{'fraction_non_alphanumeric': 0.04382164628301959, 'fraction_numerical': 0.026952421117202776, 'mean_word_length': 4.837958818263205, 'pattern_counts': {'":': 0, '<': 4, '<?xml version=': 0, '>': 3, 'https://': 0, 'lorem ipsum': 0, 'www.': 2, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Phytochemicals from Rhizophora mucronata Propagules, Its In Vitro Anti-Cancer and In Silico Drug-Likeness Potential

: This is the ﬁrst report to identify the presence of 3- O -caffeoyl quinic acid ( 1 ), 4- O -caffeoyl quinic acid ( 2 ), 5- O -caffeoyl quinic acid ( 3 ), epi-catechin ( 4 ), and procyanidin B2 ( 5 ) in the young propagules of Rhizophora mucronata . Compounds 2 - 5 were puriﬁed and then treated against breast, colorectal, and ovarian cancer cell lines for 72 h and the results of the Sulphorhodomine-B (SRB) assay were evaluated for percent cell viability and IC 50 values. Epi-catechin, 4- O -caffeoyl quinic acid, 5- O caffeoyl quinic acid and procyanidin B2 showed strong to moderate inhibitory effects when treated on breast (T47D), colorectal (HT29), and ovarian (A2780, SKOV3) cancer cell lines with IC 50 values ranging from 16.77 ± 0.58 to 28.28 ± 0.89 µ g/mL. In silico evaluation was performed to evaluate the drug-likeness and toxicological effects of these compounds using Molinspiration calculation and OSIRIS program. It was found that compounds 2 , 3 , and 4 have the potential to be orally active and have a low risk in exerting the mutagenic, tumorigenic, irritant, and reproductive effects. was monitored at 280 and 330 nm. of 3- O -caffeoyl quinic acid O -caffeoyl quinic acid O -caffeoyl quinic acid (CAS and epi-catechin (CAS 490-46-0) were used for the identiﬁcation of major peaks in the chromatogram.


Introduction
Rhizophora mucronata (Rhizophoracea) is one of the mangrove plant species that can be found in the tropical and subtropical coastal areas and river estuaries from the east coast of Africa to Asia and Australia, and its hardy wood has been popularly used as fuel and charcoal [1]. The bark, leaves, and fruits consist of tannin [2] along with other phytochemicals that were later scientifically discovered. Mangrove plants are not only ecologically important in protecting the coastal area from soil erosion and strong tidal waves, but they also have various medicinal uses. The bark and leaves of R. mucronata are used to treat diarrhea, dysentery, fever, malaria, leprosy inflammation, and angina disorders [1,3]. The plant has also been reported to be used to treat tonsillitis, pharyngeatis, hemorrhoids, and burns [4]. The bark is claimed to have astringent and anti-diabetic properties [2].
The ethanol and methanol extracts from the leaves of R. mucronata have been found to have anti-microbial properties in which these extracts inhibited the growth of Escherichia coli, Staphylococcus aureus, and Bacillus cereus [12], while the ethyl acetate extract inhibited the growth of E. coli, S. aureus, Klebsiella pneumonia, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas fluorescens, Salmonella typhii, and Bacillus subtilis [13]. The leaves have also been reported to have anti-oxidative [14], antinociceptive [8,15], and anti-HIV activities [16]. A compound from the leaves of R. mucronata identified as quinizarin showed anti-microbial activity against Bacillus cereus, Klebsiella aerogenes, anti-oxidant activity via the DPPH radical scavenging assay, and cytotoxic activity against cervical HeLa and MDA-MB231 breast cancer cell lines [17]. Taniguchi et al. 2018 [11] reported that a xanthone (jacareubin) compound isolated from the leaves showed cytotoxic potential against HeLa and HL60 cancer cell lines.
Fewer phytochemical and bioactivity studies have been reported for other parts of the plant, including roots and fruits or propagules. Beyerane diterpenoid (Rhizophorin B) and isopimarane diterpenoids have been isolated from the ethyl acetate extract of R. mucronata roots [25], while sesquiterpene (mucronatone) and pentacyclic triterpenoid esters (3β-E-caffeoyltaraxerol and 3β-Z-caffeoyltaraxerol) have been isolated from R. mucronate fruits [26]. The extract from R. mucronata stilt root has in vitro antioxidant properties [27] and larvicidal activity [28]. To our knowledge, however, scientific studies on the medicinal properties of fruits and propagules of R. Mucronata are still lacking. Therefore, the aim of this study was to isolate and identify the chemical constituents present in the propagules of R. mucronata and to evaluate their anti-cancer activities in vitro against some cancer cell lines with their drug-likeness potential.

