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PASS-predicted Vitex negundo activity: antioxidant and antiproliferative properties on human hepatoma cells-an in vitro study
© Kadir et al.; licensee BioMed Central Ltd. 2013
Received: 19 August 2013
Accepted: 29 November 2013
Published: 4 December 2013
Hepatocellular carcinoma is a common type of tumour worldwide with a high mortality rate and with low response to current cytotoxic and chemotherapeutic drugs. The prediction of activity spectra for the substances (PASS) software, which predicted that more than 300 pharmacological effects, biological and biochemical mechanisms based on the structural formula of the substance was efficiently used in this study to reveal new multitalented actions for Vitex negundo (VN) constituents.
Experimental studies based on antioxidant and antiproliferative assays verified the predictions obtained by the PASS-predicted design strategy. Antioxidant activity of VN extract was studied using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and Ferric reducing or antioxidant power (FRAP) assays. The antiproliferative activity of VN extract against WRL68 and HepG2 was investigated based on methylthiazol tetrazolium (MTT) spectrophotometric assay.
VN extract showed 79.43% inhibition of DPPH stable radical with IC50 13.31 ± 0.18 μg/ml. This inhibition was too closed to butylated hydroxyl toluene (BHT) 82.53% (IC5013.8 ± 0.14) and gallic acid 89.51% (IC50 3.1 ± 0.08). VN extract exhibited the strongest free radical scavenging power compared with two commercial antioxidants, BHT and ascorbic acid. VN increased the activities of antioxidant enzymes in normal embryonic liver cells (WRL68) including, superoxide dismutase (SOD) and glutathione peroxidase (GPX) compared with to H2O2 group. The ethanolic extract of VN showed cytotoxicity to HepG2 cells in a dose and time-dependent manner with IC50 66.46 μg/ml, 57.36 μg/ml and 65.12 μg/ml at 24, 48, and 72-hours incubation respectively, with no sensitivity in WRL68 cells. This was associated with significant elevation in lactate dehydrogenase (LDH) release in HepG2 cells. In addition, the activation of caspase-3 enzyme suggesting that the observed cytotoxicity was mediated via an intrinsic apoptosis pathway.
PASS-predicted plant activity could efficiently help in selecting a promising pharmaceutical leads with high accuracy and required antioxidant and antiproliferative properties. This is the first report on PASS-predicted VN activity.
Hepatocellular carcinoma (HCC) is a frequent tumour worldwide. It approximately accounts for 6% of cancer occurrences among human and overall, it rates as the seventh most common malignancy in males and the ninth most in females . At least, one million new cases of HCC occur annually and mortality of the disease remains high despite the treatment especially in Southeast Asia countries and tropical Africa, which show the highest incidence . Substantial advances have been applied in the chemotherapy regimen for treating patients with HCC; however, still there is an urge to discover and explore effective strategies for its treatment throughout the use of medicinal plants. Some of the most effective cancer treatments to date are natural products or compounds derived from natural products . Therefore, it is common that there was increasing to find the biological activity among plants with approved medicinal uses rather than from plants randomly selected.
Due to absence of an effective chemotherapy for liver cancer, many studies using different cell lines, animal models and human epidemiological trials have been shown to have considerable potential of herbal medicine to act as anti-proliferative agents and have received a special attention lately . To date, there have been several large (at least 7000 participants) trials testing the efficacy of antioxidant supplements in preventing cancer. A recent review of available literature suggests antioxidants function to prevent free radical damage, and that’s important in preventing our bodies from cancers, arthritis, diabetes, autoimmune diseases and 90+ other diseases . The antioxidant activity of phenolics is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors, singlet oxygen quenchers and metal chelators. Meanwhile, the strategy for research and in vitro evaluation of biological activity of natural products has changed in the past few years.
