Bioactivity-guided isolation of antioxidant and anti-hepatocarcinoma constituents from Veronica ciliata
© Yin et al. 2016
Received: 26 December 2015
Accepted: 19 April 2016
Published: 3 May 2016
Veronica ciliata Fisch., widely distributed in western China, has been traditionally used in Tibetan Medicine as a treatment for hepatitis, cholecystitis, rheumatism, and urticaria. However, V. ciliata Fisch. has not been subjected to detailed chemical constitution analysis and the bioactive studies were restricted to its crude extracts. It is necessary to investigate the active chemical components of these extracts and identify their biological effects.
Four iridoid glycosides, (veronicoside, cataposide, amphicoside, and verminoside) were isolated from the ethyl acetate fraction. Among these compounds, veronicoside and verminoside were isolated for the first time from this plant. These compounds exhibited strong antioxidant activity and inhibitory activity on HepG2 cell proliferation. The antioxidant activity of verminoside was equal to Vc. Cataposide, amphicoside and verminoside had stronger anti-hepatocarcinoma activity than 5-fluorouracil.
Four iridoid glycosides,(veronicoside, cataposide, amphicoside and verminoside) were isolated from the extract of V. ciliata Fisch. using bioassay-guided screening.Among these compounds, veronicoside and verminoside were isolated for the first time from this plant. The above results indicated that these compounds were the active chemical components responsible for the antioxidant and anti-hepatocarcinoma properties of V. ciliata Fisch. The underlying mechanism of their bioactivity is worthy of further investigation.
Liver cancer is common in sub-Saharan Africa and Southeast Asia and is currently the most common type of cancer in many countries in these regions . A large number of medicinal plants have been tested and found to contain active compounds with curative proper properties against liver cancer [2–4]. Rehmannia glutinosa and Scrophularia ningpoensis Hemsl. (Scrophulariaceae) were used for the treatment of liver diseases and have a long history [5, 6]. Picroliv is a standardized fraction of alcoholic extract from Picrorhiza kurroa (Scrophulariaceae) and significantly protects against hepatic damage . Therefore, scrophulariaceous plants are worth studying to explore their anti-hepatocarcinoma activities.
Veronica ciliata Fisch., belonging to Scrophulariaceae, is a psychrophyte from the northwest territories, northern Sichuan, and the Tibetan autonomous region (China). In China, this plant has been traditionally used in Traditional Chinese Medicine to treat hepatitis, cholecystitis, rheumatism and urticarial . The extracts of V. ciliata Fisch. were reported to have strong antioxidant activities and significantly protective effects against acute hepatotoxicity induced by CCl4 . Five iridoid glycosides and three derivatives of benzoic acid have been isolated from V. ciliata Fisch. . However, V. ciliata Fisch. has not been subjected to detailed chemical constitution analysis and the bioactive studies were restricted to its crude extracts. It is necessary to investigate the active chemical components of these extracts and identify their biological effects. Given that there are no reports of the anti-hepatocarcinoma activity of V. ciliata Fisch., this study examined the antioxidant activity and anti-hepatocarcinoma activity of crude extracts and four fractions of V. ciliata Fisch. on hepatoma cell HepG2 proliferation. Subsequently, four iridoid glycosides with these bioactivities, especially the anti-hepatocarcinoma activity on HepG2 cells, were identified from V. ciliata Fisch. Among these compounds, veronicoside and verminoside were isolated for the first time from this plant.
Results and discussion
Structure identification of the purified compounds
13C-NMR data of compouds 1–4
In vitro antioxidant activity assays
As mentioned above, the antioxidant activity of the ethanol extracts was higher than the water extract, and the antioxidant activity order of the four fractions was:water fraction <petroleum ether fraction <n-BuOH fraction <EtOAc fraction. It has been reported that free hydroxyl groups in phenoliccompounds are mainly responsible for antioxidant activity . This may also be the cause of the higher antioxidant activity of cataposide, amphicoside and verminoside which all contain multiple phenolic hydroxylgroups. Additionally, fraction E showed stronger antioxidant activity than cataposide, amphicoside and verminoside at the same concentrations. It is possible that the antioxidant activity of fraction E did not come from any one of these compounds and that it emerged from the interaction of all of these compounds simultaneously.
In vivo anti-hepatocarcinoma activity assays
Although the ethyl acetate fraction showed a lower inhibition rate than 5-fluorouracil, it was higher than the 95 % ethanol extract (Fig. 4b). This result indicated that-, after the 95 % ethanol extract was partitioned into the four fractions, the active compounds were concentrated into the ethyl acetate fraction.
