**The Extracts of** *Artemisia absinthium* **L. Suppress the Growth of Hepatocellular Carcinoma Cells through Induction of Apoptosis via Endoplasmic Reticulum Stress and Mitochondrial-Dependent Pathway**

### **Xianxian Wei †, Lijie Xia †, Dilinigeer Ziyayiding, Qiuyan Chen, Runqing Liu, Xiaoyu Xu and Jinyao Li \***

Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi, Xinjiang 830046, China; 15099141611@189.cn (X.W.); xialijie1219@163.com (L.X.); dilnigar9696@sina.com (D.Z.); m15276567620\_1@163.com (Q.C.); Lucyducy@163.com (R.L.); 15276654427@139.com (X.X.)

**\*** Correspondence: ljyxju@xju.edu.cn; Tel.: +86-991-8583259; Fax: +86-991-8583517

† These two authors contributed equally.

Academic Editor: Roberto Fabiani

Received: 27 January 2019; Accepted: 28 February 2019; Published: 5 March 2019

**Abstract:** *Artemisia absinthium* L. has pharmaceutical and medicinal effects such as antimicrobial, antiparasitic, hepatoprotective, and antioxidant activities. Here, we prepared *A. absinthium* ethanol extract (AAEE) and its subfractions including petroleum ether (AAEE-Pe) and ethyl acetate (AAEE-Ea) and investigated their antitumor effect on human hepatoma BEL-7404 cells and mouse hepatoma H22 cells. The cell viability of hepatoma cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. The apoptosis, cell cycle, mitochondrial membrane potential (Δψm), and reactive oxygen species (ROS) were analyzed by flow cytometry. The levels of proteins in the cell cycle and apoptotic pathways were detected by Western blot. AAEE, AAEE-Pe, and AAEE-Ea exhibited potent cytotoxicity for both BEL-7404 cells and H22 cells through the induction of cell apoptosis and cell cycle arrest. Moreover, AAEE, AAEE-Pe, and AAEE-Ea significantly reduced Δψm, increased the release of cytochrome c, and promoted the cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP) in BEL-7404 and H22 cells. AAEE, AAEE-Pe, and AAEE-Ea significantly upregulated the levels of ROS and C/EBP-homologous protein (CHOP). Further, AAEE, AAEE-Pe, and AAEE-Ea significantly inhibited tumor growth in the H22 tumor mouse model and improved the survival of tumor mice without side effects. These results suggest that AAEE, AAEE-Pe, and AAEE-Ea inhibited the growth of hepatoma cells through induction of apoptosis, which might be mediated by the endoplasmic reticulum stress and mitochondrial-dependent pathway.

**Keywords:** *Artemisia absinthium*; apoptosis; endoplasmic reticulum stress; mitochondrial-dependent pathway

### **1. Introduction**

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and was the sixth most commonly diagnosed cancer and the fourth leading cause of cancer death worldwide in 2018, with about 841,000 new cases and 782,000 deaths annually [1,2]. The main risk factors for HCC are chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), aflatoxin-contaminated foodstuffs, heavy alcohol intake, obesity, smoking, and type 2 diabetes [3]. Currently, therapeutic options for the treatment of HCC include liver resection, transplantation, palliative intra-arterial therapies, immunotherapy strategies, and so on [4,5]. However, the prognosis of most patients with

HCC is poor [6]. For HCC treatment, the main drugs, including oxaliplatin and sorafenib, remain unsatisfactory because of their side effects and multidrug resistance [7,8]. Therefore, it is urgent to develop novel therapeutic agents to treat HCC.

*Artemisia absinthium* L. belongs to the Asteraceae family and is commonly known as wormwood. The chemical components of *A. absinthium* include sesquiterpene lactone, sesquiterpene lactone-pinene, β-thujone, α-thujone, sabinyl acetate, and β-thujone [9]. *A. absinthium* has pharmaceutical and medicinal effects such as antimicrobial [9], insecticidal [10], antiparasitic [11], antitumor [12], antipyretic [13], hepatoprotective [14,15], and antioxidant activities [16,17]. In the present study, *A. absinthium* ethanol extract (AAEE) and its subfractions including petroleum ether (AAEE-Pe) and ethyl acetate (AAEE-Ea) were prepared, and their antitumor effects on HCC were investigated both in vitro and in vivo. AAEE, AAEE-Pe, and AAEE-Ea selectively inhibited the growth of hepatoma cells both in vitro and in vivo without cytotoxic effects on normal hepatic cells. Moreover, these extracts could arrest the cell cycle at the G2/M phase and induce apoptosis through endoplasmic reticulum (ER) stress and the mitochondrial-dependent pathway in human hepatoma BEL-7404 cells and mouse hepatoma H22 cells, and they might be used as safe and effective agents for the treatment of HCC.

### **2. Results**

### *2.1. AAEE, AAEE-Pe, and AAEE-Ea Suppress the Growth of BEL-7404 and H22 Cells In Vitro*

To investigate the anti-proliferative effects of AAEE, AAEE-Pe, and AAEE-Ea, BEL-7404 and H22 cells were treated with 25, 75, and 150 μg/mL of AAEE, AAEE-Pe, and AAEE-Ea. After 24 h, the morphology of BEL-7404 and H22 cells was observed with an inverted microscope. Compared to untreated cells, BEL-7404 and H22 cells treated with AAEE, AAEE-Pe, and AAEE-Ea became shrunk and round, and cell numbers were reduced in a dose-dependent manner (Figure 1A). Cell viability was detected by MTT assay after treatment for 24, 48, and 72 h. The viability of BEL-7404 and H22 cells was dose- and time-dependently decreased after treatment with AAEE, AAEE-Pe, or AAEE-Ea (Figure 1B). The IC50 (50% inhibitory concentration) values of AAEE, AAEE-Pe, and AAEE-Ea for BEL-7404 and H22 cells at 24, 48, and 72 h are shown in Table 1. The IC50 values of H22 cells followed the order AAEE-Pe ≤ AAEE < AAEE-Ea. The IC50 values of BEL-7404 cells followed the order AAEE ≤ AAEE-Pe < AAEE-Ea. The effect of AAEE, AAEE-Pe, and AAEE-Ea was also detected on normal liver cells NCTC1469. AAEE and AAEE-Pe showed some cytotoxicity on NCTC1469 cells, but it was much lower than that of BEL-7404 and H22 cells. AAEE-Ea has no cytotoxicity on NCTC1469 cells (Figure 1C). These results suggest that AAEE, AAEE-Pe, and AAEE-Ea selectively inhibited the growth of hepatoma cells in vitro.


**Table 1.** IC50 values of AAEE, AAEE-Pe, and AAEE-Ea for BEL-7404 and H22 cells.

**Figure 1.** The effect of *A. absinthium* ethanol extract (AAEE) and its petroleum ether (AAEE-Pe) and ethyl acetate (AAEE-Ea) subfractions on the growth of BEL-7404, H22, and NCTC1469 cells. Cells were treated with different concentrations of AAEE, AAEE-Pe, and AAEE-Ea. (**A**) After 24 h, the morphology of BEL-7404 and H22 cells was observed by inverted microscope. (**B**) After 24, 48, and 72 h, the viability of BEL-7404 and H22 cells was detected by MTT assay. (**C**) After 24 h, the viability of NCTC1469 cells was detected by MTT assay. \*\* *p* < 0.01, \*\*\* *p* < 0.001 compared to Untreated.

### *2.2. AAEE, AAEE-Pe, and AAEE-Ea Induce Apoptosis in BEL-7404 and H22 Cells*

To study whether AAEE, AAEE-Pe, and AAEE-Ea induce apoptosis, BEL-7404 and H22 cells were stained with Annexin V and propidium iodide (PI) after treatment and analyzed by flow cytometry. The frequencies of apoptotic BEL-7404 and B22 cells were significantly increased by each of AAEE, AAEE-Pe, and AAEE-Ea in a dose-dependent manner (Figure 2A,B). AAEE, AAEE-Pe, and AAEE-Ea did not induce the necrosis of BEL-7404 cells but significantly induced necrosis of H22 cells (Figure 2A,B). The pro- and anti-apoptotic members of the B-cell lymphoma-2 (BCL-2) protein family

serve important roles in the regulation of cell apoptosis. After treatment with AAEE, AAEE-Pe, and AAEE-Ea, the levels of pro-apoptotic Bax and anti-apoptotic Bcl-2 in BEL-7404 and H22 cells were upregulated and downregulated, respectively. The ratios of Bax/Bcl2 significantly increased upon AAEE, AAEE-Pe, and AAEE-Ea treatment (Figure 2C). The results indicate that AAEE, AAEE-Pe, and AAEE-Ea inhibited the growth of BEL-7404 and H22 cells through the induction of apoptosis.

