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Abstract

Purification of the petroleum ether and ethyl acetate fractions from Ludwigia hyssopifolia yielded 9 natural products, ethyl gallate (1), vanillin (2), trans-p-hydroxycinnamic acid (3), trans-p-hydroxy-ethyl cinnamate (4), ozoroalide (5), scopoletin (6), de-O-methyllasiodiplodin (7), syringaldehyde (8), and 3,3′-dimethoxy-4,4′-dihydroxy-stilbene (9). The structures of 1 to 9 were determined by spectroscopic analyses including 1D Nuclear Magnetic Resonance (NMR) (¹H NMR and ¹³C NMR) and Mass Spectrum (MS) data. Four compounds displayed cytotoxic effects on the human laryngeal cancer Hep-2 cell line. Compounds 5 and 7 were also effective against TU212 cell line and significantly inhibited cancer cell growth in a dose- and time-dependent manner on Hep-2 cell line.

Keywords

phenols macrolides cytotoxic activity

Ludwigia hyssopifolia is widely distributed in Guangxi Zhuang Autonomous Region, China. It has the effects of relieving fever, swelling, and pain, and is used to treat diseases such as dysentery, infectious hepatitis, and edema.¹ Pharmacological studies have shown that the plant exhibits a variety of biological activities including anticancer, antibacterial, antidiarrheal, and anti-ulcer activities.^2
-4 Phytochemical studies have shown that L. hyssopifolia contains piperine,³ palmitic acid, isovanillin, β-sitosterol, stigmasterol-3-O-β-d-glucopyranoside, gallic acid, ethyl gallate, oleanolic acid, 2,4,6-trihydroxybenzoic acid, ursolic acid,⁵ kaempferol, ginsenoside Rb₁,⁶ 6β,24-hydroxy tormnetic acid, xanthyletin, (+)trans-decursidinol, β-sitosterol-β-d-glucopyranoside, 6β,23-hydroxy tormentic acid, 23-hydroxy tormentic acid, and 6β,23-hydroxy tormentic acid.⁷ In the present study, 7 known phenols and 2 known macrolides were isolated from L. hyssopifolia. On the basis of spectroscopic data (Figure S1-S25), they were identified as ethyl gallate (1),⁸ vanillin (2),⁹ trans-p-hydroxycinnamic acid (3),¹⁰ trans-p-hydroxy-ethyl cinnamate (4),¹¹ ozoroalide (5),¹² scopoletin (6),¹³ de-O-methyllasiodiplodin (7),¹⁴ syringaldehyde (8)¹⁵ and 3,3′-dimethoxy-4,4′-dihydroxy-stilbene (9)¹⁶ (Figure 1). Compounds 2 to 9 were isolated from L. hyssopifolia for the first time. The cytotoxicity of all isolates was tested against human laryngeal carcinoma Hep-2 and TU212 cell lines. The antilaryngeal carcinoma activities of compounds 5 and 7 have not been previously reported. Our results showed that compounds 5 and 7 significantly inhibited the cancer cell growth in a dose-dependent manner. In addition, all the compounds did not show significant cytotoxic activity against HEK293 cell line (Table 1).

Figure 1

Structures of compounds 1 to 9.

Table 1

Cytotoxic Activity of Compounds 1 to 9 in 24 h (IC₅₀, μg/mL; mean ± SD; N = 3).

Compound	Hep-2	TU212	HEK293
1	＞100	＞100	＞100
2	＞100	＞100	＞100
3	＞100	＞100	＞100
4	42.76 ± 1.39	＞100	＞100
5	10.82 ± 0.16	17.84 ± 0.31	83.30 ± 9.67
6	57.18 ± 2.48	＞100	＞100
7	3.50 ± 0.04	4.41 ± 0.01	31.63 ± 0.47
8	＞100	＞100	＞100
9	＞100	＞100	＞100

Compounds 4 and 6 displayed weak cytotoxicity against Hep-2 cell line (IC₅₀ = 42.76 ± 1.39 and 57.18 ± 2.48 µg/mL) in our study (Table 1; Figure 2(a) and (c)). Meanwhile, compounds 5 and 7 displayed more potent inhibition against Hep-2 cell line (IC₅₀ = 10.82 ± 0.16 and 3.50 ± 0.04 µg/mL) than other compounds with less toxic effects against HEK293 than human laryngeal cancer cell lines (Table 1; Figure 2(b) and (d)). After 12, 24, and 48 hours of incubation, cell viability and cytotoxicity were evaluated, and the results were shown in Table 2 and Figure 2(e) and (f).

