**3. Results**

#### *3.1. 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 2A), corolla (Figure 2B), stamen (Figure 2C), and pistil (Figure 2D) extract in the negative ion mode are shown in 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.

### 3.1.1. 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.

**Figure 2.** The base peak chromatograms (BPCs) of the calyx (**A**), corolla (**B**), stamen (**C**), and pistil (**D**) extract in negative ion mode. Note: the peak numbers denoted were the same as those in Table 1.

**Figure 3.** Schematic diagram of the fracture site of related flavonoids in negative ion mode.

*Dihydroflavonol and flavone* Compounds **11** and **34** were identified as dihydroflavonol and flavone, respectively. Compound **11** produced a [M −H]<sup>−</sup> ion at *m*/*z* 319.05 and abundance fragment ions, such as ions at *m*/*z* 301.06, 193.01, 165.02, and 151.00. The product ion at *m*/*z* 301.06 was generated from the elimination of H2O 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 C27 H30 O15 and abundant fragment ions were generated at 285.04, 255.03, and 227.04. After removing the glycosyl and CH2 from *m*/*z* 593.15 ion, the product ion of *m*/*z* 285.04 was generated. The fragment ions of *m*/*z* 255.03 and 227.04 were formed by dropping CH2O and CO. Hence, compound **34** was proposed as 4-methoxyl-5,7-dihydroxyl flavone-[- *O*-*β*-D-xylopyranosyl-(1 →3)]- *Oβ*-D-glucopyranoside, consistent with previous studies [14].

*Hibiscus parent flavonols* Compounds **6**, **8**, and **28** were identified as hibiscus parent flavonols with the adducted ion as [M −H]<sup>−</sup> or [M+HCOO]<sup>−</sup>. The primary distinctive fragment ions of hibiscus parent flavonols were 1,2 A− (A fragment with A ring after the 1,2 bonds of C ring were broken) and 1,3 A− obtained by RDA fragmentation. Compounds **6** and **8** produced [M+HCOO]− ion at *m*/*z* 541.08 and abundance fragment ions, such as ions at *m*/*z* 333.03, 315.01, 287.02, 195.00, and 167.00. The *m*/*z* 333.03 was the product ion [M − H −glc]<sup>−</sup> due to cleavage of the glycosidic bond. The product ions at *m*/*z* 315.01 and 287.02 were generated from the elimination of H2O and CO from *m*/*z* 333.03 ion. The *m*/*z* 195.00 and 167.00 were 1,2 A− and 1,3 A− obtained by RDA fragmentation. The adducted ion of compound **28** was observed as [M −H]<sup>−</sup> and the fragment ions basic peak [M − H −glu]<sup>−</sup> was observed at *m*/*z* 333.03 by the loss of 176 Da. Meanwhile, the *m*/*z* 195.00 and 167.00 were 1,2 A− and 1,3 A− obtained by RDA fragmentation. Therefore, compounds **6**, **8**, and **28** were identified as hibiscetin-3- *O*-glucoside, floramanoside B, and floramanoside C, respectively, which is consistent with previous studies [15]. See Table 1 for details.

*Gossypetin and myricetin parent flavonols* 7 and 11 constituents were identified as flavonoids with gossypetin and myricetin parent flavonols, respectively, including **12**, **13**, **25**, **39**, **40**, **43**, **44** and **14**, **15**, **16**, **18**, **19**, **20**, **21**, **26**, **33**, **38**, **41**. We found that gossypetin and myricetin parent flavonols were more likely to lose H2O and CO fragments. In addition, the main characteristic fragments of these two flavonols were ions obtained from the cleavage of RDA. Taking compounds **39** and **20** as examples, the molecular ions were located at *m*/*z* 493.06 and 479.08, suggesting that the molecular formulas of the two compounds were C21 H18 O14 and C21 H20 O13, respectively. They all have base peaks at *m*/*z* 317.03, indicating that compounds **39** and **20** had a glycosidic acid and a glucose group, respectively. The main difference between the two was that the former fragment ions were [1,2A]− (*m*/*z* 195.00), [1,3A]− (*m*/*z* 167.00), and [1,3 A −CO]<sup>−</sup> (*m*/*z* 139.00), while the latter fragment ions were [1,2A]− (*m*/*z* 179.00), [1,3A]− (*m*/*z* 151.00), and [1,2B]− (*m*/*z* 137.02). The difference is due to the different positions of hydroxyl groups in aglycon. Therefore, compounds **39** and **20** were identified as gossypetin 8- *O*-*β*-D-glucuronide and myricetin 3- *O*-*β*-D-glucopyranoside and further confirmed by the reference substance.

