Toxicity Study of Dibutyl Phthalate of Rubia cordifolia Fruits: In Vivo and In Silico Analysis Amrita Anantharaman,1 Rajendra Rao Priya,1 Hridya Hemachandran,1 Sivaramakrishna Akella,2 Chandrasekaran Rajasekaran,1 Jai Ganesh,1 Devanand P. Fulzele,3 Ramamoorthy Siva1 1
School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu 632014, India
2
School of Advanced Sciences, VIT University, Vellore, Tamil Nadu 632014, India
3
Plant Biotechnology and Secondary Metabolites Section, NA&BTD, Bhabha Atomic Research Centre, Mumbai, Maharashtra 400 094, India
Received 19 August 2014; accepted 29 December 2014 ABSTRACT: Natural toxins from plant sources with wide ranges of biological activities reflect the upswing of drug design in the pharmaceutical industry. Rubia cordifolia L. is one of the most important red dye yielding plants. Most of the former researches have focused on the bioactive compounds from the roots of R. cordifolia, while no attention was paid towards the fruits. For the first time, here we report the presence of dibutyl phthalate in the fruits of R. cordifolia. Structural characterization was carried out using Ultraviolet–Visible spectrophotometer (UV–Vis), Fourier transform infrared (FTIR), gas chromatography– mass spectrophotometer (GC–MS), Nuclear magnetic resonance (NMR). Acute toxicity of the crude ethanolic extracts of the R. cordifolia fruits was examined in Swiss albino mice. No mortality was observed in all treated mice with 100, 500, 1000 mg/kg body weight of crude extract of R. cordifolia fruit and it indicates that the LD50 value is higher than 1000 mg/kg body weight. This study exhibited a significant change in the body weight. Alanine transaminase (ALT), total protein, triglycerides, glucose, and also the histopathological analysis of liver for all treated mice showed difference from the control group. The dibutyl phthalate was further evaluated for the toxicity study through in silico analysis. Together, the results highlighted that the toxic potential of R. cordifolia fruits extracts and also the toxicity profile of C 2015 Wiley the fruit should be essential for the future studies dealing with the long term effect in animals. V Periodicals, Inc. Environ Toxicol 00: 000–000, 2015.
Keywords: Rubia cordifolia; fruits extracts; dibutyl phthalate; acute oral toxicity; in silico
INTRODUCTION Plants produce a wide array of secondary metabolites that are toxic to herbivore as well as to pathogen and it considCorrespondence to: D. P. Fulzele; e-mail: [email protected], dfulzele@ barc.gov.in Contract grant sponsor: CSIR, New Delhi, India. Contract grant number: 37 (1451) /10/EMR-II. Contract grant sponsor: BRNS, Mumbai, India. Contract grant number: 2013/35/14/BRNS. Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22115
ered as defense molecules (Wittstock and Gershenzon, 2002). According to World Health Organization, plants are the best source for obtaining a variety of drugs and 70–80% of world populations depend upon medicinal plants (TetteyLarbi et al., 2013). Many traditional medicinal plants have proved to be toxic through in vivo toxicological studies (Atsamo et al., 2011). For example: Saleh et al. (2013) reported that high level of polyphenolics from the green tea causes liver damage. Rubia cordifolia L. (Rubiaceae), commonly known as ‘Indian madder’ was used in the ayurvedic medicinal system for the treatment of skin disease and cancer. In addition, the crude extracts have shown to possess antibacterial, antiviral, antineoplastic, and hepatoprotective
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activities (Kaur et al., 2010). The dried roots were used as herbal medicine in the Chinese Pharmacopeia for the treatment of arthritis, dysmenorrhea, hematorrhea, hemostasis, and wound healing (Chang et al., 2000; Siva, 2003). The R. cordifolia root contains significant amount of various anthraquinones like alizarin, purpurin, pseudo-purpurin, rubiadin, munjistin, and Rubiacordone A (Siva, 2007; Vankar et al., 2008; Li et al., 2009). There are reports that anthraquinones possess antioxidant, anti-inflammatory, and cytotoxic properties (Bhakta and Siva, 2009; Siva et al., 2011). There are various reports available on phytoconstitutents and therapeutic efficacies of R. cordifolia roots (Rao et al., 2006; Divakar et al., 2010; Bhatt and Kushwah, 2013). However, not much exploration on the phytochemical constituents and pharmacological activities of R. cordifolia fruits was reported. It has been observed that the fruits are edible and are available during October–November (Chen and Ehrendorfer, 2011). In this present study, isolation and characterization of dye fraction from the ethanolic extract of R. cordifolia fruit was evaluated through multispectroscopic technique. In addendum, the pharmaco-toxicological effects of the R. cordifolia fruit extract were examined through the acute toxicity study in Swiss albino mice. To comprehend, the dye fraction was further evaluated through in silico analysis. To the best of our knowledge, this is the first report on this sort of study.
MATERIALS AND METHODS Plant Material The fresh fruits of R. cordifolia were collected from Shervaroy hills of Eastern Ghats, Tamil Nadu, India. The fruits were washed with running tap water and subsequently washed with distilled water. Fruits were dried under shade and then taken up for extraction.