**Figure 2.** AAEE, AAEE-Pe, and AAEE-Ea induced apoptosis in BEL-7404 and H22 cells. Cells were treated with different concentrations of AAEE, AAEE-Pe, and AAEE-Ea for 24 h. After staining with Annexin V and PI, BEL-7404 (**A**) and H22 (**B**) cells were analyzed by flow cytometry. (**C**) Proteins were isolated, and the levels of Bax and Bcl-2 were analyzed by Western blot. \* *p* < 0.05; \*\*\* *p* < 0.001 compared to Untreated.

### *2.3. AAEE, AAEE-Pe, and AAEE-Ea Induce Cell Cycle Arrest in BEL-7404 and H22 Cells*

The morphological characteristics of apoptosis include chromatin condensation and DNA fragmentation. After treatment with AAEE, AAEE-Pe, and AAEE-Ea, BEL-7404 and H22 cells were stained with Hoechst 33258 and observed by inverted fluorescence microscopy. The nuclei of untreated cells showed homogeneous staining, while the nuclei of cells treated with AAEE, AAEE-Pe, and AAEE-Ea showed condensed chromatin (Figure 3A), suggesting that AAEE, AAEE-Pe, and AAEE-Ea induce apoptosis in BEL-7404 and H22 cells.

**Figure 3.** AAEE, AAEE-Pe, and AAEE-Ea induced cell cycle arrest in BEL-7404 and H22 cells. BEL-7404 and H22 cells were treated with different concentrations of AAEE, AAEE-Pe, and AAEE-Ea for 24 h. (**A**) BEL-7404 and H22 cells were stained with Hoechst 33258 and observed by inverted fluorescence microscope. (**B**) DNA contents in BEL-7404 cells were analyzed by flow cytometry and are shown in the upper panels. The summaries of the cell cycle distributions in BEL-7404 and H22 cells are shown in the middle and bottom panels, respectively. (**C**) Expression of Cyclin B1 was analyzed by Western blot. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001 compared to Untreated.

Next, the distribution of the cell cycle in BEL-7404 and H22 cells was detected by PI staining after treatment with AAEE, AAEE-Pe, and AAEE-Ea for 24 h. We observed that cells in the G2/M phase were significantly increased and cells in the S phases were significantly decreased upon AAEE, AAEE-Pe, and AAEE-Ea treatment (Figure 3B), indicating that AAEE, AAEE-Pe, and AAEE-Ea arrested the cell cycle of BEL-7404 and H22 cells at the G2/M phase. Consistently, AAEE, AAEE-Pe, and AAEE-Ea reduced the expression of cyclin B1 in BEL-7404 and H22 cells (Figure 3C). The results suggest that AAEE, AAEE-Pe, and AAEE-Ea induced apoptosis and cell cycle arrest in hepatoma cells.

### *2.4. AAEE, AAEE-Pe, and AAEE-Ea Reduce Mitochondrial Membrane Potential (*Δ*ψm)*

Mitochondrial membrane integrity is strictly regulated by the pro- and anti-apoptotic members of the BCL-2 protein family. After treatment with AAEE, AAEE-Pe, and AAEE-Ea for 24 h, the Δψm was measured by flow cytometry using JC-1 as a fluorescent dye. As shown in Figure 4A, AAEE, AAEE-Pe, and AAEE-Ea significantly reduced the Δψm values of BEL-7404 and H22 cells in a dose-dependent manner. Consistently, AAEE, AAEE-Pe, and AAEE-Ea promoted the release of cytochrome c in the cytosol of BEL-7404 and H22 cells. The activation of caspases is generally considered to be a key hallmark of apoptosis. We also observed that the levels of cleaved caspase-3 and caspase-9 were increased upon AAEE, AAEE-Pe, and AAEE-Ea treatment, which promoted the cleavage of poly(ADP-ribose) polymerase (PARP) in both BEL-7404 and H22 cells (Figure 4B). The results indicate that AAEE, AAEE-Pe, and AAEE-Ea induced apoptosis in hepatoma cells through the mitochondria-dependent pathway.

**Figure 4.** The mitochondria-dependent apoptosis induced by AAEE, AAEE-Pe, and AAEE-Ea. BEL-7404 and H22 cells were treated with different concentrations of AAEE, AAEE-Pe and AAEE-Ea for 24 h. (**A**) Cells were stained with JC-1 dye and analyzed by flow cytometry. (**B**) Total protein was isolated to detect the release of cytochrome c and the levels of poly(ADP-ribose) polymerase (PARP), cleaved-PARP (C-PARP), cleaved-caspase-3, caspase-3, cleaved-caspase-9, and caspase-9 by Western blot. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001 compared to Untreated.

### *2.5. AAEE, AAEE-Pe, and AAEE-Ea Promote Reactive Oxygen Species (ROS) Generation and ER Stress*

To examine the effects of AAEE, AAEE-Pe, and AAEE-Ea on oxidative stress, ROS generation was detected by flow cytometry at the indicated time points. After treatment with AAEE, AAEE-Pe, and AAEE-Ea, ROS levels were significantly increased at 6 h, reached a peak at 12 h, and then were maintained to 24 h in BEL-7404 cells (Figure 5A). Similarly, AAEE, AAEE-Pe, and AAEE-Ea dose-dependently increased ROS levels in H22 cells after treatment for 24 h (Figure 5B).

**Figure 5.** AAEE, AAEE-Pe, and AAEE-Ea induced ROS generation and ER stress. (**A**) BEL-7404 cells were treated with 150 μg/mL of AAEE, AAEE-Pe, and AAEE-Ea and the levels of ROS were analyzed by flow cytometry at indicated time points. (**B**) H22 cells were treated with different concentrations of AAEE, AAEE-Pe, and AAEE-Ea for 24 h and the levels of ROS were analyzed by flow cytometry. (**C**) BEL-7404 and H22 cells were treated with different concentrations of AAEE, AAEE-Pe, and AAEE-Ea for 24 h. Cell lysates were used to analyze the levels of CHOP by Western blot. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001 compared to Untreated.

ER stress exacerbates mitochondrial dysfunction by activating caspase-9 and increasing the release of cytochrome c [18]. CHOP, DNA damage inducible gene 153 (GADD153), is the main apoptotic factor activated by ER stress, and its overexpression promotes apoptosis in cancer [19]. After treatment with AAEE, AAEE-Pe, and AAEE-Ea for 24 h, the levels of CHOP were detected by Western blot. The results showed that AAEE, AAEE-Pe, and AAEE-Ea significantly increased the levels of CHOP in BEL-7404 and H22 cells (Figure 5C), suggesting that ER stress might be involved in the induction of apoptosis by AAEE, AAEE-Pe, and AAEE-Ea.

**Figure 6.** AAEE, AAEE-Pe, and AAEE-Ea suppressed tumor growth in vivo. A tumor mouse model was established by injection of H22 cells. After 3 days, tumor mice were treated with DMSO, cisplatin, AAEE, AAEE-Pe, and AAEE-Ea. Body weight of mice, tumor sizes (**A**), and survival rates (**B**) were monitored at the indicated time points. \* *p* < 0.05; \*\*\* *p* < 0.001 compared to control.

