New Phenolic Glycosides from Phyllanthus cochinchinensis

A investigação química das plantas Phyllanthus cochinchinensis (Euphorbiaceae) levou ao isolamento de três novos glicosídeos fenólicos, filantuosídeos A-C, juntamente com 12 compostos conhecidos. Suas estruturas foram determinadas com base em extensivas análises espectroscópicas e métodos químicos. Dentre eles, filantuosídeos A e B são dois glicosídeos fenólicos raros, com um esqueleto C6-C3-C6. A configuração absoluta dos filantuosídeos A e B foi estabelecida por dicroísmo circular elétrico calculado (ECD) usando teoria do funcional da densidade e a sua abordagem dependente do tempo (TDDFT). Os compostos isolados também tiveram sua citotoxicidade e atividade antimicrobial testadas.


Introduction
The genus Phyllanthus composing of approximate 600 species is one of the largest genera in the family Euphorbiaceae, which is distributed mainly in tropical and subtropical regions throughout the world.Thirty-three Phyllanthus species and four varieties are growing in China, particularly in the south of the Yangtze River.Among them, most species have been used in folk medicine to treat kidney and urinary bladder disturbances, intestinal infections, diabetes, and hepatitis B. 1 Phytochemcial investigation of this genus have revealed the occurrence of a large number of bioactive constituents including tannins, alkaloids, flavonoids, lignans, phenols, and terpenes. 1-7[3][4][5][6] Phyllanthus cochinchinensis mainly grows in the southern parts of China.Previous phytochemical studies carried out by our group on this plant have afforded antifeedant limonoids and sucrose esters. 5,7As a part of our continuing study on bioactive compounds from Phyllanthus species, [4][5][6][7] three new phenolic glycosides, phyllanthuosides A-C (1-3), together with twelve known compounds were isolated from the whole plants of P. cochinchinensis.Their structures were established by means of extensive spectroscopic analysis and chemical methods.Most of the isolates were tested for their cytotoxicity and antimicrobial activity.The results obtained are discussed herein.

Plant material
The whole plant of P. cochinchinensis was collected from Guangdong Province, People's Republic of China, on December 2011.A voucher specimen (KUN-1215860) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, and identified by Mr. Xiao-Ming Fang from South China Botanical Garden, Chinese Academy of Sciences.

Acid hydrolysis of compounds 1 and 2
Compounds 1 and 2 (each 3 mg) in 2 mol L -1 HCl-dioxane (1:1, v/v, 5 mL) were heated at 85 °C in a water bath for 8 h, respectively.The reaction mixtures were partitioned between H 2 O and CHCl 3 (2 mL × 3) four times.The aqueous layer was neutralized with 2 mol L −1 NaOH and then dried to give a saccharide mixture.Solutions of the sugar residues of these compounds in pyridine (2 mL) were added to L-cysteine methyl ester hydrochloride (1.5 mg) and kept at 60 °C for 1 h.Trimethylsilylimidazole (1.5 mL) was added to the reaction mixtures, which were kept at 60 °C for 30 min.The supernatants (4 µL) were analyzed by GC, and determined as D-glucose trimethylsilylated L-cysteine derivatives by comparison with a standard (retention time 21.7 min).

Quantum chemical calculations
The conformation analysis was carried out using Monte Carlo searching with molecular mechanics MMFF in Sparton'06 (Wavefunction Inc.Irvine, CA).The resulted conformers were re-optimized using DFT at the B3LYP /6-31G (d) level in vacuo.The free energies and vibrational frequencies were calculated at the same level to confirm their stability, and no imaginary frequencies were found.The optimized low energy conformers with energy < 2 kcal mol −1 were considered for ECD calculation.The TD-DFT/ B3LYP /6-311G (d, p) method was applied to calculate the ECD data in vacuo.All the calculations were run with Gaussian 09. 8 The excited energies and rotational strength were used to simulate ECD spectra of each conformer by introducing the Gaussian Function.The final ECD spectra of each compound were obtained by averaging all the simulated ECD spectra of all conformers according to their excited energies and Boltzmann distribution. 6The band shape of the calculated ECD curves were all 0.5 eV.

Cytotoxicity assay
Five human cancer cell lines, human myeloid leukemia HL-60, hepatocellular carcinoma SMMC-7721, lung cancer A-549 cells, breast cancer MCF-7, and colon cancer SW480, were used in the cytotoxic assay.All the cells were cultured in RPMI-1640 or DMEM (Dulbecco's Modified Eagle Medium) medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA).The cytotoxicity assay was performed according to the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method in 96-well microplates. 9Briefly, adherent cells (100 µL) were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug add with an initial density of 0.5 × 10 5 -1 × 10 5 cells mL −1 .Each tumor cell line was exposed to the test compound dissolved in dimethyl sulfoxide (DMSO) in triplicates for 48 h at 37 °C, with cisplatinum (DDP) and taxol (Sigma, USA) as positive controls.Then, MTT (50 µL) was added to each well, and the tumor cells were incubated for another 4 h at 37 °C.After the supernatant liquor was removed, SDS (200 µL) was added to each well.The optical density was measured at 595 nm on a microplate reader.Cell viability was detected and a cell growth curve was graphed.IC 50 values were calculated by Reed and Muench's method. 10

Antimicrobial bioassay
Susceptibility testing was performed using a modified version of the NCCLS methods 11,12 using organisms obtained from the American type culture collection (Manassas, VA) including Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Cryptococcus neoformans ATCC 90113, and Aspergillus fumigatus ATCC 90906.Detailed procedures have been described in a previous paper. 13

