Two New Cytotoxic Steroidal Alkaloids from Sarcococca Hookeriana

Two new steroidal alkaloids, named hookerianine A (1) and hookerianine B (2) were isolated from the stems and roots of Sarcococca hookeriana Baill., along with two known compounds, sarcorucinine G (3) and epipachysamine D (4). On the basis of spectroscopic methods and by comparison with literature data, their structures were determined. As well as X-ray crystallography was performed to confirm compound 4. To identify novel antitumor inhibitors, all compounds were performed a CCK-8 assay against five human cancer cell lines SW480, SMMC-7721, PC3, MCF-7 and K562 in vitro. Compound 2 exhibited moderate cytotoxic activities to all cell lines with IC50 values in the range of 5.97–19.44 μM. Compound 3 was the most effective one against SW480 and K562 cell lines with IC50 values of 5.77 and 6.29 μM, respectively.


Introduction
The Sarcococca genus (Buxaceae) consists of about 20 species, widely distributed in the southwestern region of China and other south Asian countries [1].The members of Sarcococca plants are used as TCM and traditional folk medicine for the treatment of stomach pain, rheumatism, swollen sore throat and traumatic injury [2][3][4].Previous investigations on several species of this genus indicated that steroidal alkaloids are the major chemical components with a broad spectrum of biological activities, such as cholinesterase inhibition [5][6][7], antitumor [8], antibacterial [9], antileishamanial [10], antidiabetic [11] and estrogen biosynthesis-promoting [12].
Sarcococca hookeriana, one of Sarcococca plants, is usually confusedly used by ethnic minorities in China.Although dozens of steroidal alkaloids have been discovered from S. hookeriana of Nepal [13][14][15][16][17], there were few phytochemical or biological studies on this species which grows in China.Enlightened by the diverse bioactivities of steroidal alkaloids and the use of Sarcococca plants as folk medicine, S. hookeriana was chosen for searching antitumor agent by our research group and several cytotoxic steroidal alkaloids have been reported [18].In continuation of our ongoing study on this plant, two new steroidal alkaloids, named hookerianine A (1) and hookerianine B (2), together with two known ones, sarcorucinine G (3) [19] and epipachysamine D (4) [20] (Figure 1), were characterized and their cytotoxicity were evaluated in vitro with a CCK-8 assay.Herein, we describe the isolation, structure elucidation and cytotoxicity of the isolates.
Molecules 2019, 24, 11 4 of 7 The structures of known compounds 3-4 were determined by comparing their spectral data with literature data.To further confirm the chemical structure of compound 4, a colorless crystal was obtained from CH 2 Cl 2 , and X-ray crystallography analysis with Mo Kα radiation was performed.Through structural refinement by direct method SHELX-2014 [21,22], the chemical structure of 4 was identified as shown in Figure 4.The structures of known compounds 3-4 were determined by comparing their spectral data with literature data.To further confirm the chemical structure of compound 4, a colorless crystal was obtained from CH2Cl2, and X-ray crystallography analysis with Mo Kα radiation was performed.Through structural refinement by direct method SHELX-2014 [21,22], the chemical structure of 4 was identified as shown in Figure 4.

Results of the Cytotoxicity Test
The IC50 values of four compounds against five human cancer cell lines: SW480, SMMC-7721, PC3, MCF-7 and K562 are summarized in Table 2 (DDP and 5-FU was used as the positive control).The compound 2, a new steroidal alkaloid, exhibited moderate cytotoxic activities to all cell lines with IC50 values in the range of 5.97-19.44μM.Compared to the positive control 5-FU with IC50 values of 7.65 and 4.78 μM against SW480 and K562 cell lines, the compound 3 was the most effective one against these cell lines with IC50 values of 5.77 and 6.29 μM, respectively.The structure-activity relationships of compound 1 and 4 showed that steroidal alkaloids possessed a novel phenylacetyl group instead of benzoyl group located at C-3 can increase the cytotoxicity to human cancer cell lines: SW480, SMMC-7721, PC3 and K562.Interestingly, the cytotoxicity of compound 3 is stronger than compound 4, which indicated that the presence of double bond between C-16 and C-17 can increase the cytotoxicity.Meanwhile, compared to compound 4, compound 2 possessed an epoxy group at C-16 and C-17 also showed better cytotoxicity.The results suggested that C-16 and C-17 of steroidal alkaloids play an important role in anticancer potential.a Values of IC50 expressed as mean ± SD, n = 3 for all groups.b DDP, the abbreviation of cisplatin, used as reference drug.c 5-FU, the abbreviation of 5-fluorouracil, used as reference drug.