VN is a perfect example of medicinal plant credited by numerous medicinal qualities validated by modern science and used since ancient times. It belongs to the family of Verbenceae, and the genus consists of 250 species and most of them have commercial and medicinal importance . It is a large aromatic shrub and commonly called five leaved chaste tree or Monk’s Pepper . The leaves contain an alkaloid, flavonoids like flavones, luteolin-7-glucoside, casticin, iridoid, glycosides, an essential oil and other constituents like vitamin C, carotene, glucononital, benzoic acid, β-sitosterol and glycoside . The plant has a pungent, bitter, acrid taste used in various traditional treatment of stomach-ache, disease of the eye, inflammation, enlargement of spleen, bronchitis, asthma, as an antihelmintic, to promote growth of hair and painful teething in children .
The isolated compound from the VN extract showed protection of hepatocytes, nephrocytes and pancreatic β-cells probably by its action against NF-kB and induced- nitric oxide synthase iNOS mediated inflammation in streptozotocin-induced diabetes . VN seeds extract showed analgesic, antinociceptive activity  and hepatoprotective activity [11–13].
Apoptosis is a type of programmed cell death; it allows proper development and remodeling of normal tissues, besides generating immune responses and destroying abnormal cells. It is well known that malignant transformation of human cancer cells is a multi-staged process involving mutation or deletions of various human suppressor’s genes. This can lead to activation of oncogenes and alterations in the level of expression of key regulatory genes, providing growth advantages and metastatic potential for tumor cells . Those genetic alterations result in abnormalities in cellular and nuclear morphology and signal transduction pathways. This would normally activate a suicidal pathway and induce apoptosis in the cells leading to defects and/or mutations of p53 delay cell-cycle arrest and abolish the DNA repair process, which otherwise render the cells to apoptosis . Virtual screening is of specific significance to understand the pharmacological action of the plant compounds . The prediction of activity spectra for substances (PASS) software , predicted more than 300 pharmacological effects, biological and biochemical mechanisms based on the structural formula of the substance. This was efficiently used in this study to reveal new multitalented actions for the isolated components of VN extract.
HepG2 cell line represents one of the most widely used experimental model for in vitro studies on HCC . Hence, this work was carried out to investigate the antioxidant, antiproliferative and apoptotic effect of ethanolic extract of VN extract against WRL68 and HepG2 cell lines based primarily on the rich literature review with the support of PASS prediction program.
Computational evaluation of biological activity
The biological activity spectra of the phytoconstituents for VN extract were obtained using the Prediction of Activity Spectra for Substances (PASS) software. PASS prediction tool is constructed using 20,000 principal compounds from the MDDR database (produced by Accelrys and Prous Science) .
Preparation of crude ethanol extract
Fresh leaves of VN plant were obtained from Kampung Baru, Sungai Ara, Penang, Malaysia. The plant was identified and the voucher specimen number (KLU 34968) was deposited in University Malaya (Department of Pharmacy). Dried and ground leaves of VN were weighed, then homogenized in 95% ethanol at a ratio of 1:10 of plant to ethanol and left to soak for 4 days at 25°C while shaking and stirring it occasionally. The mixture was filtered, centrifuged at 14,000 rpm for 10 min and then concentrated under reduced pressure at 45°C to obtain a dark gummy–green extract. The concentrated extracts were then frozen and finally lyophilized with freeze dryer, yielding the crude extract of the leaves of VN.
DPPH scavenging assay
Where Control A is the absorbance of the control reaction. Sample A is the absorbance in the presence of VN extract. The test was conducted in triplicate.
The FRAP assay measures the change in absorbance at 593 nm due to the formation of blue coloured Fe2+ - tripyridyltriazine (Fe - TPTZ) compound from the colourless oxidized Fe3+ form by the action of electron donating antioxidants . The experiment was conducted at 37°C under pH 3.6 condition with a blank sample in parallel. In the FRAP assay, reductants “antioxidants” in the sample reduce Fe (III)/tripyridyltriazine complex, present in stoichiometric excess, to the blue ferrous form, with an increase in absorbance at 593 nm.