Preparation of Extracts and Isolation The fresh fruits about 80 g was subjected to exhaustive dye extraction in a soxhlet apparatus using 100% ethanol (250 ml) as a solvent. The extracts obtained was then evaporated to dryness at 40 C under reduced pressure (337 mbar) € in a Rotavapor R-215 (BUCHI Labortechnik AG, Switzerland), and thereafter stored in 220 C until further use. This dried extracts, referred to as the crude extract here onwards, yielded a greenish purple dye of 2.5 g. Thereafter, 1 g of dried ethanolic crude extract was subjected to column chromatography with 30 g silica gel (60– 120 mesh). In column, hexane with silica gel was poured into the column and gradually added slurry. The fraction was eluted from the crude extract with C6H14:Ethyl acetate (4:1). Fractions obtained from column chromatography were checked individually and identical fractions were pooled together based upon the absorbance using UV–Vis spectro-
Environmental Toxicology DOI 10.1002/tox
Fig. 1. UV–Vis spectrophotometer of isolated dibutyl phthalate. The wavelength absorbed by isolated compound in chloroform solvent at 274, 406, 432, and 664 nm.
photometer (Fig. 1) with chloroform as blank. The obtained fraction was further used for phytochemical analysis.
Phytochemical Analysis The yielded green color compound is semisolid (20 mg) and the absorbance wavelength of isolated molecules was analyzed using UV–Vis spectrophotometer (Systronics AU-2401 UV–Vis double beam spectrophotometer). FTIR analysis was carried out using a Shimadzu IR Affinity-1 fourier transform infrared spectrophotometer. Spectra were collected and treated using the OMNIC software. 1H and 13C NMR spectra were determined in CDCl3 solution at 400 MHz using Bruker Ascend Model. Mass Spectra were recorded on a PerkinElmer clarus 680 (GC) and clarus 600 (EI, Mass).
ACUTE TOXICITY STUDY Animals, Housing, Diet, and Water The toxic effect of crude extracts in mice was evaluated through histopathological and biochemical parameter. Swiss albino female mice were obtained from the animal house, VIT University, Vellore, Tamil Nadu after procuring clearance from the institutional ethical Committee (VIT/IAEC/VII/22/ 2013). All animals were housed in standard cages with sawdust bedding and were illuminated on an approximate 12 h light/dark cycle. The temperature was maintained at 23 6 2 C and administrated with potable water, standardized diet ad libitum. Adult Swiss albino mice were in the weight range from 25 to 30 g on the first day of treatment. The experiments were carried out as per the Organization for Economic Cooperation and Development (OECD) guidelines.
Experimental Design The animals were divided into four groups of five female mice. The crude extracts were administrated by oral gavage
TOXICITY STUDY THROUGH IN SILICO ANALYSIS
Fig. 2. FTIR spectrum of isolated dibutyl phthalate from the Rubia cordifolia fruit in the 4000–400 cm21 region. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
at doses of 100, 500, and 1000 mg/kg body weight. Control animal received the vehicle alone (0.1% ethanol). The body weight was measured on 0, 7th, and 14th day after the administration of crude extracts. All surviving animals were sacrificed on the 14th day. The liver, kidney, and spleen were dissected quickly from each animal, cleaned with phosphate buffer saline, weighed, and preserved in 10% formalin for histopathological analysis.
Biochemical Parameter The blood sample was collected from all sacrificed mice in plastic test tubes and allowed to stand for complete clotting. The clotted blood was centrifuged at 3000 rpm for 15 min and serum sample were separated and stored at 220 C until further analysis. The serum sample from each group was analyzed using Cogent clinical chemistry division of span diagnostics kits for the determination of total protein, cholesterol, triglycerides, alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinine, urea, albumin, and glucose.
Histopathological Evaluation The organs (liver, kidney, and spleen) were examined for histopathological analysis to observe for the sign of toxicity through macroscopic method. The organs were stained with hematoxylin and eosin.
Molecular Property, Bio-Activity Score and Drug-Likeness A good oral bioavailability is an important factor for the development of bioactive molecule as a therapeutic agent, hence the physicochemical properties of dibutyl phthalate were calculated through molinspiration program. It calculates the important molecular property likes LogP, polar surface area, number of hydrogen bond donors, and acceptors and others. It also predicts the bioactivity score of most
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important drug targets such as GPCR ligands, kinase inhibitors, ion channel modulators, nuclear receptors, Protease inhibitor and enzyme inhibitor. Drug likeness is a qualitative concept used in drug design. The theoretical calculation of drug-like property and maintain of certain properties of a molecule can give us better assumption of biological activity of certain molecule. Lipinski’s rule of five is a rule of thumb to evaluate drug likeness or determine a chemical compound with a certain pharmacological or biological activity that would make it a likely orally active drug in humans (Lipinski et al., 1997). The parameters defined by Lipinski’s rule were calculated for dibutyl phthalate of R. cordifolia fruits by the online software through www.molsoft.com.
Statistical Analysis Values were expressed as Mean 6 SEM (n 5 3). Comparison between groups was done by one way ANOVA followed by Dunnett’s multiple comparison test. P < 0.05 was considered as significant. All the statistical analysis was done using software Graphpad Prism version 5 (Graphpad prism, USA).
RESULTS AND DISCUSSION Characterization of the Compound The characterization of the compound was carried out through various spectroscopic and analytical techniques. The product obtained by column chromatography was greenish semisolid compound. The UV–Vis spectrum in chloroform solvent observed at 274, 406, 432, and 664 nm (Fig. 1). Infrared spectral data of the isolated compound revealed the specific functional group of band at 1670 cm21 indicating the presence of an ester group (ACOOR) (Fig. 2). Other absorption frequencies at 3244 and 2954 cm21 for aromatic (@CAH) and aliphatic CAH stretching vibrations were observed, respectively. A band at 1384 and 1016 cm21 indicates the presence of CAC and CAO bond. The structure of the compound was further supported by mass spectral data (Fig. 3). Mass spectrum shows the molecular ion (m/z) at 279 and other main fragments at 167 and 149 (Table I). 1H and 13C NMR spectrum clearly indicated the carbon skeleton of the compound [Fig. 4(a,b)]. 1H NMR clearly demonstrated the presence of two sets of aromatic protons at d 7.50 and d 7.69 as two multiplets. This confirms that there are two different types of aromatic protons adjacent to each other. A triplet at d 4.2 shows the presence of ‘XACH2A CH2A group in the molecule, where X 5 heteroatom (N or O). The presence of aliphatic protons in the range of d 0.9– 2.0 indicates that there must be a long saturated hydrocarbon chain. 13C-NMR shows a peak at d 167.9 for the ester carbonyl carbon and other peaks in the range of d 121.9–134.5 for aromatic ring carbons. A signal at d 68.3 indicates the presence of methylene group attached to heteroatom oxygen. Finally, the structure of the molecule was confirmed as
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cordifolia fruits, which possess dibutyl phthalate component and the results were discussed below.
