Research on the origin identification of Laggera pterodonta based on UPLC fingerprint and chemometrics

doi:10.21203/rs.3.rs-2003093/v1

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Research on the origin identification of Laggera pterodonta based on UPLC fingerprint and chemometrics

https://doi.org/10.21203/rs.3.rs-2003093/v1

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Laggera pterodonta is a famous Chinese herbal medicine in China, which has the effect of treating viral infection, bronchial and sore throat. Yunnan Province of China is the main production area, and Laggera pterodonta grows in a wide range of areas in Yunnan Province. The climates of the respective growing places are also different, which may be different for the accumulation of medicinal components in it, and the evaluation of different the quality of the traditional Chinese medicine of Laggera pterodonta of origin is of great significance. At present, there was still a lack of reliable technology for the origin identification of Laggera pterodonta, especially the origin of Laggera pterodonta cannot be identified from the appearance, which was very difficult to evaluate the efficacy of Laggera pterodonta from different origins. Therefore, it is of great significance to develop a quick and easy method for the origin identification of Laggera pterodonta. Thirty-four samples of Laggera pterodonta from five main production areas of Dali, Jianshui, Lancang, Simao and Yuanjiang in Yunnan Province were used as the research objects. UPLC and PDA detector were used to collect fingerprints, the common peaks were obtained using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)", and then carry out PCA and OPLS-DA analysis by R and SIMCA-P, respectively. Laggera pterodonta from different origins can be distinguished by OPLS-DA, which was consistent with the PCA analysis results. Through the comparison of VIP values, 8 characteristic components with great differences are obtained, and the UV absorption of these characteristic components was mainly concentrated at 320-350 nm and 250-280 nm, possibly characteristic UV absorption of terpenes, sesquiterpenes, and alkaloids. Through the comparison of the standard products, it was confirmed that the two characteristic components were chrysosplenetin and artemisetin, and the quantitative analysis of these two compounds in the Laggera pterodonta from different origins was carried out. The results of variance analysis showed that the areas with the most significant difference in chrysosplenetin content were Lancang and Honghe (p=9.03×10-5), and the areas with the most significant difference in artemisetin content were Lancang and Simao (p=5.10×10-7). The Simao and Dali areas have lower levels of artemisetin than other areas, with the highest levels being the Lancang area. The content of chrysosplenetin in Dali area was lower than that in other areas, and the content was higher in Lancang area. It was speculated that Laggera pterodonta in Lancang area may be better than other origins in terms of quality and efficacy of Chinese medicinal materials. Chrysosplenetin can also be used as one of the important indicators for quality control and origin traceability of Laggera pterodonta. Using UPLC combined with chemometric methods, Laggera pterodonta from different regions of China’s Yunnan Province, can be effectively identified.

Origin identification

Laggera pterodonta

UPLC fingerprint

chemometrics

Laggera pterodonta is a famous Chinese herbal medicine, which has the effect of treating viral infection, bronchial, sore throat, etc. China's Yunnan Province is the main producing area of Laggera pterodonta ^1,2. At present, the research on Laggera pterodonta mainly focuses on the separation and purification of active small-molecule compounds ^2-5，the extraction and activity research of essential oil ^1,6,7, but there were few researches on other aspects.

Since the efficacy of Chinese herbal medicines is mainly due to the combined effect of one or more components, and these components are usually important intermediates in the metabolic process of Chinese herbal medicines or the end products of their metabolism, they are regulated by genetic factors. However, plant endogenous factors will be changed with the change of environmental factors, thereby inducing cell signal transduction and regulating the expression of related genes ⁸, followed by transcriptional processing, protein translation and modification, etc., which ultimately affect the types and quantities of secondary metabolite transcripts ⁹. Therefore, environmental factors have a greater impact on the efficacy of Chinese herbal medicines.