Briefly 50 μl from the dissolved extract was added to 1.5 ml freshly prepared and pre warmed (37°C) FRAP reagent (300 mM acetate buffer, pH3.6, 10 mM TPTZ in 40 mM HCl and 20 mM FeCl3.6H2O in the ratio of 10: 1:1) and incubated at 37°C for 10 min. The absorbance of the sample was read against reagent blank (1.5 ml FRAP reagent + 50 μl distilled water) at 593 nm. Increased absorbance of the reaction mixture indicated increased reducing power. Ascorbic acid, galic acid and BHT were used as standards. All analyses were run in triplicate and results averaged.
In vitro VN antioxidant inWRL68 cell lines
In vitro antioxidant for cell line experimental design
Oxidant agent (1000 μM H2O2)
Treatment (100 μg/ml)
Normal (untreated cell group)
No oxidant agent (10 μl medium)
10 μl solvent
H 2 O 2 (Oxidative damaged group)
10 μl solvent
VN ethanol extract (10 μl)
Two types of cells were used; (WRL68) and (HepG2). Both cell types were obtained from Department of Molecular Medicine, Faculty of Medicine, University of Malaya. Cells were cultured in the DMEM, supplemented with 10% fetal bovine serum, penicillin (100 units/ml-streptomycin (100 μg/ml), using 75 cm2 flasks in a 37°C in humidified 5% CO2 incubator.
Where A (sample) is the absorbance of wells containing different concentrations of plant extract and A (control) is the absorbance of control wells containing cell culture medium without samples. The experiment was carried out in triplicates .
Cell observation using an inverted microscope
HepG2 cell lines were cultured in 96-well plates and treated with VN ethanolic extract. The cells were then rinsed with 1× Phosphate Buffer Saline (PBS) (Sigma). Morphological and confluence changes of the cells in VN-treated group 57.36 μg/ml according to IC50 and untreated group for 48 hours were observed under × 10 magnification by a trinocular inverted phase contrast microscope (Olympus, Japan).
Acridine orange/ethidium bromide (AO/EB) staining
Dual staining with acridine orange and ethidium bromide was performed based on the protocol previously described . Cells were seeded in six well plates for 48 hours and subjected to treatment with VN in a dose of 57.36 μg/ml according to IC50. After incubation, the cells were harvested by trypsinization and rainsed with PBS, and then stained with 0.1 mg/ml acridine orange and 0.1 mg/ml ethidium bromide. Stained cell suspension (10 μl) was placed on a clean glass slide and covered with a cover slip. The cells were then observed under a fluorescence microscope (Leica) in both red channel (590 nm) and green channel (520-550 nm).
Lactate dehydrogenase (LDH) assay
To determine the effects of ethanolic extract of VN on membrane permeability in WRL 68 and HepG2 cell lines, LDH release assay was done using (Cayman Chemical Company, USA) LDH Cytotoxicity Assay Kit, (item No. 10008882). The presence of LDH enzyme in the cell culture medium is an indication of cell membrane damage . Basically, LDH cytotoxicity assay kit measures cell death in response to chemical compounds using a coupled two-step reaction. In the first step, LDH catalyzes the reduction of NAD+ to NADH and H+ by oxidation of lactate to pyruvate. In the second step of the reaction, diphorase uses the newly-formed NADH and H+ to catalyze the reduction of a tetrazolium salt (INT) to highly-coloured formazan which absorbs strongly at 490-520 nm. The amount of formazan produced is propotional to the amount of LDH released into the culture medium as a result of cytotoxicity.
The cells were seeded in a 96-well plate at a density of 104-105 cells/well in 120 μl of culture medium with or without compounds to be tested.
Detection of apoptosis of HepG2 cells by measuring caspase- 3 enzyme activity
Caspase-3 activity was assessed using the caspase-3Colorimetric Assay Kit (BioVison), following the manufacturer’s instructions is based on spectrophotometric detection of the chromophore p-nitroaniline (p NA) after cleavage of a specific substrate DEVD-pNA.