Acute Toxicity Study The biological properties of substance can be studied through toxicity analysis. The assessment of toxicity of medicinal plants is important to know their safety profile. Mapanga and Musabayane (2010) reported that the renal function can also be affected through the use of medicinal plants for the treatment of different diseases. The consumption of medicinal plants without understanding their toxicity profiles, which can lead to unexpected effects towards the function of kidney and liver. Limited information was available on the adverse effect of natural products on human health (Chandrasekaran et al., 2009). The effect of the administration of a single oral dose of crude ethanol extracts of R. cordifolia fruits (100, 500, and 1000 mg/kg body weight) to mice was analyzed through biochemical parameter and histopathological analysis (Table II and Fig. 5). Fig. 3. GC–MS of dibutyl phthalate. Mass spectrum shows the molecular ion (m/z) of isolated dibutyl phthalate at 279 and other main fragments at 167 and 149. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Biochemical Parameter
dibutylpthalate from the obtained spectroscopic as well as mass data.
TABLE I. Various major fragments interpreted as per the MS spectrum
Bioactivity of Dibutyl Phthalate
Structure of Fragment
The synthetic dibutyl phthalates are toxic because of its plasticizer effect. Moreover, it has ability to disrupt the endocrine system (Foster et al., 2000). Gray et al., (2000) found that dibutyl phthalate induce reproductive malformation and decreased androgen-dependent organ weight in adult male rats. It also induces peroxisomes proliferation in the liver of mice and rats that result in hepatomegaly and hepatocarcinogenesis (O’Brien et al., 2001). Further, the dibutyl phthalate act as drug channeling agent (Makhija and Vavia, 2003). Higuchi et al. (2003) stated that synthetic dibutyl phthalate was detected in urine and serum samples of human, because of its prevalence in the stored food, water and air etc. The essential oil from Osmanths fragrans, Phyllanthus arenarius, P. urinaria, P. niriru, Labelia pyramidalis are known to possess dibutyl phthalate (Xiangrong et al., 2008; Wang et al., 2009; Joshi et al., 2011). The Streptomyces albidoflavus produces dibutyl phthalate and proven to have antimicrobial activity (Roy et al., 2006). Hsu et al., (2011) reported that bioactive component dibutyl phthalate isolated from Typhonium blumei showed the antiproliferative activity towards human lung adenocarcinoma A549 cells. Many researchers have reported the bioactivity of dibutyl phthalate with positive and negative effects, but this study mainly focus on the toxicity evaluation of edible Rubia
Environmental Toxicology DOI 10.1002/tox
During the toxicity study, no mortality was observed and the oral LD50 of the R. cordifolia fruits extracts were above
Molecular Formula
Mass (m/z)
C16H22O4
278
C8H6O4
166
C8H4O3
148
18.40 6 9.19 34.48 6 11.48 435.69 6 112.67 51.2 6 23.69 37.54 6 4.78 500.36 6 5.15a 54.53 6 8.70 34.54 6 2.26 383.59 6 3.27a 33.86 6 14.2 34.72 6 2.66 401.49 6 6.61a
238.516 6 13.50 51.60 6 0.61a 52.94 6 1.97a 58.56 6 0.91a
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ALT, alanine transaminase; ALP, alkaline phosphatase. Values are mean 6 Standard Error Mean (n 5 3), dosage given in mg/kg body weight. a Significant difference from control P < 0.05.
ALP(IU/L) ALT (IU/L)
77.20 6 33.06 1760.99 6 82.31 259.49 6 1.78 37.78 6 0.44 7.53 6 0.16 365.38 6 35.97a 1127.288 6 34.44 353.22 6 9.56 43.56 6 1.22a 6.670 6 0.36 265.78 6 109.03a 621.95 6 20.63 374.23 6 24.44 44.56 6 0.62a 6.625 6 0.34 73.66 6 48.99a 2613.464 6 209 730.06 6 289.5 44.74 6 0.384a 6.777 6 0.08 Control 100 mg 500 mg 1000 mg
Albumin (g/dL) Total Protein (g/dL) Total Cholesterol (mg/dL)
1000 mg/kg body weight of mice. In this study, the biochemical parameters ALT, total protein, triglycerides, and glucose level showed a significant difference between control and treated groups. Liver is act as an important target for most of the drugs and xenobiotics, because it involves in the excretion and metabolism of the chemical substance. The ALT (transaminase) biomarker was a well-recognized enzyme for the liver damage (Mukinda and Eagles, 2010). In the present investigation, the level of ALT in serum of treated groups is different from the control group. The initiation of hepatoxicity by crude extracts may be direct cell stress that causes mitochondrial damage, which result in the mitochondrial membrane permeability transition and subsequent necrosis occurs that leads to the leakage of the ALT enzyme along the liver cell membrane into the circulating system (Singh et al., 2011). In addition, hepatic cells of the liver also involve in the metabolism of cholesterol and glucose (Wang et al., 2014). Therefore, the change in triglycerides and glucose level in the treated mice was observed, which indicates R. cordifolia fruits extracts had effects on lipid and carbohydrate metabolism of mice. The significant decrease in the blood glucose level, suggesting the R. cordifolia fruit may induce hypoglycemic effect by stimulating insulin in all treated mice (Chiranthanut et al., 2013). The plasma protein is synthesized in hepatocytes and act as a marker for liver
Oral Acute Toxicity Treatment
Fig. 4. (a) 1H NMR of dibutyl phthalate. The intensity of peak in the spectral region of d 0–11 was observed. (b) 13C NMR spectrum of dibutyl phthalate. The NMR signal peak of isolated compound in CDCl3. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
TABLE II. Biochemical analysis of acute toxicity study of Rubia cordifolia fruit in female mice
Creatinine (mg/dL)
Urea (mg/dL)
Triglycerides (mg/dL)
Glucose (mg/dL)
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Fig. 5. Histopathological examination included liver, kidney, and spleen section. Photomicrograph of control with vehicle, 100, 500, and 1000 mg/kg body weight of ethanol exact of fruit of Rubia cordifolia treated mice. No change was observed in spleen for all treated mice. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
capacity. The least change in total protein is due to hepatocellular damage (Rasekh et al., 2008). Table II showed a significant change in the total protein level, which indicates the disturbance of liver function. In addition, increased
Environmental Toxicology DOI 10.1002/tox
creatinine level in blood is an indication of kidney damage (Rhiouani et al., 2008). In our study, the result shows no change in the creatinine level was observed in serum in all treated mice.