### *2.6. AAEE, AAEE-Pe, and AAEE-Ea Inhibit the Growth of H22 Cells In Vivo*

To further confirm the inhibitory effect of AAEE, AAEE-Pe, and AAEE-Ea on tumor growth in vivo, an H22 tumor mouse model was established in Kunming mice. Tumor mice were treated with different doses of AAEE, AAEE-Pe, and AAEE-Ea after 3 days of H22 cell injection. Cisplatin was used as a positive control and DMSO was used as a solvent control. We observed that cisplatin significantly reduced the weight of mice, but AAEE, AAEE-Pe, and AAEE-Ea did not affect the weight of mice. The tumor growth was significantly inhibited in the cisplatin, 100 mg/kg AAEE-Pe, and 100 and 200 mg/kg AAEE-Ea groups. AAEE at a 200 mg/kg dose also suppressed tumor growth to a certain degree (Figure 6A). At the end of the tumor study, the survival rates of tumor mice in each group were calculated. All mice were dead in the control (7 out of 7) and DMSO (7 out of 7) groups. The survival rates were 37.5%, 25%, 37.5%, 50%, and 87.5% in the cisplatin (3 out of 8), 200 mg/kg AAEE (2 out of 8), 100 mg/kg AAEE-Pe (3 out of 8), and 100 (4 out of 8) and 200 mg/kg (7 out of 8) AAEE-Ea groups, respectively (Figure 6B). These data suggest that AAEE, AAEE-Pe, and AAEE-Ea effectively inhibited the growth of H22 cells in vivo and improved the survival of tumor mice without obvious toxicity.

### **3. Discussion**

Chinese herbal medicine (CHM) has a long history of use in treating cancers and provides potential antitumor remedies. *A. absinthium*, a kind of CHM, has been used as an antipyretic, antiseptic, and anti-parasitic agent for the treatment of chronic fevers and inflammation of the liver [20]. In this study, we prepared AAEE, AAEE-Pe, and AAEE-Ea and investigated their antitumor effects on hepatoma cells. We found that AAEE, AAEE-Pe, and AAEE-Ea significantly suppressed the growth of BEL-7404 and H22 cells, induced apoptosis and cell cycle arrest, reduced Δψm, increased the release of cytochrome c, activated caspases, and promoted ROS production and ER stress.

It has been reported that a number of components of CHM can inhibit the growth of tumor cells both in vitro and in vivo, such as polysaccharides, flavones, terpenoids, and phenols [21–26]. The components of polysaccharides, flavones, and triterpenes in AAEE, AAEE-Pe, and AAEE-Ea were quantified. Although the three extracts contained different concentrations of polysaccharides, flavones, and triterpenes, they had similar antitumor effects. The results suggest that polysaccharides, flavones, and triterpenes might not be the major antitumor components in AAEE, AAEE-Pe, and AAEE-Ea. We will further identify the major antitumor components in the extracts of *A. absinthium* in future study.

The BCL-2 protein family strictly controls the apoptosis of cells [27,28]. An imbalance of proteins in the BCL-2 family triggers the intrinsic apoptosis pathway that increases mitochondrial permeability and the release of cytochrome c and activates caspase-9/caspase-3 [29]. We found that the levels of Bax/Bcl2 significantly increased in BEL-7404 and H22 cells after AAEE, AAEE-Pe, and AAEE-Ea treatment, which might cause the reduction of Δψm and the release of cytochrome c to activate caspases and apoptosis. Similarly, Shafi et al. [30] reported that the methanol extract of *A. absinthium* induced the apoptosis of human breast cancer cells through the modulation of BCL-2 family proteins.

Accumulating evidence points to the role of ER stress in the induction of apoptosis in various cancer cells [31–33]. ER stress exacerbates mitochondrial dysfunction by activating caspase-9 and increasing the release of cytochrome c [34,35]. Our results showed that AAEE, AAEE-Pe, and AAEE-Ea treatment significantly increased the expression of the ER stress-related protein CHOP, indicating that AAEE, AAEE-Pe, and AAEE-Ea might induce apoptosis of BEL-7404 and H22 cells through ER stress and the mitochondria-dependent pathway.

We observed that the ROS levels in hepatoma cells treated with AAEE and AAEE-Pe were lower than that in untreated cells at the beginning of 3 h, although they were significantly upregulated after 6 h, suggesting that the extracts might have antioxidant activities. This is similar to results found in other studies [16,17]. The increased ROS might be due to the reduction of Δψm induced by AAEE and AAEE-Pe treatment.

Finally, the antitumor and side effects of AAEE, AAEE-Pe, and AAEE-Ea were evaluated in an H22 tumor mouse model. All three extracts did not affect the weight of mouse but cisplatin significantly reduced the weight of mouse, suggesting that AAEE, AAEE-Pe, and AAEE-Ea might have no side effects in vivo. Moreover, AAEE-Pe and AAEE-Ea significantly inhibited tumor growth, which was similar with cisplatin. AAEE, AAEE-Pe, and AAEE-Ea further improved the survival of H22 tumor mice, and the high dose of AAEE-Ea showed the highest survival rate.

In conclusion, AAEE, AAEE-Pe, and AAEE-Ea inhibited the growth of hepatoma cells through the induction of apoptosis that might be mediated by ER stress and the mitochondria-dependent pathway. AAEE, AAEE-Pe, and AAEE-Ea suppressed H22 tumor growth and improved the survival of H22 tumor mice without obvious side effects, indicating that the three extracts might be used to develop safe and effective antitumor agents.

### **4. Materials and Methods**

### *4.1. Preparation of AAEE, AAEE-Pe, and AAEE-Ea*

The AAEE, AAEE-Pe, and AAEE-Ea were prepared according to the following procedure. Briefly, the powder was made using the aerial parts of *A. absinthium* including stems, leaves, flowers, and seeds (Alikang Uygur medicine technology co., Ltd., Urumqi, Xinjiang, China) and extracted overnight using 10 volumes of distilled water at 4 ◦C. After centrifugation at 8000 rpm for 20 min, the pellet was collected and extracted with 10 volumes of distilled water for 2 h at 60 ◦C. After centrifugation, the pellet was extracted using 10 volumes of 85% ethanol at 60 ◦C three times (2 h/time). The supernatant was collected after filtration and concentrated by a rotary evaporator to obtain the extractum. Some extractum was dried by a vacuum freeze-dryer to obtain AAEE. The remaining extractum was dissolved in distilled water and extracted by an equal volume of petroleum ether eight times, then the upper layer was collected and dried by a vacuum freeze–dryer to obtain AAEE-Pe. The bottom layer was extracted by an equal volume of ethyl acetate eight times, then the supernatant was collected and dried by a vacuum freeze–dryer to obtain AAEE-Ea. The AAEE, AAEE-Pe, and AAEE-Ea were dissolved in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA) and filtered with a 0.22 μm filter. The contents of flavonoids, terpenoids, and polysaccharides were determined by AlCl3–KAC, vanillin–glacial acetic acid, and anthrone–sulfuric acid colorimetry, respectively, which were shown in Table 2.

**Table 2.** The contents of polysaccharides, flavonoids, and triterpenoids in AAEE, AAEE-Pe, and AAEE-Ea.


### *4.2. Cell Culture*

The NCTC1469, H22, and BEL-7404 cells were obtained from the Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, Xinjiang University (Urumqi, Xinjiang, China) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (MRC, Changzhou, China) and 1% L-glutamine (100 mM), 100 U/mL penicillin, and 100 μg/mL streptomycin (MRC, Changzhou, China) at 37 ◦C in humidified air with 5% CO2.

### *4.3. Cell Viability Assay*

The proliferation of NCTC1469, H22, and BEL-7404 cells was analyzed by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) (Sigma, Louis, MO, USA) assay. Briefly, cells (5000 cells/well) were seeded in 96-well plates and treated with various doses of AAEE, AAEE-Pe, and AAEE-Ea for 24 h, 48 h, or 72 h. DMSO (0.3%) was used as a solvent control and cisplatin (35 μg/mL) was used as a positive control. The supernatant was discarded after centrifugation at 1200 rpm for 5 min and 100 μL of MTT solution (0.5 mg/mL in PBS) was added to each well and incubated at 37 ◦C for 3 h. The formed formazan crystals were dissolved in 200 μL DMSO. The OD490 values were measured by a 96-well microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). The relative cell viability was calculated according to the formula Cell viability (%) = (ODtreated/ODuntreated) × 100%. This experiment was conducted three times independently.

