024
10 18 e3740525 1 2024
26 8 2024
3 9 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Chelidonii herba is a traditional Chinese medicinal herb with effects including antispasmodic, analgesic, antitussive, and bronchodilator properties. Alkaloids are the main bioactive ingredients in Chelidonii herba. In this study, a two-dimensional nuclear magnetic resonance (Heteronuclear Singular Quantum Correlation, HSQC-2D-NMR) technique was employed to quantitatively analyze the total alkaloid content and three major active alkaloid monomers in Chelidonii herba from eleven different sources. The quantification results of the three monomeric alkaloids were also verified using conventional quantitative control methods such as HPLC. Experimental findings indicate that the total alkaloid content is not directly correlated with the content of the three monomeric alkaloids. Furthermore, the content of any individual monomeric alkaloid does not accurately reflect the overall quality of Chelidonii herba. It was demonstrated that the 2D-Q-NMR NMR can be applied as an alternative method. While maintaining the same levels of accuracy and precision, the 2D-Q-NMR method is simpler to operate and provides more comprehensive results with higher reproducibility in some cases.

Graphical abstract

Image 1

Highlights

• D-Q-NMR are used for the quantification of Chelidonii herba.

• D-Q-NMR demonstrates consistency with HPLC in terms of quantitative reliability.

• D-Q-NMR allows for the overall quantification of alkaloids.

Keywords

2D-Q-NMR
Chelidonii herba
Quantification
==== Body
pmc1 Introduction

Chelidonii herba as a traditional Chinese medicine, is the dried whole herb of the plant Chelidonium majus L. of the genus Chelidonium L. in the family Papaveraceae. It is widely distributed in regions such as Northeast China, Shandong, Anhui, Henan, Jiangsu, and Hubei. In traditional Chinese medicine theory, Chelidonii herba is believed to have the effects of antispasmodic, analgesic, antitussive, and bronchodilator. It is commonly used to treat conditions like gastric spasms, coughs, asthma, and pertussis. Modern pharmacological studies have revealed that Chelidonii herba exhibits various activities, including antibacterial [[1], [2], [3], [4], [5]], insecticidal [6,7], antiviral [8], antitumor [[9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]], anti-inflammatory [20,21], analgesic [22,23], antispasmodic [24,25], anti-osteoporotic [26], and antioxidant [27] effects. The main components of Chelidonii herba are alkaloids, flavonoids, and phenolic acids, with alkaloids being the predominant and primary active compounds [28]. The three most significant alkaloid components are chelerythrine [1,2,[12], [13], [14], [15]], berberine [9,14,15] and chelidonine [3,13,[16], [17], [18],27].

Quantitative Nuclear Magnetic Resonance (Q-NMR) technology is a novel analytical technique based on the principles of nuclear magnetic resonance spectroscopy. This method offers several advantages, including accuracy, simplicity, high precision, good reproducibility, and non-destructiveness [29]. Q-NMR has found widespread applications in quantitative studies across various fields such as food [[30], [31], [32], [33]], pharmaceuticals [[34], [35], [36], [37], [38], [39], [40]], and the environment [41,42]. Q-NMR allows for the precise quantification of a compound's content based on its characteristic chemical shift values. It also allows for the simultaneous quantification of multiple different molecules in a single snapshot without the need for separation, derivatization, or purification. This method presents a significant advantage compared to traditional analyses.

Unlike High Performance Liquid Chromatography (HPLC), Q-NMR can also quantitatively determine the total content of a class of compounds sharing similar structural features and exhibit similar chemical shifts in NMR spectra [30,32]. This feature makes it particularly valuable for determining the most active compounds with a certain type of characteristic structure in complex chemical systems such as traditional Chinese medicine and natural products.

However, in complex systems with multiple components, 1D-Q-NMR may encounter issues with signal overlapping and shifting. In such cases, 2D-Q-NMR has been proposed as an optimized technique over 1D-Q-NMR. 2D-Q-NMR introduces an additional dimension, dispersing the overlapping signal peaks and yielding independent and reliable quantification points. This addresses challenges that 1D-Q-NMR cannot overcome [[33], [34], [35], [36], [37]]. When dealing with complex multi-component systems, 2D-Q-NMR offers greater advantages by enabling more accurate identification and quantification of various components. As a result, it is gradually gaining attention in the field of traditional Chinese medicine analysis. For example, in more than 40 known mogroside compounds from Siraitia grosvenorii (luo han guo), only 11-α-hydroxymogroside exhibits a sweet taste, while other triterpenoid compounds are tasteless or bitter. 2D-Q-NMR selectively quantifies the content of 11-α-hydroxymogroside in dried fruits, accurately predicting their sweetness potential [30]. In another wine system studied, carbonyl compounds are crucial oxidative products, but their prediction is challenging due to various complex associative reactions. 2D-Q-NMR can simultaneously and directly quantify five carbonyl compounds and their sulfite salts, hydrates, and carboxylic acid forms, comprehensively analyzing the occurrence and development of wine oxidation processes [32].

Currently, quantitative analysis of Chelidonii herba is mostly conducted using HPLC [[43], [44], [45], [46]]. Only J. Hua et al. [47] employed an acid dye colorimetric method to determine the total alkaloid content in Chelidonii herba, which marks the first study of quantifying total alkaloid content in this plant. Apart from these, thin-layer chromatography (TLC) is suitable only for qualitative analysis, and capillary electrophoresis [48] is limited to charged and polar compounds. Thus, a reliable, stable, and widely applicable quantitative analysis method for total alkaloid content in Chelidonii herba is currently lacking.