Veronicoside, cataposide, amphicoside and verminoside all strongly inhibited the proliferation of HepG2 cells (Fig. 4c), and the inhibition rate increased in a concentration-dependent manner. The inhibition rates of the compounds, except veronicoside, were much higher than that of 5-fluorouracil. Cataposide and verminoside had a similar suppressive effect on HepG2 cell proliferation and the IC50 values of veronicoside, cataposide, amphicoside, verminoside and 5-fluorouracil were 41.25 ± 0.17, 15.54 ± 0.53, 28.32 ± 0.22, 17.82 ± 0.42 and 29.62 ± 0.32 μg/mL, respectively.
The capability of the compounds to hinder proliferation of a cancerous cell line was ascertained by measuring their cytotoxicity in a hepatocarcinoma cell line. The majority of the iridoid glycosides and their derivatives have been described as having an attached aromatic ring. Aromatic rings are cited to be one of the most ‘preferred structures’ to be associated with bioactivities . Hence, as shown in Fig. 4, all the compounds possessed cytotoxic activity. Additionally, cataposide, amphicoside and verminoside inhibited cell proliferation more effectively than veronicoside.We suspected that this was because cataposide, amphicoside and verminoside have more phenolic hydroxyl groups than veronicoside, although their chemical structures are very similar.As reported previously,Picroliv is a standardized mixture obtained from P. kurroa and contains at least 60 % of iridoid glycosides. In a number of tests aimed at delineating the anti-hepatotoxic effects of picroliv, it has been shown to have similar or better activity than silymarin  0.5-fluorouracil is a broad-spectrum anti-cancer drug and our data shows that the anti-hepatocarcinoma activities of cataposide, amphicoside, and verminoside were stronger than 5-fluorouracil. Moreover, our work showed that V. ciliata Fisch. contains a high amount of iridoid glycosides, indicating that it is potentially valuable as an anti-hepatotoxic drug.
The antioxidant and anti-hepatocarcinoma activities of the ethanol extracts were stronger than those of the aqueous extract, and the ethyl acetate fraction of the 95 % ethanol extract showed the highest activities. Four iridoid glycosides (veronicoside, cataposide, amphicoside, and verminoside) were isolated from the ethyl acetate fraction. All of the compounds exhibited strong antioxidant activity and inhibitory activity on HepG2 cell proliferation. The antioxidant activity of verminoside was equal to Vc. Cataposide, amphicoside and verminoside had stronger anti-hepatocarcinoma activity than 5-fluorouracil.
Samples were dissolved in methanol, and electrospray ionization ion trap multiple mass spectrometry (ESI–MS) was performed on a MicrOTOF-Q II (Bruker Daltonics, Germany) plus LC/MS system. UV spectra were obtained using a Perkin-Elmer Lambda 35 spectrometer. 1H NMR spectra,13C NMR spectra,and 2D NMR (HMBC) spectra were recorded on a Bruker Ascend-400 spectrometer, operating at 400 and 100 MHz for 1H and 13C, respectively, using MeOD-d 4 as solvents. Chemical shifts were reported in δ (ppm) downfield from tetramethylsilane (TMS) as an internal reference, and coupling constants were reported in Hz. Column chromatography (CC) was performed using silica gel (200–300 mesh, 2.4 kg) and Sephadex LH-20. The spots on TLC plates were detected under UV light or by holding under iode vapor, and were visualized by spraying with ethanol-H2SO4 after heating. Separations by HPLC (LC-3000) were carried out using an Welchrom-C18 column (10 × 250 mm, 5 μm).Unless specified otherwise,the flow rate was 2.0 mL/min.
5-FU(purity >99 %) was purchased from Chengdu Hua Xia chemical reagent co., LTD, vitaminc(Vc)(purity >99.7 %), 2,6-ditert-butyl-4-methylphenol(BHT)(purity >99.9 %) and Penicillin sodium were purchased from Sigma-Aldrich. Acetonitrile was obtained from Merck. The solvents used for HPLC (high performance liquid chromatography) were of HPLC grade. All other chemicals and reagents used in this study were of analytical grade.
The herbs of V. ciliata Fisch. were purchased from Tibet Tibetan Medicine Group Co., Ltd., China. A voucher specimen (No. 00721478) was identified by Dr. Jie Bai, School of Life Sciences, Sichuan University, and deposited in the Herbarium of Sichuan University.
