Genotypic Assessment by RAPD Markers and Ultrastructural Characteristics of a NaCl-Tolerant Potato Cell Line

4. Conclusion and Discussion

important to the test result. The precision of the data There are some methods to detect stomach function

acquisition system is the base of others, it should be in clinical now. But most of the routine methods are

selected by experiment demand. ADuC834 is a chip invasive or dear. Bioelectrical impedance because of its

collecting multiple functions into it, especially for noninvasive, safe, cheap, simple and functional

temperature and pressure changes slowly and the information-rich features, is easy to be accepted by

measurement of DC or low frequency signals.

Parameters Analysis of Gastric Motility Signals in Time Domain and Frequency Domain

Additionally, it can sample gastric motility impedance

770-774. [2] Z. Li, C. Ren, Gastric motility measurement and

signal in high-precision 24-bit, so it is possible that we evaluation of functional dyspepsia by a bio-impedance

observe the greatest range of impedance signals based method, Physiol. Meas. 29 (6) (2008) 373-382. on changes in gastric motility. Wavelet transform

[3] J.D. Chen, X.M. Lin, M. Abo, Multi-channel gastric successfully removes the influence of the heart activity

electrical simulation for the acceleration of gastric signals. It ensures that all the information used for emptying, in: Advances in Medical Signal and Information Processing: First International Conference

analyzing is valid. Percentage of normal frequency, (IEEE Conf. PUB), 2000, pp. 60-65. percentage of normal power, frequency instability

[4] M. Kusano, H. Zai, Y. Shimoyama, H. Hosaka, S. coefficient and power instability coefficient are several

Kuribayashi, O. Kawamura, et al., Rapid gastric emptying, of the most representative parameters which could

rather than delayed gastric emptying might provoke functional dyspepsia, J. Gastroenterol. Hepatol. 26 (3)

clearly reflect the difference of CG and PG.

(2011) 75-78.

In both time and frequency domains, impedance [5] P. Du, G. O’Grady, L.K. Cheng, A.J. Pullan, A multiscale signal and synchronize EGG have some similar

model of the electrophysiological basis of the human features. However synchronize EGG cannot be totally electrogastrogram, Biophys. J. 99 (9) (2010) 2784-2792.

X.H. Xu, H.B. Zhu, J.D. Chen, Pyloric electrical instead by gastric motility, especially in morbid state,

stimulation reduces food intake by inhibiting gastric EGG is not correspondence to impedance signal. The

motility in dogs, Gastroenterology 128 (1) (2005) 43-50. gastric contraction or gastric emptying is a complex

[7] Z. Li, C. Ren, Gastric motility measurement and procedure including electrical and mechanical activity. evaluation of functional dyspepsia by a bio-impedance method, Physiol. Meas. 29 (6) (2008) 373-382.

EIP and the synchronous EGG should be analyzed [8] N.A. Hadi, A. Giouvanoudi, R. Morton, Variations in together. We will make a great of experiments to obtain

gastric emptying times of three stomach regions for simple the relationship between EIP and EGG. All the

and complex meals using scintigraphy, IEEE Transactions procedure in digestion, such as EGG, gastric on Nuclear Science 49 (2002) 2328-2331.

A. Giouvanoudi, W.B. Amaee, J.A. Sutton, Physiological contraction, peristalsis and gastric emptying, have to be

interpretation of electrical impedance epigastrography think over, the operative evaluation to gastric kinetics

measurements, Physiol. Meas. 24 (1) (2003) 45-55. can be constructed. We will add more EIP parameters

A. Keshtkar, Z. Salehnia, M.H. Somi, A.T. Eftekharsadat, to improve the performance in further research. In turn Some early results to electrical impedance of normal and abnormal gastric tissue, Phys. Med. 28 (1) (2012) 19-24.

this could lead to the development of a noninvasive

H. Ji, C. Ferm йller, Robust wavelet-based method for the continuous recording of volume

super-resolution reconstruction: Theory and algorithm, variability with gastric contraction.

IEEE Trans. Pattern. Anal. Mach. Intell. 31 (4) (2009) 649-660.

