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Activity guided chemo toxic profiling of Cassia occidentalis (CO) seeds: Detection of toxic compounds in body fluids of CO exposed patients and experimental rats. Gati Krushna Panigrahi, Ratnasekhar CH, Mohana Mudiam, Vipin M Vashishtha, Sheikh Raisuddin, and Mukul Das Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00056 • Publication Date (Web): 27 Apr 2015 Downloaded from http://pubs.acs.org on May 3, 2015

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Chemical Research in Toxicology

Activity guided chemo toxic profiling of Cassia occidentalis (CO) seeds: Detection of toxic compounds in body fluids of CO exposed patients and experimental rats.

Gati Krushna Panigrahia,d, Ratnasekhar CHb, Mohana K. R. Mudiamb, Vipin M. Vashishthac, S. Raisuddind and Mukul Dasa*

a.

Food, Drug and Chemical Toxicology Group, CSIR-Indian Institute of Toxicology Research, Mahatma Gandhi Marg, Post Box- 80, Lucknow-226001, India

b.

Analytical Chemistry Division, CSIR- Indian Institute of Toxicology Research, Mahatma Gandhi Marg, Post Box- 80, Lucknow-226001, India

c.

Mangla Hospital and Research Centre, Bijnor-246701, Uttar Pradesh, India

d.

Department of Medical Elementology and Toxicology, Jamia Hamdard, New Delhi-110062, India

*To whom all correspondence should be addressed E-mail: [email protected] Tel: 091-522-2613786 Fax: 091-522-262822

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TOC

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Abstract Our prior studies have shown the association between children death and consumption of Cassia occidentalis (CO) seeds. However, chemicals involved in the causation of CO poisoning is not known. Therefore, the present study was designed to identify the key moieties in CO seeds and their cytotoxicity in rat primary hepatocytes and HepG2 cells. The identification of toxic compounds in the CO seeds was carried out by activity guided sequential extraction and fractionation of the seeds followed by GC-MS analysis. Subsequently, these identified compounds were detected and quantified in the blood and urine of CO exposed rats and CO poisoning study cases. GC-MS analysis of different fractions of methanol extract of CO seeds revealed the presence of five anthraquinones (AQs) viz. physcion, emodin, rhein, aloe-emodin and chrysophanol. Interestingly, these AQs were detected in the serum and urine samples of the study cases and CO exposed rats. Further, cytotoxicity of the above AQs in rat primary hepatocytes and HepG2 cells revealed that rhein is the most toxic moiety followed by emodin, aloe-emodin, physcion and chrysophanol. These studies indicate that AQ aglycones are responsible for producing toxicity, which may be associated with symptoms of HME in CO poisoning cases.

Key words: Cassia occidentalis, Seeds, Hepatomyoencephalopathy, Accidental poisoning, Anthraquinone, GC-MS, Children

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1. Introduction: Hepatomyoencephalopathy (HME) in young children, with the involvement of muscle, liver and brain with high mortality rate has been plaguing several parts of northern India over the past two decades.

1, 2

It occurs during seasonal outbreaks (Sep-Dec) and was earlier diagnosed

presumptively as encephalitis due to unknown viral etiology. 1, 2 Earlier, a case control study had shown that the illness is due to the consumption of the seeds of a common weed, Cassia occidentalis (CO).

3

World Health Organization in its report on prevention of child injury has

mentioned this aspect and urged to look into the etiology of children death. 4 Recently, the association between children death and consumption of CO seeds has been extensively examined by clinical and toxicological investigations. 5 In this study, comparison of the clinical parameters, biochemical alterations and histopathological changes both in HME patients and experimental rats exposed to CO seeds revealed a close similarity in the toxic manifestation. These observations strengthen the evidence for the association of CO seeds consumption with the etiology of the disease. However, none of the studies have confirmed the association of the chemical(s) responsible for CO poisoning cases having symptoms of HME. For definitive diagnosis of a toxic disease, the toxin has to be identified. In the absence of identified toxin(s) involved, the etiological association is based only on epidemiological and observational studies. 6 CO, belongs to family Leguminosae, grows throughout the tropics and subtropics including United States, Africa, Australia and Asia. 7 Though it is widely distributed in almost all areas of Indian subcontinent, the weed density is particularly high in western Uttar Pradesh (UP), Uttarakhand, Haryana, and Punjab.

8

Several experimental studies in domestic and laboratory

animals have been carried out to evaluate the toxic effects of CO seeds in different species 4


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including chicken, 9 rabbit, 10 and rat

11

to establish the significance of CO poisoning. Recently,

encephalopathy in cattle due to consumption of CO was reported in Brazil, where the mortality rate was over 75%.