In this study, the 2D-Q-NMR technique was employed to identify characteristic structural points of total alkaloids and three individual alkaloids in Chelidonii herba. Quantitative analysis of the content of total alkaloids and three individual alkaloids in Chelidonii herba was conducted, aiming to provide a scientific basis for enhancing the quality standards of Chelidonii herba. Furthermore, this research lays the foundation for further pharmacological studies on Chelidonii herba.

2 Materials and methods

2.1 Instruments, reagents, and materials

Bruker AV 600 MHz spectrometer with a 5 mm TXI triple resonance probe, Bruker AV Ⅲ 400 MHz HD spectrometer in 298K, from Massachusetts, USA.

Waters 2695 HPLC System, Waters 2996 UV Full Wavelength Detector, XBridge Shield RP 18 chromatographic column(4.6 × 250 mm, 5 μm; Waters, Ireland), from Massachusetts, USA.

The deuterated solvent CD3OD used for NMR analysis was a product of Shanghai Haosu Chemical Technology Co., Ltd. Conventional 5 mm NMR sample tubes were obtained from Norell Inc. (Landisville, NJ, USA).

The 11 batches of Chelidonii herba samples (S1-S11) were collected from different regions, as shown in Table 1. The standard samples of chelidonine, berberine, and chelerythrine (>99.5 %, standards for HPLC) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd.Table 1 Summary of sample origin.

Table 1Sample	Origin	Sample	Origin	
S1	Anhui, China	S7	Shandong, China	
S2	Hebei, China	S8	Shanxi, China	
S3	Henan, China	S9	Zhejiang, China	
S4	Hubei, China	S10	Hunan, China	
S5	Jiangsu, China	S11	Liaonig, China	
S6	Jiangxi, China			
The source of the above-mentioned eleven batches of Chinese medicinal herbs has been authenticated by Wang Kun, the vice president of Ningxia Institute for Drug Control, confirming their origin to be genuine.

2.2 Sample preparation

2.2.1 Samples for 2D-Q-NMR

2.0 g of herbal material from each of the 11 batches was weighed and placed in a conical flask. The flask was then filled with 100 mL of 80 % methanol solvent, and it was subjected to ultrasonic extraction at a frequency of 40 kHz for 2 h. After extraction, the solution was centrifuged, and all of the supernatant(about 15 mL) was collected. The solvent was evaporated, and then it was reconstituted with methanol to a final volume of 1.0 mL in a volumetric flask. 500 μL of deuterated methanol was accurately added to dissolve the residue and transfer it to a 5 mm NMR tube. This process results in the prepared sample solution for NMR.

2.2.2 Sample for HPLC

2.0 g of herbal material from each of the 11 batches was weighed and placed in a conical flask. The flask was then filled with 100 mL of 80 % methanol solvent, and it was subjected to ultrasonic extraction at a frequency of 40 kHz for 2 h. After extraction, the solution was centrifuged, and all of the supernatant(about 15 mL) was collected. The solvent was evaporated, and then it was reconstituted with methanol to a final volume of 10 mL in a volumetric flask. This process results in the prepared sample solution for HPLC.

2.3 Preparation of standards

2.3.1 Standards for 2D-Q-NMR

15 mg of chelidonine, berberine, and chelerythrine standard substances were weighed separately. They were then dissolved in deuterated methanol and transferred into a 5 mm NMR tube to obtain the standard solution (the total alkaloids were standardized using the berberine standard solution).

2.3.2 Standards for HPLC

2 mg of the reference standard of chelidonine, 2 mg of berberine, and 1 mg of chelerythrine were weighed separately. They were accurately weighed and placed into 1 mL volumetric flasks. They were dissolved in methanol solvent and then made up to the mark to obtain the standard solution.

2.4 Analysis method

2.4.1 Analysis method for 2D-Q-NMR

In the 2D-Q-NMR quantification experiment, the influence of scan numbers on the experiment was investigated. Scans were conducted with 4, 8 and 16 repetitions. The results showed that with 4 scans, the signal-to-noise ratio of the spectra was low; with 8 scans, the signal-to-noise ratio was higher and the spectra were more stable; with 16 scans, the signal-to-noise ratio was almost consistent with 8 scans. As increasing the number of scans would extend the experimental time, a scan number of 8 was ultimately chosen for the experiment.

For the final 2D-Q-NMR quantification experiment, a Bruker Avance 600 MHz nuclear magnetic resonance (NMR) spectrometer equipped with a 5 mm TXI triple resonance probe was utilized for detection. The experiments were conducted at a temperature of 298 K. The spectroscopic acquisition parameters were set as follows: the pulse sequence employed was the Bruker standard pulse sequence hsqcedetgpsp.3, the proton (1H) and carbon (13C) spectral widths were 10.01 ppm and 200 ppm, respectively. The 1H channel acquired 1024 data points, and the 13C channel acquired 256 data points. The experiment was repeated 8 times with a blank run of 16 times, and the acquisition time was 0.14 s. The relaxation delay time was set to 1.5 s. The total acquisition time for the 2D-Q-NMR quantification experiment was 55 min.

The nuclear magnetic resonance (NMR) spectra were processed using Bruker Topspin software version 2.1.0. The following data processing steps were applied to the raw data:

Zero filling: The data were zero-filled to achieve a 1k x 1k matrix size.

Multiplication with squared sinebell function: The two dimensions were multiplied using a squared sinebell function to improve the signal-to-noise ratio.

Fourier transformation and phase correction: The processed data underwent Fourier transformation and phase correction.

Chemical shift referencing: The solvent peak signal of deuterated methanol (δH 3.3 ppm; δC 49 ppm) was used as an internal reference to calibrate the chemical shifts.

Baseline correction: Manual baseline correction was performed on both carbon and hydrogen dimensions.