Laggera pterodonta grows in a wide range of areas in Yunnan Province, and the climates where it grows are also different, which may be different for the accumulation of medicinal components, and is important for evaluating the quality of traditional Chinese medicines of Laggera pterodonta from different origins. However, at present, there was still a lack of reliable technology for the origin identification of Laggera pterodonta, especially the origin of Laggera pterodonta cannot be identified from the appearance, which brings great difficulties to the origin identification and efficacy evaluation of Laggera pterodonta from different origins.

At present, the commonly used methods to trace the origin of Chinese medicinal materials mainly include spectral analysis methods such as raman spectroscopy ¹⁰, nuclear magnetic resonance ¹¹, infrared spectroscopy ¹², near-infrared spectroscopy ¹³ and fluorescence spectroscopy ¹⁴，mass spectrometry analysis methods such as isotope ratio mass spectrometry ¹⁵, inductively coupled plasma mass spectrometry ¹⁶, liquid chromatography-mass spectrometry ¹⁷ and gas chromatography-mass spectrometry ¹⁸. However, these methods all require more professional and complex instruments and equipment, and have higher requirements for instrument operators. Although they can effectively trace the origin of Chinese medicinal materials, they do not have universal applicability.

Liquid chromatography-fingerprint method is the most common and widely used traceability method in traditional Chinese medicinal materials. The price of the required instruments and equipment is moderate, and most laboratory conditions can be met, but the similarity evaluation method used in liquid chromatography-fingerprint is less accurate ^19,20. With the continuous development of chemometrics, its combination with liquid chromatography-fingerprint can effectively improve the accuracy of identification in the traceability process, and has become one of the quick and easy methods for tracing the origin of Chinese medicinal materials.

In this study, ultra-high performance liquid chromatography was used to replace traditional liquid chromatography, which can effectively shorten the time of fingerprint data collection. Combined with the method of chemometrics, the traceability research of Laggera pterodonta in five main producing areas in Yunnan Province, China, was carried out. The research results can provide reference for the quality evaluation and discovery of active ingredients of Laggera pterodonta and other Chinese herbal medicines.

2.1. Sample Collection and Preparation

Our experimental research including the collection of plant material complies with relevant institutional, national, and international guidelines and legislation. All sample collections have been permitted by the agricultural and Chinese herbal medicine authorities in China, identified by one of the co-authors Jie Xia from Yunnan Provincial Hospital of Traditional Chinese Medicine and the voucher specimen was deposited in the publicly available herbarium, namely the Chinese herbal medicine hall of Yunnan Provincial Hospital of Traditional Chinese Medicine (No. T000200121). The representative growth area of Laggera pterodonta in Yunnan Province, China (Figure 1) was selected, including 6 samples from Dali, 7 samples from Jianshui, 9 samples from Lancang, 5 samples from Simao, 7 samples from Yuanjiang, a total of 34 samples samples. For each sample, 1 kg of the above-ground growing part of wild Laggera pterodonta was selected, transferred to the laboratory at 4°C, dried and crushed at 60°C, filtered through a 0.28 μm sample sieve, and stored at 4°C in the dark.

2.2. Chemicals and Reagents

HPLC grade methanol and formic acid were purchased from Merck KGaA (Darmstadt, Germany) and Millipore Sigma Company (St. Louis, MO, USA), and high-purity water was prepared by a Milli-Q water purification system (Bedford, MA, USA). Chrysosplenetin (CAS: 603-56-5) and artemisetin (CAS: 479-90-3) with a purity greater than 98% were purchased from Tianjin Alta Scientific., Ltd and Chengdu Must Bio-technology Co., Ltd, respectively.

2.3. Sample Preparation and Analysis

2.3.1. Sample Preparation Method

The experiment was carried out in accordance with the regulations of the agricultural and Chinese herbal medicine authorities in China. 1 g sample was weighed into a 50 mL centrifuge tube, 10 mL methanol-water solution (V:V = 90:10) was added, vortexed for 2 min, ultrasonically extracted for 10 min, centrifuged at 5000 r/min for 3 min, and the supernatant was filtered through a 0.22 μm filter before analysis.