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TABLE III. Molecular property and bioactivity score of dibutyl phthalate Molecular Property
Fig. 6. The body weight curve of control and treated mice (n 5 5) with single dose of (100, 500, and 1000 mg/kg body weight) fruit of Rubia cordifolia extract. Values are in Mean6 Standard Error Mean. W0 5 body weight on day 0 before the administration of dye extract; W7 5 body weight on day 7 after administration of dye extract; W14 5 body weight on day 14 after administration of dye extract.
Histopathological and Body Weight The histopathological changes were observed in the liver of all treated mice. The Figure 5 shows the diffuse vacuolar degenerative changes were observed in hepatocytes of liver for all survival mice treated with a single dose of different concentration of crude extracts and no change were observed in spleen for all treated mice. Apart from this, treated mice with 500 and 1000 mg/kg body weight of crude extract has showed mild degenerative changes in the renal tubular epithelial cells (Fig. 5) and this type of lesion is minimal, therefore no pathological changes were observed in the kidney that could attribute to crude extracts treatment (Gelderblom et al., 2001). El-Hilaly et al. (2004) reported that change in body weight can be a sign of adverse effect of the drug. The significant change in the body weight was observed in 500 and 1000 mg/kg body weight of the treated group as compared to the control group after 14 days of a single oral dose of acute toxicity of crude dye (Fig. 6).
Bioactivity Score
miLogP (octanol/water partition coefficient): 24.429 (5) TPSA (molecular polar surface area): 52.61 MW: 278.348 (500) nON: 4 (10) nOHNH: 0 (5) Nrotb: 10 Volume: 273.913
GPCR ligand: 20.16 Ion channel modulator: 20.09 Kinase inhibitor: 20.27 Nuclear receptor ligand: 20.12 Protease inhibitor: 20.25 Enzyme inhibitor: 20.07
partition coefficient (log P), molar refractivity, molecular weight, number of heavy atoms, number of hydrogen donor, and number of hydrogen acceptor. The rule of five states that most molecules with good membrane permeability have log P 5, molecular weight 500, number of hydrogen bond acceptors 10, and number of hydrogen bond donors 5 (Ahsan et al., 2011). The calculated molecular properties of dibutyl phthalate satisfy the Lipinski rule of five. However, the Drug-likeness score was negative (20.07) (Fig. 7) and the bioactivity score also shows that, the dibutyl phthalate is biologically inactive compound. This result proposes that dibutyl phthalate is a nondrug compound and it may contain mild toxic in their compound.
CONCLUSION The safety usage of the plant products should be first tested in animals to evaluate their toxicity profile. This study provides valuable information on the presence of dibutyl phthalate in the R. cordifolia fruit and also it has shown holistic toxicity through in silico analysis. Besides, R. cordifolia fruits extract was evaluated for the short term toxicity study. The
Molecular Characterization and Drug-Likeness of Dibutyl Phthalate The dibutyl phthalate was evaluated for the various parameters to consider as a probable drug. It is also screened for bioactivity by calculating the score for GPCR ligand, ion channel modulator, kinase inhibitor, nuclear receptor, protease inhibitor, and enzyme inhibitor, which are the six major drug classes. In general, the bioactivity score for major drug classes are active at more than 0.00, moderately active at 20.50 to 0.00 and inactive at less than 20.50 (Mazumder et al., 2009). The bioactivity score for dibutyl phthalate was inactive in all the six major drug classes, which are shown in Table III. This result suggests that, the dibutyl phthalate is not biologically active compound. Similarly, the drug likeness score was calculated by considering Lipinski’s rule of five,
Fig. 7. The compound dibutyl phthalate is a nondrug compound, which has the toxicity in it. The drug-likeness score also negative (20.07). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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crude extract has a deleterious effect on the hepatic cells, which have been evaluated through biochemical parameter and histopathological changes. Further studies are required to determine the effects of this fruit on the long term administration in animals and also in the developmental toxicity study. The toxic dose of this fruit cannot be determined through animal toxicity study, but the clinical trial should be conducted to evaluate the safety profile for the humans. The authors wish to express our sincere gratitude to Central University Llaboratory, Tamil Nadu Veterinary, and Animal Science University, Chennai, India for the histopathological analysis. They wish to thank VIT-SIF lab, SAS, Chemistry Division for FTIR, NMR, and GC-MS. Our grateful to the VIT university management of the infrastructure provided to carry out the research.