Figure 2

Compounds 4-7 inhibited human laryngeal cancer cell proliferation. (a-d) Hep-2, TU212, and HEK293 cells were treated with indicated concentrations of compounds 4-7 for 24 h and determined by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. (e, f) Hep-2 cell line was treated with indicated concentrations of compounds 5 and 7 for 12, 24, and 48 h and determined by MTT assay.

Table 2

Cytotoxic Activity of Compounds 5 and 7 Against Hep-2 (IC₅₀, μg/mL; mean ± SD; N = 3).

Time (h)	Compound 5	Compound 7
12	14.98 ± 0.42	3.83 ± 0.05
24	10.82 ± 0.16	3.50 ± 0.04
48	9.40 ± 0.23	3.17 ± 0.04

The morphological changes of Hep-2 cell line treated with different concentrations of compounds 5 and 7 were observed under an inverted-phase contrast microscope. The results (Figure 3(a) and (c)) showed that with increasing dose, the number of adherent cells decreased and the number of floating cells increased. Until at high dosage, the phenomena of shrinkage, distortion, and flotage were more obvious in Hep-2 cell line. After staining Hoechst 33258 (Figure 3(b) and (d)), compared with the treated group, the cells in the blank group were intact and uniformly colored. Apoptotic phenomena were observed in the treated group in which cells were chromatin condensed, partitioned into blocks, and appeared as bright apoptotic bodies, especially in the high-concentration group with the yellow arrows.

Figure 3

Observation of morphological changes in Hep-2 cell line. (a, c) The morphology of the cells treated with compounds 5 and 7 was photographed under an inverted-phase contrast microscope. (b, d) The morphology of the cells treated with compounds 5 and 7 was photographed under a fluorescence microscope. The cell nuclei were stained for Hoechst 33258 and the apoptotic bodies were indicated by yellow arrows.

In order to examine whether compounds 5 and 7 could induce apoptosis in the Hep-2 cell line after 24 hours, cells were stained with Annexin V-fluorescein isothiocyanate (FITC) and analyzed by flow cytometry.¹⁷ The percentage of apoptotic Hep-2 cells obtained by Fluorescence Activated Cell Sorting (FACS) (propidium iodide [PI] and Annexin V) analysis was significantly higher after incubation with different concentrations (5, 15, and 25 µg/mL) of compound 5 (Q2 + Q3: 14.16%, 26.40%, and 42.00%) than that of the control group (Q2 + Q3: 9.66%) (Figure 4(a)). The percentage of apoptotic Hep-2 cells was also significantly higher after incubation with different concentrations (3, 4, and 5 µg/mL) of compound 7 (Q2 + Q3: 17.47%, 23.91%, and 34.10%) than that of the control group (Q2 + Q3: 6.11%) (Figure 5(a)). Observation and analysis under fluorescence microscopy showed that compounds 5 and 7 induced apoptosis in the Hep-2 cell line (Figures 4(b) and 5(b)). These results suggest that compounds 5 and 7 inhibit the growth of Hep-2 cell line in a concentration-dependent manner by inducing apoptosis.

Figure 4

Compound 5 induced apoptosis of Hep-2 cell line. (a) Hep-2 cells were treated with compound 5 for 24 h, then stained with Annexin V/propidium iodide staining and evaluated by flow cytometry. (b) Hep-2 cells were treated with compound 5 for 24 h, then stained with Annexin V/propidium iodide staining and observed by fluorescence microscope. Annexin V-fluorescein isothiocyanate fluorescence was green and propidium iodide fluorescence was red.