The HepG2 cells were seeded in sterile 60 mm dishes, and at the end of VN treatment, the cells were washed with PBS and lysed in lysis buffer provided by the kit. After freezing and thawing three times, the cell lysate was centrifuged at 20,000× g at 4°C for 15 minutes. The supernatants were collected and DEVD-pNA was then added and incubated for 1–2 hours at 37°C. The concentration of the pNA released was measured at 405 nm, and the quantity of pNA was calculated from a calibration curve of pNA standard. Caspase-3 activity was expressed spectrophotemetrically compared to the control untreated cells.The experiment was carried out in triplicates .
The analysis of variance (ANOVA) was used to determine differences between treated and control groups with p < 0.05 being considered as statistically significant, using SPSS programme for Windows version 18.0 (SPSS Inc. Chicago, IL, USA).
Results and discussion
PASS prediction and assistant experimental design
In order to accelerate the research for potent natural products, computer-aided drug discovery program PASS was used to predict the antioxidant and antioproliferative properties. PASS prediction tools were constructed using 20000 principal compounds  and about 4000 kinds of biological activity on the basis of structural formula with mean accuracy about 90% . The result of prediction is presented as the list of activities with appropriate Pa and Pi ratio.
Pa and Pi are the estimates of probability for the compound to be active and inactive, respectively. It is reasonable that only those types of activities may be revealed by the compound, which Pa > Pi. If Pa > 0.3 the compound is likely to reveal this activity in experiments, but in this case the chance of being the analogue of the known pharmaceutical agents for this compound is also high. Thus, potential biological effects of the plant constituents were predicted by PASS program based on structure activity relationship (SAR) analysis of the training set containing thousands of compounds which have many kinds of biological activity. Therefore, before we started our experiments, we used PASS program to validate whether VN constituents based on SAR strategy is in agreement with the SAR of the training set of the PASS database.
Part of PASS for VN chemical compounds
Lipid peroxidation inhibitor
Free radical scavengers
Ferric Reducing antioxidant power (FRAP) of VN extract
The phenolic components most frequently represented in ethanol extracts from VN: negundoside, agnuside, vitegnoside, 7,8 dimethyl herbacetin 3-rhamnoside, 5,3′-dihydroxy—7,8,4′-trimethoxy flavanone, 5-hydroxy-3,6,7,3′,4′-pentamethoxy flavone, 5,7 dihydroxy- 6,4′ dimethoxy flavonone, and 5 hydroxy-7,4′ dimethoxy flavone, and among these, negundoside is the most active phenol acting as an antioxidant. It can protect against CCl4-induced toxicity and oxidative stress. The mechanism of protection involves decreased production of ROS and lipid peroxidation. The agnuside, vitegnoside and flavonoids present in the plants are also natural antioxidants [12, 13].
IC 50 value and percentage inhibition of the DPPH radical scavenging assay
Max. inhibition% ± SEMa
48.4 ± 0.01
2.58 ± 0.4
IC50 b value μg/l ± S.E.M
13.31 ± 0.18
13.8 ± 0.14
3.1 ± 0.08
12.9 ± 0.12
The FRAP assay measures the ferric [Fe3+-TPTZ]-to-ferrous [Fe2+-TPTZ] iron reduction in the presence of antioxidants. FRAP assay treats the antioxidants in the sample as a reductant in a redox-linked colorimetric reaction . The trend for ferric ion reducing activity of VN against BHT, gallic acid and ascorbic acid are shown in Figure 2. VN exhibited the strongest free radical scavenging power compared with two commercial antioxidants, BHT and ascorbic acid. This seems to suggest that VN extract can donate electron easily. This activity is believed to be mainly due to their redox properties. Hence VN extract should be able to donate electrons to free radicals stable in the actual biological and food system.
The ethanolic extract of VN was found to be an effective scavenger of DPPH and FRAP with a good reducing power activity. The high antioxidant activity of VN enhanced the potential interest in this plant for improving the efficacy of different products as nutraceutical and pharmacological agents.