*Quercetin parent flavonols* 17 constituents including **17**, **22**, **23**, **24**, **27**, **29**, **30**, **31**, **32**, **35**, **37**, **42**, **45**, **46**, **47**, **48**, and **49** were identified as flavonoids with quercetin parent flavonols. They were more likely to lose H2O and CO fragments and the ions obtained by RDA fragmentation were the main characteristic fragments. Taking compound **27** as example, the molecular ions was located at *m*/*z* 463.09, suggesting that the molecular formulas was C21 H20 O12. The *m*/*z* 301.03 was the product ion [M − H −glc]<sup>−</sup> due to cleavage of the glycosidic bond. The fragments ions of *m*/*z* 253.05 and 237.54 were formed by dropping CO and H2O. The *m*/*z* 179.00 and 151.00 were 1,2 A− and 1,3 A− obtained by RDA fragmentation. 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 C30H26O13. The *m*/*z* 447.09 was the fragments ion [M−H−C9H6O2]<sup>−</sup> by dropping hydroxycinnamoyl group, *m*/*z* 285.04 was the product ion [M−H−C9H6O2−glc]<sup>−</sup> due to the cleavage of the glycosidic bond. The fragments ions of *m*/*z* 257.05 and 239.03 were ions [M−H−C9H6O2−glc−CO]<sup>−</sup> and [M−H−C9H6O2−glc−CO−H2O]<sup>−</sup> formed by dropping CO and H2O. Therefore, compound **50** was identified as tiliroside which confirmed by the reference substance.

### 3.1.2. Identification of Organic Acids

The adducted ion of organic acids was observed as [M−H]<sup>−</sup> in the negative mode. The MS/MS spectra of compounds **1**, **4**, **9** usually had a basic peak at [M−H−CO2]<sup>−</sup>, and then produce [M−H−CO2−H2O]<sup>−</sup> by the loss of H2O. The basic peak of compound **7** was the removal of caffeic acid group [M−H−C9H6O3]<sup>−</sup>, followed by the loss of 2H2O and CO produce [M−H−C9H6O3−2H2O−CO]<sup>−</sup>. Compounds **1**, **4**, **7**, **9** were identified as 3,4,5-trihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, chlorogenic acid, and caffeic acid, respectively, which were further confirmed by the reference substance. The basic peak of compounds **2**, **3** were [M−H−glc]<sup>−</sup>. At the same time, the other fragments were consistent with the identified components **1**, **4**. Therefore, compounds **2**, **3** were identified as gallic acid 3-*O*-*β*-glucoside and protocatecheuic acid 3-*O*-*β*-D-glucoside. See Table 1 for details.

### 3.1.3. Identification of Ester

Compound **5** gave precursor ion [M−H]<sup>−</sup> at *m*/*z* 299.08, suggesting that its molecular formula was C13H16O8. The fragment ion was observed at *m*/*z* 137.02 by the loss of 162 Da, attributed to the loss of a glucose group. The loss of H2O from [M−H−glc]<sup>−</sup> resulted in the fragment at *m*/*z* 119.03, showing the existence of hydroxyl group. Hence, it was identified as 4-hydroxybenzoic acid *β*-D-glucosyl ester by referring to the previous studies [16].