Materials and Methods
Chemicals and general methods: All reagents and solvents used were of analytical or gradient grade for liquid chromatography. 1 H and 13 C NMR spectra were recorded in ppm (δ) in CD 3 OD and acetone-d 6 employing a Bruker DRX 300 spectrometer operating at 300 MHz for 1 H and 75 MHz for 13 C, respectively. Column chromatography was performed with MCI gel CHP 20P (75-150 µm, Supelco, Sigma-Aldrich, St. Louis, MO, USA), Chromatorex ODS (100-200 mesh, Fuji Silysia Chemical Ltd., Aichi, Japan) and Sephadex LH-20 (GE Healthcare Bio-Science AB, Uppsala, Sweden). HPLC analysis was carried out on a Waters Delta 600 system, equipped with an in-line degasser, a quaternary pump, an auto sampler, and a diode array detection (DAD) system, connected to an analytical column (Phenomenex LUNA PFP(2)). Data collection was performed using Empower software (Waters). The water used for all the solutions and dilutions was prepared with an ELGA Purelab Classic water purification system. Bransonic Series ultrasonic bath was used for extraction. TLC was performed on precoated silica gel 60 F 254 plates (0.2 mm thick, Merck) with CHCl 3 −MeOH−H 2 O (9:1:0.1 or 8:2:0.1 or 7:3:0.5 or 6:4:1), and spots were detected by UV illumination and by spraying with 10% H 2 SO 4 solution followed by heating or spraying with 2% FeCl 3 solution followed by drying with a stream of cold air.
Plant material: Young and old propagules of R. mucronata were collected from Carey Island, Selangor, in Malaysia and the samples were coded as BK(A), BK(B), and BK(C) (Figure 1). The voucher specimen was prepared and deposited with number NMY1-KEP 245125 in the herbarium of FRIM, Kepong.
Chemistry 2021, 3, FOR PEER REVIEW3 AB, Uppsala, Sweden). HPLC analysis was carried out on a Waters Delta 600 system, equipped with an in-line degasser, a quaternary pump, an auto sampler, and a diode array detection (DAD) system, connected to an analytical column (Phenomenex LUNA PFP(2)). Data collection was performed using Empower software (Waters). The water used for all the solutions and dilutions was prepared with an ELGA Purelab Classic water purification system. Bransonic Series ultrasonic bath was used for extraction. TLC was performed on precoated silica gel 60 F254 plates (0.2 mm thick, Merck) with CHCl3−MeOH−H2O (9:1:0.1 or 8:2:0.1 or 7:3:0.5 or 6:4:1), and spots were detected by UV illumination and by spraying with 10% H2SO4 solution followed by heating or spraying with 2% FeCl3 solution followed by drying with a stream of cold air.
Plant material: Young and old propagules of R. mucronata were collected from Carey Island, Selangor, in Malaysia and the samples were coded as BK(A), BK(B), and BK(C) (Figure 1). The voucher specimen was prepared and deposited with number NMY1-KEP 245125 in the herbarium of FRIM, Kepong. Preparation of the crude plant extracts: The sliced fresh samples were immediately extracted by soaking in methanol (fresh weight/volume, 1 g/10 mL) for 3 days at room temperature. After that, the solvent was filtered, and the residue was extracted again using methanol with the same weight/volume ratio for another 3 days at room temperature. The solvent was evaporated under reduced pressure using a rotary evaporator with a bath temperature of 45 °C. The extraction process was repeated for the third time. The combined extracts were then stored at −20 °C for further analysis.
Isolation, purification, and characterization of pure compounds: The concentrated methanol extract (150 g) of the hypocotyl of young propagule coded as BK(B) was fractionated through liquid column chromatography employing MCI gel CHP 20P. The resulting fractions of eluent were combined to three fractions based on the thin layer chromatographic profiles. Fraction 2, encoded as BK (2), which had the most visible spots on thin layer chromatography, was further fractionated and purified, employing a combination of Sephadex LH-20, Chromatorex ODS, and MCI gel CHP 20P to yield four thin layer chromatographically homogeneous compounds (compounds 2-5) as shown in Figure 2. The identities and structures of these compounds were deterimined by 1 H and 13 C NMR spectroscopic analyses and by comparison with values in the literature or standard compounds to be 4-O-caffeoyl quinic acid (2), 5-O-caffeoyl quinic acid (3), Epicatechin (4), and procyanidin B2 (5). Preparation of the crude plant extracts: The sliced fresh samples were immediately extracted by soaking in methanol (fresh weight/volume, 1 g/10 mL) for 3 days at room temperature. After that, the solvent was filtered, and the residue was extracted again using methanol with the same weight/volume ratio for another 3 days at room temperature. The solvent was evaporated under reduced pressure using a rotary evaporator with a bath temperature of 45 • C. The extraction process was repeated for the third time. The combined extracts were then stored at −20 • C for further analysis.