Acknowledgment

A. Bernjak, A. Stefanovska, Importance of wavelet analysis in laser Doppler flowmetry time series, in: Conf. This project is supported by National Natural

Proc. IEEE Med. Biol. Soc., 2007, pp. 4064-4067. Science foundation of China (60901045), the Science [13] Z. Li, C. Ren, Gastric motility measurement and evaluation of functional dyspepsia by a bio-impedance

and Technology Foundation of Chongqing Education method, Physiol. Meas. 29 (2008) 373-382.

Committee (KJ090505) and Science and Technology [14] M. Chacón, G. Curilem, G. Acu ňa, C. Defilippi, A.M. Research Key Projects Foundation of Chongqing,

Madrid, S. Jara, Detection of patients with functional China (CSTC, 2009AA5049). dyspepsia using wavelet transform applied to their electrogastrogram, Braz. J. Med. Biol. Res. 42 (12) (2009)

References 1203-1209.

[15] J.A. Sutton, G.R. McClelland, J.A. Sutton, Epigastric [1] R. Huerta-Franco, M. Vargas-Luna, E. Hernandez, K.

impedance: A non-invasive method for the assessment of Capaccione, T. Cordova, Use of short-term bio-impedance

gastric emptying and motility, Gut. 26 (6) (1985) for gastric assessment, Med. Eng. Phys. 31 (7) (2009)

607-614.

Journal of Life Sciences 6 (2012) 20-27

Validation of Metformin Hydrochloride in Human Plasma by HPLC-Photo Diode Array (PDA) for Application of Bioequivalence Study

Yahdiana Harahap, Krisnasari Dianpratami, Mahi Wulandari and Rina Rahmawati Department of Pharmacy, University of Indonesia, Depok, Jakarta 16424, Indonesia

Received: March 10, 2011 / Accepted: July 25, 2011 / Published: January 30, 2012.

Abstract: A sensitive and specific high performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of metformin hydrochloride (HCl) in human plasma. The HPLC method consists of isocratic eluation with

a mixture of 60% buffer (10 mM sodium dihyrogenphosphate-10 mM sodium dodecyl sulphate) and 40% acetonitrile with final pH 7.0 with flow rate of 1.0 mL/min on a Kromasil ® Akzo Nobel RP-18 (4.6 mm ID × 250 mm, 5 µm) column at an ambient temperature. Photo diode array detection was performed in program mode at 234 nm. The analyte and diazepam as internal standard (IS) were extracted from plasma using 10% trichloroacetic acid. The assay was linear over the therapeutic concentration range of 20-2,500 ng/mL for metformin HCl with correlation coefficient of r = 0.9999. Limit of quantitation was 20 ng/mL. The results obtained for intra/inter day accuracy and precision complied very well with the generally accepted criteria for bio-analytical assay. The method was applied to bioequivalence (BE) study of metformin HCl in healthy Indonesian volunteers after treatment with 750 mg XR metformin HCl. This BE study shows that the two formulations are equivalent so that they were therapeutically interchangeable for each other.

Key words: Metformin HCl, diazepam, HPLC, sodium dodecyl sulphate (SDS), photo diode array (PDA) detector, bioequivalence (BE) study.

1. Introductions peak (C max ) of 1.6 ± 0.38 g/ml are reached (T max ) at

2.6 ± 0.8 h after oral administration of a single 500 mg Metformin hydrochloride (N,N-Dimethyl-imido-di- dose [2, 6]. It is negligibly bounded to plasma proteins carbonimidic diamide hydrochloride) is a strongly and approximately 90% of the absorbed drug that is basic bisubstituted guanidine derivative with short side eliminated via the renal route within the first 24 h, with chains. An oral biguanidine antihyperglycaemic agent plasma elimination half-life of 3.6-6.2 h [5-7]. improves glucose control in patients with type 2 Metformin hydrochloride is freely soluble in water diabetes by lowering both basal and postprandial and it is practically insoluble in acetone, ether, plasma glucose levels [1-4]. Metformin HCl decreases chloroform, and most organic solvents [3], which hepatic glucose production, intestinal absorption of renders its extraction from aqueous complex plasma glucose, and improves insulin sensitivity by increasing matrix difficult [4, 8, 9]. Because metformin HCl is peripheral glucose uptake and utilization [5-6]. very polar, and is usually analyzed using normal phase Metformin hydrochloride is slowly and with Silica column [4, 8, 9], but it is not economical in incompletely absorbed from the gastrointestinal tract time consuming because of complex extraction with a bioavailability of 50% to 60%. Plasma levels procedures. Analyzing it using reversed phase (RP)

column will not provide optimum result, therefore this Corresponding author: Yahdiana Harahap, Ph.D., Prof.,

method used RP system that is combined with ion-pair. research fields: bioanalysis, BE study and DNA adducting.

