Comparative Analysis of Chemical Constituents in Different Parts of Lotus by UPLC and QToF-MS

Six parts of lotus (seeds, leaves, plumule, stamens, receptacles and rhizome nodes) are herbal medicines that are listed in the Chinese Pharmacopoeia. Their indications and functions have been confirmed by a long history of clinical practice. To fully understand the material basis of clinical applications, UPLC-QToF-MS combined with the UNIFI platform and multivariate statistical analysis was used in this study. As a result, a total of 171 compounds were detected and characterized from the six parts, and 23 robust biomarkers were discovered. The method can be used as a standard protocol for the direct identification and prediction of the six parts of lotus. Meanwhile, these discoveries are valuable for improving the quality control method of herbal medicines. Most importantly, this was the first time that alkaloids were detected in the stamen, and terpenoids were detected in the cored seed. The stamen is a noteworthy part because it contains the greatest diversity of flavonoids and terpenoids, but research on the stamen is rather limited.


Introduction
Nelumbo nucifera Gaerth., an aquatic plant in the Nymphaeaceae family, is distributed in wetlands throughout Asia. It is also known as sacred lotus; it is not only used as a food and herb, but also deeply related to Buddhism in Asia, and its flower is used as the pedestal for divine figures. The medicinal functions of lotus were recognized earlier than its edible value and were recorded for the first time in the book "Er ya" (400 B.C.) [1]. Almost all parts of this plant have been used as food as well as herbal medicine for over 2000 years, and especially the lotus seed and rhizome are more widely used due to their delicious taste and great nutritive value [2]. The seed (Nelumbinis Semen), leaf (Nelumbinis Folium), plumule (Nelumbinis Plumula), stamen (Nelumbinis Stamen), receptacle (Nelumbinis Receptaculum) and rhizome node (Nelumbinis Rhizomatis Nodus) are listed in the official Pharmacopoeia of China (CP). Their indications and functions, which were confirmed by clinical practice for thousands of years, were recorded in the Chinese Pharmacopoeia. According to the records, lotus leaves, receptacles and rhizome nodes have a hemostatic effect; stamens can prohibit pathological spermatorrhoea and frequent urination; and the seeds and plumule can mind-tranquilize and improve sleep [3] (Figure 1).
In recent decades, increasing research has focused on this special herb, especially the plumule and leaf, and many of its constituents have been found to possess extensive features of health benefits. The ingredients and activities of the different lotus parts, together with their applications in the food and healthcare area, have similarities and differences [2]. Without a doubt, the pharmacological effects of any plant or effective part are based on their phytochemicals. Flavonoids that have been found in the six parts of lotus are associated with a variety effects, including antioxidant [4][5][6][7], anti-inflammatory [8], antiviral [9,10], anti-obesity [11,12], and antimicrobial effects [13]. Alkaloids are effective ingredients used for treating cardiovascular diseases [14][15][16], regulating blood lipids [17], [8], antiviral [9,10], anti-obesity [11,12], and antimicrobial effects [13]. Alkaloids are effective ingredients used for treating cardiovascular diseases [14][15][16], regulating blood lipids [17], tranquilizing the mind [18] and treating cancer [19]. However, the chemical bases of their activities are far from clear compared with the medical history of thousands of years. For example, stamens and receptacles are reported to have anti-ischemic effects [20]; meanwhile, neferine and liensinine were confirmed to be anti-ischemic agents [21], but there is no proof that stamens contain alkaloids. Thus, just what do the stamens and receptacles contain that contributes to the anti-ischemic effect? Furthermore, the material basis for traditional uses as hemostatic agents is a "blank" area. Moreover, there are no index components for the quality control of seeds, stamens, receptacles and rhizome nodes in CP. Hence, this study focuses on the chemical constituents of the six parts of lotus. UPLC is the best chromatographic method in terms of resolution, sensitivity, and speed. QToF-MS is the most sensitive quantitative and most comprehensive qualitative detector to identify and quantify the broadest range of compounds in the most complex and challenging samples. With its combined ability of high resolution and sensitivity, UPLC-QToF-MS has been successfully used for the analyses of complex samples [22]. It can be used for the rapid differentiation of different parts of a plant [23], identification of the habitats of herbs [24], and evaluation of the quality of TCMs and processed products [25]. The UNIFI information system has the ability to incorporate scientific library into with UPLC and QToF-MS data, which streamlines the process of identifying chemical structures in complex natural products. To obtain the chemical constituent profile of the cored seeds (Sem), leaves(Fol), plumule (Plu), stamens (Sta), receptacles (Rec) and rhizome nodes (RN) of lotus, we proposed a multiple ingredients identification strategy based on UPLC-QToF-MS coupled with the UNIFI informatics platform. This method can quickly identify multiple components. In this study, the constituents of the six parts were efficiently separated by ultra-performance liquid chromatography (UPLC) and detected by quadrupole time-of-flight tandem mass spectrometry (QToF-MS). Then, the data obtained by UPLC and QToF-MS were processed by the integrated information platform UNIFI.
As a result, a total of 171 components were identified from the six parts. Moreover, the differentiating components were screened by principal component analysis (PCA) UPLC is the best chromatographic method in terms of resolution, sensitivity, and speed. QToF-MS is the most sensitive quantitative and most comprehensive qualitative detector to identify and quantify the broadest range of compounds in the most complex and challenging samples. With its combined ability of high resolution and sensitivity, UPLC-QToF-MS has been successfully used for the analyses of complex samples [22]. It can be used for the rapid differentiation of different parts of a plant [23], identification of the habitats of herbs [24], and evaluation of the quality of TCMs and processed products [25]. The UNIFI information system has the ability to incorporate scientific library into with UPLC and QToF-MS data, which streamlines the process of identifying chemical structures in complex natural products. To obtain the chemical constituent profile of the cored seeds (Sem), leaves(Fol), plumule (Plu), stamens (Sta), receptacles (Rec) and rhizome nodes (RN) of lotus, we proposed a multiple ingredients identification strategy based on UPLC-QToF-MS coupled with the UNIFI informatics platform. This method can quickly identify multiple components. In this study, the constituents of the six parts were efficiently separated by ultra-performance liquid chromatography (UPLC) and detected by quadrupole time-of-flight tandem mass spectrometry (QToF-MS). Then, the data obtained by UPLC and QToF-MS were processed by the integrated information platform UNIFI.
As a result, a total of 171 components were identified from the six parts. Moreover, the differentiating components were screened by principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). These methods provide a holistic and intuitionistic description of the chemical constituents in the six parts. Twenty-three robust biomarkers were found to distinguish the six parts. The established method can be used as a standard protocol for directly discriminating between and predicting the six parts of lotus. Most importantly, data analysis provided useful information for further study and usage of the plant.