2.3.2. UPLC Analysis Method

Waters ACQUITY UPLC H-Class and UPLC PDA detector was used for data acquisition (Waters, MA, USA) with the UV spectral scan wavelength was 345 nm. A Waters ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm, Waters, MA, USA) was selected for high-efficiency separation Laggera pterodonta, maintained at 35^∘C, and injected with a volume of 3 µL. Mobile phases A and B were methanol and ultrapure water containing 0.02% formic acid, respectively, with a flow rate of 0.2 mL/min, and the following gradient elution program was used: 0~70.0 min (5% A ~ 95% A), 70.0~80.0 min (95% A), 80.0~80.5 min (95% A ~ 5% A), 80.5~85.0 min (5% A).

2.3.3. Data processing and differential marker identification

The obtained chromatograms of 34 Laggera pterodonta samples were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" in CDF format, the time window was set to 0.1, and a common pattern was established after automatic peak matching. Using the median method and automatic matching through multi-point calibration, UPLC fingerprints were generated, and the chromatographic peaks with larger peak area and better resolution were selected as common peaks. Principal component analysis (PCA) was performed on the common peaks and SIMCA-P 14.1 software was used for OPLS-DA analysis, respectively. The VIP value of each common peak was obtained, and the characteristic components were found by the value of VIP.

3.1 Optimization of elution time in UPLC

The chemical components in Laggera pterodonta are complex, and the ideal chromatographic separation effect may not be achieved by isogradient elution or conventional high performance liquid chromatography. Therefore, in this study, UPLC with better separation effect was selected for fingerprint determination. In this study, 0–30.0 min (5%–95% methanol) was first chosen, but within 10–15 min, most of the compounds in Laggera pterodonta were not separated.

In order to better effectively separate the compounds in Laggera pterodonta, the separation effects of 0~55.0 min (5% ~ 95% methanol) and 0~70.0 min (5% ~ 95% methanol) were further compared. The results are shown in Figure 2, the gradient of 0~70.0 min (5%~95% methanol) was better than other gradients for the separation of compounds in Laggera pterodonta, but it takes a long time and still does not achieve a good baseline separation for some of the compounds. However, through the subsequent analysis of fingerprints and chemometrics, the fingerprints obtained under this gradient condition can achieve the expected traceability effect. Therefore, in order to avoid more consumption of acquisition time, this gradient was selected.

3.2 Optimization of UV wavelengths

In this study, the UV absorption of the compounds at 210, 250, 300, 345 and 390 nm were compared, respectively. From Figure 3, it can be seen that Laggera pterodonta has fewer chromatographic peaks at 390 nm and 210 nm, while there are relatively more chromatographic peaks at 250, 300 and 345 nm, especially the chromatographic peak response at 345 nm within 45-60 min was better. Therefore, 345 nm was finally selected as the best wavelength for the determination of Laggera pterodonta fingerprints.

3.3 Establishment of UPLC Fingerprint

Laggera pterodonta samples from different origins were pretreated according to section 2.3.1., UPLC analysis of the samples according to step 2.3.2., and data processing according to step 2.3.3. In the end, the fingerprint of the Laggera pterodonta (Figure 4) was obtained. Then, using the software of "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Version)", 12 common peaks (Table 1) were calibrated through peak matching, and the peak areas of the common peaks were used as different risks and R was used to PCA analyze. The results are shown in Figure 5, Laggera pterodonta from different origins can be well separated by PCA, indicating that Laggera pterodonta grown in different growth regions can be effectively distinguished by using the common peak combined with the chemometric method.

3.4. OPLS-DA analysis

SIMCA-P 14.1 software was used to perform OPLS-DA analysis on the peak areas of the common peaks of the Laggera pterodonta samples. From the OPLS-DA in Figure 6, it can be seen that Laggera pterodonta from different origins can be clearly distinguished, which was consistent with the PCA analysis results.