BK (a) BK (b) BK (c)
Isolation, purification, and characterization of pure compounds: The concentrated methanol extract (150 g) of the hypocotyl of young propagule coded as BK(B) was fractionated through liquid column chromatography employing MCI gel CHP 20P. The resulting fractions of eluent were combined to three fractions based on the thin layer chromatographic profiles. Fraction 2, encoded as BK (2), which had the most visible spots on thin layer chromatography, was further fractionated and purified, employing a combination of Sephadex LH-20, Chromatorex ODS, and MCI gel CHP 20P to yield four thin layer chromatographically homogeneous compounds (compounds 2-5) as shown in Figure 2. The identities and structures of these compounds were deterimined by 1 H and 13  Cancer cell lines, cell culture maintenance, and treatments: Ovarian (SKOV-3), (T47D), and colorectal (HT-29) cell lines were purchased from American Type C Collections, USA, and A2780 ovarian cancer cell line was purchased from Eur Collection of Cell Cultures (ECACC), UK. The cell lines were cultured and sub-cu in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 5% fetal b serum (FBS), 1% penicillin-streptomycin, 0.25% amphotericin B, and 1% gentam Approximately 4000 to 6000 cells were seeded in each well of the 96 well plate incubated in a humidified incubator at 37 °C and 5% carbon dioxide in air for 24 h cell line was then treated with the extracts and compounds obtained from R. mucron five different concentrations (0.1, 1, 10, 20, and 100 μg/mL) in triplicate. Cisplatin, a k chemo-drug, was also treated on these cell lines in different concentrations (0.25, 1, and 50 μg/mL) as in the comparative studies. The treated cells were then incubated same incubator with the mentioned conditions for 72 h. The experiment was repea least three times.
In vitro anti-cancer activities assessed via Sulphorhodamine B (SRB) assay: Th treatments were stopped by performing Sulphorhodamine B (SRB) assay [29,30]. B 50 μL of ice cold tricholoroacetic acid (TCA) was added to each well and allowed to for 30 min at room temperature, followed by rinsing each well with tap water. The μL of 0.4% SRB was added to each well to stain living cells for 30 min followed by a Cancer cell lines, cell culture maintenance, and treatments: Ovarian (SKOV-3), breast (T47D), and colorectal (HT-29) cell lines were purchased from American Type Culture Collections, USA, and A2780 ovarian cancer cell line was purchased from European Collection of Cell Cultures (ECACC), UK. The cell lines were cultured and sub-cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 1% penicillin-streptomycin, 0.25% amphotericin B, and 1% gentamycin. Approximately 4000 to 6000 cells were seeded in each well of the 96 well plates and incubated in a humidified incubator at 37 • C and 5% carbon dioxide in air for 24 h. Each cell line was then treated with the extracts and compounds obtained from R. mucronata at five different concentrations (0.1, 1, 10, 20, and 100 µg/mL) in triplicate. Cisplatin, a known chemo-drug, was also treated on these cell lines in different concentrations (0.25, 1, 5, 25, and 50 µg/mL) as in the comparative studies. The treated cells were then incubated in the same incubator with the mentioned conditions for 72 h. The experiment was repeated at least three times.
In vitro anti-cancer activities assessed via Sulphorhodamine B (SRB) assay: The 72 h treatments were stopped by performing Sulphorhodamine B (SRB) assay [29,30]. Briefly, 50 µL of ice cold tricholoroacetic acid (TCA) was added to each well and allowed to stand for 30 min at room temperature, followed by rinsing each well with tap water. Then, 100 µL of 0.4% SRB was added to each well to stain living cells for 30 min followed by a rinse with 1% acetic acid. Finally, 100 µL of Tris buffer was added to each well and the optical density (OD) of the treated and non-treated cells were read at 492 nm with a Magellan V.4 microtiter plate reader (Tecan, Switzerland). The percentage of cell viability was calculated based on (OD 492nm of the treated cells/ OD 492nm of the non-treated cells) × 100. The IC 50 values were determined from the dose-response curve of percentage of cell viability versus the concentration of the extracts (µg/mL). Cell viability assays for each treatment were performed in triplicate in at least three independent experiments, and the IC 50 values are given as the mean ± S.D.
In silico analysis: In silico analysis of drug-likeness and ADMET properties were performed based on Lipinski's Rule of Five (ROF) using a free online server. Prediction of drug-likeness and bioactivity score was conducted using Molinspiration (Molinspiration Chemiformatics, Nova ulica 61, SK-900 26 Slovensky Grob, Slovak Republic) and OSIRIS property explorer for calculation of the physicochemical properties (www.organicchemistry. org/prog/peo/, accessed on 9 May 2019). Prediction on ADMET of the compounds was performed using vNN-ADMET [31] and OCHEM [32].