Integration processing: After baseline correction, the same integration parameters were used for δC: 0–200 ppm and δH: 0–10 ppm ranges. The integral minimum threshold was set to the relative value of 0.02 (absolute value of 10830542.44) to remove signal background noise and impurities, roughly as shown in Fig. 1 above yellow.Fig. 1 Signal interception height in the extract of Chelidonii herba.

Fig. 1

This data processing workflow ensured accurate and consistent analysis of the NMR spectra for quantification and characterization purposes.

2.4.2 Analysis method for HPLC

Column: XBridge Shield RP 18 column (4.6 × 250 mm, 5 μm; Waters, Ireland).

Mobile phase: A: Acetonitrile, B: 1 % triethylamine buffer (adjusted to pH 3.0 with phosphoric acid), gradient elution program as shown in Table 2.Table 2 Gradient elution program.

Table 2t/min	ϕ(A)/%	ϕ(B)/%	
0	10	90	
5	15	85	
55	20	80	
70	25	75	

Detection wavelength: 257 nm.

Flow rate: 1.0 mL/min.

Column temperature: 30 °C.

Injection volume: 20 μL.

2.4.3 HPLC system suitability test

The analysis was performed in accordance with the chromatographic conditions outlined in 2.2.2. The separation of the chromatographic peaks for the three components of interest is greater than 1.5, and the symmetry factors are within the required specifications. The results are presented in Fig. 2.Fig. 2 Sample solution (A); HPLC chromatograms of solution of mixed reference Standards (B) 1- chelidonine; 2-berberine; 3-chelerythrine.

Fig. 2

2.5 Method validation

The developed analytical method underwent rigorous validation to ensure its accuracy, precision, reliability, and robustness. The validation process encompassed several parameters, including linearity, precision, repeatability, accuracy and stability. The detailed summary of the validation results is provided below.

In the 2D-Q-NMR quantitative method, a good linear relationship was observed between the concentration of Chelidonium alkaloids within the range of 1–60 mg/mL and the integration value of the methylenedioxy signal, represented by the equation y = 7 × 1010x - 6 × 107 with an R2 of 0.9991. Similarly, the linear relationships between concentration and characteristic quantification signal integration values were well-established for chelidonine and berberine, with equations y = 7 × 1010x + 5 × 106 (R2 = 0.9981) for concentrations within 0.5–5.5 mg/mL and y = 4 × 1011x - 3 × 108 (R2 = 0.9991) for concentrations within 0.5–5.5 mg/mL, respectively. For chelerythrine, within the range of 0.25–2.0 mg/mL, the relationship was represented by y = 6 × 1011x - 2 × 107 (R2 = 0.9992).

In the HPLC quantitative method, chelidonine demonstrated a good linear relationship between concentration and the characteristic UV absorption peak area integration value within the range of 0.02–5.5 mg/mL, represented by y = 4 × 107x + 36534 with an R2 of 0.9982. Similarly, for berberine within 0.02–5.5 mg/mL and chelerythrine within 0.01–2.0 mg/mL, the linear relationships were established with equations y = 7 × 107x + 603216 (R2 = 0.9997) and y = 8 × 107x + 126757 (R2 = 0.9991), respectively.

The precision of the instrument was evaluated by conducting 6 repeated measurements on the same standard solution. The Chelidonium alkaloids standard solution was subjected to 6 repeated measurements, resulting in an RSD value of 1.2 % for the integration value of the methylenedioxy signal in the Chelidonium alkaloids structures. Similarly, the standard solutions of chelidonine, berberine, and chelerythrine were each subjected to 6 repeated measurements using the 2D-Q-NMR and HPLC method. The RSD values for the integration values of the characteristic quantification points in 2D-Q-NMR were found to be 1.6 %, 1.0 %, and 0.38 % for chelidonine, berberine, and chelerythrine, respectively. Moreover, the RSD values for the quantification peak areas in HPLC were 1.4 %, 1.7 %, and 1.4 %. These results collectively demonstrated that the instruments exhibit good precision.

Repeatability analysis was conducted on six Chelidonii herba samples from Liaoning province, the RSD values for the quantitative points of total alkaloids using 2D-Q-NMR were 2.9 %. The RSD values for the characteristic quantitative points of the three individual alkaloids were 2.5 %, 0.70 %, and 1.1 % using 2D-Q-NMR, and 1.4 %, 1.7 %, and 1.4 % using HPLC. These results indicate good repeatability of the measurement results using this experimental method.

Stability testing involved measuring the quantification point integration values for total alkaloids, chelidonine, berberine, and chelerythrine in six Chelidonii herba samples from Liaoning province over 24 h, resulting in RSD values of 1.0 %, 1.0 %, 0.37 % and 1.6 % using 2D-Q-NMR. Chelidonine, berberine, and chelerythrine's RSD values for HPLC quantification peaks were 1.5 %, 1.1 %, and 1.6 %. This indicates stability of the sample solutions within the 24-h period.

Recovery assessment was performed by preparing nine parallel sample solutions of Chelidonii herba from Liaoning province, and after quantification by both 2D-Q-NMR and HPLC methods, corresponding standard solution concentrations (80 %, 100 %, 120 %) were added. The average recovery rate of total alkaloids in white aconitine using 2D-Q-NMR was 101.8 %, with an RSD of 2.2 %. The average recovery rates for chelidonine, berberine, and chelerythrine were 101.8 %, 102.6 % and 101.1 %, respectively, using 2D-Q-NMR, with RSD values of 2.2 %, 0.82 %, and 1.3 %. The average recovery rates for HPLC were 96.8 %, 95.3 %, and 103.3 %, with RSD values of 2.5 %, 2.1 %, and 2.1 %.

The methodological validation demonstrates that the developed analytical method is accurate, precise, reliable, and durable for quantifying Chelidonium alkaloids in various samples. The results indicate that these methods are fit for their intended purpose and provide consistent and accurate quantitative data.

