*3.2. Plant Material*

The plants of *Sarcococca hookeriana* Baill. were collected in Hezhang County, Guizhou Province, China, in April 2012 and identified by Prof. Qingwen Sun, Guiyang College of Traditional Chinese Medicine. A voucher specimen (No. 20120401401) was deposited at the Key Laboratory of Miao Medicine of Guizhou Province, Guiyang College of Traditional Chinese Medicine.

#### *3.3. Extraction and Isolation*

The air-dried and powdered roots of *S*. *hookeriana* Baill. (2.5 kg) were extracted with 95% (25 L) EtOH under reflux three times, with an extraction time of 2 h. The combined extracts (443 g) were concentrated and suspended in H2O (3 L). The suspension was extracted with CHCl3 to obtain the CHCl3 fraction (94 g). The CHCl3 fraction was subjected to silica gel column chromatography (Si CC) and eluted with petroleum ether-diethylamine (100:1, 95:5, 9:1, 8:2) to yield four fractions (Fractions A−D). Fraction B (1.6 g) was subjected to Si CC and eluted with petroleum ether-diethylamine (100:2, 9:1) to yield the fractions B1−B3. Fraction B2 (330 mg) was chromatographed using Si CC and was developed with petroleum ether-diethylamine (100:2) to yield compounds **3** (21 mg) and **4** (35 mg). Si CC was performed on Fraction C (12.3 g) with a gradient eluent of petroleum ether-diethylamine (100:5, 9:1, 8:2) to yield four fractions (fractions C1−C4). Fraction C2 (1.4 g) was first subjected to Si CC (petroleum ether-diethylamine, 100:5), before being purified on Sephadex LH-20. This yielded compounds **2** (26 mg), **5** (30 mg) and **7** (31 mg). Fraction C4 (985 mg) was purified on Sephadex LH-20, before Si CC was used (petroleum ether-diethylamine, 9:1) to separate compounds **1** (33 mg) and **6** (19 mg).

## 3.3.1. Sarchookloide A (**1**)

This was a white amorphous powder with a HR-ESI-MS *m*/*z* of 461.3731 [M + H]<sup>+</sup> (calculated for C28H49N2O3, 461.3738). The [α] 22 <sup>D</sup> was +52.99 (*c* 0.58, MeOH); UV (MeOH) had a λmax of 209.4 nm; and IR (KBr) had a νmax of 3424, 2931, 2867, 1662 and 1623 cm–1. The 1H and 13C NMR data are shown in Table 1.

#### 3.3.2. Sarchookloide B (**2**)

This was a white amorphous powder with a HR-ESI-MS *m*/*z* of 445.3775 [M +H]<sup>+</sup> (calculated for C28H49N2O2, 445.3789). The [α] 22 <sup>D</sup> was +30.18 (*c* 0.57, MeOH); UV (MeOH) had a λmax of 205.8 nm; and IR (KBr) had a νmax of 3442, 2930, 2966, 1664 and 1629 cm–1. The 1H and 13C NMR data are shown in Table 1.

#### 3.3.3. Sarchookloide C (**3**)

This was a white amorphous powder with a HR-ESI-MS *m*/*z* of 429.3829 [M + H]<sup>+</sup> (calculated for C28H49N2O, 429.3839). The [α] 22 <sup>D</sup> was +7.10 (*c* 0.62, MeOH); UV (MeOH) had a λmax of 207.2 nm; and IR (KBr) had a νmax of 3454, 2930, 2853, 1665 and 1625 cm–1. The 1H and 13C NMR data are shown in Table 1.

#### *3.4. Cytotoxicity Assay*

The cytotoxicity of compounds **1**–**7** was tested on the human cervical cancer cell line Hela, lung adenocarcinoma cell line A549, breast cancer cell line MCF-7, colon cancer cell line SW480 and leukemia CEM cells. All cells were cultured in a RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), which was supplemented with 10% fetal bovine serum (Hyclone) in 5% CO2 at 37 ◦C. The cytotoxicity assay was performed using the MTT method in 96-well microplates [22]. Briefly, the adherent cells (100 μL) were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before the addition of the drug. The suspended cells were seeded just before the addition of the drug at an initial density of 1 × <sup>10</sup><sup>5</sup> cells/mL. Each tumor cell line was exposed to the tested compound at different concentrations for 48 h. The experiments were performed in triplicate. Adriamycin (Sigma, St. Louis, MO, USA) was used as a positive control. After treatment, cell viability was measured and the cell growth curve was plotted. The IC50 values were calculated by the Reed and Muench method [23].

#### **4. Conclusions**

We obtained three new pregnane-type steroidal alkaloids, sarchookloides A–C (**1**–**3**), along with four known compounds, pachysamine G (**4**), pachysamine H (**5**), sarcovagine B (**6**), and pachyaximine A (**7**), from the roots of *Sarcococca hookeriana*. The new compounds, sarchookloides A–C (**1**–**3**), were shown to possess a 3*α* substituent, which has rarely been reported. By performing a cytotoxic assay on Hela, A549, MCF-7, SW480 and CEM cell lines in vitro, all three amide substituted compounds

exhibited significant cytotoxic activities on all cells, which suggests that the three amide group of these compounds was the necessary group for the cytotoxicity. The most active compound, pachysamine H (**5**), inhibited all cancer cells with IC50 values in the range of approximately 1.05–2.23 μM. The results suggested that these types of steroidal alkaloids merit further biological evaluation of their cytotoxic activities and might have the potential to be studied for anticancer activity.

**Supplementary Materials:** The following 1H NMR, 13C NMR, 2D NMR, HR-ESI-MS spectra and the RAW data of the new compounds are available as supporting data. Supplementary materials are available online.

**Author Contributions:** K.H. and J.D. designed the experiments and revised the paper; K.H. and J.W. performed the experiments, analyzed the data, and wrote the paper; J.W. and J.Z. contributed to bioassay reagents and materials and analyzed the data; J.W., S.H. and J.D. revised the paper. All authors read and approved the final manuscript.

**Acknowledgments:** This research work was financially supported by the Science and Technology Foundation of Guizhou Province [grant number J (2013) 2071], the Science and Technology Cooperation Program of Guizhou Province [grant number LH (2015) 7277], the Youth Science and Technology Talent Project of Guizhou Province [grant number (2017) 5618], and the Open Project of the Key Laboratory of Miao Medicine of Guizhou Province [grant number K (2017) 004].

**Conflicts of Interest:** We wish to confirm that there are no known conflicts of interest associated with this publication and that there has been no significant financial support for this work that could have influenced its outcome.