E-mails: yahdiana03@yahoo.com; diana@farmasi.ui.ac.id. This method used simple sample preparation steps. To

Validation of Metformin Hydrochloride in Human Plasma by HPLC-Photo Diode Array (PDA) for

Application of Bioequivalence Study

ensure that the used method shall give accurate and metformin was prepared by diluting aliquots of the reliable result for bioequivalence study, a bioanalytical

stock solution with the distilled water to make up final validation must be performed on the method [10].

concentration of 100, 25, 10, 2.5, 1, 0.5 and 0.2 µg/mL. The objective of this research is to obtain the validity

These were used to prepare for plasma calibration of metformin analysis method using RP system which

standard in the linear dynamic range covering 20 to is combined with ion-pair in order to be applied for

2,500 ng/mL. Three quality control (QC) plasma bioequivalence study.

samples containing 100, 1,000 and 2,000 ng/mL were

2. Experimental prepared. The QC samples were used to determine

stability: long term, short term, stock solution, freeze

2.1 Chemicals and thaw stability and auto sampler stability. Accuracy Metformin hydrochloride and the internal standard

and precision were also evaluated using the above QC diazepam were obtained from the United State

samples. For the precision was checked by calculating, Pharmacopeia reference standard and Menjangan Sakti

the variation of the measured value and the accuracy Company. Sodium dihydrogen phosphate, sodium

was checked by calculating the difference between the hydroxide, trichloroacetic acid, sodium dodecyl measured values and the actual values. Precision and sulphate, as well as HPLC-grade methanol, acetonitrile

accuracy should be measured by using minimum of were purchased from Merck (Germany) and five determinations per concentration and a minimum

aquabidestilata from Ikapharmindo (Indonesia). three concentrations in the range that expected Human plasma was used as the assay blank and for the

concentration is recommended. The acceptance limit preparation of spiked plasma standards was obtained

for coefficient of variation (CV) and % differentiation from the Indonesian Red Cross (Palang Merah

(Diff) is should not exceed ± 15%, except for the lower Indonesia, Jakarta).

limit of quantification (LLOQ) where it should not exceed ± 20% of the CV and Diff.

2.2 Chromatographic Condition The internal standard diazepam stock standard

The concentration of metformin HCl in plasma is solution was prepared by dissolving 10 mg in 10 mL determined using a high performance liquid methanol to make a final concentration of 1,000 µg/mL. chromatography with PDA detection. The HPLC

2.4 Sample Preparation

system is Waters 2695 equipped with auto sampler. The separation was performed on Kromasil ® C-18, 250

As much as 600 µL of human plasma which contains × 4.6 mm, 5 µm column from Akzo Nobel. The

metformin hydrochloride will be mixed in a 1.5 mL wavelength was set at 234 nm. The mobile phase was a

effendorf vial with 30 µL internal standard (1,000 mixture of 40% acetonitrile, 0.01 M sodium dodecyl

µg/mL in distilled water) and 600 µL trichloroacetic sulphate, 0.01 M sodium dihydrogen phosphate and

acid. The sample will be shaken with vortex for 120 distilled water to 100%, adjusted to pH 7 at a flow rate

seconds and centrifuged at 10,000 rpm for 5 min. After of 1 mL/min.

that 1,000 µL supernatant was separated in a clean vial before adding 60 µL of 4 N NaOH. The mixture was

2.3 Matrix Based Standard Solutions and Quality vortexed (5 seconds). A 100 µL aliquot of sample was Control Samples injected into the equilibrated HPLC system.