Identification of Components
A total of 171 compounds were identified or tentatively characterized from the six parts of lotus, including alkaloids, flavonoids, terpenoids, steroids, organic acids, etc. Among them, 84 compounds were identified in positive mode, and 127 compounds were identified in negative mode. The base peak intensity (BPI) chromatograms marked with the number of compounds are shown in Figure 2. The compound identification data were listed in Table 1. The chemical structures of the compounds are shown in Figure 3. More specifically, 86, 56, 89, 87, 60 and 19 compounds were identified from Sem, Fol, Plu, Sta, Rec and RN, respectively. By comparing the numbers of the detected compounds and signal strength of the chromatographic peak observed in the UPLC-QToF-MS assay, it seems that ESI − mode is better than ESI + for this test. However, running the ESI + mode is still necessary because some compounds display better responses in ESI + mode than in ESI − .     As listed in Table 1, the compounds were determined according to their characteristic MS fragmentation patterns, or the retention times of reference standards. Take an alkaloid (17)   As listed in Table 1, the compounds were determined according to their characteristic MS fragmentation patterns, or the retention times of reference standards. Take an alkaloid (17) and a flavonoid (56) as examples to illustrate the resolution process of the compounds. Compound 17 is a benzylisoquinoline alkaloid (t R = 2.10 min, C 19 H 23 NO 3 ) yielded [M + H] + ion at m/z 314.1754, and produced fragment ion at m/z 283.0286 due to parent ion peak losing CH 3 NH 2 . m/z 206.1181 and m/z 107.0875 are fragment ion peaks formed by benzyl cleavage. By comparison with literature information, this component was identified as armepavine [27]. The mass spectrogram is shown in Figure 4a. Compound 56 is an oxygen glycosides flavonoid (t R = 5.70 min, C 21 H 20 O 12 ) yielded [M − H] − ion at m/z 463.0343, and produced fragment ion at m/z 300.9948 due to parent ion peak losing glucose moiety. By comparison with literature and reference standard information, this component was identified as isoquercetin [28]. The mass spectrogram is shown in Figure 4b.