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reported in Argentina.

A similar toxic encephalopathy in cattle causing high mortality was 13

The mechanism of toxicity of CO seeds in experimental animals

involves multiple pathways including impairment of antioxidant defense and xenobiotic metabolism, impairment in glucose/glycogen metabolism, down regulation of protective molecules leading to cell cycle arrest and apoptosis. 14 Despite these mortality data on domestic and experimental animals in addition to the accidental poisoning reports both in cattle and children, no study has been carried out to identify the key toxic ingredients of the CO seeds. Hence, the present study was designed to characterize the toxic moieties in CO seeds by systematic activity guided extraction, fractionation and GCMS analysis so as to provide the chemical evidence to the association of HME with CO seeds consumption.

2. Materials and methods: 2.1. Chemicals and reagents Aloe-emodin, Chrysophanol, Emodin, Physcion, Rhein, N,O-Bistrifluoroacetamide (BSTFA) in 1% trimethylchlorosilane (TMCS), anhydrous pyridine, RPMI-1640, Modified Eagle’s medium (MEM), Sodium pyruvate, Glutamine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Neutral red, collagenase from Clostridium histolyticum, collagen type IV from rat tail, antibiotic antimycotic solutions, trypsin, trypan blue were purchased from Sigma Aldrich (St Louis, MO). All the other chemicals and solvents used were of the highest purity available from commercial sources. 2.2. Collection, extraction and fractionation of CO seed sample

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CO seeds were collected from the Bijnor district of Uttar Pradesh, India and authenticated by one of us (VMV). One voucher specimen of the seed sample has been kept in Food Toxicology Division, CSIR-Indian Institute of Toxicology Research, Lucknow, India. The seeds were dried and grinded to fine powder. Sieving was carried out to obtain a homogenous yellowish powder. Samples were stored in amber colored bottles. Extraction of CO seed powder was carried out with increasing order of polarity of the solvents. In brief, sequential extraction was carried out with hexane (1:5, w/v), ethyl acetate (1:5, w/v), methanol (1:5, w/v) and water (1:5, w/v) to obtain respective extracts (Figure 1A). All these extracts were tested for cytotoxicity both in rat primary hepatocytes and HepG2 cells. Methanol extracts being the most cytotoxic one, was evaporated to dryness and further fractionated sequentially with hexane (1:5, w/v), chloroform (1:5, w/v), butanol (1:5, w/v) and water (1:5, w/v) to obtain respective fractions. The detailed outline of the method for preparation of above fractions of methanol extract is shown in Figure 1B. Subsequently, as that of extracts, these fractions of methanol extract were utilized for in vitro cytotoxicity studies in rat primary hepatocytes and HepG2 cells. 2.3. HepG2 cell line HepG2 cells (human hepatoma cell line) were obtained from the National Centre for Cell Science, Pune, India. Cells were grown in MEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (10,000 units of penicillin and 10 mg of streptomycin in 100ml of 0.9% NaCl) in a humidified atmosphere of 5% CO2; 95% air at 37◦C. The passage number for the cell line ranged between 10 to 15. For experimental purposes, a fully confluent 75 cm2culture flask containing the cells was trypsinized (500 µl of 0.25% trypsin). The cell viability and counting of live cells was carried out using trypan blue. Finally, 1x104cells/ well (in 200 µl media) were transferred to a 96 well plate. Cells were allowed to attach for 24 h prior to the treatment with different extracts or fractions of CO seeds. 6


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2.4. Rat primary hepatocytes culture Hepatocytes were isolated from the liver of overnight fasted male wistar rat (150-200g) according to the two step collagenase perfusion method described earlier with minor modifications. 15 In brief, perfusion was done initially by injecting calcium and magnesium-free HEPES buffer containing EGTA (0.4 g/L) at 80-100 mL/min for 10-15 min. The liver was then perfused with 0.5g/L collagenase solution at 50-70 mL/min for 10 min. After perfusion, the liver capsule was incised, the fibrous tissue was discarded and cell suspensions were harvested. The released cells were filtered through 100 µm cell strainer and washed three times with incomplete media (RPMI-1640) with concomitant centrifugations (50 g x 3min). The hepatocyte viability greater than 95% as determined by trypan blue exclusion was used for culture. Hepatocytes were maintained in RPMI-1640 media supplemented with 10% FBS and 1% of penicillinstreptomycin solution, 1mM sodium pyruvate, 2 mM glutamine under an atmosphere of 5% CO2; 95% air in an incubator (Eppendorf, Hamburg, Germany) with controlled humidity at 370C. The cells were seeded at a density of 5×104 cells/well in 0.1% collagen pre-coated 96 well plates. After 6 hrs, the media was changed to remove unattached cells followed by exposure to different extracts or fractions of CO seeds. 2.5. Cytotoxicity assessment of different extracts and fractions of CO seeds 2.5.1. MTT assay MTT dye uptake in HepG2 cells or rat primary hepatocytes in the presence and absence of CO seeds extracts and fractions were determined by the method described earlier.