A stock standard solution of metformin (1,000 µg/mL)

2.5 Application to Bioequivalence Study was prepared by dissolving 10 mg metformin HCl in

10 mL by distilled water. Working standard of Twelve healthy volunteers were included in this

22 Validation of Metformin Hydrochloride in Human Plasma by HPLC-Photo Diode Array (PDA) for Application of Bioequivalence Study

study. The study protocol was approved by the Ethics obtained values were within ± 20% of the actual value Committee of Medical Faculty of University Indonesia

and the reproducibility was within the CV value 20%. and written informed consent was obtained from the

In this research, we observed that the concentration of volunteers. Metformin was administered in a single

101.7 ng/mL gives CV value of 2.69% and % Diff dose of 750 mg to the volunteers after over-night

between -6.11% to 1.26%, concentration of 50.85 fasting. Plasma samples were collected at several

ng/mL gives CV value of 1.48% and % Diff between intervals after dosing until 30 hours and freeze

-2.37% to 1.37%, and concentration of 20.34 ng/mL immediately at -20ºC until assayed.

gives CV value 2.46% and % Diff 9.77% to 16.86%. Therefore, the limit of quantitation of this procedure

3. Results and Disscusions

was established at 20.34 ng/mL, because the CV and %

3.1 Bioanalytical Validation-Calibration Curves Diff values are not more than 20%. The standard substance was dissolved in water to

3.4 Precision

make standard stock solution of 1,000 µg/mL The precision was checked by calculating the metformin HCl. Certain amount of standard stock variation of the measured value. The acceptance limit

solution was diluted with blank plasma to make the for the precision was within ± 15% for low, medium

following concentrations: 0 (blank), zero (blank + and high concentrations and within ± 20% for the lower

internal standard), 20, 50, 100, 250, 500, 1,000 and limit of quantification (LLOQ) concentration of the

2,500 ng/mL, calibration curve was prepared by least

actual value.

square linear regression (y = a + bx), where x was the

3.4.1 Intra-Assay Precision (Within Day Variation) concentration of metformin HCl, and y was the peak

Three concentrations of the test samples (low, area ratio of metformin HCl to diazepam (internal

medium, high) were measured in 5 replicates each standard). The standard calibration curve had the

within the same determination and the CV values were following regression equation:

calculated. The CV of the observed values for the three Y = 0.0005 x + 0.0031 with r = 0.9999, as the result

concentrations of the test samples (low, medium, and of calculation of the slope and intercept.

high) ranged between 1.20-6.91 %, which fulfilled the

3.2 Linearity

criteria.

3.4.2 Inter-Assay Precision (Day-to-Day Variation) The linearity of the standard calibration curve was

Three concentrations of the test samples (low, evaluated by calculating the linear correlation medium, high) were measured in 5 replicates each in 5

coefficient of the curve. The linearity of the standard different days and the CV values were calculated. The calibration curve was shown by the linear correlation

CV of the observed values for the three concentrations coefficient (r) of 0.9999.

of the test samples were 8.05%, 7.5% and 7.14% for

3.3 Limit Lower of Quantitation (LLOQ) low, medium and high concentration respectively

which fulfilled the criteria.

The lowest concentration of the test substance by

3.5 Accuracy

which the reliability of assay result can be ensured was determined. The low concentration of the test sample

The accuracy was checked by calculating the was diluted with blank plasma to 1/2 or 1/4, and the

difference between the measured values and the actual diluted concentrations were measured (in) 5 replicates

values. The acceptance limit for the accuracy was each within the same determination. The limit of

within ± 15% for low, medium and high concentrations quantitation was the lowest concentration by which the

and within ± 20% for the lower limit of quantification

Validation of Metformin Hydrochloride in Human Plasma by HPLC-Photo Diode Array (PDA) for

Application of Bioequivalence Study

(LLOQ) concentration of the actual value. 12.20%-6.96% for low, medium and high

3.5.1 Intra-Assay Accuracy (Within Day Variation) concentrations respectively, which fulfilled the criteria. Three concentrations of the test samples (low,

3.5 Selectivity

medium, high) were measured in 5 replicates each within the same determination and the difference

The LLOQ concentrations of the test samples in 6 between the measured values and the actual values

different blank samples of human plasma were were calculated. The intra-assay accuracy as shown by

determined and the CV value was calculated. Blank the differences between the measured values and the

samples of human plasma were obtained from the actual values (% diff) for the three concentrations of