Results and Discussion
The air-dried and powdered whole plants of P. cochinchinensis were extracted with MeOH under reflux.Evaporation of the solvent under vacuum gave a MeOH residue, which was suspended in water and then extracted sequentially with CHCl 3 and n-BuOH.
Compound 1 was isolated as a colorless amorphous powder.Its molecular formula was determined to be C 24 H 30 O 11 , on the basis of positive HRESIMS (m/z 517.1686 [M+Na] + , calcd.517.1680) and its 13 C NMR (DEPT) spectra.The IR spectrum showed the occurrence of hydroxyl (3425 cm −1 ) and benzene rings (1600-1400 cm −1 ) groups.Twenty-four carbon resonances were well resolved in the 13 C NMR spectrum (Table 1) and further assigned by DEPT and HSQC experiments as one carbonyl (d C 199.3), six quaternary aromatic carbons (d C 111-155), one aliphatic methine (d C 53.8) and six aromatic methines, an oxygenated methylene at d C 75.9 (C-9), and three methoxyls, in addition to one hexosyl moiety.The 1 H NMR spectrum of 1 (Table 1) displayed two sets of characteristic ABX coupled aromatic protons at d H 7.16 (1H, d, J = 8.6 Hz), 7.69 (1H, dd, J = 8.6, 1.8 Hz) and 7.59 (1H, d, J = 1.8 Hz), and d H 6.71 (1H, d, J = 8.2 Hz), 6.75 (1H, dd , J = 8.2, 1.9 Hz) and 6.90 (1H, d, J = 1.9 Hz), suggesting the existence of two 1,3,4-trisubstituted benzene rings.Additionally, two aromatic methoxys (d H 3.82, 3.87, each 3H, s), one aliphatic methoxy (d H 3.34, 3H, s), and an anomeric proton signal (d H 5.01, 1H, d, J = 7.6 Hz) were observed apparently.The aforementioned NMR data for 1 were closely resembled to those of evofolin B, 4-hydroxy-1,2-bis(4-hydroxy-3methoxyphenyl)butan-1-one, a phenolic compound isolated from Tetradium glabrifolium. 26The differences were merely the presence of an additional aliphatic methoxy [d H 3.34, d C 59.3] and one more hexosyl moiety in 1, related to evofolin B. Acid hydrolysis of 1 afforded D-glucose as sugar residue, which was confirmed by GC analysis of its corresponding trimethylsilylated L-cysteine adduct.In the HMBC spectrum of 1 (Figure 1), correlation of the glucosyl anomeric proton (d H 5.01, H-1") with C-4 (d C 152.3) indicated the additional β-D-glucosyl unit located on C-4 position.Moreover, the HMBC correlations of the aliphatic methoxy at d H 3.34 with d C 75.9 (C-9) and H-9 (d H 4.12) with C-7 (d C 199.3, carbonyl carbon)/C-8 (d C 53.8)/9-OMe (d C 59.3) confirmed the location of the aliphatic methoxy on C-9.The ROESY correlations of the glucosyl H-1" with H-5 (d H 7.16/7.17),the 3-OMe with H-2 (d H 7.59) and the 3'-OMe with H-2' (d H 6.90) further confirmed the positions of the β-D-glucosyl and the aromatic methoxy.The absolute configuration of 1 was determined by comparison of the time dependent density functional theory (TDDFT) 6 calculated ECD curve with the experimental results (Figure 3).The positive Cotton effects at 200 nm, 250 nm, and 260 nm of 1 agreed well with the calculated ECD curve of the aglycon of 1 with 8S configuration.Thus, the structure of compound 1 was established as shown in Figure 1 and named as phyllanthuoside A.
Compound 2, a colorless amorphous powder, had the same molecular formula C 24 H 30 O 11 as 1, on the basis of the positive HRESIMS (m/z 517.1688 [M+Na] + , calcd.517.1680) and 13 C NMR (DEPT) spectra (Table 1).Acid hydrolysis of 2 afforded D-glucose as sugar residue, which was confirmed by GC analysis of its corresponding trimethylsilylated L-cysteine adduct.The 1 H and 13 C NMR spectroscopic data (Table 1) of 2 showed close resemblance to those of 1. Extensive analysis of the 1D and 2D NMR data suggested that 2 and 1 had the same C6-C3-C6 skeleton, while the only difference was the position of the β-D-glucosyl group.All the proton and carbon signals of compound 2 could be assigned unambiguously by HSQC, and HMBC analysis (Figure 2).In the HMBC experiment, the glucosyl H-1'' (d H 4.85/4.86) of 2 was correlated with the aromatic carbon at d C 147.38/147.41(C-4'), indicating that the β-D-glucosyl group in 2 was located on C-4'.This was further confirmed by the ROESY correlation between H-1" with H-5' (d H 7.08/7.09).Other key 1 H-1 H COSY, HMBC, and ROESY (Figure 2) correlations confirmed the planar structure of 2 as shown in Figure 1.The experimental ECD curve of 2 displayed the mirror image of that of compound 1 with 8S configuration (Figure 3), which suggested its 8R configuration.Thus, the structure of compound 2 was constructed and named as phyllanthuoside B.
It was noted that some aromatic protons and carbons of the new phenolic glycosides 1-3 appeared to be very close pairs of signals in the 1 H and 13 C NMR spectra acquired at room temperature, due to the equilibrium between two conformational isomers resulting from the slow rotation of the sugar unit around the glycosidic linkage. 32The energy barrier around the glycosidic bond was sufficiently high to prevent fast exchange between the two rotamers at room temperature. 33ll of the isolated compounds, except compounds 7 and 9, were evaluated for their cytotoxicity against

Figure 3 .
Figure 3.Comparison of the calculated ECD with the experimental ECD of 1 and 2.

Table 1 .
13 NMR and13C NMR spectroscopic data of compounds 1 a and 2 a (CD 3 OD, d in ppm)