Atalantums H-K from the peels of Atalantia monophylla and their cytotoxicity

Abstract Four new benzoyltyramines, atalantums H-K (1–4) and seven known compounds were isolated from the peels of Atalantia monophylla. All compounds were tested for cytotoxicity against HeLa, HCT116 and MCF-7 cell lines, as well as normal cells (Vero cells). Compound 5 showed cytotoxicity against HeLa, HCT116 and MCF-7 cell lines with IC50 values ranging from 16-25 μg/mL but was inactive against Vero cells. Compound 6 also showed interesting results as compound 5 with IC50 values ranging from 15-18 μg/mL and an IC50 value of 80.20 μg/mL against Vero cells. This means compounds 5 and 6 can be used as lead compounds for anticancer agents. Graphical Abstract


Introduction
Atalantia monophylla is a plant in the family Rutaceae. It is a small woody climber which has a brown stem full of thorns. The fruits and leaves look and smell similar to lime, although the fruits are not edible. It can be found in South India, Sri Lanka, East Bengal, Ceylon, and Southeast Asia including Thailand, where it is called ''Ma Nao Phee'' (Bunyapraphatsara 1999;Panda 2004). Various parts of this plant have been used in traditional medicine for several purposes. For instance, this plant is used as an antispasmodic, and is also used to cure paralysis, hemiplegia, and chronic rheumatism (Basa 1975). Oil from the fruits can be used to treat rheumatism and respiratory diseases (Basa 1975). Pathogenic fungi and itching can also be treated using the oil from its leaves (Panda 2004). There are many groups of compounds that have been isolated from A. monophylla comprising limonoids, acridone alkaloids, furoquinoline alkaloids (Kumar et al. 2010), coumarins, flavonoids (Posri et al. 2018) and benzoyltyramines (Govindachari et al. 1970;Sribuhom et al. 2017). In this study, eleven N-benzoyltyramine derivatives including four new compounds (1 -4) and seven known compounds (5 -11), were isolated. All isolated compounds were evaluated for cytotoxicity against HeLa (cervical cancer), HCT116 (colon cancer), MCF-7 (breast cancer), and Vero cell lines (Kumnerdkhonkaen et al. 2018).
Compound 2 showed a molecular ion at m/z 702.4700 [M þ Na] þ (calcd. 702.4710) indicative of the molecular formula C 42 H 65 O 6 N and corresponding to 11 indices of hydrogen deficiency. The 1 H and 13 C NMR spectra showed the same patterns as those of 1, except for the presence of a palmitoyloxy moiety instead of a hydroxyl group at the C-4 position. The 13 C NMR spectrum also displayed an additional ester-type carbonyl carbon at d 172.9 (C-1 000 ). In the HMBC spectrum, cross-peaks between H-4 and C-1 000 were observed. The rest of the proton and carbon signals were consistent with the hydrocarbon [(CH 2 ) 14 CH 3 ] chain of palmitic acid. Therefore, compound 2, atalantum I, was characterized as rac-N-{2-[4-(4-palmitoyloxy-6-hydroxy-7-methoxy-3,7dimethyl-2-octen-1-yl)oxy]phenyl}ethylbenzamide ( Figure 1).

Biological activity
All isolated compounds were evaluated for cytotoxicity against HeLa (cervical cancer), HCT116 (colon cancer), MCF-7 (breast cancer) and Vero cells (normal cells) by MTT assay. The results at 72 hr. showed compound 5 exhibited cytotoxicity against HeLa, HCT116 and MCF-7 cell lines with IC 50 values ranging from 16 to 25 lg/mL and displayed an IC 50 value of more than 100 lg/mL against normal cells (Table  S3). This indicates this compound can be used as a lead compound for an anticancer agent. Comparing between 5 and 2, compound 2 showed inactivity against these cell lines. The results indicate that the hydroxyl group may play an important role for cytotoxicity. This evidence can be observed in the cases of compounds 6 and 4. The IC 50 values of 6 were 15-18 lg/mL against HeLa, HCT116 and MCF-7 cell lines and showed an IC 50 value of 80.20 lg/mL against Vero cells. These data confirmed that the hydroxyl group may play an important role for cytotoxicity. In the case of cytotoxicity against Vero cells of compounds 6 and 7, compound 7 showed stronger cytotoxicity with an IC 50 value of 25.20 lg/mL. These results mean the palmitoyloxy group at the C-7 position selected to Vero cells. Comparing between compounds 9 and 10, compound 10 displayed stronger cytotoxicity against all cell lines than 9. This evidence shows that the tetrahydrofuran moiety was necessary for the activity.