General Experimental Procedures
Optical rotations were measured with a Rudolph Autopol І automatic polarimeter (Rudolph, Hackettstown, NJ, USA).UV spectra were obtained on a Shimadzu UV-2401PC spectrophotometer (Shimadzu, Kyoto, Japan).IR spectra were measured with a Bruker TENSOR-27 spectrophotometer (Bruker, Bremerhaven, Germany) using KBr pellets.The 1D and 2D NMR spectra were recorded on

Results of the Cytotoxicity Test
The IC 50 values of four compounds against five human cancer cell lines: SW480, SMMC-7721, PC3, MCF-7 and K562 are summarized in Table 2   a Values of IC 50 expressed as mean ± SD, n = 3 for all groups.b DDP, the abbreviation of cisplatin, used as reference drug.c 5-FU, the abbreviation of 5-fluorouracil, used as reference drug.

General Experimental Procedures
Optical rotations were measured with a Rudolph Autopol I automatic polarimeter (Rudolph, Hackettstown, NJ, USA).UV spectra were obtained on a Shimadzu UV-2401PC spectrophotometer (Shimadzu, Kyoto, Japan).IR spectra were measured with a Bruker TENSOR-27 spectrophotometer (Bruker, Bremerhaven, Germany) using KBr pellets.The 1D and 2D NMR spectra were recorded on JEOL ECX 500 MHz spectrometers (JEOL Ltd, Kyoto, Japan) with TMS as an internal standard.Chemical shifts (δ) were expressed in ppm with reference to solvent signals.High-Resolution Electrospray Ionization Mass Spectrometry (HR-ESI-MS) was recorded on a Bruker Daltonics micrOTOF-Q II spectrometer (Bruker, Bremerhaven, Germany).Column chromatography (CC) was performed on Silica gel (200-300 and 300-400 mesh, Qingdao Marine Chemical Ltd., Qingdao, China).Fractions were monitored by TLC (GF 254, Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), and spots were visualized by Dragendorff's reagent.Solvents were distilled prior to use for extraction and isolation.

Plant Material
The plants of S. hookeriana were collected from Hezhang Country, Guizhou Province of China, in July 2015 and identified by Prof. JunHua Zhao, Guiyang College of Traditional Chinese Medicine.A voucher specimen (No. 150708) was deposited at College of Pharmacy, Guiyang College of Traditional Chinese Medicine.
Hookerianine A (1) was obtained as white amorphous powder, positive to Dragendorff's reagent.The molecular formula of C 31 H 48 N 2 O was determined by HR-ESI-MS (m/z 465.3833, [M + H] + ).
(DDP and 5-FU was used as the positive control).The compound 2, a new steroidal alkaloid, exhibited moderate cytotoxic activities to all cell lines with IC 50 values in the range of 5.97-19.44µM.Compared to the positive control 5-FU with IC 50 values of 7.65 and 4.78 µM against SW480 and K562 cell lines, the compound 3 was the most effective one against these cell lines with IC 50 values of 5.77 and 6.29 µM, respectively.The structure-activity relationships of compound 1 and 4 showed that steroidal alkaloids possessed a novel phenylacetyl group instead of benzoyl group located at C-3 can increase the cytotoxicity to human cancer cell lines: SW480, SMMC-7721, PC3 and K562.Interestingly, the cytotoxicity of compound 3 is stronger than compound 4, which indicated that the presence of double bond between C-16 and C-17 can increase the cytotoxicity.Meanwhile, compared to compound 4, compound 2 possessed an epoxy group at C-16 and C-17 also showed better cytotoxicity.The results suggested that C-16 and C-17 of steroidal alkaloids play an important role in anticancer potential.