Isolation, Purification, and Identification of Compound
The methanol extract, BK(B), exhibited IC 50 values of ≤20 µg/mL for three of the cancer cell lines used in the in vitro anti-cancer study, indicating very potent anti-cancer effects [33,34]. with 1% acetic acid. Finally, 100 μL of Tris buffer was added to each well and the optical density (OD) of the treated and non-treated cells were read at 492 nm with a Magellan V.4 microtiter plate reader (Tecan, Switzerland). The percentage of cell viability was calculated based on (OD492nm of the treated cells/ OD492nm of the non-treated cells) × 100. The IC50 values were determined from the dose-response curve of percentage of cell viability versus the concentration of the extracts (μg/mL). Cell viability assays for each treatment were performed in triplicate in at least three independent experiments, and the IC50 values are given as the mean ± S.D.
In silico analysis: In silico analysis of drug-likeness and ADMET properties were performed based on Lipinski's Rule of Five (ROF) using a free online server. Prediction of drug-likeness and bioactivity score was conducted using Molinspiration (Molinspiration Chemiformatics, Nova ulica 61, SK-900 26 Slovensky Grob,Slovak Republic) and OSIRIS property explorer for calculation of the physicochemical properties (www.organicchemistry.org/prog/peo/, accessed on 9 May 2019). Prediction on ADMET of the compounds was performed using vNN-ADMET [31] and OCHEM [32].

Isolation, Purification, and Identification of Compound
The methanol extract, BK(B), exhibited IC50 values of ≤20 μg/mL for three of the cancer cell lines used in the in vitro anti-cancer study, indicating very potent anti-cancer effects [33,34]. The isolation and subsequent purification activities of the BK(B) extract identified four compounds known as 4-O-caffeoyl quinic acid (2) (3)

In Vitro Anti-Cancer Studies
The in vitro anti-cancer activities of extracts, fractions, and compounds of Rhizop mucronata were evaluated on the basis of theirIC50 values (

In Vitro Anti-Cancer Studies
The in vitro anti-cancer activities of extracts, fractions, and compounds of Rhizophora mucronata were evaluated on the basis of theirIC 50 values ( Table 1). Phytochemicals that give an IC 50 value ≤ 20 µg/mL indicate very potent anti-cancer activities [33,34]. The results indicate that the four compounds have inhibitory effects with IC 50 values of less than 100 µg/mL. 4-O-caffeoyl quinic acid was found to be the most active of the four compounds in the treatment of ovarian (SKOV3) and colorectal (HT29) cancer cell lines, followed by epicatechin and 5-O-caffeoyl quinic acid, which are also active against ovarian (A2780) cancer cell lines. The compound procyanidin-B2 showed moderate to almost very potent anti-cancer activities in all cancer cell lines tested.