Extraction and isolation
The spectroscopic data were listed below
Veronicoside (compound 1) was obtained as a white amorphous powder. ESI–MS (positive) m/z: 489[M + Na]+; ESI–MS (negative)m/z: 465[M-H]−; 1H-NMR (400 MHz, CH3OH-d 4 ) δ: 2.48(1H, dd, J = 9.0, 7.0 Hz, H-9), 2.59 (1H, m, H-5), 3.0 ~ 3.24 (4H, m, H-2′,3′,4′,5′), 3.46 (1H, m, H-6′b), 3.74 (1H, brs, H-7), 3.77 (1H, m, H-6′a), 3.77 (1H, m, H-10b), 3.95 (1H, dd, J = 13.3, 4.8 Hz, H-10a), 4.65 (1H, d, J = 8.0 Hz, H-1′), 5.03 (1H, m, H-4), 5.14 (1H, m, H-6), 5.14 (1H, d, J = 9.5 Hz, H-1), 6.45 (1H, d, J = 6.5 Hz, H-3), 7.58 (2H, t, J = 8.0 Hz, H-3′′, 5′′), 7.72 (1H, t, J = 7.5 Hz, H-4′′), 8.04 (2H, d, J = 8.5 Hz, H-2′′, 6′′); 13C-NMR (100 MHz, CH3OH-d 4 ): see Table 1.
Cataposide (compound 2) was obtained as a white amorphous powder. ESI–MS (positive) m/z: ESI–MS m/z: 483 [M + H]+; 1H-NMR (400 MHz, CH3OH-d 4 ) δ: 2.49 (1H, m, H-9), 2.57 (1H, m, H-5), 3.0-3.23 (4H, m, H-2′, 3′, 4′, 5′), 3.42 (1H, dd, J = 11.8, 6.8 Hz, H-6′b), 3.68 (1H, d, J = 1.5 Hz, H-7), 3.71 (1H, dd, J = 11.8, 1.8 Hz, H-6′a), 3.72 (1H, d, J = 13.0 Hz, H-10b), 3.92 (1H, d, J = 13.5 Hz, H-10a), 4.63 (1H, d, J = 8.0 Hz, H-1′), 4.97 (1H, dd, J = 6.0, 4.5 Hz, H-4), 5.07 (1H, dd, J = 8.0, 1.0 Hz, H-6), 5.12 (1H, d, J = 9.5 Hz, H-1), 6.43 (1H, dd, J = 5.5, 1.5 Hz, H-3), 6.86 (2H, d, J = 9.0 Hz, H-3′′, 5′′), 7.86 (2H, d, J = 8.5 Hz, H-2′′, 6′′); 13C-NMR (100 MHz, CH3OH-d 4 ): see Table 1.
Amphicoside (compound 3) was obtained as a white amorphous powder. ESI–MS (positive) m/z: 535 [M + Na]+; ESI–MS (negative)m/z: 511[M-H]−; 1H-NMR (400 MHz, CH3OH-d 4 ) δ: 2.63 (1H, m, H-9), 2.68 (1H, m, H-5), 3.23–3.43 (4H, m, H-2′,3′, 4′, 5′), 3.65 (1H, dd, J = 12.0, 6.5 Hz, H-6′b), 3.75 (1H, d, J = 1.0 Hz, H-7), 3.85 (1H, d, J = 13.0 Hz, H-10b), 3.90 (3H, s, OCH3), 3.93 (1H, dd, J = 12.0, 2.0 Hz, H-6′a), 4.21 (1H, d, J = 13.5 Hz, H-10a), 4.80 (1H, d, J = 8.0 Hz, H-1′), 5.01 (1H, dd, J = 5.8, 4.3 Hz, H-4), 5.11 (1H, dd, J = 8.3, 1.3 Hz, H-6), 5.20 (1H, d, J = 9.5 Hz, H-1), 6.38 (1H, dd, J = 6.0, 1.5 Hz, H-3), 6.87 (1H, d, J = 8.5 Hz, H-5′′), 7.57 (1H, d, J = 2.0 Hz, H-2′′), 7.60 (1H, dd, J = 8.5, 2.0 Hz, H-6′′); 13C-NMR (100 MHz, CH3OH-d 4 ): see Table 1.