On the other hand, by analyzing the VIP of each common peak (Figure 7), the VIP value was used as the screening basis. When the VIP value was larger, it indicates that the difference between the common peaks was greater and the difference was more significant. The eight characteristic components with large differences have peak times of 21.805, 33.911, 37.678, 39.317, 30.434, 40.753, 44.351 and 30.984 min according to their contribution rates.

3.5 Analysis of characteristic components

Through the UV spectral analysis of each characteristic component, as shown in Figure 8, the main UV absorption was mainly in the range of 320-350 nm and 250-280 nm, which was consistent with the characteristic UV absorption of terpenes, sesquiterpenes and alkaloids.

According to the above analysis of the ultraviolet absorption spectrum of the characteristic components, combined with the reports on Laggera pterodonta in the literature ^21-23, the standard materials of the main active ingredients of Laggera pterodonta reported in the literature including pterodontic acid, pterodondiol, chrysosplenetin, artemisetin were purchased to carry out the qualitative and quantitative analysis of characteristic components. The results show that only chrysosplenetin and artemisetin were consistent with the two peaks of 37.678 min and 40.753 min in the characteristic components of Laggera pterodonta. Unfortunately, the other characteristic components were not pterodontic acid and pterodondiol reported in the literature. The chemical structure confirmation of other characteristic components also needs to be determined by phytochemical methods such as isolation, purification, MS and NMR.

In this study, two standard materials, chrysosplenetin and artemisetin, were used for quantitative analysis of Laggera pterodonta from different origins, by UPLC and the fingerprinting method. The boxviolin was drawn by R as shown in Figure 9, the results of variance analysis showed that when the confidence level was 0.95, there was no significant difference in the content of chrysosplenetin between Dali and Honghe, Dali and Simao, Honghe and Simao, Lancang and Yuanjiang (p>0.05), while the content of chrysosplenetin in Dali and Lancang, Dali and Yuanjiang, Honghe and Lancang, Honghe and Yuanjiang, Lancang and Simao, Simao and Yuanjiang were significantly different (p<0.05), the most significant difference was Lancang and Honghe (p=9.03×10^-5). On the other hand, there was no significant difference in the content of artemisetin between Dali and Honghe, Dali and Simao, Lancang and Yuanjiang (p>0.05), while Dali and Lancang, Dali and Yuanjiang, Honghe and Lancang, Honghe and Simao, Honghe and Yuanjiang, Lancang and Simao, Simao and Yuanjiang had significant differences (p<0.05), and the largest difference was Lancang and Simao (p=5.10×10^-7).

The content of chrysosplenetin and artemisetin in Laggera pterodonta from different origins varies greatly. The content of chrysosplenetin in Dali area was lower than that in other areas, and the content of chrysosplenetin was higher in Lancang area. On the other hand, the content of artemisetin in Simao and Dali area was relatively low, and the content in Lancang area was the highest. Therefore, the content of chrysosplenetin and artemisetin in Laggera pterodonta grown in Lancang area was higher than that in other areas, and may be better than Laggera pterodonta in other areas in terms of quality and efficacy of Chinese medicinal materials.

Chrysosplenetin and artemisetin are the active ingredients in Laggera pterodonta, with topoisomerase I and II inhibition properties ²⁴, inhibition of enterovirus²⁵, blood pressure lowering ²⁶，antitumor and other biological activities ²⁷. The Chinese Pharmacopoeia stipulates that artemisetin was an index for quality control of Laggera pterodonta, and requires that the content of artemisetin in Laggera pterodonta must be greater than 0.1% ²⁸. The Chinese Pharmacopoeia stipulates that artemisetin is an index for quality control of Laggera pterodonta, and requires that the content of artemisetin in Laggera pterodonta must be greater than 0.1%. As one of the characteristic components of Laggera pterodonta, chrysosplenetin has a higher content in Laggera pterodonta according to the content determination results in this study. Due to its good biological activity, it was predicted that chrysosplenetin can also be used as one of the important indicators for quality control and origin traceability of Laggera pterodonta.