Metabolomics Analysis of Six Different Parts of Lotus
Metabolomics analyses of six parts of lotus included PCA and OPLS-DA. First, to separate the parts and obtain the maximum variables, PCA was used to obtain the score plots ( Figure 5a) and loading plots (Figure 5b). In the score plots, the green QC points are closely gathered together to form a cluster, which indicates that the system is stable. It can be seen from the figure that the samples from the Sem, Rec, Plu, Sta, Fol and RN groups could be easily divided into six clusters, and the six parts had achieved obvious separation, indicating that the six parts could be easily distinguished. In the loading plots, 23 variables that can be distinguished among the six clusters were found.
Second, to further evaluate the differences between the six parts, one was distinguished from the others, the maximum separation of the six parts was achieved, the potential biomarkers that may lead to the differences were found, and OPLS-DA was carried out. Then, for the visualization of the OPLS-DA and convenient interpretation of the model, S-plots were created. At the same time, to screen the different components, the variable importance of the projection (VIP) was introduced. The metabolites with VIP values above 1.0 and p-values below 0.05 were considered as potential biomarkers [29][30][31]. Based on these two important parameters and the identification of the components from six parts (Table 1), 23 reliable known biomarkers were found to distinguish the six parts and were labeled in the S-plots ( Figure 6). In addition, a heatmap (Figure 7) was drawn to systematically evaluate these biomarkers and visually display the intensity of these biomarkers. For Sem, there were three potential biomarkers, including flavonoids (110, 125) and a quinone (99). For Fol, there were three potential biomarkers, including terpenoids (105, 108) and an alkaloid (102). For Plu, there were nine potential bi-

Metabolomics Analysis of Six Different Parts of Lotus
Metabolomics analyses of six parts of lotus included PCA and OPLS-DA. First, to separate the parts and obtain the maximum variables, PCA was used to obtain the score plots ( Figure 5a) and loading plots (Figure 5b). In the score plots, the green QC points are closely gathered together to form a cluster, which indicates that the system is stable. It can be seen from the figure that the samples from the Sem, Rec, Plu, Sta, Fol and RN groups could be easily divided into six clusters, and the six parts had achieved obvious separation, indicating that the six parts could be easily distinguished. In the loading plots, 23 variables that can be distinguished among the six clusters were found.
Second, to further evaluate the differences between the six parts, one was distinguished from the others, the maximum separation of the six parts was achieved, the potential biomarkers that may lead to the differences were found, and OPLS-DA was carried out. Then, for the visualization of the OPLS-DA and convenient interpretation of the model, S-plots were created. At the same time, to screen the different components, the variable importance of the projection (VIP) was introduced. The metabolites with VIP values above 1.0 and p-values below 0.05 were considered as potential biomarkers [29][30][31]. Based on these two important parameters and the identification of the components from six parts (Table 1), 23 reliable known biomarkers were found to distinguish the six parts and were labeled in the S-plots ( Figure 6). In addition, a heatmap (Figure 7) was drawn to systematically evaluate these biomarkers and visually display the intensity of these biomarkers. For Sem, there were three potential biomarkers, including flavonoids (110, 125) and a quinone (99). For Fol, there were three potential biomarkers, including terpenoids (105, 108) and an alkaloid (102). For Plu, there were nine potential biomarkers, including flavonoids (29, 47, 55, 86), alkaloids (28, 38, 44) an organic acid (10) and an organic acid ester (85). For Sta, there were four potential biomarkers, including steroids (133, 153), a terpenoid (131) and an organic acid (158). For Rec, there were three potential biomarkers, including an alkaloid (106), a steroid (135), and an amide (126). For RN, there was only one potential biomarker-a terpenoid (130). These robust biomarkers enabling the differentiation among Sem, Fol, Plu, Sta, Rec and RN can be used for the rapid identification of six parts of lotus.