Figure 5

Compound 7 induced apoptosis of Hep-2 cell line. (a) Hep-2 cells were treated with compound 7 for 24 h, then stained with Annexin V/propidium iodide staining and evaluated by flow cytometry. (b) Hep-2 cells were treated with compound 7 for 24 h, then stained with Annexin V/propidium iodide staining and observed by fluorescence microscope. Annexin V-fluorescein isothiocyanate fluorescence was green and propidium iodide fluorescence was red.

According to the anticancer mechanisms, regulating apoptotic proteins is an attractive therapeutic strategy for laryngeal cancer.¹⁸ Therefore, we investigated the effects of compounds 5 and 7 on the regulatory proteins involved in apoptosis. Caspase-3 plays the executioner role in apoptosis, and its activation depends on the precursor form cleaved by the caspase cascade.¹⁹ Western blot analysis showed that the treatment of Hep-2 cell line with compounds 5 and 7 concentration dependently enhanced the expression levels of cleaved caspase-3 (Figure 6). Overall, these data indicated that compounds 5 and 7 have significant potential to induce human laryngeal carcinoma Hep-2 cell line apoptosis.

Figure 6

Effects of compounds 5 and 7 on the apoptosis regulatory protein of Hep-2 cell line. (a, b) The related expression of cleaved caspase-3 was detected in Hep-2 cells treated with compounds 5 and 7 by western blotting. β-actin was used as a control (*P < 0.05, **P < 0.01, or ***P < 0.001).

In conclusion, 7 known phenols and 2 known macrolides were isolated from L. hyssopifolia. On the basis of the spectroscopic data, they were identified as ethyl gallate (1), vanillin (2), trans-p-hydroxycinnamic acid (3), trans-p-hydroxy-ethyl cinnamate (4), ozoroalide (5), scopoletin (6), de-O-methyllasiodiplodin (7), syringaldehyde (8), and 3,3′-dimethoxy-4,4′-dihydroxy-stilbene (9). Compounds 2 to 9 were isolated from L. hyssopifolia for the first time. Compounds 5 and 7 significantly inhibited cancer cell growth in a dose-dependent manner and induced apoptosis by regulating caspase-3.

Experimental

General Experimental Procedures

Semipreparative High Performance Liquid Chromatography (HPLC) was carried out on a Waters 2535 HPLC connected with a 2998 Photodiode Array Detector, a 2707 Autosampler (Waters) and a Waters Sunfire C₁₈ column (5 µm, 10 × 150 mm) (Waters, Ireland). Optical rotations were measured on a Perkin-Elmer polarimeter 341. Infrared Spectrophotometry (IR) spectra were determined on a Nicolet Magna FT-IR 750 spectrometer (ν _max in cm⁻¹). The NMR spectra were recorded on the Bruker DRX-600 NMR spectrometer for 1D and 2D NMR. Direct injection ESIMS and HP High Performance Liquid Chromatography-Photo-Diode Array-Electrospray Ionization Mass Spectrometry (HP LC PDA-ESIMS) analyses were recorded on a Waters ACQUITY SQD MS system (Waters, United States) connected to a Waters 1525 HPLC with a 2998 Photodiode Array Detector (Waters, United States). Commercial silica gel (Qing Dao Hai Yang Chemical Group Co., 200-300 and 300-400 meshes) was used for column chromatography. Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co., GF-254) were used for analytical Thin Layer Chromatography (TLC).

Plant Material

The whole plants of L. hyssopifolia were collected from Luchuan County, Yulin, Guangxi Zhuang Autonomous Region, China, in August 2016. The plant was identified as L. hyssopifolia (G. Don) Exell by Professor Songji Wei of Guangxi University of Chinese Medicine, China. A voucher specimen (No. SC0864) has been deposited in the Herbarium of South-Central University for Nationalities, Wuhan, China.