### *4.4. Observation of Cell Morphology*

H22 and BEL-7404 cells were seeded in 96-well plates and were treated with different concentrations of AAEE, AAEE-Pe, and AAEE-Ea for 24 h. After treatment, the morphology of H22 and BEL-7404 cells was observed by inverted fluorescence microscope (Nikon Eclipse Ti-E, Tokyo, Japan).

### *4.5. Analysis of Apoptosis*

H22 and BEL-7404 cells were treated with different concentrations of AAEE, AAEE-Pe, and AAEE-Ea for 24 h, and then stained with an Annexin V-FITC/propidium iodide (PI) Apoptosis Detection Kit (YEASEN, Shanghai, China) according to the manufacturer's instructions. Cisplatin and DMSO were used as positive and negative controls, respectively. Samples were analyzed by flow cytometry (BD FACSCalibur, San Jose, CA, USA). This experiment was conducted three times independently.

### *4.6. Hoechst 33258 Staining*

H22 and BEL-7404 cells were seeded in 6-well plates at the concentration of 1 × <sup>10</sup><sup>5</sup> cells/well in 2 mL medium. After 60%~70% confluence, the cells were treated with AAEE, AAEE-Pe, and AAEE-Ea for 24 h. The cells were collected and fixed with 4% ice-cold Paraformaldehyde at 4 ◦C for 10 min. After washing with PBS, cells were stained with Hoechst 33258 (Beyotime, Shanghai, China) at 4 ◦C for 10 min. Samples were observed using an inverted fluorescence microscope.

### *4.7. Analysis of the Cell Cycle*

H22 and BEL-7404 cells were inoculated in 60 mm culture dishes and treated with different concentrations of AAEE, AAEE-Pe, and AAEE-Ea for 24 h. All cells were collected and washed twice with PBS, then fixed in 70% ice-cold ethanol overnight at 4 ◦C. After washing twice with PBS, cells were re-suspended in 250 μL propidium iodide/RNase staining buffer (BD Biosciences, San Jose, CA, USA). After 10 min at room temperature, samples were collected by flow cytometry and the cell cycle distribution was analyzed using ModFit LT 3.0 software (BD FACS Calibur, San Jose, AC, USA). This experiment was conducted three times independently.

### *4.8. Analysis of* Δ*ψm*

H22 and BEL-7404 cells were treated with different concentrations of AAEE, AAEE-Pe, and AAEE-Ea for 24 h and then stained with the membrane-permeable JC-1 dye (Beyotime, Shanghai, China) for 30 min at 37 ◦C. Samples were analyzed by flow cytometry. This experiment was conducted three times independently.

### *4.9. Analysis of ROS*

BEL-7404 cells were treated with AAEE, AAEE-Pe, and AAEE-Ea for 2, 4, 6, 12, and 24 h. H22 cells were treated with AAEE, AAEE-Pe, and AAEE-Ea for 24 h. Cells were stained by 10 mM of fluorescent probe 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime, Shanghai, China) for 20 min at 37 ◦C. After washing three times with ice-cold PBS, samples were analyzed by flow cytometry. This experiment was conducted two times independently.

### *4.10. Western Blot*

The antibodies against caspase-9, Bax and Bcl-2, and anti-mouse IgG-HRP and anti-rabbit IgG-HRP were purchased from BBI Life Sciences (Shanghai, China). The antibodies against caspase-3, PARP, cytochrome c, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibodies against CHOP and CyclinB1 were bought from Beyotime (Shanghai, China).

H22 and BEL-7404 cells were treated with AAEE, AAEE-Pe, and AAEE-Ea for 24 h. After washing twice with PBS, cell lysates were prepared with RIPA Lysis Buffer (Beijing ComWin Biotech Co., Ltd., Beijing, China) and protein concentrations were detected using a BCA Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Proteins were separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. After incubation with primary and secondary antibodies, target proteins were detected by chemiluminescence (Beyotime, Shaghai, China). Signals were quantified using ImageJ digitizing software (ImageJ 1.50, National Institutes of Health, Bethesda, MD, USA). This experiment was conducted three times independently.

### *4.11. Animals and Ethics Statement*

Six- to eight-week-old male Kunming mice were purchased from Animal Laboratory Center, Xinjiang Medical University (Urumqi, Xinjiang, China). Mice were kept in a standard temperature-controlled, light-cycled animal facility at Xinjiang University. All animal experiments were approved by the Committee on the Ethics of Animal Experiments of Xinjiang Key Laboratory of Biological Resources and Genetic Engineering (BRGE-AE001) and performed under the guidelines of the Animal Care and Use Committee of College of Life Science and Technology, Xinjiang University.

### *4.12. Tumor Mouse Study*

For establishment of a tumor mouse model, male Kunming mice were injected with 1 × <sup>10</sup><sup>6</sup> H22 cells in 100 μL PBS subcutaneously. After 3 days, mice were randomly divided into seven groups (7 mice/group for Control and DMSO, 8 mice/group for the other five groups). The solvent control group intraperitoneally received 0.1 mL DMSO daily. The positive group was intraperitoneally injected with 5 mg/kg cisplatin at intervals of five days. The experimental groups were intraperitoneally injected with 200 mg/kg AAEE, 100 mg/kg AAEE-Pe, or 100 mg/kg or 200 mg/kg AAEE-Ea in 0.1 mL DMSO every two days. Tumor sizes were measured using calipers and tumor volume was calculated according to the formula tumor volume (mm3) = (length × width2)/2.

### *4.13. Statistical Analysis*

The data are expressed as mean ± standard error of the mean (SEM). Statistical significance was analyzed using one-way analysis of variance (ANOVA) by Tukey's Multiple Comparison Test. *p* < 0.05 was considered statistically significant.

**Author Contributions:** X.W., D.Z., Q.C., R.L. and X.X. performed the experiments; L.X. and J.L. designed the experiments, analyzed the data, and wrote the paper.

**Funding:** This work was supported by the Chinese National Natural Science Foundation Grant [U1803381 to Jinyao Li and 31860258 to Lijie Xia], the 1000 Young Talents Program of China to Jinyao Li and the "Tianshan Youth Project" Young Ph.D. Science and Technology talents Project [no. 2017Q077] to Lijie Xia.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


**Sample Availability:** Samples of the compounds are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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### **Johanna Willer 1, Karin Jöhrer 2, Richard Greil 2,3, Christian Zidorn <sup>1</sup> and Serhat Sezai Çiçek 1,\***


### Academic Editor: Roberto Fabiani

Received: 30 January 2019; Accepted: 25 February 2019; Published: 28 February 2019

**Abstract:** In our continuing search for new cytotoxic agents, we assayed extracts, fractions, and pure compounds from damiana (*Turnera diffusa*) against multiple myeloma (NCI-H929, U266, and MM1S) cell lines. After a first liquid-liquid solvent extraction, the ethyl acetate layer of an acetone (70%) crude extract was identified as the most active fraction. Further separation of the active fraction led to the isolation of naringenin (**1**), three apigenin coumaroyl glucosides **2**–**4**, and five flavone aglycones **5**–**9**. Naringenin (**1**) and apigenin 7-*O*-(4"-*O*-*p*-*E*-coumaroyl)-glucoside (**4**) showed significant cytotoxic effects against the tested myeloma cell lines. Additionally, we established a validated ultra-high performance liquid chromatography diode array detector (UHPLC-DAD) method for the quantification of the isolated components in the herb and in traditional preparations of *T. diffusa*.

**Keywords:** multiple myeloma; quality control; naringenin; flavonoids; traditional preparation

### **1. Introduction**

*Turnera diffusa* Willd., Passifloraceae, commonly referred to as damiana, is a shrub occurring in north-eastern Brazil, Mesoamerica, the Caribbean, Mexico, and Texas [1]. The traditional use of *T. diffusa* in Latin America encompasses usage as an aphrodisiac, a tonic, and for the treatment of diabetes [2,3]. Damiana extracts with tequila are allegedly used as love potions [3]. Due to the long history of *T. diffusa* as an aphrodisiac, both the stimulating effect as well as the underlying mechanisms are relatively well investigated. In animal testing, the aqueous extract of *T. diffusa* was found to increase the sexual activity of rats [4]. The flavonoids obtained by percolation with methanol, and here especially the flavanone pinocembrin, were identified as aromatase inhibitors resulting in increased testosterone levels and improved libido [5]. Regarding the antidiabetic activity, conflicting results were obtained. Whereas Alarcon-Aguilar et al. could not find any hypoglycemic effect of an ethanol-water extract [6], Parra-Naranjo et al. demonstrated hypoglycemic effects of a methanol extract and identified teuhetenone A, a nor-sesquiterpene, as the active principle [7].