Indonesia Red Cross (Palang Merah Indonesia). No the test samples ranged between 5.55%-12.95%,

endogenous peaks from plasma were found to interfere 12.28%-3.35%, 12.20%-9.66% for low, medium and

with the elution of metformin or diazepam as shown at high concentrations, respectively, which fulfilled the

Fig. 1A. The LLOQ concentration of the test samples criteria.

in six different blank samples of human plasma was

3.5.2 Inter-Assay Accuracy (Day-to-Day Variation) determined within the same determination, and the Three concentrations of the test samples (low,

coefficient of variation was 5.44% with % Diff ranged medium, high) were measured in 5 replicates each in 5

between 4.51%-19.52%.

different and the difference between the measured

3.6 Recovery

values and the actual values were calculated. The inter-assay accuracy as shown by the differences

The recovery absolute and relative was determined between the measured values and the actual values (%

using the concentration of the test samples (low, diff) for the three concentrations of the test samples

medium, and high). The recovery relative throughout ranged between 12.75%-13.81%, 14.80%-10.00%,

the validation period determined. The relative recovery

Fig. 1 Representative chromatograms for extracts of plasma samples containing. (A) a blank plasma sample, (B) a spiked sample containing 100 ng/mL metformin (low quality control sample), (C) a spiked sample containing 1,000 ng/mL metformin (medium quality control sample), (D) a spiked sample containing 2,000 ng/mL metformin representing the high quality control sample. All samples were spiked with the 30 µL solution of diazepam 1,000 μg/mL as the internal standard.

24 Validation of Metformin Hydrochloride in Human Plasma by HPLC-Photo Diode Array (PDA) for Application of Bioequivalence Study

for three concentrations of the test samples ranged The results of the test determination for three between 89.87%-113.08% (low), 87.22%-110.00%

freeze/thaw cycles were good as shown by the accuracy (medium), 87.80%-106.96% (high). And the absolute

value (% diff) ranged between -11.02%-(-6.95)%, recovery for three concentrations of the test samples

-9.18%-(-7.57)% for low and high concentrations ranged between 64.25%-86.77% (low), 50.52%-59.77%

respectively.

(medium), and 51.18%-55.98% (high). Absolute

3.7.4 Stock Solution Stability

recovery was evaluated by measuring the response of The stability testing was performed under room processed spike plasma standard expressed as percentage

temperature condition for 6 hours and under refrigerator of the responds of pure standard in distilled water.

storage condition for 30 days. The results of the test determination for stock solution stability were good as

3.7 Stability shown by the accuracy value (% Diff) of

Stability was studied during sample collection -0.44%-(-0.42)% for 6 hours at room temperature, and storage and processing. All stability studies were

-0.15%-0.14% for 30 days at refrigerator storage conducted using freshly prepared stock solution in the

condition.

distilled water. Stability experiments extended

4. Discussion

throughout the analysis duration and until the last test sample was assayed.

For validation of bioanalytical methods, the

3.7.1 Short Term Stability guidance for industry bioanalytical method validation Using the two concentrations of the test samples

[10] have recommended the accomplishment of (low and high) in plasma, the stability testing was

accuracy test, precision, specificity, and linearity of the performed under room temperature for 0, 6 and 24

method. Because metformin HCl is very polar, it is hours. The result of the test determination for short

usually analyzed using normal phase with Silica term stability (24 hours at room temperature) was as

column, but it is not economical in terms of cost. good as shown by the accuracy value (% Diff) ranged

Analyzing it using RP column will not provide between -2.35%-2.35% (low) and -6.69%-(-2.34)%

optimum result, therefore this method use RP system (high). Therefore metformin HCl in plasma was stable

that is combined with ion-pair. In this research SDS as for 24 hours at room temperature.

ion-pair was used. The counter-ion in the aqueous

3.7.2 Long Term Stability mobile phase is used to regulate the retention of Using the two concentrations of the test samples

metformin, and influence the selectivity and also (low and high) in plasma, the stability testing was

ensure conditions for efficient chromatographic performed under freezing (-20 °C) storage condition

performance [4, 8].