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Environmental Toxicology DOI 10.1002/tox
School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu 632014, India
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School of Advanced Sciences, VIT University, Vellore, Tamil Nadu 632014, India
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Plant Biotechnology and Secondary Metabolites Section, NA&BTD, Bhabha Atomic Research Centre, Mumbai, Maharashtra 400 094, India
Received 19 August 2014; accepted 29 December 2014 ABSTRACT: Natural toxins from plant sources with wide ranges of biological activities reflect the upswing of drug design in the pharmaceutical industry. Rubia cordifolia L. is one of the most important red dye yielding plants. Most of the former researches have focused on the bioactive compounds from the roots of R. cordifolia, while no attention was paid towards the fruits. For the first time, here we report the presence of dibutyl phthalate in the fruits of R. cordifolia. Structural characterization was carried out using Ultraviolet–Visible spectrophotometer (UV–Vis), Fourier transform infrared (FTIR), gas chromatography– mass spectrophotometer (GC–MS), Nuclear magnetic resonance (NMR). Acute toxicity of the crude ethanolic extracts of the R. cordifolia fruits was examined in Swiss albino mice. No mortality was observed in all treated mice with 100, 500, 1000 mg/kg body weight of crude extract of R. cordifolia fruit and it indicates that the LD50 value is higher than 1000 mg/kg body weight. This study exhibited a significant change in the body weight. Alanine transaminase (ALT), total protein, triglycerides, glucose, and also the histopathological analysis of liver for all treated mice showed difference from the control group. The dibutyl phthalate was further evaluated for the toxicity study through in silico analysis. Together, the results highlighted that the toxic potential of R. cordifolia fruits extracts and also the toxicity profile of C 2015 Wiley the fruit should be essential for the future studies dealing with the long term effect in animals. V Periodicals, Inc. Environ Toxicol 00: 000–000, 2015.
Keywords: Rubia cordifolia; fruits extracts; dibutyl phthalate; acute oral toxicity; in silico
INTRODUCTION Plants produce a wide array of secondary metabolites that are toxic to herbivore as well as to pathogen and it considCorrespondence to: D. P. Fulzele; e-mail: [email protected], dfulzele@ barc.gov.in Contract grant sponsor: CSIR, New Delhi, India. Contract grant number: 37 (1451) /10/EMR-II. Contract grant sponsor: BRNS, Mumbai, India. Contract grant number: 2013/35/14/BRNS. Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22115
ered as defense molecules (Wittstock and Gershenzon, 2002). According to World Health Organization, plants are the best source for obtaining a variety of drugs and 70–80% of world populations depend upon medicinal plants (TetteyLarbi et al., 2013). Many traditional medicinal plants have proved to be toxic through in vivo toxicological studies (Atsamo et al., 2011). For example: Saleh et al. (2013) reported that high level of polyphenolics from the green tea causes liver damage. Rubia cordifolia L. (Rubiaceae), commonly known as ‘Indian madder’ was used in the ayurvedic medicinal system for the treatment of skin disease and cancer. In addition, the crude extracts have shown to possess antibacterial, antiviral, antineoplastic, and hepatoprotective
C 2015 Wiley Periodicals, Inc. V
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activities (Kaur et al., 2010). The dried roots were used as herbal medicine in the Chinese Pharmacopeia for the treatment of arthritis, dysmenorrhea, hematorrhea, hemostasis, and wound healing (Chang et al., 2000; Siva, 2003). The R. cordifolia root contains significant amount of various anthraquinones like alizarin, purpurin, pseudo-purpurin, rubiadin, munjistin, and Rubiacordone A (Siva, 2007; Vankar et al., 2008; Li et al., 2009). There are reports that anthraquinones possess antioxidant, anti-inflammatory, and cytotoxic properties (Bhakta and Siva, 2009; Siva et al., 2011). There are various reports available on phytoconstitutents and therapeutic efficacies of R. cordifolia roots (Rao et al., 2006; Divakar et al., 2010; Bhatt and Kushwah, 2013). However, not much exploration on the phytochemical constituents and pharmacological activities of R. cordifolia fruits was reported. It has been observed that the fruits are edible and are available during October–November (Chen and Ehrendorfer, 2011). In this present study, isolation and characterization of dye fraction from the ethanolic extract of R. cordifolia fruit was evaluated through multispectroscopic technique. In addendum, the pharmaco-toxicological effects of the R. cordifolia fruit extract were examined through the acute toxicity study in Swiss albino mice. To comprehend, the dye fraction was further evaluated through in silico analysis. To the best of our knowledge, this is the first report on this sort of study.
MATERIALS AND METHODS Plant Material The fresh fruits of R. cordifolia were collected from Shervaroy hills of Eastern Ghats, Tamil Nadu, India. The fruits were washed with running tap water and subsequently washed with distilled water. Fruits were dried under shade and then taken up for extraction.
Preparation of Extracts and Isolation The fresh fruits about 80 g was subjected to exhaustive dye extraction in a soxhlet apparatus using 100% ethanol (250 ml) as a solvent. The extracts obtained was then evaporated to dryness at 40 C under reduced pressure (337 mbar) € in a Rotavapor R-215 (BUCHI Labortechnik AG, Switzerland), and thereafter stored in 220 C until further use. This dried extracts, referred to as the crude extract here onwards, yielded a greenish purple dye of 2.5 g. Thereafter, 1 g of dried ethanolic crude extract was subjected to column chromatography with 30 g silica gel (60– 120 mesh). In column, hexane with silica gel was poured into the column and gradually added slurry. The fraction was eluted from the crude extract with C6H14:Ethyl acetate (4:1). Fractions obtained from column chromatography were checked individually and identical fractions were pooled together based upon the absorbance using UV–Vis spectro-
Environmental Toxicology DOI 10.1002/tox
Fig. 1. UV–Vis spectrophotometer of isolated dibutyl phthalate. The wavelength absorbed by isolated compound in chloroform solvent at 274, 406, 432, and 664 nm.
photometer (Fig. 1) with chloroform as blank. The obtained fraction was further used for phytochemical analysis.
