Structure and Cytotoxicity of Novel Lignans and Lignan Glycosides from the Aerial Parts of Larrea tridentata

Previously, the authors conducted phytochemical investigations of the aerial parts of Larrea tridentata and reported triterpene glycosides and lignan derivatives. In continuation of the preceding studies, 17 lignans and lignan glycosides (1–17) were isolated, including seven new compounds (1–7). Herein, the structure of the new compounds was determined based on spectroscopic analysis and enzymatic hydrolysis. The cytotoxicity of 1–17 against HL-60 human promyelocytic leukemia cells was examined. Compounds 4–11 and 14–16 were cytotoxic to HL-60 cells, with IC50 values in the range of 2.7–17 μM. Compound 6, which was the most cytotoxic among the unprecedented compounds, was shown to induce apoptotic cell death in HL-60 cells.


Introduction
Larrea tridentata is an evergreen small shrub belonging to the Zygophyllaceae family. It grows in the desert areas of the southwestern United States and Mexico, and is commonly called 'Creosote bush'. It has been used as a medicinal plant for the treatment of a variety of illnesses-including infertility, rheumatism, arthritis, diabetes, gallbladder and kidney stones, pain, and inflammation [1]. The phytochemical constituents of L. tridentata have been extensively investigated with the aim of discovering new biologically active secondary metabolites. In this quest, bioactive compounds with antibacterial [2,3], antimicrobial [3], cytotoxic [2,4], anti-inflammatory [5], anti-tuberculosis [6], anti-fungal [7], and anti-protozoal [8] potential have been isolated and identified. Previously, we reported 25 triterpene glycosides and three lignan derivatives, including larrealignans A and B, from the aerial parts of L. tridentata, and their cytotoxicity against HL-60 human promyelocytic leukemia cells [9,10]. Some plant lignans are promising seed compounds for new anticancer agents, and etoposide, a clinically applied anticancer medicine, is a chemically modified plant lignan. Acute promyelocytic leukemia is a cancer of the white blood cells, and there will be approximately 14,287 new cases and 8809 deaths in 2018 in Japan [11,12]. It is relatively sensitive to chemotherapy agents, often successfully leading to remission. However, when leukemia recurs, the present anticancer agents are not satisfactorily effective against leukemia. Thus, the development of new antileukemia agents is still expected to overcome leukemia. Our phytochemical investigation of this plant with the focus on lignan constituents resulted in the isolation of 17 lignan derivatives (1-17), seven (1-7) of which were previously undescribed. This study deals with the structural characterization of novel lignan derivatives based on spectroscopic analysis and hydrolysis. The cytotoxicity of the isolated compounds against HL-60 cells is evaluated.

Apoptosis Inducing Activity of 6
The apoptosis-inducing activity of 6 was evaluated. Among the unprecedented compounds, 6 exerted the highest cytotoxicity in HL-60 cells. HL-60 cells were treated with 40 µM of 6 for 24 h, stained with Annexin V and propidium iodide (PI), and analyzed by flow cytometry. The cell populations of early (Q4 area) and late (Q2 area) apoptotic cells, for which the vehicle control was 1.9 ± 0.033% and 4.5 ± 0.38%, increased to 6.5 ± 0.033% and 24 ± 0.87%, respectively ( Figure 5). HL-60 cells treated with 40 µM of 6 were stained with 4 ,6-diamidino-2-phenylindole (DAPI), and their morphology was observed under a fluorescence microscope. As shown in Figure 6, the HL-60 cells exhibited chromatin condensation and nuclear disassembly, which are representative phenomena in apoptosis. In addition, the cell cycle distribution of HL-60 cells was analyzed using a flow cytometer. When the cells were treated with 40 µM of 6 for 24 h, the sub-G1 population increased to 25 ± 0.58%, compared to 5.5 ± 0.088% in the vehicle control populations (Figure 7). Notably, the results showed that, for HL-60 cells treated with 6, the population of cells in the G 0 /G 1 phase (P3 area) increased (control: 48 ± 0.35%; 6: 51 ± 0.71%), whereas those in the S phase declined (P4 area) (control: 21 ± 0.38%; 6: 13 ± 0.29%) compared with the vehicle control.

Plant Material
The aerial parts of L. tridentata were purchased from Richters Herbs (Ontario, Canada) in 2007. A voucher specimen was kept at the herbarium of the Tokyo University of Pharmacy and Life Sciences.

Extraction and Isolation Procedures
The aerial parts of L. tridentata (dry weight, 3.0 kg) were extracted with hot MeOH  For 1 H-NMR spectral data of the sugar moiety, see Table 2. For 13 C-NMR spectral data, see Table 3. For NMR data, see Supplementary Materials. The 1 H-NMR spectra of 1 and 2 were recorded at 500 MHz in CD 3 OD and 3 was recorded at 600 MHz in C 5 D 5 N.  Table 2. For 13 C-NMR spectral data, see Table 3 1H, m, H-2), 0.79 (3H, d, J = 6.7 Hz, Me-11), 0.69 (3H, d, J = 6.7 Hz, Me-12). For 1 H-NMR spectral data of the sugar moieties, see Table 2. For 13 C-NMR spectral data, see Table 3. For NMR data, see Supplementary Materials.  of which resulted in less than 50% cell growth at a concentration of 20 µM, and the exact concentration at which 50% inhibition (IC 50 ) of cell growth occurred was calculated.

Detection of Apoptosis
HL-60 cells (1 × 10 6 cells/well) were pre-incubated for 4 h in a 6 well plate, and then treated with either EtOH/H 2 O (1:1) (control), 20 µM of cisplatin, or 40 µM of 6. After 24 h treatment, HL-60 cells were collected and washed with PBS, and incubated for 15 min at 28 • C in 1 × Annexin V binding buffer containing Annexin V-FITC and propidium iodide (PI) as provided by the manufacturer (15342, Nacalai Tesque). Apoptotic cells were analyzed by a BD FACSCelesta TM flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Cell Cycle Distribution Analysis
HL-60 cells (1 × 10 6 cells/well) were seeded for 4 h in a 6 well plate, and then treated with either EtOH/H 2 O (1:1), 20 µM of cisplatin, or 40 µM of 6. HL-60 cells were collected using PBS and fixed EtOH/H 2 O (7:3) overnight at −20 • C. Following procedures were carried out same as previously described [23]. Analysis of cell cycle distribution was performed by a BD FACSCelesta TM flow cytometer (BD Biosicences).

Statistical Anlysis
Statistical analysis was carried out one-way analysis of variance (ANOVA) followed by Dunnett s test. A probability (p) value of less than 0.001 or 0.05 was considered to represent a statistically significant difference.

Conclusions
A phytochemical investigation of the aerial parts of L. tridentata was performed to obtain 17 lignans and lignan glycosides (1-17), including seven novel compounds (1-7). Compound 3 has a unique structure with the D-glucosyl moiety on three different aromatic hydroxy groups. Compounds 4-11 and 14-16 are cytotoxic to HL-60 cells, with IC 50 values in the range of 2.7-17 µM. Additionally, 6 induced apoptotic cell death in HL-60 cells. The apoptosis-inducing mechanism of 6 is currently under investigation.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Isolated compounds are not available from the authors.