In this study, a total of 34 Laggera pterodonta samples from 5 regions of Dali, Jianshui, Lancang, Simao and Yuanjiang in Yunnan Province, China, were taken as the research objects. UPLC and PDA detector were used to collect fingerprints and then obtained the common peaks by" Chinese medicine chromatographic fingerprint similarity evaluation system (2012 Edition)” . The PCA analysis was performed on the common peaks using R, and the OPLS-DA analysis was performed using the SIMCA-P 14.1 software to obtain the VIP value of each common peak, and the characteristic components were found by the VIP value. Laggera pterodonta from different origins can be distinguished by OPLS-DA, which was consistent with the PCA analysis results. According to the size of the VIP value, 8 characteristic components with large differences were obtained, and their peak times were 21.805, 33.911, 37.678, 39.317, 30.434, 40.753, 44.351, 30.984 min. The UV absorption of these characteristic components was mainly concentrated in the range of 320-350 nm and 250-280 nm, which was in line with the characteristic UV absorption of terpenes, sesquiterpenes and alkaloids.

Through the comparison of standard materials, it was confirmed that the two characteristic components were chrysosplenetin and artemisetin, and the quantitative analysis of chrysosplenetin and artemisetin from different origins was carried out. The results of variance analysis showed that the areas with the largest difference in chrysosplenetin content were Lancang and Honghe (p=9.03×10^-5), the areas with the greatest difference in artemisetin content are Lancang and Simao (p=5.10×10^-7). The content of artemisetin in Simao and Dali area was lower than other areas, and the highest content was in Lancang area, while the content of chrysosplenetin in Dali area was relatively low, and the content in Lancang area was higher. Therefore, the contents of chrysosplenetin and artemisetin in Laggera pterodonta in Lancang were higher than those in other regions, and may be better than those in other origins in terms of quality and efficacy of Chinese herbal medicines. Due to the content and good biological activity of chrysosplenetin, it was predicted that chrysosplenetin can also be used as one of the important indicators for quality control and origin traceability of Laggera pterodonta.

Author Contributions: An Wang and Song-Mei Li carried out the data collection and analysis, developed the core idea of the manuscript and contributed to all parts. Xiaoyun Tong supervised the data analysis process, Jia Qiua and Dianfei Ma collected the samples. Jie Xia revised the manuscript.

Declaration of competing interest: The authors declare no conflict of interest.

Acknowledgements: The work was supported by Yunnan University of Chinese Medicine School-College Joint Fund Project (XYLH202004) and Yunnan Provincial Department of Science and Technology Basic Research on Traditional Chinese Medicine Project (202101AZ070001-298).

Data availability statement: The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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Table 1. Retention time and peak area of the common peaks of Laggera pterodonta