3 Results

3.1 Quantification of total alkaloids with 2D-Q-NMR

The attribution of qHSQC signals corresponding to the common structural motif (methylenedioxy) of the 11 alkaloid standards selected based on their relative content and activity in Chelidonii herba is summarized in Fig. 3. Meanwhile, a comparative analysis was conducted by comparing the qHSQC spectra of the 11 alkaloid standards (Fig. 4) with the qHSQC spectrum of the crude extract of Chelidonii herba (Fig. 5). It was observed that the resonance signal peaks of the methylenedioxy group appeared at δH 6.1 ± 0.5 ppm and δC 102.6 ± 1 ppm positions for all 11 alkaloids, without any other interfering signals. Hence, the methylenedioxy signal peak can be chosen for quantifying the content of total alkaloids.Fig. 3 Common structural characteristic signal point of 11 alkaloids in Chelidonii herba.

Fig. 3

Fig. 4 Comparison of the methylenedioxy signal peaks among the 11 alkaloid standard compounds.

Fig. 4

Fig. 5 Key HSQC correlations of total alkaloids with a methylene dioxy structure in the extract of Chelidonii herba.

Fig. 5

Precisely weighed 2.0 g of each of the 11 batches of Chelidonii herba samples were taken. The test samples were prepared according to the method described under "2.2.1". The determinations were conducted under the experimental conditions outlined in "2.3.1″, and the peak area integration values of the quantification signal peak for total alkaloids in Chelidonii herba were recorded. Subsequently, the content of total alkaloids was calculated using the standard curve method. The results are presented in Fig. 6.Fig. 6 Contents of total alkaloids in Chelidonii herba.

Fig. 6

From Fig. 6, it can be inferred that the batch S8 from Shanxi has the highest total alkaloid content among all batches. The batch S5 from Jiangsu and S11 from Liaoning follow next in terms of content. The batch S3 from Henan has the lowest content, being only one-third of S8's content. The alkaloid content in the remaining regions is approximately two-thirds of that in S8.

3.2 Quantification of three individual alkaloids with 2D-Q-NMR

By comparing the attributions of HSQC signals of the alkaloids obtained from the Chelidonii herba (as shown in Table 3), it can be determined that by the 2D-Q-NMR spectrum of Chelidonii herba crude extract (Fig. 8), including the δH 6.84 (H-11)/δC 121 (C-11) HSQC cross-peak signal generated by chelidonine, the δH 8.70 (H-12)/δC 121.5 (C-12) HSQC cross-peak signal generated by berberine, and the δH 7.52 (H-1)/δC 107 (C-1) HSQC cross-peak signal generated by chelerythrine. These signal does not overlap with other peaks and exhibit good separation, which can be utilized for quantifying the content of chelidonine, berberine and chelerythrine. The quantitative points of the three compounds are shown in Fig. 7. Moreover, through the comparison of the HSQC spectra of Chelidonii herba alkaloids with the standards of chelidonine, berberine and chelerythrine (Fig. 9, Fig. 10, Fig. 11), the accuracy of the quantification signal points in the Chelidonii herba crude extract has been further confirmed.Table 3 HSQC Signal comparison of 11 alkaloids.

Table 3position	HSQC (δC/δH ppm)	
stylopine	tetrahydroberberine	berberine	berberrubine	corysamine	chelidonine	chelerythrine	dihydrosanguinarine	7-hydroxydihydro
sanguinarine	protopine	allocryptopine	
1	105.6/6.73	105.5/6.73	106.5/7.66	106.4/8.59	109.2/7.02	110.0/6.67	107/7.52	104.3/7.10	104.6/7.69	106.8/6.64	109.2/6.95	
2	145.9	145.8	149.9	149.9	149.2	149.7	151.7	148.1	147.3	146.0	148	
3	146.1	146.1	152.0	151.9	151.2	147	150.9	147.4	148.0	148.0	147.3	
4	108.3/6.59	108.3/6.59	109.3/6.96	109.3/6.94	111.8/7.33	112.9/6.78	105/8.15	100.7/7.66	100.5/7.12	108.0/6.90	110.5/6.63	
4a	127.6	127.6	131.8	131.6	121.9	126.9	121.7	126.5	126.8	135.6	132.8	
5	29.3	29.5	28.1	28.3	29.0	63.9/3.66	133.3	142.5	138.1	31.3	32.3	
6	51.1	51.3	57.1	56.9	58.5	–	–	–	–	57.6	57.5	
7	52.8	53.9	146.4/9.76	145.9/9.77	143.8/9.72	54.3	152/9.95	48.4/4.19	85.7/5.37	51.1/3.60	50.1	
7a	116.5	127.8	123.3	121.1	134.7	118.1	119.8	113.6	113.1	117.3	128.5	
8	143.2	150.2	145.7	147	148.5	144.4	147.4	144.6	145.2	146.2	151.5	
9	144.9	145	152.1	146.6	146.6	146.9	150.7	147	147.2	146	146	
10	106.7/6.69	110.9/6.79	128.0/8.12	119.3/7.72	112.8/7.90	108.3/6.76	127.3/8.19	107.1/6.85	108.8/6.94	110.3/6.69	110.4/6.80	
11	121/6.64	123.8/6.87	124.5/8.00	126.3/8.01	120.6/8.00	121/6.84	120.8/8.64	116.1/7.30	116.3/7.77	124.9/6.66	127.7/6.91	
11a	128.3	128.6	135.1	134.3	134.4	132.3	129.9	127.2	125.7	128.6	129.5	
12	36.2	36.4	121.5/8.70	119.2/8.59	132.7	42.5/3.06	127.0	124.3	122.7	45.9	46.2	
12a	59.7/3.59	60.1/3.54	139.7	138.6	137.4	–	–	–	–	–		
13	130.4	130.8	121.8	122.1	121.3	74.1/4.20	119.4/8.60	120.3/7.69	120.1/7.42	194.2	193.3	
14	–	–	–	–	–	39.9	132.5/8.16	123.9/7.48	123.7/7.49	132.2	136	
14a	–	–	–	–	–	129.5	134.2	130.7	131.0	–	–	
-OCH2O-	101.8/5.96	101.7/5.92	103.6/6.10	103.6/6.09	103.6/6.10	102.6/5.97	103.3/6.26	101.6/6.05	101.8/6.05	101.6/5.95	101.7/5.94	

Fig. 7 Quantitative signal point of chelidonine, berberine and chelerythrine.