Apart from traditional usage, an aqueous extract of *T. diffusa* was found to inhibit the monoamine oxidase A with IC50 values of 130 mg/mL as well as the acetyl- and butyrylcholinesterase with IC25 values of 0.352 and 0.370 mg/mL, respectively [8]. Additionally, cytotoxic effects of a methanolic extract against breast carcinoma cell line MDA-MD-231 were demonstrated [9]. Though the mechanism behind the observed cytotoxicity remains unsolved, the compounds responsible for cell death induced by the methanolic extract were identified as arbutin and apigenin.

The antioxidative effects of flavonoids are well documented and linked to certain structural features such as a dihydroxylated B-ring, a double bond located between C2 and C3, and a 4-oxo function at ring C [10]. Moreover, flavonoids are known to influence the metabolism, e.g., by inhibiting oxidases or activating antioxidative enzymes [11,12]. However, for flavonoids isolated from *T. diffusa* a variety of additional activities were reported. Velutin (**7**), a dimethoxylated hydroxyflavone known from the açaí berry (*Euterpe oleracea* Mart., Arecaceae), has been shown to possess strong anti-inflammatory effects by inhibiting NF-κB activation as well as p38 and JNK phosphorylation and hence by down-modulation of the expression of TNF-α and IL-6 [13]. At low doses, velutin (**7**) is more potent than established anti-inflammatory agents such as apigenin. Brito et al. found that the anti-inflammatory effect of velutin (**7**) against periodontitis is caused by an inhibition of HIF-1α expression [14]. Besides, the compound was reported to possess cytotoxic effects against human nasopharynx carcinoma (KB) cells with an IC50 value of 4.8 μM [15]. Acacetin 7-*O*-methyl ether (**9**), another methoxylated flavonoid, showed moderate cytotoxic effects on HeLa cells. Subsequent testing focused on the influence of aminoalkylation of acacetin 7-*O*-methyl ether (**9**) and the resulting antiproliferative activity against three human cancer cell lines (HeLa, HCC1954, SK-OV-3) [16]. The Mixe Indians (Oaxaca, Mexico) use an aqueous extract of *Calea zacatechichi* Schltdl. (Asteraceae) as a remedy for malaria. In subsequent experiments, the flavone genkwanin (**6**) was identified as active principle [17]. Boege et al. reported acacetin (**5**) to inhibit the topoisomerase I [18], though the compound lacks the structural feature (3-hydroxy group) supposed necessary for topoisomerase activity [19].

In the present study, the effect of extracts, fractions, and pure compounds from *T. diffusa* on multiple myeloma (MM) cell lines was investigated. Flavones, e.g., apigenin, chrysin, and luteolin, have been shown to block proteasome catalytic activities in tumor cells [20] and to induce cell death in myeloma cells [21]. Proteasome inhibitors are state-of-the-art in the therapy of multiple myeloma, but most patients develop resistance over time and new drugs are urgently needed. In addition, compounds of damiana are expected to induce reactive oxygen species [22], presumably adding to the expected cytotoxicity in myeloma cells. Our bioactivity-guided approach led to the isolation of seven flavonoids and a mixture of acacetin and genkwanin (Figure 1, **1**–**9**). In a second step, a validated ultra-high performance liquid chromatography (UHPLC) diode-array detector (DAD) method for the quantification of phenolic constituents in extracts and preparations of *T. diffusa* has been established.

**Figure 1.** Chemical structures of flavonoids isolated from the aerial parts of *T. diffusa*. Naringenin (**1**), apigenin 7-*O*-(6"-*O*-*p*-*E*-coumaroyl)-glucoside (**2**), apigenin 7-*O*-(6"-*O*-*p*-*Z*-coumaroyl)-glucoside (**3**), apigenin 7-*O*-(4"-*O*-*p*-*E*-coumaroyl)-glucoside (**4**), acacetin (**5**), genkwanin (**6**), velutin (**7**), gonzalitosin I (**8**), and acacetin 7-*O*-methyl ether (**9**).

### **2. Results**

### *2.1. Bioactivity of Tested Fractions*

After an acetone extract (70%) of *T. diffusa* showed cytotoxic potential in an initial screening against MM cell lines (Figure 2a,b (positive control)), the crude extract was fractionated with organic solvents of increasing polarity. Subsequently, the obtained fractions TD-1 (ethyl acetate), TD-2 (*n*-butanol), TD-3 (acidified *n*-butanol), and TD-4 (aqueous layer) were evaluated for their cytotoxicity. As shown in Figure 2c,d, the cytotoxic activity was mainly retained in the ethyl acetate fraction (TD-1), resulting in decreased viability in all tested concentrations after both 24 and 48 h of treatment, respectively. In contrast, treatment with TD-2 decreased the amount of viable cells only moderately after 48 h of treatment, whereas no effects were observed for fractions TD-3 and TD-4.

**Figure 2.** *Cont*.

**Figure 2.** Viability of multiple myeloma (MM) cell lines NCI-H929, U266, and MM1S (**a**) 48 h after treatment with an acetone 70% extract of *T. diffusa* and after treatment with bortezomib (**b**). Viability of NCI-H929 cells 24 and 48 h after treatment with ethyl acetate (TD-1), *n*-butanol (TD-2), acidified *n*-butanol (TD-3) fractions and the remaining water layer (TD-4) derived from an acetone (70%) extract of *T. diffusa* (**c**,**d**). Three concentration levels (25, 50, 100 μg/mL) are shown.

In a second approach, the crude acetone extract was fractionated in smaller polarity steps using *n*-hexane (TD-1a), diethyl ether (TD-1b), and ethyl acetate (TD-1c). The resulting fractions as well as TD-1 were then evaluated for their cytotoxic potential against MM cell lines MM1S, U266, and NCI-H929 after 24 h of incubation (Figure 3). As expected, TD-1 decreased viable cells significantly at a concentration of 100 μg/mL, but not at lower concentrations. Unlike before, partitioning in smaller polarity steps did not result in a single active fraction but revealed different activities for TD-1a to TD-1c. At a concentration of 100 μg/mL, TD-1a and TD-1b showed pronounced effects against MM1S cell lines, which were higher in TD-1b and lower in TD-1a, compared to TD-1. TD-1b moreover showed higher activity against NCI-H929 cell lines than TD-1 and slightly stronger effects against U266 cell lines. In contrast, TD-1a was less active against these two cell lines. TD-1c was only moderately active against all three tested cell lines.

**Figure 3.** Viability of MM cell lines after treatment with fractions TD-1, TD-1a, TD-1b, and TD-1c. Three concentration levels (25, 50, 100 μg/mL) and 24 h incubation times are shown.

### *2.2. Chromatographic Analyses of Tested Fractions*

The tested fractions were analyzed by UHPLC in order to eventually attribute the observed effects to specific peaks (Figure 4). Comparison of fractions TD-1 and TD-2, which were obtained in the first separation step, clearly shows that the active fraction TD-1 contains a number of peaks that are missing in fraction TD-2. These peaks, which are eluting after 25 min comprise, amongst other, the later isolated compounds **2**, **3** and **5**–**9**. UHPLC analysis of the three fractions of the second partitioning step (TD-1a to TD-1c) resulted in similar findings. Here, fraction TD-1c, which was significantly less active than TD-1a and TD-1b, was lacking compounds **5**–**7** and showed clearly lower amounts of compounds **8** and **9**. However, compound **4** was present in more or less the same concentration and compounds **2**, **3** and various polar constituents were present in higher amounts. The difference between fraction TD-1a and the slightly more active fraction TD-1b was less significant. Apart from the presence of a few peaks in the more polar region, TD-1b showed higher amounts of compounds **5**–**8** and lower concentrations of compounds **2** and **9**. Thus, the isolation of peaks eluting after 25 min was considered most promising for the identification of the cytotoxic principle(s) present in *T. diffusa*. As the respective compounds were quantitatively extracted in the first separation step, but not in the second one, fraction TD-1 was chosen for isolation of the desired substances.