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In brief, a

desired number of cells (1x104cells/well for HepG2 and 5x104cells/well in case of primary hepatocytes) in 200 µl media were dispensed in a 96 well plate on day 1, followed by exposure to different concentrations of extracts or fractions of CO seeds on day 2. After 24 hr of treatment, 7



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20 µl MTT (5 mg/ml PBS) was added to each well. The plates were further incubated for 2 hr. Following completion of the incubation time the media was decanted and the formazone formed was dissolved in 100 µl DMSO. Finally the absorbance was taken at 550 and 660 nm after 10 min, on a micro plate reader (Synergy HT, BIO-TEK International, Winooski). The percentage viability was calculated by comparing the absorbance of control and treated cells. 2.5.2. Neutral red uptake assay The neutral red uptake (NRU) assay was carried out following the method described earlier to determine the accumulation of neutral red dye in the lysosome of viable cells. 17 In brief, desired number of cells (as mentioned in section 2.5.1) were seeded in a 96 well plate on day 1 followed by exposure to different concentrations of extracts or fractions of CO seeds on the next day. After 24 hr of treatment, the media was removed from the wells and washed with PBS, followed by addition of 100 µl NR medium [media and NR stock solution (4 mg/ml PBS) in a ratio of 100:1, v/v]. The plates were then incubated at 370C for 2 hr. Subsequently, the NR medium was removed followed by washing with PBS. Finally, 100 µl of NR destaining solution (50% ethanol, 1% acetic acid and 49% distilled water) was added to each well with proper mixing. The accumulation of NR dye in the lysosome was measured by reading the absorbance at 540 and 660 nm after 5 min in a micro plate reader. The percentage viability was calculated by comparing the absorbance of control and treated cells. 2.6. Collection and preparation of serum and urine samples from accidental poisoning study cases Blood and urine samples were collected from patients after the approval of the Institutional Ethics Committee of Mangla Hospital, Bijnor, India (Ref. no. 2012/IEC/02). Patient blood samples were collected in heparin coated vials, centrifuged at 3000 g x 10 min and the serum was stored at -80°C until analysis. Preparation of serum samples for GC-MS analysis was done 8


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following the method described earlier. 18 In brief, 500 µl of serum sample was deproteinized by adding 50 µl of 3.5% perchloric acid. The aliquot was subsequently extracted with 2 ml of ethyl acetate and the process was repeated three times. The pooled ethyl acetate extracts were dried and further used for GC-MS analysis. Urine samples were collected in a 50 ml centrifuge tubes and stored at -80°C until analysis. Acid hydrolysis was performed as reported earlier. 19 Briefly, one ml urine sample was treated with 150 µl of 6N HCL and incubated at 900C for 30 min. The hydrolyzed samples were further extracted with 2 ml ethyl acetate and the process was repeated three times. The pooled ethyl acetate extracts were evaporated and further utilized for GC-MS analysis. 2.7. Preparation of serum and urine samples from CO seeds exposed rats Male wistar rats (150-200 g) used in this study were procured from the breeding colony of CSIRIndian Institute of Toxicology Research, Lucknow, India and were housed in air-conditioned room in stainless steel metabolic cages, maintained at 25±3oC under standard laboratory conditions of light/dark cycle (12-12 hr) and have free access to food (Provini Animal Nutrition India Pvt Ltd, Bangalore) and water ad libitum. All animal handling procedures were performed following the regulations of Institutional Animal Ethics Committee with prior approval for using the animals (Ref. no. ITRC/IAEC/16/2009). After five days of acclimatization, five overnight fasted rats were orally given a single dose of aqueous suspension of CO seeds (2000 mg/kg) while control animals were treated with the vehicle in a similar fashion. Following treatment, urine was collected for 48 hr and blood was collected through cardiac puncture after sacrificing the animal by cervical dislocation after 48 hr. The collected urine and serum samples were processed and extracted in a similar manner as mentioned above in section 2.6. 2.8. Derivatization and gas chromatography mass spectrometry (GC-MS) analysis