Fig. 7

Fig. 8 Key HSQC correlations of chelidonine, berberine and chelerythrine in the extract of Chelidonii herba.

Fig. 8

Fig. 9 Key HSQC correlations of chelidonine standard.

Fig. 9

Fig. 10 Key HSQC correlations of berberine standard.

Fig. 10

Fig. 11 Key HSQC correlations of chelerythrine standard.

Fig. 11

Each of the 11 batches of Chelidonii herba samples was precisely weighed with 2.0 g. The sample solutions were prepared according to the method described under section "2.2.1". The quantitative experiments were conducted under the conditions specified in section "2.3.1″, and the peak area integration values of the quantification signals for chelidonine, berberine, and chelerythrine were recorded. Subsequently, the content of these three components was calculated using the standard curve method. The results are presented in Table 4.Table 4 Contents of chelidonine, berberine and chelerythrine in Chelidonii herba by 2D-Q-NMR.

Table 4No.		Component (mg/g)		
chelidonine	berberine	chelerythrine	
S1	1.875	1.301	0.397	
S2	2.175	1.374	0.351	
S3	1.794	1.294	0.420	
S4	1.918	1.381	0.489	
S5	2.376	1.365	0.429	
S6	1.921	1.490	0.423	
S7	2.027	1.302	0.378	
S8	2.537	1.405	0.419	
S9	2.237	1.345	0.572	
S10	1.996	1.267	0.409	
S11	2.267	1.302	0.476	

Among the eleven batches of Chelidonii herba samples, the batch S8 exhibited the highest chelidonine content, followed by batches S5, S9, S11, and S2, while the remaining batches had similar content. In terms of berberine content, batch S6 had the highest content, followed by batches S8, S4, S5, S9, and S2, while the remaining batches had similar content. Regarding chelerythrine content, batch S9 had the highest content, followed by batches S4 and S11. Batches S2 and S7 had the lowest content, while the content of the other batches was relatively similar.

3.3 Quantification of three individual alkaloids with HPLC

2.0 g of 11 batches of Chelidonii herba samples from different origins were precisely weighed. The samples were prepared into test solutions following the method described in "2.2.2". Then, the analysis should be carried out according to the experimental conditions outlined in '5.4.2,' and the peak areas of chelidonine, berberine, and chelidonine should be recorded. The content of these three components should be calculated using the standard curve method. The results will be presented in Table 5.Table 5 Contents of chelidonine, berberine and chelerythrine in Chelidonii herba by HPLC.

Table 5No.		Component (mg/g)		
chelidonine	berberine	chelerythrine	
S1	1.862	1.294	0.397	
S2	2.180	1.372	0.475	
S3	1.803	1.291	0.351	
S4	1.922	1.381	0.423	
S5	2.380	1.365	0.420	
S6	1.922	1.499	0.409	
S7	2.016	1.301	0.419	
S8	2.542	1.409	0.573	
S9	2.240	1.351	0.429	
S10	1.992	1.257	0.377	
S11	2.278	1.300	0.489	

The comparison between the quantitative content determined by 2D-Q-NMR and HPLC as shown in Fig. 12. Paired t-tests were conducted to compare the data obtained from 2D-Q-NMR. The results showed that for chelidonine, berberine, and chelerythrine, a p > 0.1 coefficient was found. Therefore, it can be concluded that there is no significant difference among them.Fig. 12 Comparison between the quantitative content determined by 2D-Q-NMR and HPLC.

Fig. 12

4 Discussion

The experimental results from this study indicate that the current method for quantifying the content of chelerythrine in Chelidonii herba using HPLC may not completely reflect the total content of alkaloids, the effective component in Chelidonii herba. For example, in batch S5 from Jiangsu province, the content of chelerythrine is not high, but its total alkaloid content ranks second among the eleven samples, and batch S6 from Jiangxi province, although the contents of total alkaloids, chelidonine, and chelerythrine contents are not remarkable, it has the highest content of berberine. Particularly noteworthy is in batch S3 from Henan province, the content of all three individual alkaloids isn't significantly lower than other batches, but its total alkaloid content is 2–3 times lower than that of other batches. Overall, batches S8 from Shanxi province, S11 from Liaoning province, and S5 from Jiangsu province have relatively higher contents of total alkaloids and individual alkaloid. This provides valuable reference for quality control and practical application of Chelidonii herba. This also suggests that the current method of using single standard substances for quality control of complex composition in herbal medicines exist limitations.

