*2.1. Structure Elucidation*

A previous study using a NMR-based metabolomics method to investigate the effects of processing on the Chinese herbal medicine *Flos Lonicerae* revealed that the NMR approach can provide not only a holistic view on the change of chemical composition during processing, but also the information on identity of individual components [5]. In the present study, with the aid of information obtained from the NMR-based metabolomic analyses of RAM before and after processing, we could narrow the scope of isolation targets to the hexane partition fraction and its sub-fractions based on new signals of interest observed in their NMR spectra.

Compound **1** was obtained as a white amorphous powder. Its molecular formula was established as C31H40O2 based on its high-resolution atmospheric pressure chemical ionization mass data (HR-APCI-MS, *m*/*z* = 445.3134 [M + H]+). From its 13C-NMR spectrum, only 16 resonance signals were observed, suggesting **1** might contain two symmetrical sub-structures with 15 carbons for each structure. Referring to the multiplicity-edited HSQC spectrum, the signals in the 13C spectrum were assigned to two methyls, six methylenes, one exocyclic methylidene, one aliphatic methine, one aliphatic quaternary, and five olefinic carbons (Table 1). The 1H-NMR spectrum showed signals of methyl groups at δ<sup>H</sup> 1.87 (s) and 0.76 (s), exocyclic methylidene at δ<sup>H</sup> 4.68 and 4.84, aliphatic methine at δ<sup>H</sup> 2.10 (m), five methylenes in the range of δ<sup>H</sup> 1.40–2.50, and one methylene at δ<sup>H</sup> 3.82 (s). The integral value of the peak of methylene at δ<sup>H</sup> 3.82 was found to be only 1/3 of the value for the singlet peak of methyl at δ<sup>H</sup> 0.76 or 1.87, indicating that this methylene is the group bridging the two symmetrical moieties together with each moiety containing 15 carbons (Figure 1). The HMBC spectrum of **1** reveals the cross-peaks of the methyl signal at δ<sup>H</sup> 0.76 (H-14,14- ) with the carbon signals at δ<sup>C</sup> 42.0 (C-1,1- ), δ<sup>C</sup> 45.7 (C-5,5- ), δ<sup>C</sup> 39.2 (C-9,9- ) and δ<sup>C</sup> 36.7 (C-10,10- ). Additionally, the cross-peaks of the methyl signal at δ<sup>H</sup> 1.87 (H-13,13- ) with the carbon signals at δ<sup>C</sup> 116.6 (C-7,7- ), δ<sup>C</sup> 114.3 (C-11,11- ), and δ<sup>C</sup> 145.0 (C-12,12- ), and the cross-peaks of the exocyclic methylidene at δ<sup>H</sup> 4.68 and 4.84 (H-15,15- ) with the carbon signals at δ<sup>C</sup> 37.3 (C-3,3- ), δ<sup>C</sup> 150.0 (C-4,4- ), and δ<sup>C</sup> 45.7 (C-5,5- ) were observed in the HMBC spectrum. These observations suggested that the 15-carbon moiety had a furanoeudesmane carbon skeleton in the structure as in atractylon, which is a known furanosesquiterpene isolated from RAM in significant quantity [15]. The analysis of the 1H-1H COSY spectrum further confirmed the assignment. The nuclear Overhauser effect (NOE) interactions between H-14 (14- ) and H-2β (2- β)/H-6β (6- β), but no NOE interaction between H-14 (14- ) and H-5 (5- ), were observed in the NOESY spectrum, indicating that the *trans*-fused A/B ring junction and α,β-orientation of H-5 (5- ) and Me-14 (14- ) in the furanoeudesmane skeleton was the same as atractylon. Observations of HMBC correlations of the methylene signal at δ<sup>H</sup> 3.82 (H-16) to C-11 (11- ) at δ<sup>C</sup> 114.3 and C-12 (12- ) at δ<sup>C</sup> 145.0 in the HMBC spectrum indicated that the two symmetrical moieties were linked together through the methylene group attaching to C-11 and C-11- . Accordingly, **1** was identified to be bis(3,8α-dimethyl-5-methylene-4,4α,5,6,7,8,8α,9-octahydronaphtho-[2,3-β]-furan-2-yl)methane, trivially named methylene-biatractylon.