Phytochemical Analysis The yielded green color compound is semisolid (20 mg) and the absorbance wavelength of isolated molecules was analyzed using UV–Vis spectrophotometer (Systronics AU-2401 UV–Vis double beam spectrophotometer). FTIR analysis was carried out using a Shimadzu IR Affinity-1 fourier transform infrared spectrophotometer. Spectra were collected and treated using the OMNIC software. 1H and 13C NMR spectra were determined in CDCl3 solution at 400 MHz using Bruker Ascend Model. Mass Spectra were recorded on a PerkinElmer clarus 680 (GC) and clarus 600 (EI, Mass).
ACUTE TOXICITY STUDY Animals, Housing, Diet, and Water The toxic effect of crude extracts in mice was evaluated through histopathological and biochemical parameter. Swiss albino female mice were obtained from the animal house, VIT University, Vellore, Tamil Nadu after procuring clearance from the institutional ethical Committee (VIT/IAEC/VII/22/ 2013). All animals were housed in standard cages with sawdust bedding and were illuminated on an approximate 12 h light/dark cycle. The temperature was maintained at 23 6 2 C and administrated with potable water, standardized diet ad libitum. Adult Swiss albino mice were in the weight range from 25 to 30 g on the first day of treatment. The experiments were carried out as per the Organization for Economic Cooperation and Development (OECD) guidelines.
Experimental Design The animals were divided into four groups of five female mice. The crude extracts were administrated by oral gavage
TOXICITY STUDY THROUGH IN SILICO ANALYSIS
Fig. 2. FTIR spectrum of isolated dibutyl phthalate from the Rubia cordifolia fruit in the 4000–400 cm21 region. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
at doses of 100, 500, and 1000 mg/kg body weight. Control animal received the vehicle alone (0.1% ethanol). The body weight was measured on 0, 7th, and 14th day after the administration of crude extracts. All surviving animals were sacrificed on the 14th day. The liver, kidney, and spleen were dissected quickly from each animal, cleaned with phosphate buffer saline, weighed, and preserved in 10% formalin for histopathological analysis.
Biochemical Parameter The blood sample was collected from all sacrificed mice in plastic test tubes and allowed to stand for complete clotting. The clotted blood was centrifuged at 3000 rpm for 15 min and serum sample were separated and stored at 220 C until further analysis. The serum sample from each group was analyzed using Cogent clinical chemistry division of span diagnostics kits for the determination of total protein, cholesterol, triglycerides, alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinine, urea, albumin, and glucose.
Histopathological Evaluation The organs (liver, kidney, and spleen) were examined for histopathological analysis to observe for the sign of toxicity through macroscopic method. The organs were stained with hematoxylin and eosin.
Molecular Property, Bio-Activity Score and Drug-Likeness A good oral bioavailability is an important factor for the development of bioactive molecule as a therapeutic agent, hence the physicochemical properties of dibutyl phthalate were calculated through molinspiration program. It calculates the important molecular property likes LogP, polar surface area, number of hydrogen bond donors, and acceptors and others. It also predicts the bioactivity score of most
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important drug targets such as GPCR ligands, kinase inhibitors, ion channel modulators, nuclear receptors, Protease inhibitor and enzyme inhibitor. Drug likeness is a qualitative concept used in drug design. The theoretical calculation of drug-like property and maintain of certain properties of a molecule can give us better assumption of biological activity of certain molecule. Lipinski’s rule of five is a rule of thumb to evaluate drug likeness or determine a chemical compound with a certain pharmacological or biological activity that would make it a likely orally active drug in humans (Lipinski et al., 1997). The parameters defined by Lipinski’s rule were calculated for dibutyl phthalate of R. cordifolia fruits by the online software through www.molsoft.com.
Statistical Analysis Values were expressed as Mean 6 SEM (n 5 3). Comparison between groups was done by one way ANOVA followed by Dunnett’s multiple comparison test. P < 0.05 was considered as significant. All the statistical analysis was done using software Graphpad Prism version 5 (Graphpad prism, USA).
RESULTS AND DISCUSSION Characterization of the Compound The characterization of the compound was carried out through various spectroscopic and analytical techniques. The product obtained by column chromatography was greenish semisolid compound. The UV–Vis spectrum in chloroform solvent observed at 274, 406, 432, and 664 nm (Fig. 1). Infrared spectral data of the isolated compound revealed the specific functional group of band at 1670 cm21 indicating the presence of an ester group (ACOOR) (Fig. 2). Other absorption frequencies at 3244 and 2954 cm21 for aromatic (@CAH) and aliphatic CAH stretching vibrations were observed, respectively. A band at 1384 and 1016 cm21 indicates the presence of CAC and CAO bond. The structure of the compound was further supported by mass spectral data (Fig. 3). Mass spectrum shows the molecular ion (m/z) at 279 and other main fragments at 167 and 149 (Table I). 1H and 13C NMR spectrum clearly indicated the carbon skeleton of the compound [Fig. 4(a,b)]. 1H NMR clearly demonstrated the presence of two sets of aromatic protons at d 7.50 and d 7.69 as two multiplets. This confirms that there are two different types of aromatic protons adjacent to each other. A triplet at d 4.2 shows the presence of ‘XACH2A CH2A group in the molecule, where X 5 heteroatom (N or O). The presence of aliphatic protons in the range of d 0.9– 2.0 indicates that there must be a long saturated hydrocarbon chain. 13C-NMR shows a peak at d 167.9 for the ester carbonyl carbon and other peaks in the range of d 121.9–134.5 for aromatic ring carbons. A signal at d 68.3 indicates the presence of methylene group attached to heteroatom oxygen. Finally, the structure of the molecule was confirmed as
Environmental Toxicology DOI 10.1002/tox
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cordifolia fruits, which possess dibutyl phthalate component and the results were discussed below.