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Methanol extract and its further fractions byhexane, chloroform, butanol and water); and ethyl acetate extract of serum as well as urine of patients and experimental rats were dried and treated with 150 µl of pyridine and 200µl of BSTFA+1%TMCS followed by incubation at 70◦C in a sealed 2 mL glass vial for 60 min. Analysis was performed using Trace Gas Chromatography coupled with Quantum XLS mass spectrometer (Thermo Scientific, FL, USA). Briefly, the analytical conditions were: temperature of injector 250°C; temperature of ion source 220°C; temperature of transfer line 2900C. The oven temperature was initially held at 1000C for 2 min and then increased to 2100C at a rate of 200C per min and hold time for 1min, followed by 3000C at a rate of 100C per min and finally hold for 20 min. A 1 µL aliquot of the sample was injected into an Elite-5MS capillary column (60 m x 0.25µm film thickness of 5% phenyl and 95% methyl polysiloxane as stationary phase) (Perkin Elmer, Waltham, MA) in the splitless mode. Mass detection was achieved using electron impact ionization at 70 eV. Full scan mass spectra were acquired at the mass range of 45- 800 Da with an initial solvent delay for 6 min. In this study the standard anthraquinones (AQs) were prepared, derivatized and analyzed by GC-MS by the same method described above. 2.9. Identification and quantification of AQs by GC-MS The five major AQs in CO seeds were identified by comparing the mass spectra of standard compounds available in the NIST library and by matching the retention times with the standard compounds. Limit of detection (LOD), Limit of quantification (LOQ) were calculated as per the recommendations of Eurachem guide.

20

Five replicates were used to determine LOD and LOQ

for each AQ. All the quantifications were performed according to an earlier established method. 21

Briefly, calibration curves of 0.1 to 50µg/ml of all the standard AQs were constructed.

Subsequently, the concentrations of AQs in the extracts/ fractions/ serum/ urine were calculated by linear regression analysis from the calibration curve equation mentioned in Table 4. 10


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2.10. Evaluation of cytotoxicity of AQs The relative cytotoxic potential of the five AQs (aloe- emodin, chrysophanol, emodin, physcion and rhein) identified in CO seed and further detected in the serum and urine of patients and experimental rats, were examined by MTT and NRU assay in primary rat hepatocytes and HepG2 cells as described earlier in section 2.5. 2.11. Statistical analysis The statistical analysis was carried out by the software Prism 5. A value of p < 0.05 was used as the level of significance. The 50% lethal concentration (LC50) of different solvent extracts, subsequent fractions and purified AQs were calculated following non linear regression analysis by Prizm 5 software.

3. Results: 3.1. Cytotoxicity profile of CO seed extracts and fractions Different extracts of CO seeds were prepared for cytotoxicity assessment in HepG2 cells and rat primary hepatocytes. It was observed that among all the extracts, methanol extract of CO seeds showed maximum toxicity in both the cell types. A minimum concentration of 125µg/ml and 250µg/ml of methanol extract significantly reduced (20-30%) the viability of HepG2 cells and rat primary hepatocytes, respectively. The similar doses of other extracts (hexane, ethyl acetate and water) exhibited no significant cytotoxicity (Figure 2A, 2B, 2C and 2D). The LC50 values of all the extracts of CO seeds in HepG2 cells and rat primary hepatocytes are depicted in Table 1. The LC50 of methanol extract was found to be 728.4+2.1 and 812.7+1.1 µg/ml in HepG2 cells and rat primary hepatocytes, respectively, whereas other extracts exhibited the LC50 values more than 1000µg/ml (Table 1). Due to higher cytotoxicity of methanol extract, this was further partitioned into different fractions using hexane, chloroform, butanol and water. The resulted fractions were further studied for cytotoxicity. The data showed that the chloroform fraction of 11



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methanol extract exhibited maximum cytotoxicity in both types of cells as compared to other fractions (Figure 3A, 3B, 3C and 3D). Concentrations of 62.5µg/ml and 125µg/ml of chloroform fraction significantly reduced (20-30%) the viability of HepG2 cells and rat primary hepatocytes respectively, followed by hexane, butanol and water fractions. The LC50 values of all the fractions of methanol extract of CO seeds in HepG2 cells and rat primary hepatocytes are given in Table 1. The LC50 of chloroform fraction was found to be 242.9+1.1 and 439.9+2.9 µg/ml in HepG2 cells and rat primary hepatocytes, respectively, whereas other fractions exhibited higher LC50 values. 3.2. Identification of compounds in CO seed extract/fractions by GC-MS To identify the bioactive compounds in the methanol extract and its fractions, GC-MS analysis was carried out. The representative total ion chromatograms (TIC) of methanol extract is shown in Figure 4, whereas the TIC for hexane, chloroform, butanol and water fractions of methanol extract of CO seeds are depicted in Figure 5A-D respectively. Initially, each peak of TIC of methanol extract was analyzed in detail and found to contain a number of compounds including an AQ derivative (Physcion) at retention time 17.6min (Figure 4). Further, this compound was also detected in the TIC of chloroform and hexane fractions (Figure 5A and 3B). The detailed lists of all the compounds with their retention time detected in the methanol extract are mentioned in Table 2. Due to the presence of AQ derivatives in the methanol extract and its chloroform and hexane fractions, we focused our attention in identifying other AQ derivatives found in genus Cassia in these fractions. Hence, all the fractions were analyzed in specific ion monitoring (SIM) mode with m/z 383, 413, 471,471 and 485 as target ions for chrysophanol, physcion, emodin, aloe-emodin and rhein, respectively, as reported in different plants.