for 0, 7, 40 and 90 days. The result of the test The IS and analyte were well separated under the determination for long term stability (90 days at -20 °C)

described chromatographic condition at retention time were good as shown by the accuracy value (% diff)

of 3.3 and 7 min, respectively. No endogenous ranged between -9.78%- (-7.14)% (low) and -12.60%-

component eluted at the retention time of IS and (-9.94)% (high). Therefore metformin HCl in plasma

metformin. The total run time was 10 min. Fig. 1 shows was stable for 90 days at -20°C.

the representative chromatograms of blank plasma,

3.7.3 Freeze and Thaw Stability plasma sample spiked with metformin at 100 ng/mL, Using the two concentrations of the test samples

1,000 ng/mL and 200 ng/mL as QC samples with 30 µL (low and high) in plasma, the influence of freeze/thaw

solution diazepam 1,000 ng/mL as the internal standard. cycles (3 cycles, each in duplicates) was investigated.

The peaks were good shape, completely resolved one

Validation of Metformin Hydrochloride in Human Plasma by HPLC-Photo Diode Array (PDA) for

Application of Bioequivalence Study

from another at therapeutic concentrations of sample low), 87.22%-110.00% (QC sample medium), metformin. No interference with constituents from the

87.80%-106.96% (QC sample high) and high enough plasma was observed.

to fit for quantification. This easy and rapid sample The composition of mobile phase is a critical factor

preparation was routinely applied for the for separating metformin and is from endogenous

bioequivalence study.

substances. A solution of a mixture of 60% buffer Stability result for metformin in plasma are shown in phosphate and 40% acetonitril with final pH 7.0

Table 1, indicating that metformin was stable in plasma achieved good resolution and symetrical peak shapes

samples under different storage condition: immediately, of the analytes and IS as well as a short run time.

after 24 h at ambient temperature, after sample processing and being on the autosampler for 24 h, after

4.1 Assay Validation three freeze-thaw cycles, and after 90 days stored at

The peak area ratio of metformin to IS in human -20°C. The stock solutions of metformin and IS were plasma was linear with respect to the analyte

also stable after storage at 2°C for 30 days. This concentration over the range of 20-2,500 ng/mL. The

suggests that metformin and IS were stable in LLOQ of metformin was established as 20 ng/mL. The

experimental condition.

linear regression equation of calibration curve for the

4.2 Bioequivalence Study

analye was y = 0.0005x + 0.0031 with r = 0.9999, where y was the peak area ratio of the analyte to the IS

The validated method was employed for and x was the concentration of the analyte. Precision

determination of metformin in human plasma sample and accuracy of metformin in QC samples fell within

collected over 30 hours for bioequivalence study under the limit acceptability. All values were less then 15%.

fasting condition. All 12 volunteers successfully This validation demonstrated the realiability of our

completed the trial according to the protocol. Both method.

metformin HCl formulations were well-tolerated at the

The direct precipitate extraction procedure administered dose and no serious adverse clinical warranted high sensitivity, good accuracy and events were observed. In this study, plots of individual precision, relatively high recovery and least time for

plasma profiles for both formulations and the mean sample preparation. Metformin HCl is freely soluble in

metformin HCl concentration versus time profiles for water, and this solvent with trichloroacetic acid was

both formulations are shown in Fig. 3. From the selected as protein-precipitation to deprotein the

bioequivalence study, the results of pharmacokinetic plasma samples. It was found that the extraction

parameters of C max , AUC 0-30h , AUC 0- ∞, T max and recovery of metformin were 89.87%-113.08% (QC

t 1/2 were summarized in Table 2. The parametric 90%

Table 1 Stability of metformin concentration in plasma samples.

Diff/Bias (%) Short term stability after 24 h at 101.7

Stability study

Added concentration (ng/mL)

Found (ng/mL)

ambient temperature

Three freeze-thaw cycles

Autosampler stability for 24 h

Stability at -20 °C for 90 days

26 Validation of Metformin Hydrochloride in Human Plasma by HPLC-Photo Diode Array (PDA) for Application of Bioequivalence Study

Fig. 2 Plasma sample of a human subject, 3 h after the administration of 750 mg XR metformin HCl caplet in bioequivalence study.

Fig. 3 Mean plasma concentration-time course of metformin HCl in 12 subjects following the administration of each product (time scale 0-30 hours).

Table 2 Summary the pharmacokinetic parameters of the test and reference products after dosing.