In terms of quantitative techniques, 2D-Q-NMR, as an advanced technology that integrates nuclear magnetic resonance with quantitative analysis, holds tremendous potential in the field of quality control of herbal medicine. In the quantification of major components in Chelidonii herba, its key advantages include three aspects. First, simplified sample preparation: Unlike HPLC, there is no need for trial-and-error exploration of separation conditions, which can significantly reduce the workload for establishing large-scale multi-index quantification systems for herb medicines. Second, high precision and excellent reproducibility: NMR spectra are not influenced by various factors of HPLC, such as mobile phase ratios, column types and conditions, elution programs, column pressures, and temperatures. With the proper usage of deuterated reagents, correct quantification procedures, and well-maintained instruments, highly consistent reproducible spectra can be easily obtained. Third, multicomponent quantification: 2D-Q-NMR can simultaneously detect the total content of specific types of compounds and multiple compounds, enabling multi-dimensional quantification of complex herbal medicine samples. These advantages show that 2D-Q-NMR has the potential to revolutionize the quality control of herbal medicines, offering reliable and accurate results for component analysis and quality evaluation.

It is essential to recognize that 2D-Q-NMR also has its shortcomings in certain aspects. The most notable is the significant difference in LOD (Limit of Detection) and LOQ (Limit of Quantitation) compared to HPLC. Taking this study as an example, the LOD and LOQ for 2D-Q-NMR can be determined at the 0.x mg level, whereas conventional HPLC can achieve LOD and LOQ at the μg level, indicating an order of magnitude difference. Currently, 2D-Q-NMR is still suitable for quantitative analysis at the mg level. Secondly, the high purchase price of NMR instruments and the equally high cost of subsequent maintenance limit the widespread adoption of this technology. Although in China, for instance, the number of NMR installations has increased in recent years, it is important to see that the installation and maintenance costs have not significantly decreased, and it is also difficult to foresee them reaching the level of popularity of HPLC in the foreseeable future. Fortunately, once linear calibration is performed, the quantitative results of Q-NMR on different instruments show almost no difference. It is not necessary, as with HPLC, to conduct a system suitability test before quantification. This makes it a viable option for third-party testing institutions, especially for organizations that do not possess an NMR spectrometer but wish to conduct Q-NMR research, and it can yield convincing and consistent results. Lastly, the cost of deuterated reagents, which is higher compared to that of chromatographic purity reagents, is also considered one of the barriers to scaled application. However, when considering the amount of mobile phase required for the entire process of chromatographic system checks, column equilibration, and injection, the use of 500 μL of deuterated reagent per injection may not necessarily be significantly higher than the former.

Of course, beyond the aforementioned issues, research on 2D-Q-NMR is not yet widespread, and the specific application scenarios and scope still require further clarification. It is foreseeable that not all traditional Chinese medicinal herbs or complex systems will have sufficient independent quantification points for systematic quantitative research. Additionally, not all similar types of compounds will have structurally identical overlapping signals for overall quantification. In such cases, 2D-Q-NMR cannot achieve quantitative purposes like HPLC by adjusting separation conditions. It may be necessary to consider using pre-treatment methods such as HPLC, TLC, etc., to appropriately process the samples before conducting quantitative research.

5 Conclusion

The 2D-Q-NMR technique accurately determined the content of total alkaloids, as well as the individual alkaloids including chelidonine, berberine, and chelerythrine, in 11 batches of samples. The methodological validation of 2D-Q-NMR exhibits good precision and accuracy. Moreover, there was no significant difference between the quantification results of the individual alkaloids obtained using 2D-Q-NMR and those obtained using HPLC, indicating that the 2D-Q-NMR technique is a reliable method for quantifying these alkaloids in Chelidonii herba.

Ethics approval and consent to participate

Not applicable.

Statement of originality

The authors declare that this manuscript is original, has not been published before, and is not currently being considered for publication elsewhere. We confirm that all sources of data and ideas presented in this work, which are not our own, have been appropriately cited and referenced in accordance with academic standards. The work presents original research not previously submitted or published, and all authors have read and approved the manuscript.

Data availability

Data will be made available on request.

CRediT authorship contribution statement

Huanbang Zhang: Writing – original draft, Visualization, Investigation, Data curation. Mengjie Chen: Resources, Methodology, Investigation, Formal analysis. Yang Xu: Writing – review & editing, Supervision. Kun Wang: Validation, Resources. Hua Li: Writing – review & editing, Supervision, Project administration. Lixia Chen: Writing – review & editing, Validation, Supervision. Chao Huang: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) used ChatGPT 3.5 in order to improve grammar and enhance readability. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors thank the 10.13039/100014717 National Natural Science Foundation of China (NSFC) (No. 82141216 ), Chunhui Program-Cooperative Research Project of the Ministry of Education, Liaoning Province Natural Science Foundation (No. 2022-MS-241 ), and Shenyang Young and Middle-aged Innovative Talents Support Program (No. RC210446 ) for financial supports.
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References

