*2.2. Structural Elucidations of Camphoratins K (1) and N (2) and Benzocamphorins G (3) and I (4)*

Camphoratin K (**1**) was isolated as a white powder, and its sodiated molecular formula, C33H54O4Na, was established from a sodium adduct ion peak at *m*/*z* 537.3917 in high-resolution electrospray ionization mass spectrometric (HR-ESI–MS) analysis. The infrared (IR) absorption bands at 3427, 1714, 1643, 1455, and 891 cm−<sup>1</sup> were in agreement with the presence of a hydroxyl group, an ester, and a terminal double bond. In its 1H-NMR spectrum, there were proton signals for five methyl singlets at δ 0.80 (6H, s, CH3-18, 29), 0.97 (3H, s, CH3-19), 0.99 (3H, s, CH3-30), 1.01 (3H, s, CH3-31), 2.04 (3H, s, CH3-33), two methyl doublets at δ 1.01 (3H, d, *J* = 6.8 Hz, CH3-27) and 1.02 (3H, d, *J* = 6.8 Hz, CH3-26), and four protons at δ 2.22 (1H, sept, *J* = 6.8 Hz, H-25), 3.22 (1H, dd, *J* = 4.4 Hz, 11.6 Hz, H-3), 3.69 (2H, m, H-21), 5.05 (1H, dd, *J* = 5.8, 9.4 Hz, H-15). The 13C-, DEPT- and HMQC NMR spectra showed 33 carbon signals composed of 8 methyls at δ 15.4, 16.4, 18.3, 19.1, 21.4, 21.8, 21.9, 28.0; 9 methylenes at δ 18.2, 20.8, 26.4, 27.7, 28.2, 30.6, 31.4, 35.5, 36.0; 1 oxygenated methylene at δ 62.0; 2 oxygenated methines at δ 76.0, 78.9, and 1 terminal olefinic carbon at δ 106.4, which indicated a triterpene skeleton. An acetyl group was assigned to link to C-15 from HMBC spectral correlations of H-15 (δ 5.05) to C-16 (δ 36.0) and C-32 (δ 171.1). A terminal olefinic group and an isopropyl group were built up via <sup>2</sup>*J*, <sup>3</sup>*J*-HMBC correlations of CH3-26 (δ 1.02) to C-24 (δ 156.1), C-25 (δ 33.7), and C-27 (δ 21.9) and H-28 (δ 4.73 and 4.67) to C-23 (δ 31.4), C-24 (δ 156.1), and C-25 (δ 33.7). Other HMBC correlations indicated a sulphurenic acid skeleton with a hydroxyl methylene [H-21 (δ 3.69) to C-17 (δ28.0)/C-20 (δ 43.2)] instead of an acid connected to C-20. This C-21 hydroxyl substitution is novel and rare among all isolates from *T. camphoratus* (Figure 1).

**Figure 1.** Structure of camphoratin K (**1**) (**a**) and its key COSY (**b**), HMBC (**c**), and NOESY (**d**) correlations.

Camphoratin N (**2**) appeared as a pale yellow solid with sodiated molecular formula C30H42O6Na (*m*/*z* 521.2876). The presence of an 8(9)-ene-7,11-dione moiety was proposed along with those of a carboxyl group and a hydroxyl group, according to a UV maximum at 267 nm and IR absorptions at 3495, 1736, 1713, 1674, 1458, and 901 cm<sup>−</sup>1, respectively. Comparison of its 13C-NMR data with those of antcamphin I [18] indicated the presence of an additional oxygenated methyl group. Assignment of the oxygenated methyl group attached to a terminal carboxylate was based on the HMBC correlation of CH3-26 (δ 3.67) to C-26 (δ 175.0). Also, 12α-OH and 29α-CH3 were suggested via the NOE enhancements of H-12 (δ 4.11)/CH3-18 (δ 0.67) and CH3-19 (δ 1.54)/H-4 (δ 2.43) (Figure 2). According to a previous study, camphoratin N could include a pair of epimers (25 *S*/*R*) that have identical NMR data [18].