Parameter

Drug C max (ng/mL)

t 1/2 (h) Test

AUC 0-30h (ng.hr/mL) AUC 0- ∞ (ng.hr/mL)

T max (hr)

confidence interval on the mean of the difference and AUC 0- ∞ respectively. The results indicate that the (test-reference) between log-transformed values of the

two formulations can be considered equivalent in the two formulations were 97.00% to 115.98%, 94.78% to

extent of absorption and can be used interchangeably. 109.50% and 93.77% to 109.87% for C max , AUC 0-30h A highly sensitive and specific HPLC method for the

27

Validation of Metformin Hydrochloride in Human Plasma by HPLC-Photo Diode Array (PDA) for

Application of Bioequivalence Study

determination of metformin HCl in human plasma has Company, Inc., New Jersey, USA, 2003.

D. Stepensky, M. Friedman, W. Srour, I. Raz, A. been developed and validated, with a lower Hoffman, Preclinical evaluation of

[4]

quantitation limit 20 ng/mL. Validation experiments pharmacokinetic-pharmacodynamic rationale for oral have shown that the assay has good precision and

metformin formulation, J. Control. Rel. 71 (2001) accuracy over a wide concentration range (20-2,500 107-115. [5] Glucophage ® Metformin Hydrochloride Product

ng/mL). This BE study shows that the two formulations Information, Merck Sante S.A.S. and Associate of Merck, are equivalent so that they can therapeutically

Darmstadt, Germany, Bristol-Myers Squib Company, July interchangeable for each other.

2002. [6] T.A. Hutchinson, D.R. Shahan, Dtugdex ® System,

Acknowledgments

Thomson Micromedex, Edition Expired, Greenwood Village, CO, USA, 2004.

The authors thank to Ferron Par Pharmaceutical [7] N. Najib, N. Idkaidek, M. Beshtawi, M. Bader, I. Admour, Company, Indonesia for financial support of the

S. Alam, et al., Bioequivalence evaluation of two brands of metformin 500 mg tablets (Dialon® & Glucophage®)

project. in healthy human volunteers, Biopharm Drug Dispos. 23

References (7) (2002) 301-306.

C. Cheng, C. Chou, Determination of metformin in human [1]

[8]

G. Balan, P. Timmins, D.S. Green, P.H. Marather, In plasma by high-performance liquid chromatography with vitro-in vivo correlations (IVIV) models for metformin

spectrophotometric detection, J. Chromatogr. B. 762 (1) after administration of modified release (MR) oral dosage

(2001) 51-58.

forms to healthy volunteers, J. Pharm. Sci. 90 (8) (2001) [9] J. Song, H. Chen, S. Tian, Z. Sun, Determination of 1176-1185.

metformin by capillary electrophoresis using [2]

G. Gusler, J. Gorsline, G. Levy, S. Zhang, I.E. Weston, D. field-amplified sample stacking technique, J. Chromatogr. Naret, et al., Pharmacokinetics of metformin

B. 708 (1998) 277-283.

gastroretentive tablets in healthy volunteers, J. Clin. [10] US Dept. of Health and Human Services, Food and Drug Pharmacol. 41 (2001) 655-661.

Administration, Guidance for Industry, Bioanalytical [3] Physicians’ Desk Reference, Vol. 57, Medical Eonomics

Method Validation, Mei, 2001.

Journal of Life Sciences 6 (2012) 28-35

Rotation Thromboelastography for Assessment of Hypercoagulation and Thrombosis in Patients with Cardiovascular Diseases

1 1 1 1 Antoaneta Dimitrova-Karamfilova 2 , Yuliana Patokova , Tania Solarova , Irina Petrova and Gencho Natchev 1. Clinical Laboratory, Univesity Hospital “St. Ekaterina”, Sofia 1431, Bulgaria

2. Department of Cardiac Surgery, Univesity Hospital “St. Ekaterina”, Sofia 1431, Bulgaria

Received: July 25, 2011 / Accepted: September 13, 2011 / Published: January 30, 2012.