In vitro antioxidant of VN for WRL68 cell lines
Effects of H 2 O 2 , VN extract on the antioxidant enzymes and MDA level on H 2 O 2 – induced WRL-68 cell line
SOD (U/mg protein )
GPX (nmol/ mg protein )
MDA (nmol/ g protein )
10.61 ± 0.010
27.14 ± 0.30
17.00 ± 1.019
H 2 O 2
10.00 ± 0.065
10.98 ± 0.18a
46.94 ± 1.730a
10.37 ± 0.150
22.05 ± 0.19b
23.17 ± 2.165b
The role of oxidative stress and tissue damage in diseases, such as atherosclerosis, heart failure, neurodegenerative disorders, aging diabetes mellitus, hypertension and other several diseases are gaining a lot of recognition . Reactive oxygen species (ROS) are prospective carcinogenic substances because of the generating free radicals including hydroxyl, superoxide, peroxyl, hydroperoxyl, and alkoxyl radicals, which participate in tumor promotion, mutagenesis and progression. If there is no effective regulation, the excess ROS will damage proteins, lipids or DNA and in turn inhibition of the normal function through the modulation of gene expression, cell cycle, cell metabolism, cell adhesion and cell death .
Glutathione is the major endogenous antioxidant scavenger that protects cells from oxidative stress through its ability to bind to and reduce ROS . Thus, preserving the glutathione-mediated antioxidant defense is critical for cell survival. Glutathione is formed by γ-glutamate cysteine ligase (γ -GCL) and glutathione synthetase . The ethanol extract of VN increased the GPX and SOD activity, which indicates that this extract can effectively scavenge H2O2. The effects of the ethanol extract of VN on cell viability might involve dual actions: the direct action of oxygen radical scavenging, as shown by the DPPH radical scavenging by ethanol extract (Figure 1) and the indirect action via the induction of the antioxidant enzymes of SOD and GPX (Table 4). Furthermore, the level of lipid peroxidation was significantly higher in the cells exposed to H2O2, while the treatment with VN extract apparently attenuated the MDA level. This might reflect an idiosyncrasy of the in vitro system used in this study.
Cytotoxic effect of VN extract on HepG2 and WRL 68 cell lines
Comparison of IC 50 values for HepG2 and WRL 68 cells obtained from MTT assay following exposure to VN extract for 24,48 and 72 hours
66.46 ± 2.8
57.36 ± 1.3
65.12 ± 1.8
The cytotoxicity or anticancer activity of the crude extract expressed as the inhibitory of concentration (IC50). The sensitivity of HepG2 cells to VN is characterized by IC50. The lower the IC50 value indicated the higher anticancer effect of the sample. These results indicate that elevated anticancer effects strengthened with dose time of exposure (Table 5).
Until now, no ideal cytotoxicity test has been developed; hence, in this study, we have screened this type of plant which is native to South Eastern Asian countries for treating a variety of ailments, including cancer by using two-cell lines, WRL68 and HepG2 cells. The micro-culture tetrazolium salt (MTT) assay was used in this study to measure the amount of cell viability. This method is based on the quantification of purple-coloured formazan, which was formed by the reduction of MTT [3-(4, 5-dimethylthiazole-2-yl)-2,5-diphenyl. The potential antiproliferative effect of ethanolic crude extract of VN was investigated, determining their effect on the viability of (HepG2) and (WRL 68). The reduction of MTT is proportional to the number of active mitochondria in the live cells. The results indicated that VN extract caused significant inhibition of HepG2 cells in a dose and time-dependent manner. Generally, it was found that VN extract at IC50 57.36 μg/ml was cytotoxic for HepG2 cell lines after 48 hours of exposure, meanwhile, there was no IC50 value of VN extract against WRL 68 cell lines in all concentrations and incubation periods as shown in Table 5.
Morphological observation by inverted microscope
Morphological observation by acridine orange and ethidium bromide (AO/EB) staining
On the other hand, HepG2 cells were more sensitive to VN as compared to WRL 68 cells, LDH was released in a time and dose dependent manner.
