Comparative Study on Chemical Constituents of Medicinal and Non-Medicinal Parts of Flos Abelmoschus manihot , Based on Metabolite Profiling Coupled with Multivariate Statistical Analysis

: According to Chinese Pharmacopoeia (2020 edition), Abelmoschi Corolla (AC) is the dried corolla of Flos Abelmoschus manihot (FAM). Market research has found that AC is often mixed with the non-medicinal parts in FAM, including calyx, stamen, and pistil. However, previous studies have not clariﬁed the relationship between the medicinal and non-medicinal parts of FAM. In this study, in order to investigate whether there is any distinction between the medicinal and non-medicinal parts of FAM, the characterization of the constituents in calyx, corolla, stamen, and pistil was analyzed by UFLC-Triple TOF-MS/MS. Multivariate statistical analysis was used to classify and screen differential constituents between medicinal and non-medicinal parts of FAM, and the relative contents of differential constituents were compared based on the peak intensities. Results showed that 51 constituents in medicinal and non-medicinal parts of FAM were identiﬁed, and the fragmentation pathways to different types of constituents were preliminarily deduced by the fragmentation behavior of the identiﬁed constituents. Furthermore, multivariate statistical analysis revealed that the medicinal and non-medicinal parts of FAM differed signiﬁcantly; 20 differential constituents were screened out to reveal the characteristics of metabolic differences. Among them, the relative contents of 19 differential constituents in the medicinal part were signiﬁcantly higher than those in non-medicinal parts. This study could be helpful in the quality evaluation of AC as well as provide basic information for the improvement of the market standard of AC.


Introduction
Abelmoschi Corolla (AC) is derived from the dried corolla of Flos Abelmoschus manihot (FAM), which has the functions of eliminating dampness and heat, subduing swelling, and detoxicating [1]. It is a traditional Chinese medicine (TCM) with a long medicinal history in China [2]. The research of pharmacology showed that AC has multiple pharmacological activities, such as anti-inflammatory, antioxidant [3,4], antitumor [5], anticonvulsant, antidepressant, and neuroprotective activities [6,7], as well as therapeutic actions on renal tubular injury and diabetic nephropathy [8][9][10].
As the main raw material of the Chinese patent medicine Huangkui capsule, AC has significant medicinal value and a huge market demand. Market research found that commercial medicinal material of AC was often mixed with non-medicinal parts of FAM, including calyx, stamen, and pistil. The reason for this phenomenon is that the nonmedicinal parts of FAM are not removed during the collection and processing of corolla. However, the chemical constituents of non-medicinal parts of FAM were not characterized in previous studies, so the comparative study between medicinal and non-medicinal parts of FAM was not clear-cut. The mixing of non-medicinal parts may alter the composition of AC, reduce its quality stability, and then affect its efficacy. Therefore, it is of practical value to study the chemical constituents of medicinal and non-medicinal parts of FAM and reveal the characteristics of metabolic differences.
Because it combines the separation powers of liquid chromatography with the very sensitive detection qualities of mass spectrometry, the LC-MS equipment has been frequently employed in TCM research in recent years [11]. Among them, ultra-fast liquid chromatography coupled with triple quadrupole-time of flight tandem mass spectrometry (UFLC-Triple TOF-MS/MS) is efficient and rapid in the determination of the molecular weight and characteristic fragment ions, by which the structure of multiple constituents in TCM can be identified quickly [12]. Hence, the characterization of the constituents in calyx, corolla, stamen, and pistil was analyzed by UFLC-Triple TOF-MS/MS. We integrated metabolic profiling and multivariate statistical analysis to separate the medicinal and nonmedicinal parts of FAM and to define their chemical markers. The strategy for comparative analysis on chemical constituents of medicinal and non-medicinal parts of FAM was shown in Figure 1. The study could determine the differential constituents of medicinal and non-medicinal parts of FAM, so as to provide basic data for standardizing the harvest and market standards of AC. Our investigation will not only contribute to the quality evaluation of AC, but also has great significance in the quality stability improvement of AC.