Extraction and Isolation

Dried whole plants of L. hyssopifolia (16.5 kg) were ground and then extracted sequentially by maceration with 80% EtOH 3 times (16 L each, 4 days) at room temperature. The solvent was evaporated under reduced pressure to obtain a crude extract (1403.4 g). The extract was suspended in warm water and then partitioned successively with petroleum ether, ethyl acetate, and n-butyl alcohol to afford PE fraction (120.4 g), EtOAc fraction (474.6 g) and n-BuOH fraction (251.9 g), respectively. The PE fraction (85.0 g) was subjected to D101 macroporous resin column chromatography (2500 g, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) eluted with 10%, 30%, 50%, 70%, 80% and 95% aq. ethanol in a gradient manner to give 8 fractions (A1-A8). A2 (720 mg) was purified on a Sephadex LH-20 column eluting with MeOH (containing 0.1% formic acid) to afford 5 fractions (A2.1-A2.5). A2.3 (252 mg) was further purified by semipreparative HPLC (CNCH₃-H₂O, 15%:85% → 70%:30%, 20 minutes, containing 0.1% formic acid in both phases) to afford compounds 1 (98.4 mg) and 2 (4.1 mg), respectively. A3 (2.0 g) was purified on a Sephadex LH-20 column eluting with MeOH (containing 0.1% formic acid) to afford 8 fractions (A3.1-A3.8). A3.4 (129 mg) was further purified by semipreparative HPLC (CNCH₃-H₂O, 25%:75% → 30%:70%, 20 minutes, containing 0.1% formic acid in both phases) to afford compounds 3 (4.7 mg). A5 (10.2 g) was chromatographed over silica gel with CH₂Cl₂-MeOH (15:1 → 12:1 → 10:1 → 8:1 → 5:1 → 3:1, containing 0.1% formic acid) to afford 9 fractions (A5.1-A5.9). A5.2 (500 mg) was further purified by semipreparative HPLC (CNCH₃-H₂O, 45%:55% → 80%:20%, 20 minutes, containing 0.1% formic acid in both phases) to afford compounds 4 (33.7 mg) and 5 (25.5 mg), respectively. The EtOAc fraction (400 g) was subjected to D101 macroporous resin column chromatography (8000 g, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) eluted with 10%, 30%, 50%, 70%, 80%, and 95% aq. ethanol in a gradient manner to give 6 fractions (B1-B6). B3 (55.2 g) and B4 (78.3 mg) were further subjected to the column chromatography over silica gel with CH₂Cl₂-MeOH (50:1 → 30:1 → 25:1 → 20:1 → 15:1 → 10:1 → 8:1 → 5:1 → 3:1, containing 0.1% formic acid) to afford 4 fractions (C1-C4). C4 (200 mg) was further purified by HPLC (CNCH₃-H₂O, 25%:75% → 70%:30%, 20 minutes, containing 0.1% formic acid in both phases) to afford compounds 6 (33.7 mg) and 7 (25.5 mg), respectively. C1 (1.13 g) was purified on a Sephadex LH-20 column eluting with MeOH (containing 0.1% formic acid) to afford 6 fractions (C1.1-C1.6). C1.4 (80 mg) was further purified by semipreparative HPLC (CNCH₃-H₂O, 10%:90% → 30%:70%, 14 minutes, 32%:48% → 100%:0%, 20 minutes, containing 0.1% formic acid in both phases) to afford compound 8 (6 mg). C1.6 (56 mg) was further purified by HPLC (CNCH₃-H₂O, 30%:70% → 45%:55%, 20 minutes, containing 0.1% formic acid in both phases) to afford compounds 9 (4.7 mg).

Cell Culture

Human laryngeal cancer cell lines Hep-2 and TU212 and human embryonic kidney cell line HEK293 were obtained from American Type Culture Collection (ATCC). Hep-2, TU212, and HEK293 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) medium (Sigma-Aldrich, St. Louis, MO, United States), supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin (Hyclone, Logan, UT, United States). Cells were cultured in an incubator containing a humidified atmosphere with 5% CO₂ at 37°C.