1 Cheng R.B. Chen X. Liu S.J. Experimental study of the inhibitory effects of Chelidonium majus L. extractive on Streptococcus mutans in vitro Shang Hai Kou Qiang Yi Xue 15 2006 318 320
2 Cheng R.B. Chen X. Liu S.J. Effect of Chelerythrine on cell surface hydrophobicity and adherence of Streptococcus mutans Shang Hai Kou Qiang Yi Xue 16 2007 68 72
3 Pan J. Yang Y. Zhang R. Enrichment of chelidonine from Chelidonium majus L. using macroporous resin and its antifungal activity J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 1070 2017 7 14 10.1016/j.jchromb.2017.10.029
4 Zielińska S. Wójciak-Kosior M. Dziągwa-Becker M. The activity of isoquinoline alkaloids and extracts from Chelidonium majus against pathogenic bacteria and Candida sp Toxins 11 2019 10.3390/toxins11070406
5 Meng F. Zuo G. Hao X. Antifungal activity of the benzo[c]phenanthridine alkaloids from Chelidonium majus Linn against resistant clinical yeast isolates J. Ethnopharmacol. 125 2009 494 496 10.1016/j.jep.2009.07.029 19647059
6 Madjeed Haddao K. Dawood Saleem H. Hameed N.M. Investigation of in vitro cytotoxicity of Chelidonium majus against leishmania major Arch Razi Inst 77 2022 1211 1214 10.22092/ari.2022.358758.2301 36618311
7 Zou C. Lv C. Wang Y. Larvicidal activity and insecticidal mechanism of Chelidonium majus on Lymantria dispar Pestic. Biochem. Physiol. 142 2017 123 132 10.1016/j.pestbp.2017.04.009 29107235
8 Lozjuk R.M. Lisnyak O.I. Lozjuk L.V. Theoretical grounds and experimental confirmation of the antiviral effect of the preparation Ukrain Drugs Exp. Clin. Res. 22 1996 213 217 10.2165/00003495-199651010-00010 8899334
9 Och A. Zalewski D. Komsta Ł. Cytotoxic and proapoptotic activity of sanguinarine, berberine, and extracts of Chelidonium majus L. And berberis thunbergii DC. Toward hematopoietic cancer cell lines Toxins 11 2019 10.3390/toxins11090485
10 Xie Y.J. Gao W.N. Wu Q.B. Chelidonine selectively inhibits the growth of gefitinib-resistant non-small cell lung cancer cells through the EGFR-AMPK pathway Pharmacol. Res. 159 2020 104934 10.1016/j.phrs.2020.104934
11 Deljanin M. Nikolic M. Baskic D. Chelidonium majus crude extract inhibits migration and induces cell cycle arrest and apoptosis in tumor cell lines J. Ethnopharmacol. 190 2016 362 371 10.1016/j.jep.2016.06.056 27350008
12 Habermehl D. Kammerer B. Handrick R. Proapoptotic activity of Ukrain is based on Chelidonium majus L. alkaloids and mediated via a mitochondrial death pathway BMC Cancer 6 2006 14 10.1186/1471-2407-6-14 16417634
13 Hohenwarter O. Strutzenberger K. Katinger H. Selective inhibition of in vitro cell growth by the anti-tumour drug Ukrain Drugs Exp. Clin. Res. 18 Suppl 1992 1 4
14 Basu P. Bhowmik D. Suresh Kumar G. The benzophenanthridine alkaloid chelerythrine binds to DNA by intercalation: photophysical aspects and thermodynamic results of iminium versus alkanolamine interaction J. Photochem. Photobiol., B 129 2013 57 68 10.1016/j.jphotobiol.2013.09.011 24177205
15 Noureini S.K. Esmaeili H. Abachi F. Selectivity of major isoquinoline alkaloids from Chelidonium majus towards telomeric G-quadruplex: a study using a transition-FRET (t-FRET) assay Biochim. Biophys. Acta Gen. Subj. 1861 2017 2020 2030 10.1016/j.bbagen.2017.05.002 28479277
16 Noureini S.K. Wink M. Transcriptional down regulation of hTERT and senescence induction in HepG2 cells by chelidonine World J. Gastroenterol. 15 2009 3603 3610 10.3748/wjg.15.3603 19653337
17 Panzer A. Joubert A.M. Bianchi P.C. The antimitotic effects of Ukrain, a Chelidonium majus alkaloid derivative, are reversible in vitro Cancer Lett. 150 2000 85 92 10.1016/s0304-3835(99)00375-4 10755391
18 Havelek R. Seifrtova M. Kralovec K. Chelidonine and homochelidonine induce cell death through cell cycle checkpoints and MAP kinase pathways Nat. Prod. Commun. 12 2017 1934578X1701200910 10.1177/1934578X1701200910
19 Li M.-X. Yang Y.-Y. Liu Y. Progress of traditional Chinese medicine treating COVID-19 World Journal of Traditional Chinese Medicine 7 2021 167 183 10.4103/wjtcm.wjtcm_68_20
20 Liao W. He X. Yi Z. Chelidonine suppresses LPS-Induced production of inflammatory mediators through the inhibitory of the TLR4/NF-κB signaling pathway in RAW264.7 macrophages Biomed. Pharmacother. 107 2018 1151 1159 10.1016/j.biopha.2018.08.094 30257328
21 Park J.E. Cuong T.D. Hung T.M. Alkaloids from Chelidonium majus and their inhibitory effects on LPS-induced NO production in RAW264. 7 cells Bioorg. Med. Chem. Lett 21 2011 6960 6963 10.1016/j.bmcl.2011.09.128 22024033
22 Huang K.C. The Pharmacology of Chinese Herbs 1998 CRC press 10.1201/9781420048261
23 Mikołajczak P. Kędzia B. Ożarowski M. Evaluation of anti-inflammatory and analgesic activities of extracts from herb of Chelidonium majus L Cent. Eur. J. Immunol. 40 2015 400 410 10.5114/ceji.2015.54607 26862303
24 Boegge S.C. Kesper S. Verspohl E.J. Reduction of ACh-induced contraction of rat isolated ileum by coptisine, (+)-caffeoylmalic acid, Chelidonium majus, and Corydalis lutea extracts Planta Med. 62 1996 173 174 10.1055/s-2006-957845 8657755
25 Hiller K.O. Ghorbani M. Schilcher H. Antispasmodic and relaxant activity of chelidonine, protopine, coptisine, and Chelidonium majus extracts on isolated Guinea-pig ileum Planta Med. 64 1998 758 760 10.1055/s-2006-957576 9933996