Plant Materials
Five batches of FAM samples were collected from Xinghua City (Jiangsu Province, China 32 • 98 17 N, 119 • 90 44 E) in October 2019. Each batch was carefully divided into four parts, calyx, corolla, stamen, and pistil, which were separately dried in the oven. The drying temperature was set at 50 • C. The samples were authenticated by Professor Xunhong Liu as the flower of Abelmoschus manihot (L.) Medic. of Malvaceae family and the voucher specimens were deposited in the laboratory of Chinese medicine identification, Nanjing University of Chinese Medicine. The voucher numbers of the samples were as follows:

Preparation of Standard and Sample Solutions
A mixed standard stock solution of 18 standard substances was prepared with 70% (v/v) methanol. The diluted solutions were stored at 4 • C for further UFLC-Triple TOF-MS/MS analysis.
The 0.5 g of calyx, corolla, stamen, and pistil powder were properly weighed and ultrasonically extracted with 20 mL 70% (v/v) methanol for 30 min, respectively. To compensate for the weight lost during extraction, the same solvent was added after cooling to room temperature. The extract was then filtered, and the filtrate was centrifuged at 12,000 rpm/min for 10 min. Afterwards, the supernatant was filtered via a 0.22 µm membrane before UFLC-Triple TOF-MS/MS analysis.

Identification of the Constituents
A database of the chemical constituents of AC was formed based on previous research and the data were imported into the PeakView Software V.1.2 (AB SCIEX, Framingham, MA, USA). The chemical constituents of different parts of FAM were comprehensively characterized by comparing the retention time (t R ), accurately measuring mass and multistage MS/MS fragmentation information with standard substances, databases, and related literatures.

Multivariate Statistical Analysis
The data of UFLC-Triple TOF-MS/MS were processed by PeakView Software V.1.2 (AB SCIEX, Framingham, MA, USA) and MarkerView 1.2.1 software (AB Sciex). Principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed using SIMCA-P 13.0 software (Umetrics AB, Umea, Sweden). PCA was used to categorize and identify different parts of FAM. OPLS-DA was performed to differentiate medicinal part and non-medicinal parts of FAM, as well as to identify the common differential constituents that cause the differences in each group of comparison by variable importance in the projection (VIP).

Relative Content Comparison of Differential Constituents
The relative contents of differential constituents in medicinal and non-medicinal parts of FAM were compared according to the peak intensities. To visualize and validate the distribution regularity of differential constituents among medicinal and non-medicinal parts of FAM, one-way analysis of variance (one-way ANOVA) was applied. Diagram of relative content comparison was charted by GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA).

Identification of the Constituents in Medicinal and Non-Medicinal Parts of FAM
It was found that the best analytical selectivity and sensitivity was obtained in the negative ionization mode by comparing the data acquired in the two ion modes. As a result, we decided to collect data in the negative ion mode. The base peak chromatograms (BPCs) of the calyx ( Figure 2. Eventually, 51 constituents were identified, including 43 flavonoids, 6 organic acids, 1 ester, and 1 alkaloid. A total of 18 constituents were clearly identified by comparison with reference standards. Detailed information of the characteristic constituents is summarized in Table 1, with their structures presented in Figure S1.

Identification of Flavonoids
As the main active substance of AC, flavonoids have always been the research hotspot. In this study, a total of 43 flavonoids were identified from the extract of calyx, corolla, stamen, and pistil, including flavone, flavonols, and dihydroflavonol. At the same time, flavonols can be carefully divided into hibiscus parent flavonols, gossypetin parent flavonols, myricetin parent flavonols, quercetin parent flavonols, and kaempferol parent flavonols, respectively. The common substituents group on the A and B rings in flavonoids were hydroxyl, methoxy, and acetyl, and it was also extremely common for saccharides or glucuronic acids to interact with hemiacetal hydroxyl groups to form flavonoid glycosides. The basic fracture paths of flavonoids were the loss of these neutral pieces and Retro-Diels-Alder (RDA) cleavage of the C ring [13]. Figure 3 depicts several RDA cleavage mechanisms of related flavonoids. The molecular ion peak intensity of flavonoid glycosides was often modest, and the base peak was frequently the fragment peak of aglycon.  The product ion at m/z 301.06 was generated from the elimination of H 2 O from the molecular ion. The m/z 193.01 was the basic peak due to the removal of the B ring. The m/z 165.02 and 151.00 were 0,2 A − (A fragment with A ring after the 0,2 bonds of C ring were broken) and 1,3 A − (A fragment with A ring after the 1,3 bonds of C ring were broken). Therefore, compound 11 was identified as dihydromyricetin and further confirmed by the reference substance. The molecular ion peak of compound 34 was generated at m/z 593.15, suggesting that the molecular formula of the compound was C 27  , and floramanoside C, respectively, which is consistent with previous studies [15]. See Table 1  Therefore, compound 27 was identified as isoquercitrin and confirmed by the reference substance. The specific fragment information was shown in Table 1.
Kaempferol parent flavonols Compounds 36, 50, and 51 were identified as kaempferol parent flavonols. Taking compound 50 as an example, the molecular ion was located at m/z 593.13, suggesting that the molecular formula was C 30