22

The

analysis revealed the presence of physcion, emodin and rhein in chloroform fraction; physcion, chrysophanol and aloe- emodin in hexane fraction; chrysophanol and physcion in butanol 12


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fraction (Figure S1, S2 and S3). Further, none of the above AQs were detected in water fraction. The data of gas chromatogram and mass spectra of the identified AQ derivatives in hexane, chloroform and butanol fractions are depicted in Figure S1, S2 and S3. Further, these identified AQ derivatives in different fractions were confirmed with the m/z of respective standards. The mass spectra, structure and target ions of the standard compounds are depicted in Figure 6, whereas the retention time and identified ions are given in Table 3. 3.3. Quantification of AQs in different fractions of CO seed by GC-MS The identified AQs were quantified in different fractions of methanol extract of CO seeds using GC-MS selective ion monitoring (SIM) mode by constructing the calibration graph in the range of 0.1-50 µg/ml. The calibration equation, coefficient of regression, limit of detection and limit of quantification of different AQs are mentioned in Table 4. The concentrations of the above AQs in the respective fractions are mentioned in Table 5. Finally, the amount of different AQs in CO seeds were calculated and depicted in Table 6. The concentrations of chrysophanol, physcion, emodin, aloe-emodin and rhein in seed sample were found to be 68.7, 4134, 269, 52.5 and 132.0 µg/g, respectively. Additionally, the amount of AQ aglycones was calculated to be 0.46% in CO seeds on dry weight basis. 3.4. Detection of AQs in patient’s serum and urine Serum samples from eight patients and urine samples from four patients were analyzed by GCMS to ascertain the presence of AQs that were detected in different fractions of methanol extract of CO seeds. GC-MS analysis of serum from patients showed the presence of physcion in all the samples, whereas three samples (HS6, HS7 and HS8) showed the presence of all the five AQs (chrysophanol, physcion, emodin, aloe-emodin and rhein), that were found in different fractions of the CO seeds (Table 7 and Figure 7). It was observed that the amount of different AQ in the patient serum ranges from 0.7 to 1.26 µg/ml. In addition, it was also observed that amongst all 13



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the AQs, the concentration of rhein was maximum (0.8 to 1.26 µg/ml) in the serum of three patients that died during the course of treatment (Table 7). Among the four urine samples, one or more AQs were detected in three samples (HU1, HU2 and HU3). Interestingly, rhein was detected in all the 3 samples. In addition, emodin and aloe-emodin were also detected in HU1, whereas physcion, emodin, aloe emodin, chrysophanol and rhein were detected in HU3. The concentration of different AQs varies approximately from 0.3 to 0.6 µg/ml. The concentration of rhein was found to be the highest (0.5 to 0.6 µg/ml) in all the three samples of urine compared to other AQs (Table 7). The concentration of rhein detected in serum was found to be 2-3 times higher as compared to that of urine. 3.5. Detection of AQs in serum and urine of CO seeds treated rats In animal experimental study, the serum and urine samples from CO treated rats were collected during the first 48 hr. GC-MS analysis of the serum and urine samples from experimental rats showed the presence of all the five AQs that were detected in the CO seeds (Figure 7). The concentration of different AQs ranged from 0.6 to 0.8 µg/ml in serum and 0.3 to 0.4 µg/ml in urine samples (Table 7). As in the case of serum and urine of patients, the concentration of rhein was also found to be the highest in both serum and urine of experimental rats. In addition, the concentration of rhein detected in serum was found to be two times higher as compared to that of urine of experimental rats. 3.6. Cytotoxicity of standard AQs All the five AQs (chrysophanol, physcion, emodin, aloe-emodin and rhein) identified in different fractions of CO seeds and detected in the body fluids of patients and experimental rats were examined for their relative cytotoxicity by MTT and NRU assay in rat primary hepatocytes and HepG2 cells. From the in vitro cytotoxicity data, it was observed that rhein at a minimum concentration of 3.125µg/ml and 12.5µg/ml significantly decreased the viability in HepG2 cells 14


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and rat primary hepatocytes, respectively. However, other AQs were found to be toxic at higher concentration as compared to rhein in both the cell types (Figure 8). The LC50 of all the AQs are presented in Table 1. The LC50 of rhein was found to be 14.1+0.8 and 29.1+1.6 µg/ml in HepG2 cells and rat primary hepatocytes, respectively, whereas other AQs exhibited LC50 more than that of the rhein. The increasing order of cytotoxicity of the AQs was observed to be chrysophanol < physcion < aloe- emodin < emodin < rhein, based on their LC50 values (Table 1).