Discussion
Herbal medicines usually play a holistic role in maintaining health through multiple targets because they contain multiple constituents. Being a traditional Chinese herb, lotus has been used to treat various diseases. In the last decades, chemoinformatics and systems pharmacology have been successfully applied in the discovery of the active

Discussion
Herbal medicines usually play a holistic role in maintaining health through multiple targets because they contain multiple constituents. Being a traditional Chinese herb, lotus has been used to treat various diseases. In the last decades, chemoinformatics and systems pharmacology have been successfully applied in the discovery of the active

Discussion
Herbal medicines usually play a holistic role in maintaining health through multiple targets because they contain multiple constituents. Being a traditional Chinese herb, lotus has been used to treat various diseases. In the last decades, chemoinformatics and systems pharmacology have been successfully applied in the discovery of the active component of traditional Chinese medicines and their mechanisms of action. It is well known that the process of fully understanding the ingredients of herb using traditional methods is labor intensive, difficult and time-consuming. Fortunately, the combining of UPLC-QToF-MS technology and UNIFI platform helps researchers reveal the containing compounds in herbs in an efficient way.
The present study analysed the principal components of the lotus six parts by UPLC-QToF-MS. Combined the related literatures [6,32,33] with our previous experiments, heat reflux extraction with 80% ethanol was chosen for the samples extraction. And the detecting conditions, mobile phase elution solutions of UPLC, positive and negative ion detection modes of QToF-MS, were optimized by quality control samples. MS and MS/MS data were collected simultaneously to improve the efficiency and accuracy of data collection in the MS E model. The tolerance of t R was ± 0.1 min. The isotopic pattern was included in the peak identification. The permutation testing was the scoring function for identification and statistical analysis parameter p-value need to below 0.05. As a result, 171 compounds were identified or tentatively characterized from the six parts of lotus.
It was found that flavonoids were the most common compounds: 56 of 171 were flavonoids. Sta was ranked first due to the 31 kinds of flavonoids being detected, followed by Plu (24 kinds), Sem (23 kinds), Fol (20 kinds), and Rec (20 kinds). By comparing the species of flavonoids distributed in the six parts, it was found that 18 of the 20 in Rec are consistent with those in Sta, and half of the species in Sem are the same as those in Plu. Luteolin is the only flavonoid detected from RN, and it is also available in the other five parts.
Alkaloids are also important active components in lotus. In this experiment, 22 kinds of alkaloid compounds were detected, including isoquinoline alkaloids, aporphine alkaloids and so on. Plu contained 15 kinds, followed by Fol with 11 kinds. Armepavine and nuciferine are available in all six parts. Terpenoids were the most abundant in lotus Sta, containing 13 species. Statistical analysis was conducted on the compounds detected from the six parts, and the structure types and the numbers of compounds in the six parts are shown in Figure 8. It is well known that the process of fully understanding the ingredients of herb using traditional methods is labor intensive, difficult and time-consuming. Fortunately, the combining of UPLC-QToF-MS technology and UNIFI platform helps researchers reveal the containing compounds in herbs in an efficient way. The present study analysed the principal components of the lotus six parts by UPLC-QToF-MS. Combined the related literatures [6,32,33] with our previous experiments, heat reflux extraction with 80% ethanol was chosen for the samples extraction. And the detecting conditions, mobile phase elution solutions of UPLC, positive and negative ion detection modes of QToF-MS, were optimized by quality control samples. MS and MS/MS data were collected simultaneously to improve the efficiency and accuracy of data collection in the MS E model. The tolerance of tR was ± 0.1 min. The isotopic pattern was included in the peak identification. The permutation testing was the scoring function for identification and statistical analysis parameter p-value need to below 0.05. As a result, 171 compounds were identified or tentatively characterized from the six parts of lotus.
It was found that flavonoids were the most common compounds: 56 of 171 were flavonoids. Sta was ranked first due to the 31 kinds of flavonoids being detected, followed by Plu (24 kinds), Sem (23 kinds), Fol (20 kinds), and Rec (20 kinds). By comparing the species of flavonoids distributed in the six parts, it was found that 18 of the 20 in Rec are consistent with those in Sta, and half of the species in Sem are the same as those in Plu. Luteolin is the only flavonoid detected from RN, and it is also available in the other five parts.