Recent studies have shown that LDH is an accurate and more reliable marker of cytotoxicity, because damaged cells are fragmented completely during the higher concentration of VN extract and the path of prolonged incubation with substances [36, 37]. In this report, LDH amount from HepG2 cells was released in a time and dose dependent manner as shown in Figure 8 and this indicted that VN extract is less cytotoxic on WRL 68 cell lines and the highest concentration of VN (200 μg/ml) showed the highest toxicity on both cell lines. Based on MTT spectrophotometric assay, VN showed high antiproliferative activities toward HepG2 cell lines in a dose and time-dependent manner Figure 4A. The sensitivity of HepG2 cells to VN is characterized by IC50 value. These results indicate that elevated antiproliferative effects strengthened with the dose and time of exposure. This was based on the average of three sets of experiments. To prove that the apoptosis has been influenced by VN ethanolic extract, HepG2 cells were examined in the presence of acridine orange and ethidium bromide staining (AO/EB staining). Acridine orange is a vital dye that will stain both live and dead cells, whereas ethidium bromide will stain only those cells that have lost their membrane integrity . Cells stained green stand for viable cells, whereas reddish or orange staining illustrates late apoptotic cells. As shown in Figure 7, HepG2 cells treated with 200 μg/ml of ethanolic extract showed changes in cellular morphology, including chromatin condensation, membrane blebbing, and fragmented nuclei. Therefore, we can assume that stronger apoptosis is associated with high concentration of VN extract.
This high antiproliferative effect of VN was related to the presence of bioactive compounds such as alkaloid, flavonoids luteolin-7-glucoside, casticin, iridoid, glycosides, an essential oil and other constituents like ascorbic acid, carotene, glucononital, benzoic acid, β-sitosterol and glycoside . These results are consistent with previous study which indicated that glycosides and flavones compounds possessing potent anticancer properties against MCF-7 human breast cancer cells .
Apoptosis represents an effective way to alleviate damaged cells through the activation of caspase and to balance the cellular proliferation . Human caspase cascade is involved in chemical-induced apoptosis, caspase-3 may cleave essential cellular proteins or activate additional caspases by proteolytic cleavage .
To understand the molecular mechanism of VN induced growth inhibition, we found that there was a marked increase in the activation of caspase-3, suggesting that caspase dependent apoptotic death could be another mechanism for the beneficial effects of VN, because it is well established that activation of caspase lead to degradation of cellular proteins, cell shrinkage, DNA fragmentation, loss of plasma membrane potential and membrane blebbing . The activation of caspase-3 induced chromosomal DNA break and finally the occurrence of apoptosis [41, 42].
In the present investigation, VN extract showed the activation of caspase-3 enzyme mediated apoptosis in HepG2 cells, and this might due to the presence of glycosides and flavones. This result is in agreement with a previous report which showed that certain products from plants can induce apoptosis in cancerous cells like OCM-1, MCF-7 and HT-29 .
PASS-prediction of VN activity has successfully applied and efficiently helped in selecting the most promising pharmaceutical leads with required properties and high accuracy. It would save unnecessary wastage of chemicals and time by avoiding random plant selection methods. It can be seen from the results of PASS that most probable activities are antioxidant, antiproliferative and hepatoprotectants. Therefore, the present approach can be very useful in plant prediction activity according to their required properties without undesirable side effects. This study strongly suggests that VN extract significantly enhanced antioxidant activity and proposed a tumour preventive action against HepG2 cell lines at a dose and time-dependent manner but with lower toxicity toward WRL68 cells. In addition, the morphological analysis using AO/EB staining procedure revealed that the growth and proliferation inhibition is through proteolytic cleavage of caspase-3 protein and intrinsic apoptosis pathway. However, further studies are needed to determine the molecular mechanisms of the active components and to evaluate the potential in vivo anticancer activity of VN extract.
The financial support from (PG087-2012B) Grant, University of Malaya, Malaysia is gratefully acknowledged.
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