In Silico Analysis on Drug-Likeness and Toxicity of Selected Compounds
The evaluation of the drug-likeness properties of four compounds isolated from BK(B) showed that all compounds, except procyanidin B2, fulfilled Lipinski's Rule of Five (RoF) ( Table 2). Procyanidin B2 violated three of the RoF, namely molecular weight (578.5 > 500 Da), number of hydrogen acceptors (12 > 10), and number of hydrogen donors (10 > 5). The bioactivity score indicated that all compounds were active and moderately active against normal human receptors (Table 3). Interestingly, all of the compounds were active against nuclear receptor ligands, GPCR ligands, protease inhibitors, and enzyme inhibitors. Epicatechin is the only compound that was active against all of the predicted receptors, with the highest score for the nuclear receptor ligand (0.60), but the highest score for this receptor was 5-O-caffeoyl quinic acid (0.74). Prediction of toxicity showed that not all compounds have mutagenic, tumorigenic, reproductive, and irritant potential, except procyanidin B2, which displayed a high risk of reproductive effects (Table 4). However, as shown in Table 5, not all compounds were cytotoxic. The results of ADMET prediction are summarized in Table 5. All compounds showed no inhibition of cytochrome P450 metabolism (CYP3A4, CYP2D6, CYP2C19, CYP2C6, and CYP1A2), as predicted by OCHEM and vNN-ADMET. Table 2. Physicochemical properties of compounds isolated from BK(B).   Table 4. Toxicity prediction of compounds isolated from BK(B).