MTT Assay

The MTT method was used to test the cytotoxicity of compounds 1 to 9 on Hep-2, TU212, and HEK293 cell lines. The cells were seeded in 96-well plates and incubated for 24 hours to confluence, then exposed to different concentrations of all the compounds. Following 12, 24 and 48 hours of drug treatment, 100 µL MTT (5 mg/mL) was added to each well. After incubating for 30 minutes, the liquid in the wells was discarded and 150 µL Dimethyl sulfoxide (DMSO) was added to each well. The OD values were measured at 562 nm with a spectrophotometer (Thermo Fisher Scientific Oy, Vantaa, Finland). Percentage of cell viability ratio = $[1 - (O D_{t r e a t m e n t g r o u p} - O D_{b l a n k g r o u p}) / (O D_{c o n t r o l g r o u p} - O D_{b l a n k g r o u p})] \times 100 %$ .²⁰ The inhibition rates and IC₅₀ values were calculated by GraphPad Prism 6.0 and plotted. According to the results, compound 5 and 7 were selected for further analysis with 3 different times (12, 24, and 48 hours).

Morphological Changes of Apoptosis

The cells were seeded in 6-well plates and incubated for 24 hours to confluence, then exposed to different concentrations of compound 5 (0, 5, 15, and 25 µg/mL) and compound 7 (0, 3, 4, and 5 µg/mL) for 24 hours. After discarding the supernatant, 1.0 mL of stationary liquid (methanol:acetic acid = 3:1) were covered for about 15 minutes after which a Hoechst 33258 solution (5 µg/mL) was added to the wells and on a dark condition for 30 minutes. The cells were then examined under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

Annexin V-FITC/PI Double Staining

The cells were seeded in 6-well plates and incubated for 24 hours to confluence, then exposed to different concentrations of compound 5 (0, 5, 15, and 25 µg/mL) and compound 7 (0, 3, 4, and 5 µg/mL). Following 24 hours of drug treatment, the cells were washed twice with Phosphate Buffered Saline (PBS) and centrifuged for 6 minutes at 300 g. Then, 400 μL 1× binding buffer was added to resuspended cells and incubate with 5 μL of annexin V-FITC for 15 minutes and 5 μL of PI for 5 minutes at room temperature in the dark. A flow cytometer (Guava easyCyte, Darmstadt, Germany) and a fluorescence microscope (Leica Microsystems, Wetzlar, Germany) were used to explore and observe the cell apoptosis.

Western Blotting

The cells were seeded in 10 cm cell culture dishes and incubated for 24 hours to confluence, then exposed to different concentrations of compound 5 (0, 5, 15, and 25 µg/mL) and compound 7 (0, 3, 4, and 5 µg/mL). After 24 hours of drug treatment, the cells were mechanically separated into single cell suspensions and protein was extracted. Following the denaturation of the protein, the protein concentrations were determined by a bicinchoninic acid kit (Beyotime, Shanghai, China). Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel electrophoresis was performed at 120 V voltage until bromophenol blue was terminated at the lower edge of the electrophoresis chamber. After transferring for 1 hour and 20 minutes under 300 mA current, the membranes were incubated in 5% skim milk for 2 hours at room temperature, and then the polyvinylidene fluoride (PVDF) membranes were washed 3 times in tris buffered saline Tween. Primary antibodies were applied overnight and secondary antibodies were applied for 2 hours at room temperature. Under the gel imager (Bio-Rad, CA, United States), the protein appeared on the PVDF membrane.

Supplemental Material

Supplementary data - Supplemental material for Cytotoxic Compounds From Ludwigia hyssopifolia

Supplemental material, Supplementary data, for Cytotoxic Compounds From Ludwigia hyssopifolia by Jinyan Zhang, Chang Liu, Jianhua Wei, Bing Li, Xin Zhan, Yunqing Li, Ji Hao, Rumei Lu and Xinzhou Yang in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The work was financially supported by the grants of Innovation Project of Graduate Education of Guangxi University of Chinese Medicine(No. YCSZ20190014), Guangxi First-class discipline (No. 2019XK114), the Applied Basic Research Programs of Science and Technology Commission Foundation of Wuhan (No. 2017060201010217), and National Natural Science Foundation of China grants (81774000).

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