Retention time (min)	21.805	30.434	30.984	33.911	34.543	35.077	35.713	37.678	39.317	40.753	42.694	44.351
Sample 1	234.579	461.922	823.87	206.878	182.046	402.485	162.638	1579.94	409.048	564.376	215.593	254.768
Sample 2	154.677	384.49	663.648	175.092	136.495	328.931	106.847	1247.623	360.65	457.335	181.658	222.053
Sample 3	187.097	147.382	413.577	85.314	73.657	208.643	50.686	631.552	137.063	220.575	50.243	75.508
Sample 4	169.081	188.197	467.729	103.154	85.069	238.604	65.545	777.025	186.316	281.322	65.648	100.827
Sample 5	168.411	244.088	527.466	146.682	95.146	264.957	83.719	878.47	219.096	299.958	85.127	135.281
Sample 6	95.028	148.839	336.7	85.044	71.353	192.135	49.534	651.865	135.79	236.69	56.513	77.46
Sample 7	31.396	442.402	661.325	158.193	182.837	330.162	157.619	1296.567	380.488	522.291	170.15	211.743
Sample 8	51.918	497.59	748.8	120.372	208.783	394.928	174.904	1535.9	404.78	645.739	200.344	240.641
Sample 9	27.19	668.1	971.942	161.651	260.814	493.58	233.696	1834.707	539.809	750.449	238.421	295.113
Sample 10	72.997	548.215	817.721	205.703	221.772	389.747	185.485	1555.771	452.175	619.223	217.551	263.151
Sample 11	34.189	801.955	1138.775	203.487	293.791	553.613	282.998	2177.065	645.617	906.504	281.77	369.446
Sample 12	29.053	546.221	844.39	170.136	220.383	431.074	193.877	1636.588	458.169	672.276	204.046	252.521
Sample 13	67.378	624.514	929.477	155.57	243.41	469.827	215.009	1809.723	528.927	732.244	248.939	303.885
Sample 14	168.401	502.736	1343.013	345.082	316.117	673.913	258.423	3550.802	397.168	1411.991	268.012	449.102
Sample 15	248.977	680.955	1726.895	423.477	389.819	787.706	351.541	4205.108	489.264	1669.872	319.847	549.529
Sample 16	338.409	479.043	1378.569	338.882	304.361	692.15	254.757	3862.076	394.51	1678.875	305.751	486.456
Sample 17	405.736	635.177	1851.205	511.415	399.436	888.455	344.603	4790.911	499.331	1823.683	346.565	580.67
Sample 18	115.486	739.928	1811.326	576.799	417.76	845.682	390.565	5124.011	551.997	1976.016	372.425	647.382
Sample 19	315.081	613.678	1598.581	473.708	309.797	671.081	351.507	4067.427	448.84	1661.318	281.71	522.737
Sample 20	83.342	677.098	1894.366	719.265	437.976	916.857	350.667	5944.377	463.375	2123.241	360.853	594.057
Sample 21	127.25	624.655	1636.573	440.143	349.213	731.379	323.373	4446.243	446.436	1760.476	292.786	528.157
Sample 22	182.019	543.104	1418.145	422.784	305.227	688.726	272.121	3831.811	386.718	1411.925	264.721	420.46
Sample 23	261.965	72.685	593.522	305.539	52.716	116.192	36.157	1151.734	73.006	240.028	42.661	53.517
Sample 24	316.699	94.178	735.263	358.543	67.834	148.939	49.598	1462.571	91.793	324.409	57.783	67.577
Sample 25	415.677	111.293	717.563	300.545	72.214	170.155	59.51	1481.037	112.284	351.309	53.333	73.914
Sample 26	397.448	139.667	917.439	436.017	108.988	234.728	76.751	2038.069	102.117	481.591	87.15	116.375
Sample 27	275.022	70.514	573.592	295.796	52.124	114.014	35.331	1107.161	72.081	230.927	41.706	51.062
Sample 28	196.166	1297.924	2431.259	372.185	478.136	1054.578	411.764	3542.807	1147.703	1527.461	398.235	492.763
Sample 29	178.061	1924.042	3378.98	537.908	653.095	1452.39	699.037	4593.041	1841.431	2242.426	550.447	740.091
Sample 30	230.353	2027.977	3544.716	506.519	740.068	1496.831	658.31	4701.829	1891.395	2123.18	627.685	741.995
Sample 31	188.333	1490.249	2815.139	424.238	565.45	1241.071	542.224	3879.781	1406.578	1858.655	446.127	562.699
Sample 32	260.375	2340.261	3785.399	564.792	661.655	1440.89	764.699	5188.971	1943.822	2229.426	542.578	740.422
Sample 33	280.097	1982.466	3232.472	445.302	573.551	1325.78	614.8	4259.633	1827.066	1867.633	772.978	652.259
Sample 34	273.001	1662.143	2914.245	448.781	559.5	1247.967	511.978	4042.735	1541.624	1709.239	477.163	551.828

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Research on the origin identification of Laggera pterodonta based on UPLC fingerprint and chemometrics

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Introduction

Materials And Methods

Results And Discussion

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