**Figure 4.** Ultra-high performance liquid chromatography (UHPLC) chromatograms of TD-1, TD-2, and TD-1a to TD-1c.

### *2.3. Isolation of Natural Compounds*

Separation of fraction TD-1 was accomplished by silica gel flash chromatography, Sephadex LH-20 gel permeation chromatography, as well as preparative medium- and semi-preparative high-performance liquid chromatography and yielded seven flavonoids and a mixture of two compounds. Using analytical reference standards, mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy, the isolated compounds were identified as naringenin (**1**), apigenin 7-*O*-(6"-*O*-*p*-*E*-coumaroyl)-glucoside (**2**), apigenin 7-*O*-(6"-*O*-*p*-*Z*-coumaroyl)-glucoside (**3**), apigenin 7-*O*-(4"-*O*-*p*-*E*-coumaroyl)-glucoside (**4**), acacetin (**5**), genkwanin (**6**), velutin (**7**), gonzalitosin I (**8**), and acacetin 7-*O*-methyl ether (**9**).

### *2.4. Bioactivity of Isolated Compounds*

Cytotoxic assays of the pure compounds resulted in the identification of two compounds (**1** and **4**) with significant impact on cell viability (Figure 5). Of these two compounds, naringenin (**1**) was found cytotoxic even at low concentrations, showing a decreased viability of NCI-H929 and U266 cell lines of 25.5 ± 12.5 and 79.6 ± 15.2%, respectively, after 24 h incubation time. At the same incubation time and at a concentration of 50 μM, compound **4** decreased the viability of the same cell lines (NCI-H929 and U266) to 66.1 ± 17.4 and 84.4 ± 3.7%, respectively. Peripheral blood mononuclear cells (PBMC) from healthy donor (HD) were affected by the treatment with compounds **1** and **4**, if however, in different extent than the cancer cell lines.

**Figure 5.** Viability of MM cell lines (U266, NCI-H929) (**a**,**b**) and healthy donor (HD) cells (**c**,**d**) 24 h after treatment with compounds **1** and **4**, respectively.

### *2.5. Validation of UHPLC-DAD Assay*

Of the isolated flavonoids, compounds **4** to **8** were chosen as calibration standards, using compound **4** to co-quantify compounds **2** and **3** and compound **8** to co-quantify compound **9**. Linearity was achieved by five-point calibration with a coefficient of determination of >0.99 (Table 1). Repeatability and precision measurements were performed using a dimethyl sulfoxide (DMSO) extract (Figure 6). Repeatability experiments revealed relative standard deviations (RSD) below 2.3% (Table 2). Intra-day precision showed RSD values below 6.9% for all compounds on day 1. The relative standard deviation on day 2 showed similar results for most compounds except for compounds **5**–**7**, which had higher deviations. Also, inter-day precision was higher for these compounds, with values of 6.9 and 7.8%, whereas the other compounds showed RSD values below 6%. Spiking experiments over four calibration levels conducted for compounds **4** to **8** showed recovery rates ranging from 98.0 to 127% (Table 3).

**Table 1.** Regression curves, coefficients of determination, limit of detection (LOD), and limit of quantitation (LOQ) of the UHPLC method.


<sup>1</sup> Concentrations are given in μg/mL.


**Table 2.** Repeatability and precision of the UHPLC method.

<sup>1</sup> Concentrations are given in μg/mL, relative standard deviations are given in parentheses and are stated in percent.



<sup>1</sup> Concentrations are given in μg/mL. <sup>2</sup> Amounts are given in μg. <sup>3</sup> Recovery is stated in percent.

**Figure 6.** Chromatogram of a dimethyl sulfoxide (DMSO) extract prepared from *T. diffusa*. UHPLC was performed using a solvent mixture of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) with the following gradient: 15% B to 25% B in 15 min, to 29% B in 9 min, to 29% B in 11 min, to 36% B in 1 min, to 36% B in 19 min, to 95% B in 0.1 min, to 95% B in 9.9 min. Post-run was set to 10 min, temperature to 32 ◦C. The injection volume was 5 μL. The UV trace was recorded at 330 nm. The flow was 0.2 mL/min.

### *2.6. Chromatographic Analyses of a Traditional Preparation*

The established method was additionally used for the analysis of a traditional preparation of *T. diffusa* (Figure 7). The sample of a traditional preparation of *T. diffusa* revealed minor compounds in the medium to low polarity range and a high content of polar compounds. By comparison with MS data and retention times of isolated compounds from *T. diffusa* compounds **4** to **9** were identified and their yield quantified using the method described above. Ultraviolet (UV) spectroscopy experiments indicated the presence of phenolic compounds in the polar range. Apigenin (**e**) was identified using reference standards. Previous investigations of an aqueous extract of *T. diffusa* by Bernardo et al. revealed a variety of apigenin, luteolin, and quercetin glycosides [8]. By comparison with ultraviolet (UV) and MS data larycitrin-3-*O*-(6-glucosyl)glucoside (**a**), apigenin 8-*C*-(2-rhamnosyl)glucoside (**b**), (luteolin 8-*C*-(2-rhamnosyl)ketodeoxihexoside (**c**), apigenin 7-*O*-(2-rhamnosyl)ketodeoxihexoside (**d**) were detected.

**Figure 7.** Chromatogram of a liquor prepared from *T. diffusa*. UHPLC was performed using a solvent mixture of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) with the following gradient: 15% B to 25% B in 15 min, to 29% B in 9 min, to 29% B in 11 min, to 36% B in 1 min, to 36% B in 19 min, to 95% B in 0.1 min, to 95% B in 9.9 min. Post-run was set to 10 min, temperature to 32 ◦C. The injection volume was 5 μL. The ultraviolet (UV) trace was recorded at 330 nm. The flow was 0.2 mL/min. **4** to **9** refer to the isolated compounds (see Figure 1), compounds **a** to **d** were identified by UV spectroscopy and MS spectrometry experiments as larycitrin-3-*O*-(6-glucosyl)glucoside (**a**), apigenin 8-*C*-(2-rhamnosyl)glucoside (**b**), (luteolin 8-*C*-(2-rhamnosyl)ketodeoxihexoside (**c**), apigenin 7-*O*-(2-rhamnosyl)ketodeoxihexoside (**d**), and **e** was identified as apigenin by an authentic standard.

### **3. Discussion**

### *3.1. Bioactivity of Tested Fractions*

A crude acetone (70%) extract of *T. diffusa* displayed promising cytotoxicity against MM cell lines. Partitioning of the acetone extract located the cytotoxic compounds in the ethyl acetate (TD-1) fraction. In a second approach, using smaller polarity steps, the activity was found in the *n*-hexane (TD-1a) and the diethyl ether (TD-1b) fraction, while the remaining ethyl acetate fraction (TD-1c) was only slightly active. UHPLC experiments of the active fractions revealed similar peak patterns in the low polarity range. This peak pattern was absent in the inactive fraction TD-2, and different in TD-1c. Thus, further purification steps with fraction TD-1 focused on this polarity range and afforded the isolation of naringenin (**1**), apigenin 7-*O*-(6"-*O*-*p*-*E*-coumaroyl)-glucoside (**2**), apigenin 7-*O*-(6"-*O*-*p*-*Z*-coumaroyl)-glucoside (**3**), apigenin 7-*O*-(4"-*O*-*p*-*E*-coumaroyl)-glucoside (**4**), acacetin (**5**), genkwanin (**6**), velutin (**7**), gonzalitosin I (**8**), and acacetin 7-*O*-methyl ether (**9**).