4. Discussion: World Health Organization in 2008, in its report on child injury prevention mentioned two plant toxin induced children death.

4

One of the tragedies was in Haiti, where 65 children died after

eating unripe ackee fruit. 23 The second one was the consumption of CO seeds, the possible cause of recurrent outbreaks of encephalopathy among children in western Uttar Pradesh, India.

3, 5

Among these two incidences, the later draws attention because of its re-occurrence during the past several years with high mortality rate. Our prior studies have shown the association between children death and consumption of CO seeds.

5

However, chemicals involved in CO poisoning

are not known. Hence, in the present study we have attempted to identify the toxic moieties in the CO seeds by simple bioassay guided extraction and fractionation, followed by detection of toxicants in the body fluid of patients and experimental animals by GC-MS, so as to provide the chemical evidence for this association. For in vitro screening of plant extracts/fractions, cytotoxicity assays were carried out as these cell based assay systems are often used to predict human toxicity and for the general screening of chemicals.

24

In order to avoid inconsistency of

results and to get reliable data, more than one assay should be used to determine toxic potential of the compounds. 25 Hence, in the present study, both MTT and NRU assays were employed for in vitro cytotoxicity assessment. Since, earlier studies have reported the hepatotoxic potential of 15



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CO seeds,

5, 26

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in this study liver cells both from human and rodent origin e.g. HepG2 cells and

rat primary hepatocytes were used for in vitro cytotoxicity assessment of different extracts, fractions, as well as purified AQs. Initially CO seeds were extracted with four different solvents of varying polarity to extract most of the compounds. For this purpose four solvents viz hexane, ethyl acetate, methanol and water were selected for extraction of compounds from CO seeds. 27, 28 Later, these four extracts were tested for in vitro cytotoxicity. Of which, methanol extract showed the maximum cytotoxicity in HepG2 cells and primary rat hepatocytes. Since methanol extract is a crude plant extract, it needs to be fractionated for bioactivity or chemical characterization. Hence, further fractionation of methanol extract was carried out using four other solvents like hexane, chloroform, butanol and water.

28

The cytotoxicity of different fractions of methanol

extract showed chloroform fraction to be the most toxic followed by hexane, butanol and water fractions. Methanol extract along with all the four fractions of this extract were analyzed by GCMS to identify the chemical components. The mass spectrum of each and every peak of the TIC of methanol extract was thoroughly examined and compared with the existing mass spectra of reference compounds in the NIST library. The similarity index of more than 90% was used as a criterion to validate the compounds. The TIC of methanol extract was found to contain a number of compounds such as amino acids, sugar alcohols, carbohydrates, fatty acids and organic acids including some steroids like stigmasterol and campesterol, none of which possess toxicity reports. Interestingly, during the analysis, an AQ aglycone referred to as physcion was detected in the TIC of methanol extract, which was also detected in the TIC of chloroform and hexane fractions. Hence, we focused our attention on this group of compounds due to their toxicity reports. 29, 30 Our subsequent results suggest that four other AQs viz chrysophanol, emodin, aloe16


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emodin and rhein apart from physcion are present in the hexane, butanol and chloroform fractions of methanol extract of CO seeds. Above results, indicate that the toxicity of extracts/fractions of CO seeds is possibly due to the AQs. To ascertain this, the level of anthraquinones in the methanol extract and its fractions were quantified. Then the doses of anthraquinones in the fractions/extracts were calculated and finally compared to the doses of the pure compounds causing the toxicity. The levels of AQs in chloroform, hexane, butanol and water fractions were calculated to be 120mg/g, 65mg/g,10.5mg/g and 0 respectively. Interestingly, the cytotoxicity of the fractions revealed chloroform fraction to be the most toxic followed by hexane, butanol and water fractions. Chloroform fraction contains maximum AQs (120mg/g) being highest toxic, whereas water fraction contains no AQs was least toxic. Hence, the order of toxicity of fractions was found to be in increasing order of AQ content, which supports the fact that toxicity of the CO seeds may be due to these AQs. To validate this observation, the cytotoxicity of all the standard AQs were evaluated in HepG2 cells & rat primary hepatocytes and compared to that of extracts/fractions. The in vitro cytotoxicity potential