Multivariate Statistical Analysis
PCA was conducted to classify the different parts of FAM. The first two principal components accounted for more than 75% of the total variance, could be used to represent overall information of samples (R 2 X [1] = 0.554, R 2 X [2] = 0.235). The PCA scores plot indicated that the medicinal part and non-medicinal parts of FAM were divided into two clusters ( Figure 4). Corolla were gathered in the positive axis, non-medicinal parts of FAM were distributed in the negative axis, indicating that there was a significant difference between the medicinal and non-medicinal parts of FAM.
OPLS-DA was used to further distinguish the medicinal and non-medicinal parts of FAM, and to find out the important constituents that cause the differences with VIP values. The OPLS-DA score scatter plot, VIP plot, and S-Plot for comparison of the medicinal and non-medicinal parts of FAM were shown in Figure 5A-C. The OPLS-DA model demonstrated good adaptability (R 2 X = 0.938, 0.949, and 0.937, respectively, R 2 Y = 0.999, 0.999, and 0.999, respectively) and predictability (Q 2 = 0.999, 0.998, and 0.998, respectively). Calyx and corolla, stamen and corolla, pistil and corolla were all separated into two clusters along PC1 axis. The result revealed that the difference between the medicinal and non-medicinal parts of FAM was significant, which was completely consistent with the result of PCA.

Relative Content Comparison of Differential Constituents
The relative contents of differential constituents in medicinal and non-medicinal parts of FAM were compared based on peak intensity. Furthermore, the one-way ANOVA followed by least significant difference test (variance homogeneity) or Tamhane's test (variance heterogeneity) was carried out to illustrate the abundance variation of 20 differential constituents. As shown in Figure 6. The relative contents of 19 differential constituents

Discussion
The clinical efficacy of TCM is determined by its chemical constitution [40]. The Chinese Pharmacopoeia officially recorded that the medicinal part of AC was only the corolla of FAM. However, the phenomenon of doping non-medicinal parts in AC was common. Therefore, the chemical constituents of medicinal and non-medicinal parts of FAM were analyzed. A total of 20 differential constituents, including 1 organic acid and 19 flavonoids, were screened out by multivariate statistical analysis and their relative contents were compared. Among them, the relative content of 19 differential constituents in the corolla of FAM was significantly higher than that in the non-medicinal parts. Many studies have shown that the flavonoids of AC such as hyperin and isoquercitrin possess anti-inflammatory and renal injury protective properties [41][42][43], which is consistent with the clinical efficacy of AC [44,45]. Due to the great difference in chemical constituents between the medicinal part and the non-medicinal parts, the latter may not have the same therapeutic effect as the medicinal part. The screened differential constituents might be the pharmacodynamic substances of AC, which provide ideas for the research of pharmacodynamic substance basis of AC.
In summary, 51 constituents from medicinal and non-medicinal parts of FAM were identified and their metabolic profiles were compared. 20 differential constituents were screened to distinguish the medicinal part and non-medicinal parts of FAM. The great difference in the relative content of them indicates that the non-medicinal parts of FAM are hardly a substitute for the corolla part. Our study could be conducive to the quality evaluation and quality stability improvement of AC and provide a scientific basis for strictly regulating the harvest and market standards of AC.