**Figure 2.** Structure of camphoratin N (**2**) (**a**) and its key HMBC (**b**) and NOESY (**c**) correlations.

Benzocamphorin G (**3**), a colorless syrup, was isolated via thin-layer chromatography and has the pseudomolecular formula of C13H12O3Na, constructed from the sodiated peak at *m*/*z* 239.0686 in HR-ESI–MS analysis. Characteristic absorption bands in its IR spectrum revealed alkynes (2205 cm<sup>−</sup>1), conjugated carbonyls (1667 cm<sup>−</sup>1), and alkenes (1607 cm−1). UV absorption maxima were at 268 and 296 nm. A comparison of its NMR spectra data with those of antrocamphin A [18] indicated similar proton peaks at δ 5.44 (1H, s, terminal alkene), 5.55 (1H, s, terminal alkene), 3.82 (3H, s, OCH3), 2.22 (3H, s, CH3-3), and 2.01 (3H, s, CH3-3- ) as well as downfield shift of one aromatic proton (δ 5.99, 1H, s, H-6) and loss of two methoxy signals. 13C- and DEPT-135 NMR spectra revealed a similar pattern to that of antrocamphin A, except for a pair of *ortho*-carbonyls (δ 181.1 and 181.3), a downfield shift of C-6 (δ 107.0), and a loss of two methyl carbons. The positions of the *ortho*-carbonyls were assigned to be at C-1 and C-2 via HMBC correlations of H-6 (δ 5.90)/ with C-1 (δ 183.1), with C-2 (δ 181.3), with C-4 (δ 129.4), with C-5 (δ 158.8); and of CH3-3 (δ 2.22) with C-2 (δ 181.3), with C-3 (δ 144.6), with C-4 (δ 129.4), with C-1- (δ 81.7), respectively. Therefore, the structure of benzocamphorin G was established and is shown in Figure 3.

**Figure 3.** Structure of benzocamphorin G (**3**) (**a**) and its key HMBC (**b**) and NOESY (**c**) correlations.

Benzocamphorin I (**4**) was also isolated via thin-layer chromatography as colorless syrup. HR-ESI–MS analysis indicated its pseudomolecular formula as C18H18O8Na (*m*/*z* 385.0898). No alkynes and alkene characteristic peaks were detected in its IR spectrum. Three pairs of proton signals revealed two methyls (δ 1.99, 2.05), two methylenedioxy groups (δ 5.98, 6.00), and two methoxys (δ 3.89, 3.90). Further 13C-, DEPT, and HSQC spectra indicated a benzocamphorin D skeleton [19] with an oxygen linkage between two phenyl groups. However, one methoxy signal was lacking, and an oxygenated aromatic carbon was present compared to benzocamphorin D [19], showing the methoxy group was

replaced by a hydroxyl group. Thus, the structure of benzocamphorin I was determined and is shown in Figure 4.

**Figure 4.** Structure of benzocamphorin I (**4**) (**a**) and its key HMBC (**b**) and NOESY (**c**) correlations.

#### *2.3. P-gp Inhibitory E*ff*ects of the Extract of T. camphoratus*

A pilot study using methanol as a solvent was done to evaluate the P-gp inhibitory effects of the extract of *T. camphoratus* (Figure 5). The methanol extract was further partitioned using water and EtOAc. Therefore, the methanol extract (TAM), the EtOAc layer (TAE), and the water layer (TAW) were tested using human stably P-gp-expressing cells (ABCB1/Flp-InTM-293) in a calcein AM (acetoxymethyl) uptake assay [26]. The increased intracellular calcein fluorescence corresponded to the inhibition level of P-gp efflux function. The methanol extract as well as the EtOAc layer and the water layer exhibited P-gp inhibitory activities at concentrations of 10 and 20 μM. The methanol extract and the EtOAc layer exhibited inhibition in a dose-dependent manner. Moreover, the methanol extract at 20 μM (TAM 20) showed P-gp inhibition comparable to that of the first-generation P-gp inhibitor verapamil at a concentration of 2.5 μM.

**Figure 5.** The inhibitory effects of the methanol extract at 10 and 20 μM concentrations (TAM 10, TAM 20), the EtOAc layer (TAE 10, TAE 20), the water layer (TAW 10, TAW 20) and verapamil at 2.5 μM concentration (VER 2.5) on P-glycoprotein (P-gp) in *ABCB1*/Flp-InTM-293 cells. \* denotes *p* < 0.05 compared with the intracellular calcein fluorescence in the control group. The numbers, 2.5, 10, 20, indicate the μM concentrations.