of

these

AQs

was

found

to

be

in

the

following

order;

chrysophanol1000

Water

>1000

>1000

Anthraquinones standards

LC50(µg/ml) HepG2 cells

Primary rat hepatocytes

Rhein

14.1+0.8

29.1+1.6

Emodin

23.8+1.2

43.7+2.1

Aloe emodin

26.1+1.4

52+2.6

Physcion

32.6+1.8

59.6+2.5

Chrysophanol

49.3+2.2

80.3+4.0

Values in the parenthesis indicate the total amount of different anthraquinones (µg/ml) in the given dose

30


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Table 2: List of the compounds identified in methanol extract of CO seeds by GC-MS

SL. No.

RT

Name of the compound

25

8.77

3- Glycerophosphate

1

4.87

L-Valine

26

8.96

2 Keto 1-gluconic acid

2

5.49

L-Isoleucine

27

9.16

D-Fructose

3

5.53

D- Proline

28

9.22

Citric acid

4

5.60

Succinic acid

29

9.29

Methyl galactopyranoside

5

6.18

L-Threonine

30

9.60

Glucose

6

6.85

DL- malic acid

31

9.79

L-Gluconic acid

7

6.95

Threitol

32

9.90

Mannose

8

7.01

Erythritol

33

9.98

Galactose

9

7.07

L-Aspartic acid

34

10.19

L-Tyrosine

10

7.12

Pyroglutamic acid

35

10.25

Glucitol

11

7.24

Hydroxy Norvaline

36

10.36

Galactitol

12

7.27

Pentitol

37

10.46

Myo-inositol

13

7.34

L-Theonic acid

38

10.66

Pantothenic acid

14

7.59

D- Erythro-pentitol

39

10.84

Galactonic acid

15

7.61

L-Tartaric acid

40

10.94

Hexadecanoic acid

16

7.67

Glutamic acid

41

11.02

Galactaric acid

17

7.79

L-Phenylalanine

42

11.42

Ferulic acid

18

7.82

Xylonic acid

43

11.64

Inositol

19

7.84

Dodecanoic acid

44

12.37

9,12- octadecadienoic acid

20

8.16

Trans Aconitic acid

45

12.40

Trans 9-octadecenoic acid

21

8.25

Xylulose

46

12.63

Myo inositol monophosphate

22

8.35

1,6- Anhydro β D-glucose 47

13.77

Myo inositol diphosphate

23

8.38

Xylitol 48

13.96

D-Glucuronic acid

24

8.47

D-Arabitol

31



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Page 32 of 45

49

14.13

Uridine

56

16.30

Lactose

50

14.20

Eicosanoic acid

57

17.59

Physcion

51

14.41

Inositol phosphate

58

20.26

D-Melibiose

52

15.24

Sucrose

59

21.30

Campesterol

53

15.41

1- Monopalmitin

60

21.69

Stigmasterol

54

15.46

Turanose

61

24.69

Raffinose

55

15.94

Adenosine

32


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Table 3. Retention time and important ions present in the mass spectra of silylated anthraquinones Anthraquinones

Retention timea (min)

Identified ionsb (m/z)

Chrysophanol

15.72

383,311,253,184,73

Physcion

17.36

413,341,298,200,73

Emodin

17.56

471,355,327,228,73

Aloe-emodin

17.78

471,367,339,220,73

Rhein

18.38

485,413,295,235,73

a. Characteristic peaks of TMC derivatives detected by GC-FID b. Characteristic peak of TMC derivatives detected by GC-MS

33



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Page 34 of 45

Table 4. Calibration curve, LOD and LOQ of TMC derivatives of anthraquinones

Anthraquinone

Calibration curve equation

Coefficient(R )

Limit of detection(ng/ml)

Limit of quantification(ng/ml)

2

Chrysophanol

Y=36998730.86X12007935.83

0.997

3.725

12.292

Physcion

Y=37766164.01X13111558.50

0.996

3.216

10.612

Emodin

Y=54144137.61X16902455.83

0.997

3.169

10.457

Aloe-emodin

Y=47448325.03X17,072747.28

0.996

3.198

10.553

Rhein

Y=35174093.53X14902588.72

0.995

4.489

14.813

34


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Table 5. Identification and quantification of anthraquinones present in different fractions of the methanol extract of CO seed* Fractions

Compound identified

Target ion

RSD (%)