Alkaloids are also important active components in lotus. In this experiment, 22 kinds of alkaloid compounds were detected, including isoquinoline alkaloids, aporphine alkaloids and so on. Plu contained 15 kinds, followed by Fol with 11 kinds. Armepavine and nuciferine are available in all six parts. Terpenoids were the most abundant in lotus Sta, containing 13 species. Statistical analysis was conducted on the compounds detected from the six parts, and the structure types and the numbers of compounds in the six parts are shown in Figure 8. The established method can be used as a standard protocol for directly discriminating between and predicting the six parts of lotus. Traditional Chinese herbal therapy can be characterized by the use of a large number of multi-herb formulae. Chinese patent medicines (CPM), which come from traditional Chinese classical prescriptions, are usually prepared by modern pharmaceutical techniques with various herbs as raw material. After processing, the unique morphological characteristics of the original herbs disappeared, and the active chemical constituents were successfully preserved. So the chemical compositions and characters of CPM have been considered a reliable index of quality control. Being used as both delicious food and empirical medicine, the formula- The established method can be used as a standard protocol for directly discriminating between and predicting the six parts of lotus. Traditional Chinese herbal therapy can be characterized by the use of a large number of multi-herb formulae. Chinese patent medicines (CPM), which come from traditional Chinese classical prescriptions, are usually prepared by modern pharmaceutical techniques with various herbs as raw material. After processing, the unique morphological characteristics of the original herbs disappeared, and the active chemical constituents were successfully preserved. So the chemical compositions and characters of CPM have been considered a reliable index of quality control. Being used as both delicious food and empirical medicine, the formulation and preparation conditions must be improved to achieve better delivery of nutritional ingredients and increased bioactivities of the food and medicinal products. Lotus is one of the commonly-used herbal drugs. Its two parts or more appeared in one prescription for some disease treatment. For example, Bai Dai Wan from Hui Zhi Tang Jing Yan Fang (Experiential Prescriptions from Hui Zhi Clinic) is comprised of eight herbs including lotus seed, stamens and rhizome nodes. The biomarkers found in this paper can provide bases for examining the raw herbs types in the finished preparations and improving the quality of products.
Most importantly, data analyses provided useful information for the further study and usage of the plant. The results of this phytochemical profile study are far more comparable because the same procedure was used during sample analysis, including during sample treatment, the detection procedure, data processing and the interpretation of testing data. Compared with the previous studies [2,[33][34][35], this was the first time that alkaloids (8, 17, 20, 24, 63, 95) were detected in Sta, and terpenoids (97, 124, 139, 140, 148, 154, 159, 160,  170) were detected in Sem. Sta is a more noteworthy part than the others, for it has the greatest diversity of flavonoids and terpenoids, but relatively few studies on Sta can be found compared to the other parts.
Being a traditional Chinese herb, lotus has been used for more than 2,000 years. However, the material basis of a number of folk applications has not been illustrated clearly. For instance, being the hemostatic agent in traditional Chinese medicine, Rec and RN have the same indications. After comparison, we found that 15 of the 19 compounds identified in RN were also presented in Rec. They were three alkaloids (17, 95, 127), three terpenoids (138, 152, 154), one flavonoid (91), three steroids (147, 149,1 62), and five organic acids and esters (6, 117, 165, 167, 171). They may be the material basis of hemostatic activity of RN and Rec.
Compound 41 (liensinine) and 59 (neferine) play a major role in anti-Alzheimer disease agents [36]. However, none of them was detected in the receptacle, although Rec was reported to have an anti-Alzheimer effect [37]. In this paper, 41 and 59 were identified in Sem, Fol and Plu, as well as 41 were identified in Rec. The result provided useful information for the research and utilization of lotus seed, leaf and receptacle. Meanwhile, the discoveries of alkaloids in Sta (8, 17, 20, 24, 63, 95) and Rec (17, 41, 43, 95, 103, 106, 127) may be useful for explaining their anti-ischemic effect.
In summary, the holistic and intuitionistic description of the chemical constituents in lotus six parts in this paper contribute new information to the phytochemical research of lotus. The results will be helpful in illustrating the chemical basis of herbs activities. The established method and identified biomarkers provide valuable data and references for quality control of the CPMs who contain lotus different parts in the prescriptions.
The results of this research may be limited by the capacity of the identification database. Not compared with an analytical standard, identifications of compounds are presumptive. Therefore, the identification relies more on standard secondary spectra database. Only a fraction of compounds were included in the HMBD database, so relatively few are detected although compounds are varied and abundant in the six parts of lotus. In addition to the difference ingredients, differences in the content of common ingredients of herbs also affect their pharmacological activity. In the future, more efforts should be devoted to research on the concentrations of biological compounds and biomarkers of the herbs.