Discussion
To the best of our knowledge, these are the first reported results for all four compounds, namely epi-catechin, 4-O-caffeoyl quinic acid, 5-O-caffeoyl quinic acid, and procyanidin B2, which were isolated from the propagules of R. mucronata. Although these compounds have newly been isolated and identified in the species, they can be found in other plant species, including many types of fruits and vegetables. The 4-O-caffeoyl quinic acid and 5-O-caffeoyl quinic acid are chlorogenic acids (CGA) that are found in many species of plants, and the most notable one that is rich in CGAs is the green coffee bean [42]. CGAs are phenolic acids which are obtained from the esterification of cinnamic acids with quinic acid. CGAs exhibit several biological properties, including antioxidant, anti-inflammatory, antidiabetic, anticarcinogenic, and antibacterial activities. 5-O-caffeoyl quinic acid is the main CGA of arabica and robusta green coffee beans [42]. Chlorogenic acids, which include 4-O-caffeoyl quinic acid and 5-O-caffeoyl quinic acid, and caffeic acid have been shown to have an additive anti-proliferative effect with cisplatin on ovarian cancer cells [43].
Epi-catechin is one of the catechin polyphenols, which is one of the main compounds in green tea and cocoa [44,45]. Cocoa powder, which is rich in epi-catechin, has been found to be active and kill pancreatic cancer cells through apoptosis [46]. However, another in vitro anti-cancer study suggested that epi-catechin was less active than other catechins, such as epigallocatechin-3-gallate, when tested in prostate (DU-145) and ovarian cancer (HH639) cells [44].
Assessment of the drug-likeness properties of four compounds isolated from young propagules of R. mucronata uses the criteria of Lipinski's Rule of Five (RoF). The criteria are often used in drug design and development to predict the oral bioavailability of potential lead or drug molecules. According to Lipinski's RoF, a compound is more likely to be orally active if it has no more than one violation of the following criteria: Log P is less than 5; molecular weight is less than 500 Da; hydrogen bond donor is less than 5; and hydrogen bond acceptor is less than 10 [47]. Among the four compounds, epi-catechin, 5-O-caffeoyl quinic acid, and 4-O-caffeoyl quinic acid fulfilled Lipinski's RoF, with only one violation being observed for the latter two compounds. Epi-catechin was found to have no violation to the rule, with a TPSA below 160Å 2 , molecular weight < 500 Da, number of hydrogen donors < 5, number of hydrogen acceptors < 10, and number of rotatable bonds < 10.
Bioactivity score, as shown in Table 3, indicated that all compounds were active and moderately active against normal human receptors. A molecule with a bioactivity score greater than 0.00 is considerably active, while values from −5.00 to 0.00 are moderately active, and if the score is less than −5.00 it is considered to be inactive. Epi-catechin was active against all receptors, i.e., GPCR ligands, ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor, and enzyme inhibitor. 5-O caffeoyl quinic acid and 4-O caffeoyl quinic acid were also active against these biological targets, but were moderately effective against the kinase inhibitor. Procyanidin B2 was only active against three biological targets, namely GPCR ligand, nuclear receptor, protease inhibitor, and enzyme inhibitor, and moderately active against the other two receptors.
The ADME assessment was conducted using OCHEM and vNN-ADMET webservers. The results are summarized in Table 5. All compounds showed no inhibition of cytochrome P450 metabolism, which was predicted using both servers, with the exception of procyanidin B2, which showed conflicting results for the CYP1A2 inhibitor. Cytochrome P450 enzymes are essential for the metabolism of drugs. While there are more than 50 CYP450 enzymes, CYP3A4, CYP2D6, CYP2C19, CYP2C9, CYP1A2, and CYP3A5 enzymes metabolize 90 percent of drugs [48,49]. The human liver microsomal (HLM) stability assay is commonly used to identify and exclude compounds that are too rapidly metabolized. In order to achieve the effective therapeutic concentrations in the body, he molecule or drug cannot be metabolized too rapidly by the liver. Compounds with half-life of 30 min or longer in an HLM assay are considered to be stable; otherwise, they are considered unstable. All compounds have been classified as stable in the liver.
All compounds were classified as BBB non-permeable. BBB is a highly selective barrier that separates the circulating blood from the central nervous system. Recently, many methods have been explored to improve the permeability of BBB, including strategies in the field of physical, chemical, biological, and various nanoparticle systems [50]. P-glycoprotein (P-gp) is an essential cell membrane protein and plays an important role in the absorption and disposition of drugs. This drug transporter is also involved in the metabolism, distribution, and interaction of drugs. In this study, it was shown that all compounds are not P-gp inhibitors, however epi-catechin and procyanidin B2 were classified as substrates of P-gp. P-gp is not only important in ADMET issues, but is also extended to a field of multidrug resistance cancer [51]. Identification of compounds that are either transported out of the cell by P-gp (substrates) or impair the function of P-gp (inhibitors) is of great interest.
In this study, several toxicity parameters were predicted with OSIRIS, OCHEM, and vNN-ADMET. The prediction of the toxicity of compounds with the OSIRIS software revealed that all compounds have no mutagenic, tumorigenic, reproductive, or irritant potential, however procyanidin B2 has shown a high risk of reproductive effects (Table 4). In terms of cytotoxicity, mitochondrial toxicity (MP), and mutagenicity (AMES test), all compounds were found to be non-toxic, except for procyanidin B2, which was predicted by OCHEM to be active (Table 5). Epi-catechin and procyanidin B2 were classified as hERG blockers, while the other two compounds were classified as non-blockers. In the vNN-ADMET prediction program, compounds with an IC 50 cutoff value of 10 µM or less were classified as blockers, while compounds with an IC 50 greater than 10 µM were classified as non-blockers. It was predicted that only procyanidin B2 did not cause drug-induced liver injury damage (DILI), while the other three compounds were at high risk of causing DILI.

Conclusions
Propagules of R. mucronata have anti-cancer properties and four compounds have been identified. 4-O-caffeoyl quinic acid, epi-catechin and 5-O-caffeoyl quinic acid were shown to be active, but procyanidin B2 showed moderate inhibition of ovarian cancer cell proliferation. Of the four compounds, epi-catechin met the ADMET criteria and showed the highest drug score (0.871). Toxicological risk and metabolic prediction show that all four compounds were non-corrosive, with the exception of epi-catechin, which was believed to be irritating or corrosive to the skin. Epi-catechin can be seen as one of the most important potential compounds for the development of new drugs, e.g., as an anti-cancer agent, but efficacy, mechanism of action, and toxicity studies need further validation at the in vitro and in vivo levels.