26 Song J.Y. Yang H.O. Shim J.Y. Radiation protective effect of an extract from Chelidonium majus Int. J. Hematol. 78 2003 226 232 10.1007/bf02983799 14604281
27 Paul A. Das J. Das S. Poly (lactide-co-glycolide) nano-encapsulation of chelidonine, an active bioingredient of greater celandine (Chelidonium majus), enhances its ameliorative potential against cadmium induced oxidative stress and hepatic injury in mice Environ. Toxicol. Pharmacol. 36 2013 937 947 10.1016/j.etap.2013.08.008 24035924
28 Gilca M. Gaman L. Panait E. Chelidonium majus--an integrative review: traditional knowledge versus modern findings Forsch Komplementmed 17 2010 241 248 10.1159/000321397 20980763
29 Schenk D.J. Dormer P.G. Hesk D. NMR-based approach to the analysis of radiopharmaceuticals: radiochemical purity, specific activity, and radioactive concentration values by proton and tritium NMR spectroscopy J. Label. Compd. Radiopharm. 58 2015 291 298 10.1002/jlcr.3295
30 Çiçek S.S. Esposito T. Girreser U. Prediction of the sweetening effect of Siraitia grosvenorii (Luo han guo) fruits by two-dimensional quantitative NMR Food Chem. 335 2021 127622 10.1016/j.foodchem.2020.127622
31 Becerra-Martínez E. Florentino-Ramos E. Pérez-Hernández N. 1H NMR-based metabolomic fingerprinting to determine metabolite levels in serrano peppers (Capsicum annum L.) grown in two different regions Food Res. Int. 102 2017 163 170 10.1016/j.foodres.2017.10.005 29195936
32 Nikolantonaki M. Magiatis P. Waterhouse A.L. Direct analysis of free and sulfite-bound carbonyl compounds in wine by two-dimensional quantitative proton and carbon nuclear magnetic resonance spectroscopy Anal. Chem. 87 2015 10799 10806 10.1021/acs.analchem.5b01682 26348554
33 Tardieu A. De Man W. This H. Using one-dimensional (1D) and two-dimensional (2D) quantitative proton (1H) nuclear magnetic resonance spectroscopy (q NMR) for the identification and quantification of taste compounds in raw onion (Allium cepa L.) bulbs and in aqueous solutions where onion tissues are soaked Anal. Bioanal. Chem. 398 2010 3139 3153 10.1007/s00216-010-4255-x 20972556
34 Koso T. Rico del Cerro D. Heikkinen S. 2D Assignment and quantitative analysis of cellulose and oxidized celluloses using solution-state NMR spectroscopy Cellulose 27 2020 7929 7953 10.1007/s10570-020-03317-0
35 Mauri L. Boccardi G. Torri G. Qualification of HSQC methods for quantitative composition of heparin and low molecular weight heparins J. Pharm. Biomed. Anal. 136 2017 92 105 10.1016/j.jpba.2016.12.031 28068519
36 Tang F. Hatzakis E. NMR-based analysis of pomegranate juice using untargeted metabolomics coupled with nested and quantitative approaches Anal. Chem. 92 2020 11177 11185 10.1021/acs.analchem.0c01553 32659075
37 Dufour G. Evrard B. de Tullio P. 2D-Cosy NMR spectroscopy as a quantitative tool in biological matrix: application to cyclodextrins AAPS J. 17 2015 1501 1510 10.1208/s12248-015-9806-9 26304859
38 Bingol K. Brüschweiler R. Multidimensional approaches to NMR-based metabolomics Anal. Chem. 86 2014 47 57 10.1021/ac403520j 24195689
39 Miura T. Sugimoto N. Bhavaraju S. Collaborative study to validate purity determination by 1H quantitative NMR spectroscopy by using internal calibration methodology Chem. Pharm. Bull. (Tokyo) 68 2020 868 878 10.1248/cpb.c20-00336 32565492
40 Li N. Zhang Y. Lv J. Protective effects of ginsenoside CK against oxidative stress-induced neuronal damage, assessed with 1 H-NMR-based metabolomics Acta Materia Medica 1 2022 392 399 10.15212/AMM-2022-0009
41 Weber M. Hellriegel C. Rück A. Using high-performance quantitative NMR (HP-qNMR®) for certifying traceable and highly accurate purity values of organic reference materials with uncertainties< 0.1 Accred Qual. Assur. 18 2013 91 98 10.1007/s00769-012-0944-9
42 Ben H. Ragauskas A.J. Heteronuclear single-quantum correlation–nuclear magnetic resonance (HSQC–NMR) fingerprint analysis of pyrolysis oils Energy Fuel. 25 2011 5791 5801 10.1021/ef201376w
43 Kursinszki L. Sárközi Á. Kéry Á. Improved RP-HPLC method for analysis of isoquinoline alkaloids in extracts of Chelidonium majus Chromatographia 63 2006 S131 S135 10.1365/s10337-006-0808-8
44 Gañán N.A. Dias A.M. Bombaldi F. Alkaloids from Chelidonium majus L.: fractionated supercritical CO2 extraction with co-solvents Separ. Purif. Technol. 165 2016 199 207 10.1016/j.seppur.2016.04.006
45 Paulsen J. Yahyazadeh M. Hänsel S. 13, 14-dihydrocoptisine–the genuine alkaloid from Chelidonium majus Phytochemistry 111 2015 149 153 10.1016/j.phytochem.2015.01.006 25666130
46 Seidler-Łozykowska K. Kędzia B. Bocianowski J. Content of alkaloids and flavonoids in celandine (Chelidonium majus L.) herb at the selected developmental phases Acta Scientiarum Polonorum Hortorum Cultus 15 2016 161 172
47 Hua J. Jun L. Qiang X. Determination of total alkaloids in chelidonii herba by acid dye colorimetry Lishizhen Medicine and Materia Medica Research 28 2017 854 856
48 Sun Y. Li Y. Zeng J. Microchip electrophoretic separation and fluorescence detection of chelerythrine and sanguinarine in medicinal plants Talanta 142 2015 90 96 10.1016/j.talanta.2015.04.008 26003696