Materials and Reagents
The products of Sem, Fol, Plu, Sta, Rec and RN were collected from their respective cultivation areas or purchased from herbal markets in China. A total of 48 batches (each part 8 batches) were gathered and identified by Professor Ping-Ya Li (School of Pharmaceutical Sciences, Jilin University, Changchun, China). The voucher specimens (No. 2019224-2019272) had been deposited at the Research Center of Natural Drug, School of Pharmaceutical Sciences, Jilin University, Changchun, China. A site list of the samples collected is given in Table 2.

Species Hunan Shandong Hubei
Hebei Anhui Fujian Jiangxi Guangdong

Data Analysis
For the screening analysis, the MS raw data were imported into the Waters' UNIFI 1.7.0 platform to quickly identify the chemical components. Two hundred was set as the minimum peak area for two-dimensional peak detection. For three-dimensional peak detection, the low-energy peak intensity was over 1000 counts, and the high-energy peak intensity was over 200 counts. The mass error of the compound was within ± 5 ppm, and the retention time (t R ) was within ± 0.1 min. The negative adducts +COOH and -H and positive adducts +H and +Na were selected.
For metabonomics analysis, the original MS E data were processed by using Waters MarkerLynx XS V4.1 software, and a table of the m/z-t R pairs with the corresponding intensities of all the peaks was obtained. The same t R and m/z values in different batches of samples were regarded as the same component. The main parameters included the following: t R range, 0-30 min; minimum intensity, 5%; mass range, 100-1500 Da; mass tolerance, 0.10. Multivariate statistical analysis was performed, including PCA and OPLS-DA.