Compound **2** was obtained as a white amorphous powder. As determined from the [M + H]<sup>+</sup> peak at *m*/*z* = 459.3267 in HR-ACPI-MS, **2** has a molecular formula of C32H42O2 with 12 degrees of unsaturation (DOU). Compound **2** has the same number of DOU as compound **1**, but has an additional CH2 in molecular formula. The 1H NMR and HSQC spectra of **2** exhibit similar signal patterns as those of **1**, respectively, i.e., the signals of methyl groups at δ<sup>H</sup> 1.82/1.76 (s) and 0.75/0.77 (s), exocyclic methylidene at δ<sup>H</sup> 4.68 and 4.84, aliphatic methine at δ<sup>H</sup> 2.10 (m), six methylenes in the range of δ<sup>H</sup> 1.40–2.50. A significant difference lies in that one methylene at δ<sup>H</sup> 3.82 (s) was observed for **1**, but one aliphatic methine at δ<sup>H</sup> 4.17 (q) and one methyl at δ<sup>H</sup> 1.57 (d) were observed for **2** (Table 1). The 13C-NMR spectrum of **2** also displays a similar signal pattern as that of **1**. A significant difference between **2** and **1** is the presence of one methine carbon at δ<sup>C</sup> 30.1 and one methyl carbon at δ<sup>C</sup> 18.1 in **2**, corresponding, respectively, to the proton signals at δ<sup>H</sup> 4.17 and δ<sup>H</sup> 1.57. The analyses of the COSY and HSQC spectra confirmed the presence of an ethylidene unit in **2**, instead of the presence of a methylene in **1**. Comparing **2** to **1**, another difference in their 13C spectra is that slight splitting of carbon signals for the pair groups of C-1/1- , C-5/5- , C-9/9- , C-11/11- , C-13/13- , and C-14/14 were observed for **2** but not for **1** (Table 1). Analyses of the HMBC and COSY spectra of **2** revealed that these pair of signals were attributed to the two atractylon moieties presented in **2**. Furthermore, the *trans*-fused A/B ring junction in the two atractylon moieties was confirmed by the NOE observations from the NOESY spectrum. The HMBC correlations of the methyl protons (Me-17) in the ethylidene unit at δ<sup>H</sup> 1.75 with the carbons C-16 at δ<sup>C</sup> 30.1, C-11 (11- ) at δ<sup>C</sup> 112.9/113.0, and C-12 (12- ) at δ<sup>C</sup> 149.1, and the methine proton (H-16) in the ethylidene unit at δ<sup>H</sup> 4.17 with the carbons C-17 at δ<sup>C</sup> 18.1, C-11 (11- ) at δ<sup>C</sup> 112.9/113.0, and C-12 (12- ) at δ<sup>C</sup> 149.1 indicated that the two atractylon moieties were linked together through the methine in the ethylidene unit connecting at C-11 and C-11- . On the basis of the aforementioned evidence, the structure of **2** was established to be 2,2- -(ethane-1,1-diyl)-bis(3,8α-dimethyl-5-methylene-4,4α,5,6,7,8,8α,9-octahydronaphtho-[2,3-β]-furan), trivially named ethylidene-biatractylon.


**Table 1.** 1H-(500 MHz) and

13C-(125 MHz) NMR data of compounds **1**–**5**

(recorded in CDCl3, ppm).

174

**Figure 1.** Structures of compounds **1**–**15**.

Compound **3** was isolated as a white amorphous powder. Its molecular formula was determined as C35H42O3 based on the HR-ACPI-MS data for the [M + H]<sup>+</sup> peak at *m*/*z* = 511.3221, accounting for 15 degrees of unsaturation. Both the 1H and 13C signals of **3** in the upfield NMR chemical shift range showed close similarities to those of **1** and **2**, however, differences were observed in the downfield range. Four additional carbon signals at δ<sup>C</sup> 153.1, 141.4, 110.2, and 107.2 in the 13C spectrum, and three additional proton signals at δ<sup>H</sup> 7.35 (d), 6.30 (dd), and 6.07 (d) in the 1H spectrum were observed for **3**, as compared to **1** or **2**. These signals were assigned to a furan ring (3 degrees of unsaturation) by the analyses of COSY and HMBC spectra. Apart from these differences, the other signals displayed almost the same features as in **1** or **2.** The analyses of the 2D NMR spectra confirmed that **3** possessed a similar biatractylon skeleton in its structure as was found in **1** and **2**. Clear HMBC correlations of the methine proton (CH-16, with proton and carbon signals at δ<sup>H</sup> 5.47 (s) and δ<sup>C</sup> 36.1, respectively) to C-17 at δ<sup>C</sup> 153.1, C-18 at δ<sup>C</sup> 107.2, C-12 at δ<sup>C</sup> 144.7, and C-11 δ<sup>C</sup> 115.2 were observed, indicating that the furan ring and the two atractylon moieties were linked together through this methine. Hence, the structure of **3** was determined to be 2,2- -(furan-2-yl-methylene)-bis(3,8α-dimethyl-5-methylene-4,4α,5,6,7,8,8α,9-octahydronaphtho-[2,3-β] -furan), trivially named furan-2-methanetriyl-biatractylon.