Regarding the isolated compounds, compound **4** was abundant in all active fractions (TD-1, TD-1a to TD-1c) but was absent in TD-2. Treatment with compound **4** (50, 100 μM) led to decreased viability of NCI-H929 and in a lesser extent of U266 after incubation for 24 h. This indicates that compound **4** contributes to the cytotoxic effect observed for the tested fractions. The corresponding aglycone, the flavone apigenin, was reported repeatedly for its cytotoxic activity [9,20,21,23,24]. However, the moderately active fraction TD-1c contained less of lipophilic compounds **5** to **9**, suggesting that these compounds might at least contribute to the observed cytotoxic effects.

The most active compound of our study, the flavanone naringenin (**1**), is well investigated for its ability to induce apoptosis against HL-60 cells via the activation of caspase-3, a member of the caspase-cascade that plays an important role in apoptosis. Interestingly, the cytotoxic effect of naringenin (**1**) was found to be weakened if C7 is substituted with a sugar moiety (rutinoside) [25,26]. In the present work, naringenin (**1**) was found to possess the highest activity of all tested substances and showed a pronounced decrease of viability especially in NCI-H929 cells. Nevertheless, the active principle resulting in the lowered viability in this study requires further testing.

The experiments performed under the same conditions with HD cells indicate a negative influence on cells of the immune system after treatment with compound **1** or **4** (Figure 5). Interestingly, Chen et al. (2003) did not observe apoptosis in polymorphonuclear neutrophils (PMN) after their treatment with naringenin (**1**). However, testing conditions differed from the ones used in this study. Nevertheless, though flavonoids are generally assumed safe due to regular uptake with fruits and vegetables, they were found to evoke cytotoxic effects at higher doses [27,28].

### *3.2. Validation*

By testing different columns (Synergi 4μm Polar-RP 80 Å 150 × 2.00 mm, Luna Omega 1.6 μm C18 100 Å 100 × 2.1 mm, Kinetex 1.7 μm XB-C18 100 Å 100 × 2.1 mm) the best separation was achieved for the C18 Luna Omega column. Consequently, it was chosen as starting point for method development. Different compositions of pure water or formic acid in water as well as methanol or acetonitrile were tested for their influence on the separation of the compounds of the DMSO extract. Thereby, 0.1% formic acid and acetonitrile were identified as suitable eluents and a column temperature of 32 ◦C was found to give a good resolution of the peaks. Due to the complex mixture and a variety of similar compounds within the crude extract, isocratic steps were performed at different solvent concentrations as part of the gradient. Because of the absorption maxima around 330 nm of compounds **4** to **8**, this wavelength was chosen for detection. An injection volume of 5 μL was found to provide a good repeatability with an acceptable peak resolution.

The calibration curves for quantified constituents **4** to **8** were obtained on five levels each regarding the concentration of the corresponding analyte in the extract. The established calibration curves had determination coefficients of more than 0.99 and were thus accepted for quantification purposes. Spiking experiments showed acceptable recovery rates over a broad calibration range (Table 3).

Interday precision revealed good relative standard deviation (RSD) values for peaks **5** to **8**; compound **7** however varies in a bigger extent. These findings also account for the co-quantified peaks with acacetin 7-*O*-methylether (**9**) and the compounds **2** and **3** having similar deviations. Displaying structural isomers, the latter two compounds were co-quantified by compound **4**, while acacetin and genkwanin (**5** + **6**) were chosen to co-quantify acacetin 7-*O*-methylether (**9**) since the molecules only differ in the amount of one methyl group.

Limit of quantification (LOQ) for all quantified compounds **4**–**8** was set to the lower limit of the calibration curve. Those values provide acceptable standard deviation (data not shown). Commonly, limit of detection (LOD) is defined as one third of LOQ. Therefore, LOD was calculated from the LOQ (Table 2).

### *3.3. Traditional Preparation*

*T. diffusa* is used as remedy for various diseases in the traditional medicine of Latin America. Additionally, a liquor of *T. diffusa* is used to increase sexual activity [3]. In literature, tequila is described as extraction agent while in this study Kornbrand (Bauerndank/Edeka/Hamburg, Germany) was used. Both liquors contain 38% ethanol and thus were considered comparable. Due to the contained water, the liquor was expected to contain a high number of polar compounds. This assumption was verified by UHPLC analysis, which revealed most of the peaks eluting in the polar range. Compounds **4** to **9** were present, however, at low concentrations. In the present study, compound **4** showed moderate cytotoxicity in an in vitro assay against myeloma cell lines and healthy donor (HD) cells at higher concentrations of 50 and 100 μM. This corresponds with 28.9 mg/mL or 57.8 mg/mL, a concentration unlikely to be met in traditional preparations.

### **4. Materials and Methods**

### *4.1. General Experimental Procedures*

Solvents and reagents for isolation were of analytical quality. Solvents used for UHPLC were of LC-MS grade quality. Column chromatography was performed with silica gel (40–63 μm particle size) (Merck, Darmstadt, Germany) or with Sephadex LH-20 (GE Healthcare, Chicago, IL, USA). Thin layer chromatography (TLC) was performed on silica gel 60 F254 plates (Merck) using ethyl acetate-methanol-water (10:1:1) or *n*-hexane-ethyl acetate-methanol-formic acid (7:4:1.5:0.1) as mobile phase and vanillin-sulphuric acid for detection. Preparative medium-performance liquid chromatography (MPLC) was accomplished using a Büchi PrepChrom C-700 equipped with a Büchi PrepChrom MPLC column C18 (250 × 30 mm, 15 μm) (Büchi, Flawil, Switzerland). Semi-preparative high-performance liquid chromatography (HPLC) was carried out on Waters a Alliance e2695 Separations Module with Alliance 2998 photodiode array (PDA), 2410 RI, and WFC III fraction collector (Waters, Milford, MA, USA), and either an Aqua 5 μ C18 column (250 × 10 mm, 5 μm particle size, Phenomenex, Aschaffenburg, Germany) or a VP Nucleodur C18 (250 × 10 mm, 5 μm particle size, Macherey-Nagel, Düren, Germany). Extracts, fractions, and pure compounds were analyzed by a Hitachi ChromasterUltra RS System (VWR, Darmstadt, Germany) connected to an autosampler, column heater, PDA and a Sederé Sedex 100 evaporative light scattering detector (ELSD), using a Phenomenex Synergi Polar-RP column (150 × 2 mm, 4 μm particle size). Pure compounds were additionally analyzed by Nexera X2 system (Shimadzu, Kyoto, Japan) connected to an autosampler, column heater, PDA and a Shimadzu LCMS 8030 Triple Quadrupole Mass Spectrometer with electron spray ionization. Quantification was performed on the same instrument using a Phenomenex Luna Omega C18 column (100 × 2.1 mm, 1.6 μm particle size) and NMR spectra were recorded on an Avance III 300 NMR spectrometer (Bruker, Billerica, MA, USA) connected to a BACs-autosampler (Bruker). Centrifugation was performed on a Heraeus Megafuge 16 (Thermo Fisher Scientific Inc., Waltham, MA, USA). The authentic standard of Apigenin >98% HPLC was purchased from TransMIT (Gießen, Germany).

### *4.2. Plant Material*

Dried and cut aerial parts of *T. diffusa* (HAB-2014 quality) were obtained from Caesar & Loretz GmbH (Caelo), Hilden, Germany (art.-No.: 257a, lot number: 15294206).

### *4.3. Extraction and Fractionation*

Dried herb (1.00 kg) was ground and extracted five times with 2 liters of acetone 70% undergoing sonification. The solvent was evaporated under reduced pressure to afford 85 g of crude extract. For isolation the crude extract was repeatedly partitioned between ethyl acetate and water and the ethyl acetate layer was evaporated to dryness, yielding 32.6 g (TD-1). Subsequently, this procedure was repeated with butanol (14.6 g, TD-2). After acidification of the water layer with 2.5 mL formic acid, the solution was again extracted with butanol (5.40 g, TD-3) and the aqueous layer was then evaporated to dryness, yielding 32.4 g (TD-4).

In a second approach, 5.0 g of crude extract were suspended in water and extracted with *n*-hexane. The fraction was evaporated to dryness, yielding 0.53 g (TD-1a). The procedure was repeated with diethyl ether (0.21 g, TD-1b) and ethyl acetate (0.22 g, TD-1c).