### 3.1.4. Identification of Alkaloid

Compound **10** gave precursor ion [M−H]<sup>−</sup> at *m*/*z* 252.09, suggesting that its molecular formula was C12H15NO5. The fragment ions basic peak [M−H−H2O]<sup>−</sup> was observed at *m*/*z* 234.08 by the loss of 18 Da, indicating the presence of hydroxyl group. Afterwards, the loss of CO2 from [M−H−H2O]<sup>−</sup> produced the fragment at *m*/*z* 190.09. Thus, it was identified as acortatarine A, consistent with previous studies [17].

### *3.2. 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 (R2X [1] = 0.554, R2X [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 (R2X = 0.938, 0.949, and 0.937, respectively, R2Y = 0.999, 0.999, and 0.999, respectively) and predictability (Q<sup>2</sup> = 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.







rhamnos. MS1: quasi-molecular ion, MS2: product fragment ion.

**Figure 4.** The principal component analysis scores scatter plot (**A**) and loading scatter plot (**B**) of medicinal and non-medicinal parts of FAM.

The identification of potential chemical markers to distinguish the medicinal and nonmedicinal parts of FAM was the focus of this study. Based on their VIP values (i.e., larger than 1.0), 20 constituents were screened out to discriminate the medicinal and non-medicinal parts of FAM, including protocatecheuic acid 3-*O*-*β*-D-glucoside (**3**), hibiscetin-3-*O*-glucoside (**6**), floramanoside B (**8**), gossypetin 3-*O*-*β*-glucopyranoside-8-*O*-*β*-glucuronopyranoside (**12**), gossypetin 3-*O*-*β*-glucuronopyranoside-8-*O*-*β*-glucopyranoside (**13**), myricetin 3-*O*-*β*-D-galact opyranoside (**18**), myricetin 3-*O*-*β*-D-glucopyranoside (**20**), myricetin 3-*O*-rutinose (**21**), quercetin 3-*O*-*β*-D-xylopyranosyl-(1→2)-*O*-*β*-D-galactopyranoside (**23**), floramanoside C (**28**), quercetin 3-*O*-*β*-D-robinobioside (**29**), hyperin (**30**), rutin (**31**), isoquercitrin (**32**), myricetin 3-*Oβ*-D glucopyranoside (**33**), 6-acetylhyperin (**35**), gossypetin 8-*O*-*β*-D-glucuronide (**39**), myricetin (**41**), quercetin <sup>3</sup>-*O*-*β*-D-glucoside (**46**), and floramaroside E (**48**). Therefore, these constituents could be selected as differential constituents to distinguish the medicinal and non-medicinal parts of FAM.

### *3.3. 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 (protocatecheuic acid 3-*O*-*β*-D-glucoside, hibiscetin-3-*O*-glucoside, floramanoside B, gossypetin 3-*O*-*β*glucopyranoside-8-*O*-*β*-glucuronopyranoside, gossypetin 3-*O*-*β*-glucuronopyranoside-8-*O*-*β*glucopyranoside, myricetin 3-*O*-*β*-D-galactopyranoside, myricetin 3-*O*-*β*-D-glucopyranoside, myricetin 3-*O*-rutinose, floramanoside C, quercetin 3-*O*-*β*-D-robinobioside, hyperin, rutin, isoquercitrin, myricetin <sup>3</sup>-*O*-*β*-D glucopyranoside, 6-acetylhyperin, gossypetin 8-*O*-*β*-Dglucuronide, myricetin, quercetin <sup>3</sup>-*O*-*β*-D-glucoside, floramaroside E) in corolla were significantly higher than those in non-medicinal parts, only quercetin 3-*O*-*β*-D-xylopyranosyl- (1→2)-*O*-*β*-D-galactopyranoside in calyx of non-medicinal part was higher than that of medicinal part.

**Figure 5.** *Cont*.

**Figure 5.** The orthogonal partial least squares discriminant analysis score scatter plot, VIP plot, and S-Plot of calyx and corolla (**A**), stamen and corolla (**B**), pistil and corolla (**C**).

**Figure 6.** The relative contents of 20 differential constituents in medicinal and non-medicinal parts of FAM. (Different letters indicate significant differences, *p* < 0.05).