Compound **4** was obtained as a white amorphous powder. The [M + H]<sup>+</sup> ion at *m*/*z* = 541.3299 in the HR-ACPI-MS spectrum revealed its molecular formula as C36H44O4, with the same 15 degrees of unsaturation but with one more carbon, two more protons, and one more oxygen when compared to **3**. The 1H-NMR spectrum of **4** showed almost the same features as that of **3**, except for having one more singlet signal at δ<sup>H</sup> 4.55. The 13C-NMR spectrum of **4** also displayed close similarity to that of **3**, except for the addition of an oxygenated methylene signal at δ<sup>C</sup> 57.6 (corresponding to the above signal at δ<sup>H</sup> 4.55 in the HSQC spectrum) and the significant downfield shift of the olefinic carbon signal from δ<sup>C</sup> 114.4 to 153.0. In the HMBC spectrum of **4**, the protons of this oxygenated methylene demonstrated clear correlations to the downfield-shifted carbons C-20 at δ<sup>c</sup> 153.0 and C-19 at δ<sup>c</sup> 108.7, indicating its connection at C-20. No other significant differences were observed when comparing the NOESY spectra of **4** and **3**. Accordingly, the structure of **4** was established to be (5-(bis(3,8α-dimethyl-5-methylene-4,4α,5,6,7,8,8α,9-octahydronaphtho-[2,3-β]-furan-2-yl)methyl) furan-2-yl)methanol, trivially named 5-furanmethanol-2-methanetriyl-biatractylon.

Compound **5** gave the [M + H]<sup>+</sup> ion at *m*/*z* = 739.4719 in its HR-APCI-MS spectrum, which was consistent with a molecular formula C51H62O4 possessing 21 degrees of unsaturation. As compared with **4**, compound **5** has six more degrees of unsaturation, 15 more carbons, and 18 more hydrogens in the molecular formula. Both its 1H and 13C-NMR spectra exhibit similar signal patterns to those of **4**, respectively, except for the occurrence of additional peaks which were attributed to atractylon moieties. Given that an atractylon moiety possesses 6 degrees of unsaturation, 15 carbons, and 19 hydrogens; it is implied that **5** should contain three atractylon moieties in its structure. Further analyses of the 1D and 2D NMR spectra confirmed the assignment, with the supporting data listed in Table 1. The methine proton (H-16) at δ<sup>H</sup> 5.42 displayed clear HMBC correlations to the carbon pairs C-11/11- at δ<sup>C</sup> 115.1/115.1, C-12/12 at δ<sup>C</sup> 144.8/144.8, as well as C-17 at δ<sup>C</sup> 151.5 and C-18 at δ<sup>C</sup> 107.9, indicating two atractylon moieties were linked through this methine to the furan unit. The methylene protons (H-21) at δ<sup>H</sup> 3.86 showed clear HMBC correlations to the carbon C-11" at δ<sup>C</sup> 114.9, C-12" at δ<sup>C</sup> 144.3, C-19 at δ<sup>C</sup> 106.5, and C-20 at δ<sup>C</sup> 151.4, revealing that the third atractylon moiety is linked through this methylene to the furan unit. On the basis of the above information, the structure of **5** was assigned as 2,2- -((5-((3,8α-dimethyl-5-methylene-4,4α,5,6,7,8,8α,9-octahydronaphtho-[2,3-β]-furan-2-yl)methyl) furan-2-yl)methylene)-bis(3,8α-dimethyl-5-methylene-4,4α,5,6,7,8,8α,9-octahydronaphtho-[2,3-β] furan), trivially named furan-5-methanediyl-2-methanetriyl-triatractylon.

In addition to the aforementioned five new compounds, ten known compounds (**6**–**15**) were also isolated from the processed RAM in the present study (Figure 1). They were identified as 5-(hydroxymethyl)furfural (**6**) [16], 5-(Hydroxymethyl)-2-(dimethoxymethyl)furan (**7**) [17], 2,3-dihydro-3,5-dihydroxy-6-methyl-4-pyranone (**8**) [18], atractylon (**9**) [19], atractylenolide I (**10**) [20], atractylenolide II (**11**) [20], atractylenolide III (**12**) [20], atractylenolide VII (**13**) [21], γ-selinene (**14**) [22], and selina-4(14),7(11)-dien-8-one (**15**) [23] with comparing their spectroscopic data with those in the literature. Compound **7** is likely an artifact from **6** since methanol was used in the isolation process. Compound **8** was isolated from wheat [18], indicating that it might come from the wheat which was used during the process.