For the validated UHPLC-DAD assay, sieved plant material (800 μm mesh width) was extracted threefold with DMSO, centrifuged, and the supernatants were collected and diluted in a 20 mL volumetric flask.

The traditional liquor from *T. diffusa* was prepared by maceration of 35 g of ground drug material with 0.7 liters of 38% alcohol (Bauerndank) for four weeks. Subsequently, the extract was filtered.

### *4.4. Isolation*

TD-1 was subjected to silica gel column chromatography (40 × 8 cm) and eluted in a gradient manner with petroleum ether-ethyl acetate-methanol-water (10:0:0:0 to 0:0:19:10) yielding 160 fractions. After characterization by TLC the obtained subfractions were combined to 20 fractions (TD-1\_1 to TD-1\_20).

From TD-1\_2 compound **1**, a two to one mixture of **5** and **6**, and compound **8** were obtained after column chromatography with Sephadex LH20 and acetone–dichloromethane (15:85) as eluting solvent. The thereby eluted subfraction TD-1\_2\_D was subjected to semi-preparative HPLC using 0.025% formic acid in water and methanol in a gradient matter, yielding 14.2 mg of velutin (**7**) and 27.8 mg of linoleic acid. 27 mg of acacetin 7-*O*-methylether (**9**) and another 58 mg of linoleic acid were obtained from TD-1\_4 by preparative column chromatography using 0.025% formic acid in water and methanol in a gradient matter. TD-1\_8 (196 mg) was submitted to further separation by Sephadex LH20 (using acetone as eluent) to give 11.7 mg of compound **4**. TD-1\_12 was subjected to semi-preparative HPLC using 0.025% formic acid in water and acetonitrile in an isocratic matter, yielding 6.3 mg of compound **3**. TD-1\_14 yielded 10.0 mg of sitosterol and TD-1\_ 20 9.0 mg of compound **2**. Structure elucidation of the isolated compounds was accomplished by comparison of MS- and NMR-spectra with literature data [29–36]. MS and NMR spectra (1H, HSQC, HMBC) are provided in the Supplementary Materials (**S1** to **S15**).

### *4.5. Cytotoxic Assays*

Cytotoxicity was assessed for TD-1 to TD-4, TD-1a to TD-1c as well as compounds **1** and **3** to **9**. Induction of apoptosis was measured in myeloma cell lines NCI-H929, MM1S, and U266 as well as in PBMCs of healthy donors by flow cytometry using established protocols [37] thereby staining the cells with Annexin-fluorescein isothiocyanate and propidium iodide. Bortezomib (Eubio, Vienna, Austria) was used as positive control. Cell lines were purchased from DSMZ (Braunschweig, Germany) and routinely fingerprinted and tested for mycoplasma negativity. All cells (cell lines and PBMC) were grown in RPMI-1640 medium (Life Technologies, Paisley, UK) supplemented with 10% fetal calf serum (FCS; PAA, Linz, Austria), L-glutamine 100 μg/ml, and penicillin-streptomycin 100 U/ml. PBMCs from healthy donors were utilized after obtaining written informed consent at the University Hospital Salzburg (ethics committee approval 415-E/1287/6-2011). Cells were subjected to Ficoll separation (Ficoll PaqueTM, VWR, Darmstadt, Germany), and incubated in RPMI-1640 Media with supplements as above. In brief, 0.5 × 106 myeloma cells/mL or similar numbers of PMBCs were incubated for 24 h and 48 h with or without the tested compounds dissolved in DMSO in different concentrations. At least three analysis in triplicates were performed for each cell line and a solvent control was included. The extent of non-apoptotic cells (AnnexinV/propidium iodide negative) was calculated as percentage of viable cells in respect to the untreated control. Data are shown as mean percentage of viable cells and standard deviation (error bars).

### *4.6. Chromatographic Analyses*

Solvents used for UHPLC analyses during isolation steps were 0.1% formic acid in water and methanol using a gradient from 40% of methanol to 95% in 80 min with a flow of 0.2 mL/min. Post-run was 10 min, injection volume 5 μL, temperature 30 ◦C. UV traces were detected at 210 nm, 254 nm, and 280 nm. Additionally, an evaporative light scattering signal was recorded.

For quantification a solvent mixture of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) was used with the following gradient: 15% B to 25% B in 15 min, to 29% B in 9 min, to 29% B in 11 min, to 36% B in 1 min, to 36% B in 19 min, to 95% B in 0.1 min, to 95% B in 9.9 min. Post-run was set to 10 min, temperature to 32 ◦C. The injection volume was 5 μL. The UV trace was recorded by at 330 nm. The flow was 0.2 mL/min.

### *4.7. Method Validation*

The UHPLC-DAD method was validated for linearity, LOD and LOQ, accuracy, precision, and repeatability. For the determination of linearity calibration curves were established by serial dilution of compounds **4** to **8**. Thus, calibration ranges of 5 μg/mL to 50 μg/mL (**4**), 0.75 μg/mL to 7.5 μg/mL (**7**), and 0.5 μg/mL to 5 μg/mL (**5** + **6** and **8**) were obtained. Corresponding regressions curves, coefficients of determination as well as LOD and LOQ are given in Table 1.

Repeatability was determined by measuring one sample six-fold while intra-day precision was studied by measuring six different samples once. Interday precision of the method was verified by assessing six samples on two different days. Consistency of compound concentrations was thereby investigated (Table 2).

Spiking experiments were performed on four levels for each quantified compound. For compound **5** + **6**, **7**, and **8** stock solutions of 0.04 mg/mL were prepared. From these, 0.75 mL, 0.5 mL or 0.25 mL were taken and mixed with 0.25 mL, 0.5 mL or 0.75 mL of the plant extract. Additionally, for all quantified constituents 0.25 mL of the lowest level of the calibration curve was added to 0.75 mL of the extract. Of these resulting solutions 5 μL were injected three-fold. Results are given in Table 3**.**

Acacetin 7-*O*-methylether (**9**) was co-quantified by acacetin and genkwanin (**5** + **6**), compounds **2** and **3** by compound **4**.

### **5. Conclusions**

Investigation of the cytotoxic properties of *T. diffusa* revealed significant effects for different apolar extracts against the myeloma cell lines MM1S, U266 and NCI-H929. Systematic evaluation of the active extracts by UHPLC led to the reduction of the complex metabolite to a range of possible candidates, which were subsequently isolated. Of these compounds, naringenin (**1**) and apigenin 7-*O*-(4"-*O*-*p*-*E*-coumaroyl)-glucoside (**4**) were identified as two components responsible for the observed activity. The cytotoxicity of naringenin is in line with previous findings, if however, observed for other cell lines [25,26]. Up to the best of our knowledge, compound **4** is described as cytotoxic for the first time. Nevertheless, its aglycone apigenin has been found active against cancer cell lines before [9,20,21,23,24]. Interestingly, only one of the two tested apigenin coumaroyl glucosides (compounds **3** and **4**) showed activity in the conducted assays, indicating steric effects to play a pivotal role for the cytotoxicity of these compounds.

Furthermore, the present study describes the first validated UHPLC-DAD method for the quantification of phenolic constituents in *T. diffusa*. The established assay allows the quantitation of eight flavonoids in both, the herb and the traditional preparation of *T. diffusa*, and coupled to mass spectrometry gives information on the abundance of another five flavonoids occurring in hydroethanolic damiana extracts.

**Supplementary Materials:** The following are available online, Figures S1–S15.

**Author Contributions:** Conceptualization, S.S.Ç. and C.Z.; validation, J.W.; investigation, J.W. and K.J.; resources, R.G. and C.Z.; writing—original draft preparation, J.W. and K.J.; writing—review and editing, C.Z. and S.S.Ç.; supervision, S.S.Ç.; project administration, S.S.Ç.

**Funding:** We acknowledge financial funding support by Land Schleswig-Holstein within the funding programme Open Access Publikationsfonds.

**Acknowledgments:** The authors thank Matthias Mayr for conducting the cytotoxicity assays.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


**Sample Availability:** Samples of the compounds **5** + **6** and **7** are available from the authors.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*