Acute Toxicity Study The biological properties of substance can be studied through toxicity analysis. The assessment of toxicity of medicinal plants is important to know their safety profile. Mapanga and Musabayane (2010) reported that the renal function can also be affected through the use of medicinal plants for the treatment of different diseases. The consumption of medicinal plants without understanding their toxicity profiles, which can lead to unexpected effects towards the function of kidney and liver. Limited information was available on the adverse effect of natural products on human health (Chandrasekaran et al., 2009). The effect of the administration of a single oral dose of crude ethanol extracts of R. cordifolia fruits (100, 500, and 1000 mg/kg body weight) to mice was analyzed through biochemical parameter and histopathological analysis (Table II and Fig. 5). Fig. 3. GC–MS of dibutyl phthalate. Mass spectrum shows the molecular ion (m/z) of isolated dibutyl phthalate at 279 and other main fragments at 167 and 149. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Biochemical Parameter
dibutylpthalate from the obtained spectroscopic as well as mass data.
TABLE I. Various major fragments interpreted as per the MS spectrum
Bioactivity of Dibutyl Phthalate
Structure of Fragment
The synthetic dibutyl phthalates are toxic because of its plasticizer effect. Moreover, it has ability to disrupt the endocrine system (Foster et al., 2000). Gray et al., (2000) found that dibutyl phthalate induce reproductive malformation and decreased androgen-dependent organ weight in adult male rats. It also induces peroxisomes proliferation in the liver of mice and rats that result in hepatomegaly and hepatocarcinogenesis (O’Brien et al., 2001). Further, the dibutyl phthalate act as drug channeling agent (Makhija and Vavia, 2003). Higuchi et al. (2003) stated that synthetic dibutyl phthalate was detected in urine and serum samples of human, because of its prevalence in the stored food, water and air etc. The essential oil from Osmanths fragrans, Phyllanthus arenarius, P. urinaria, P. niriru, Labelia pyramidalis are known to possess dibutyl phthalate (Xiangrong et al., 2008; Wang et al., 2009; Joshi et al., 2011). The Streptomyces albidoflavus produces dibutyl phthalate and proven to have antimicrobial activity (Roy et al., 2006). Hsu et al., (2011) reported that bioactive component dibutyl phthalate isolated from Typhonium blumei showed the antiproliferative activity towards human lung adenocarcinoma A549 cells. Many researchers have reported the bioactivity of dibutyl phthalate with positive and negative effects, but this study mainly focus on the toxicity evaluation of edible Rubia
Environmental Toxicology DOI 10.1002/tox
During the toxicity study, no mortality was observed and the oral LD50 of the R. cordifolia fruits extracts were above
Molecular Formula
Mass (m/z)
C16H22O4
278
C8H6O4
166
C8H4O3
148
18.40 6 9.19 34.48 6 11.48 435.69 6 112.67 51.2 6 23.69 37.54 6 4.78 500.36 6 5.15a 54.53 6 8.70 34.54 6 2.26 383.59 6 3.27a 33.86 6 14.2 34.72 6 2.66 401.49 6 6.61a
238.516 6 13.50 51.60 6 0.61a 52.94 6 1.97a 58.56 6 0.91a
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ALT, alanine transaminase; ALP, alkaline phosphatase. Values are mean 6 Standard Error Mean (n 5 3), dosage given in mg/kg body weight. a Significant difference from control P < 0.05.
ALP(IU/L) ALT (IU/L)
77.20 6 33.06 1760.99 6 82.31 259.49 6 1.78 37.78 6 0.44 7.53 6 0.16 365.38 6 35.97a 1127.288 6 34.44 353.22 6 9.56 43.56 6 1.22a 6.670 6 0.36 265.78 6 109.03a 621.95 6 20.63 374.23 6 24.44 44.56 6 0.62a 6.625 6 0.34 73.66 6 48.99a 2613.464 6 209 730.06 6 289.5 44.74 6 0.384a 6.777 6 0.08 Control 100 mg 500 mg 1000 mg
Albumin (g/dL) Total Protein (g/dL) Total Cholesterol (mg/dL)
1000 mg/kg body weight of mice. In this study, the biochemical parameters ALT, total protein, triglycerides, and glucose level showed a significant difference between control and treated groups. Liver is act as an important target for most of the drugs and xenobiotics, because it involves in the excretion and metabolism of the chemical substance. The ALT (transaminase) biomarker was a well-recognized enzyme for the liver damage (Mukinda and Eagles, 2010). In the present investigation, the level of ALT in serum of treated groups is different from the control group. The initiation of hepatoxicity by crude extracts may be direct cell stress that causes mitochondrial damage, which result in the mitochondrial membrane permeability transition and subsequent necrosis occurs that leads to the leakage of the ALT enzyme along the liver cell membrane into the circulating system (Singh et al., 2011). In addition, hepatic cells of the liver also involve in the metabolism of cholesterol and glucose (Wang et al., 2014). Therefore, the change in triglycerides and glucose level in the treated mice was observed, which indicates R. cordifolia fruits extracts had effects on lipid and carbohydrate metabolism of mice. The significant decrease in the blood glucose level, suggesting the R. cordifolia fruit may induce hypoglycemic effect by stimulating insulin in all treated mice (Chiranthanut et al., 2013). The plasma protein is synthesized in hepatocytes and act as a marker for liver
Oral Acute Toxicity Treatment
Fig. 4. (a) 1H NMR of dibutyl phthalate. The intensity of peak in the spectral region of d 0–11 was observed. (b) 13C NMR spectrum of dibutyl phthalate. The NMR signal peak of isolated compound in CDCl3. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
TABLE II. Biochemical analysis of acute toxicity study of Rubia cordifolia fruit in female mice
Creatinine (mg/dL)
Urea (mg/dL)
Triglycerides (mg/dL)
Glucose (mg/dL)
TOXICITY STUDY THROUGH IN SILICO ANALYSIS
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Fig. 5. Histopathological examination included liver, kidney, and spleen section. Photomicrograph of control with vehicle, 100, 500, and 1000 mg/kg body weight of ethanol exact of fruit of Rubia cordifolia treated mice. No change was observed in spleen for all treated mice. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
capacity. The least change in total protein is due to hepatocellular damage (Rasekh et al., 2008). Table II showed a significant change in the total protein level, which indicates the disturbance of liver function. In addition, increased
Environmental Toxicology DOI 10.1002/tox
creatinine level in blood is an indication of kidney damage (Rhiouani et al., 2008). In our study, the result shows no change in the creatinine level was observed in serum in all treated mice.