383 413 471

Concentration in pyrridin and BSTFA mixture (µg/ml) 0.45 16.55 1.6

2.22 1.83 1.27

Amount in 100µg of fraction 0.1575 5.7925 0.560

Hexane

Chrysophanol Physcion Aloe- emodin

CHCl3

Physcion Emodin Rhein

413 471 485

30.25 2.54 1.25

0.92 2.12 1.6

10.59 0.889 0.4375

BuOH

Chrysophanol Physcion

383 413

0.35 2.66

0.98 0.57

0.1225 0.931

H 20

-

-

-

-

-

Concentration of anthraquinones in respective fraction (mg/g) 1.575 57.925 5.60 Total AQ: 65 mg/g 105.9 8.89 4.375 Total AQ: 120 mg/g 1.225 9.31 Total AQ: 10.5 mg/g -

Total content in respective fractions(mg) of MeoH extract(15g) 1.47(0.93g) 53.85(0.93g) 5.25(0.93g) 320.8(3.03g) 26.9(3.03g) 13.2 (3.03g) 5.4 (4.38g) 40.7(4.38g)

(6.0g)

* Calculations for quantification of specific anthraquinones: 100µg of each fraction was taken for GCMS study (100µl of 1mg/ml fraction was evaporated). 350µl of Pyridine and BSTFA reagents was added. 1µl of above was injected to GC. The peak area was calculated. Quantification was done as per the calibration curve equation for each anthraquinones as mentioned in Table 4. Then the concentration in pyrridin and BSTFA mixture was calculated as µg/ml. Finally, the concentration was calculated for respective fractions of methanol extract. It may be noted that extraction with methanol yield 15% of the extract. Subsequent fractionation of 15g methanol extract yield 0.93g hexane fraction, 3.03g CHCl3 fraction, 4.38g butanol fraction and 6g aqueous fraction.

35



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Page 36 of 45

Table 6. Concentration of five anthraquionone in methanol extract and CO seed*

Anthraquinone

Total content(mg) of anthraquinones in MeoH extract (15g)

Content of anthraquinones in CO seed (µg/g)*

Chrysophanol

6.87

68.7

Physcion

415.35

4153.5

Emodin

26.9

269.0

Aloe-emodin

5.25

52.5

Rhein

13.2

132.0

Total

467.57

4675.7 (0.46% of seed)

*Calculations of concentration of AQs have been made taking in to consideration of data from Table 5.

36


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Table 7. Average concentration of anthraquinones present in patient and experimental rat body fluids (µg/ml) Sample

Aloe-

Chrysophanol

Physcion

Emodin

HS1

ND

0.744

ND

ND

ND

HS2

ND

0.726

ND

ND

ND

HS3

ND

0.702

ND

ND

ND

HS4

ND

0.968

ND

ND

ND

HS5

ND

0.764

ND

ND

ND

HS6

0.762

0.82

0.845

0.807

1.262

HS7

0.745

0.803

0.904

0.808

1.247

HS8

0.665

0.704

0.64

ND

0.868

HU1

ND

ND

0.395

0.381

0.614

HU2

ND

ND

ND

ND

0.506

HU3

0.26

0.39

0.35

0.36

0.497

HU4

ND

ND

ND

ND

ND

RS1

0.653

0.705

0.626

0.724

0.844

RS2

0.644

0.708

0.624

0.727

0.848

RS3

0.643

0.703

0.628

0.725

0.846

RU1

0.334

0.358

0.325

0.391

0.439

RU2

0.336

0.366

0.323

0.414

0.439

RU3

0.335

0.365

0.328

0.416

0.435

code

emodin

Rhein

Each sample was run in triplicate. HS- Patient sera, HU- Patient urine, RS- rat sera, RU- rat urine, ND- not detected

37



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Extraction and fractionation of CO seeds 184x120mm (300 x 300 DPI)


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Cytotoxicity profile of different extracts of CO seeds after 24hr 202x166mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Cytotoxicity profile of different fractions of methanol extracts of CO seeds after 24hr 196x170mm (300 x 300 DPI)


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Total ion chromatogram of methanol extract of CO seeds 201x162mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Total ion chromatogram (TIC) of different fractions of methanol extract of CO seeds 191x145mm (300 x 300 DPI)


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Mass spectra, target ion and structure of chrysophanol, physcion, emodin, aloe- emodin and rhein. 203x159mm (300 x 300 DPI)



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Detection of different AQs in the body fluids of human patients and experimental animals 188x137mm (300 x 300 DPI)


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Cytotoxicity profile of standard AQs of CO seeds after 24hr 201x171mm (300 x 300 DPI)


Cassia occidentalis - ACS Publications - American Chemical Society

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