General experimental procedures
The NMR spectra were recorded on a Varian Mercury plus spectrometer operating at 400 MHz ( 1 H) and at 100 MHz ( 13 C). IR spectra were recorded as thin films, using a Perkin Elmer Spectrum One FT-IR spectrophotometer. Mass spectra were determined on a Micromass Q-TOF 2 hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer with a Z-spray ES source (Micromass, Manchester, UK). UV spectra were measured on an Agilent 8453 UV-Visible spectrophotometer. Melting points were determined on a SANYO Gallenkamp melting point apparatus and were uncorrected. Thin layer chromatography (TLC) was carried out on MERCK silica gel 60 F254 TLC aluminum sheet. Column chromatography was done with silica gel 0.063-0.200 mm or less than 0.063 mm and RP-18 column chromatography was also used. Preparative layer chromatography (PLC) was carried out on glass supported silica gel plates using silica gel 60 PF254 for preparative layer chromatography. All solvents were routinely distilled prior to use.

Plant material
The peels of A. monophylla were collected in June 2016 from Phuwieng District, Khon Kaen Province, Thailand. The plant was identified by Prof. Dr. Pranom Chantaranothai, Faculty of Science, Khon Kaen University, Thailand. A botanically identified voucher specimen (KKU022015) was deposited at Faculty of Science, Khon Kaen University.

Cytotoxic activity assay
Cytotoxic effect on cancer cells was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay. Cells (8 Â 10 3 cells/well) were seeded onto 96-well plates and incubated for 24 hours to adhere. Human cervical cancer (HeLa) cell line was obtained from Dr. C. Pientong (Khon Kaen University, Khon Kaen, Thailand). Human breast adenocarcinoma (MCF-7) and human colon cancer (HCT116) cell lines were kindly provided by Dr. O. Tetsu (University of California, San Francisco, U.S.A.). A non-cancer (Vero) cell line was kindly provided by Dr. S. Barusrux (Khon Kaen University, Khon Kaen, Thailand), respectively. All cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL) (Gibco-BRL, USA) and incubated at 37 C in a humidified atmosphere of 5% CO 2 . For preliminary testing, cells were exposed to the selected compounds at a concentration of 100 mg/mL for 24, 48 and 72 hours. The compounds that caused cell viability less than 50% were further evaluated for their half maximal inhibitory concentration (IC 50 ) values. To evaluate IC 50 values, cells were exposed to increasing concentrations (10 -100 mg/mL) of selected compounds for 24, 48 and 72 hours. Control groups were treated with a solvent (a mixture of DMSO and ethanol; 1:1). After incubation for the indicated times, the medium was replaced with 110 mL of fresh medium containing MTT (0.5 mg/mL in PBS) (Sigma Chemical Co., St Louis, MO, USA) and incubated for 2 h. Formazan formed after conversion of MTT was dissolved in DMSO. The absorbance of formazan was measured with a microplate reader (Bio-Rad Laboratories, USA) at the wavelength of 550 nm using 655 nm as a reference wavelength. Each assay was replicated four times. The percentage of viable cells which corresponds to the production of formazan was calculated as previously described (Kumnerdkhonkaen et al. 2018

Conclusion
Chemical investigation of the methanol extract of the peels of Atalantia monophylla led to the isolation of four new benzoyltyramines, atalantums H-K (1-4) and seven known compounds. Cytotoxicity against HeLa, HCT116 and MCF-7 cell lines, as well as normal cells (Vero cells) was evaluated using MTT assay. The results showed that 5 exhibited cytotoxicity against HeLa, HCT116 and MCF-7 cell lines with IC 50 values ranging from 16-25 lg/mL but was inactive against Vero cells. In addition, 6 also showed IC 50 values ranging from 15-18 lg/mL and an IC 50 value of 80.20 lg/mL against Vero cells. From all data, it means the hydroxyl group may play an important role for cytotoxicity.