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TABLE III. Molecular property and bioactivity score of dibutyl phthalate Molecular Property
Fig. 6. The body weight curve of control and treated mice (n 5 5) with single dose of (100, 500, and 1000 mg/kg body weight) fruit of Rubia cordifolia extract. Values are in Mean6 Standard Error Mean. W0 5 body weight on day 0 before the administration of dye extract; W7 5 body weight on day 7 after administration of dye extract; W14 5 body weight on day 14 after administration of dye extract.
Histopathological and Body Weight The histopathological changes were observed in the liver of all treated mice. The Figure 5 shows the diffuse vacuolar degenerative changes were observed in hepatocytes of liver for all survival mice treated with a single dose of different concentration of crude extracts and no change were observed in spleen for all treated mice. Apart from this, treated mice with 500 and 1000 mg/kg body weight of crude extract has showed mild degenerative changes in the renal tubular epithelial cells (Fig. 5) and this type of lesion is minimal, therefore no pathological changes were observed in the kidney that could attribute to crude extracts treatment (Gelderblom et al., 2001). El-Hilaly et al. (2004) reported that change in body weight can be a sign of adverse effect of the drug. The significant change in the body weight was observed in 500 and 1000 mg/kg body weight of the treated group as compared to the control group after 14 days of a single oral dose of acute toxicity of crude dye (Fig. 6).
Bioactivity Score
miLogP (octanol/water partition coefficient): 24.429 (5) TPSA (molecular polar surface area): 52.61 MW: 278.348 (500) nON: 4 (10) nOHNH: 0 (5) Nrotb: 10 Volume: 273.913
GPCR ligand: 20.16 Ion channel modulator: 20.09 Kinase inhibitor: 20.27 Nuclear receptor ligand: 20.12 Protease inhibitor: 20.25 Enzyme inhibitor: 20.07
partition coefficient (log P), molar refractivity, molecular weight, number of heavy atoms, number of hydrogen donor, and number of hydrogen acceptor. The rule of five states that most molecules with good membrane permeability have log P 5, molecular weight 500, number of hydrogen bond acceptors 10, and number of hydrogen bond donors 5 (Ahsan et al., 2011). The calculated molecular properties of dibutyl phthalate satisfy the Lipinski rule of five. However, the Drug-likeness score was negative (20.07) (Fig. 7) and the bioactivity score also shows that, the dibutyl phthalate is biologically inactive compound. This result proposes that dibutyl phthalate is a nondrug compound and it may contain mild toxic in their compound.
CONCLUSION The safety usage of the plant products should be first tested in animals to evaluate their toxicity profile. This study provides valuable information on the presence of dibutyl phthalate in the R. cordifolia fruit and also it has shown holistic toxicity through in silico analysis. Besides, R. cordifolia fruits extract was evaluated for the short term toxicity study. The
Molecular Characterization and Drug-Likeness of Dibutyl Phthalate The dibutyl phthalate was evaluated for the various parameters to consider as a probable drug. It is also screened for bioactivity by calculating the score for GPCR ligand, ion channel modulator, kinase inhibitor, nuclear receptor, protease inhibitor, and enzyme inhibitor, which are the six major drug classes. In general, the bioactivity score for major drug classes are active at more than 0.00, moderately active at 20.50 to 0.00 and inactive at less than 20.50 (Mazumder et al., 2009). The bioactivity score for dibutyl phthalate was inactive in all the six major drug classes, which are shown in Table III. This result suggests that, the dibutyl phthalate is not biologically active compound. Similarly, the drug likeness score was calculated by considering Lipinski’s rule of five,
Fig. 7. The compound dibutyl phthalate is a nondrug compound, which has the toxicity in it. The drug-likeness score also negative (20.07). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Environmental Toxicology DOI 10.1002/tox
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crude extract has a deleterious effect on the hepatic cells, which have been evaluated through biochemical parameter and histopathological changes. Further studies are required to determine the effects of this fruit on the long term administration in animals and also in the developmental toxicity study. The toxic dose of this fruit cannot be determined through animal toxicity study, but the clinical trial should be conducted to evaluate the safety profile for the humans. The authors wish to express our sincere gratitude to Central University Llaboratory, Tamil Nadu Veterinary, and Animal Science University, Chennai, India for the histopathological analysis. They wish to thank VIT-SIF lab, SAS, Chemistry Division for FTIR, NMR, and GC-MS. Our grateful to the VIT university management of the infrastructure provided to carry out the research.
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Environmental Toxicology DOI 10.1002/tox