Verminoside (compound 4) was obtained as a white amorphous powder. ESI–MS (positive) m/z: 547[M + Na]+; ESI–MS (negative)m/z: 523[M-H]−; 1H-NMR (400 MHz, CH3OH-d 4 ) δ: 2.61 (1H, m, H-5), 2.63 (1H, m, H-9), 3.26 ~ 3.45 (4H, m, H-2′,3′, 4′, 5′), 3.66 (1H, dd, J = 12.0, 6.5 Hz, H-6′b), 3.70 (1H, brd, J = 1.0 Hz, H-7), 3.83 (1H, d, J = 13.0 Hz, H-10b), 3.92 (1H, dd, J = 11.8, 1.8 Hz, H-6′a), 4.16 (1H, d, J = 13.0 Hz, H-10a), 4.80 (1H, d, J = 8.0 Hz, H-1′), 5.1 (1H, dd, J = 6.0, 4.0 Hz, H-4), 5.02 (1H, dd, J = 7.8, 1.3 Hz, H-6), 5.16 (1H, d, J = 9.0 Hz, H-1), 6.31 (1H, d, J = 15.5 Hz, H-8′′), 6.38 (1H, dd, J = 6.0, 1.5 Hz, H-3), 6.81 (1H, d, J = 8.0 Hz, H-5′′), 6.98 (1H, dd, J = 8.5, 2.0 Hz, H-6′′), 7.07 (1H, d, J = 2.0 Hz, H-2′′), 7.61 (1H, d, J = 16.0 Hz, H-7′′); 13C-NMR (400 MHz, CH3OH-d 4 ): see Table 1.
Assays for antioxidant activity
The antioxidant activities of the 95 % ethanol extract, and water extract of V. ciliata Fisch. were measured. Next, the 95 % ethanol extract was further divided into petroleum ether, ethyl acetate, n-butanol, and water fractions, and the antioxidant activities of each fraction were compared. The activities of 9 fractions and 4 pure compounds isolated from the ethyl acetate fraction were also determined.
DPPH radical scavenging assay
The scavenging activity of the DPPH radical was evaluated according to an improved DPPH assay  with slight modifications. Briefly, 2 mL of the samples at different concentrations (3.25–100 μg/mL, dissolved in ethanol) were mixed with 2 mL of DPPH solution (0.1 mM, in ethanol). VC was used as a comparison. Then, the mixtures were shaken evenly and allowed to stand at room temperature in the dark for 30 min before the absorbance was measured at 517 nm. The radical scavenging activity was calculated as follows: DPPH radical scavenging activity (%) = [1 − (Ai − As)/Ac] ×100, where Ac is the absorption of the negative control, Ai represents the absorption of the experiment group and As represents the absorption of the sample background. The concentration of samples reducing 50 % of free radical DPPH (IC50) was determined by plotting the percentage of inhibition against the sample concentrations.
Reducing power assay
The reducing power of the samples was measured using a previous method . Briefly, 1.0 mL of samples solutions at different concentrations(3.25–100 μg/mL, dissolved in ethanol) was mixed with 2.5 mL of phosphate buffer saline (0.2 M, pH 6.6) and 2.5 mL of 1 % (w/v) K3Fe (CN)6 solution. After incubation at 50 °C for 30 min, 2 mL of 10 % trichloroacetic acid (TCA) was added. Then 2.0 mL of the upper layer was combined with 2.0 mL of distilled water and 1 mL of 0.1 % (w/v) FeCl3 solution. The absorbance was analyzed at 700 nm (BHT was used as a positive control). Increased absorbance of the reaction mixture indicates a greater reducing power.
Human hepatocellular carcinoma HepG2 cells were obtained from the cell bank of the Chinese Academy of Sciences. The cells were cultured in RPMI 1640 medium (Gibco BRL) supplemented with 100 IU/mL penicillin, 100 IU/mL streptomycin, and 0.01 mg/mL fetal bovine serum (FBS) and were incubated at 37 °C in a humidified incubator with an atmosphere of 5 % CO2.
Cell proliferation inhibition assay
All of the results were expressed as mean ± standard deviation (SD). Statistical differences of experimental data among groups were tested using one-way ANOVA (n = 3) analysis or paired two-sample t test (n = 3) analysis (SPSS 15.0, SPSS Inc., Chicago, IL, USA). Statistical significance was set at p < 0.05.
Four iridoid glycosides, (veronicoside, cataposide, amphicoside and verminoside) were isolated from the extract of V. ciliata Fisch. using bioassay-guided screening. Among these compounds, veronicoside and verminoside were isolated for the first time from this plant. The above results indicated that these compounds were the active chemical components responsible for the antioxidant and anti-hepatocarcinoma properties of V. ciliata Fisch. The underlying mechanism of their bioactivity is worthy of further investigation.
LY, LD, FC, LT designed the experiments. LY, QL compeled the extraction and isolation of the material and identified the of four compounds, LY and ST the DPPH Radical Scavenging, Reducing Power and Cell Proliferation inhibition assay. All authors read and approved the final manuscript.
This work was supported by The National Natural Science Foundation of China (No.: 31570351).
The authors declare that they have no competing interests.
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