Abstarct: Hypercoagulation is not detected in clinical practice with routinely performed blood coagulation tests. More advanced laboratory analyses to detect or monitor hypercoagulation have not yet been introduced into routine clinical management. Thromboelastography assesses the influence of plasma factors and platelets during all phases of haemostasis, thus permits evaluation of hypo- and hyper- coagulation status. This prospective study included assessment of 35 patients with thrombotic complications (II-nd group), compared with 34 healthy controls (I-st group). Haemostasis was analyzed with routine clotting tests: protrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, platelets and rotation thromboelastography (ROTEM ® ) with measuring time to 20 min. All data are presented as mean and standard deviation (SD). Statistical comparisons of samples were performed by student’s t-test. The sensitivity, specificity, positive and negative predictive value of the parameters was calculated by using the receiver operator characteristic (ROC) curves for two groups. There was significant difference (P < 0.05) observed in the parameters of ROTEM: clot formation time (CFT), α-angle, maximum clot firmness (MCF) and thrombodynamic potential index (TPI) in the patient’s population compared to the healthy controls. No significant difference was observed in CT (ROTEM ® ) and routine coagulation tests when the two groups were compared. Rotation thromboelastography analysis demonstrated to be a reliable method for diagnosis of hypercoagulable state.

Key words: Rotation thromboelastography, hypercoagulation, TPI, routine clotting tests.

1. Introduction environmental interaction [1-3]. Still, many cases of venous thromboembolism remain “idiopathic”. In

The major cause of death and disability in the some cases, risk factors for both venous and arterial developed world are thromboembolic (TE) disease may be present, making the clinical picture complications, such as deep venous thrombosis (DVT),

more complex [4].

pulmonary embolism (PE), cerebro-vascular insult Haemostasis is a very complex and well regulated (CVI) and myocardial infarction (MI). process. The three “traditional” components of Hypercoagulable states can present themselves as haemostasis are endothelium, platelets and coagulation arterial or venous thromboembolic disease.

system.

Thrombophilia (the tendency to develop thrombosis) Several laboratory assays have been designed to can be inherited, acquired, or both. Recent family measure haemostasis. The conventional coagulation based studies indicate that TE is highly inheritable and tests as an activated partial thromboplastin time (APTT), follows a complex mode of inheritance involving prothrombin time (PT), fibrinogen, platelets are now

Corresponding author: Antoaneta Dimitrova-Karamfilova, widely used in routine clinical practice to mainly assess M.D., head of the Clinical Laboratory, research field:

the hypocoagulation. Hypercoagulation is not detected haemostasis. E-mail: aidimitrovakar@gmail.com.

Rotation Thromboelastography for Assessment of Hypercoagulation and

Thrombosis in Patients with Cardiovascular Diseases

in clinical practice with routinely performed blood study was approved by the local ethics committee. coagulation tests. More advanced laboratory tests or

Written informed consent was obtained from the experimental methods to monitor hypercoagulation,

patients or their relatives.

including D-dimers, prothrombin fragments F1 and F2, Blood was collected from 34 healthy volunteers – thrombin-antithrombin (TAT), thrombomodulin I-st group (age: 47 ± 20 years; male:female = 18:16) expression, have not yet been introduced into routine

and 35 consecutive patients – II-nd group (age: 55 ± 25 clinical management [5].

years; male:female = 22:13) with thrombotic In the process of learning more about physiological

complications at the time with the admission in haemostasis, there is a tendency to develop new

hospital. Diagnosis of the thrombotic complications methods, which can better model the physiological

was confirmed by one of the following methods: conditions, such as flow conditions and shear. The use

tomography angiography, ultrasound and Doppler of whole blood for development of the new methods is

imaging, electrocardiography and biochemical markers preferred. In addition, methods using whole blood

of myocardial necrosis, computerized tomography testing are good candidates to be used even in acute

percutaneous transluminal angioplasty (PTA) and in situations. Some of this new method is patient with implantation left cardiac assist device thromboelastografhy.

systems (LVAD Berlin heart INCOR), thrombus Thromboelastography (TEG) is a technique that

formation surrounding the canula of the left ventricular allows rapid global assessment of haemostatic function

assist device was detected. The characteristics of the using whole blood [6]. It has been used in clinical

study population are shown in Table 1. settings to detect and quantify hypo- and

Routine coagulation laboratory measurements (PT, hyper-coagulability, fibrinolysis, clot strength, and

APTT, Fibrinogen) were performed using the STA anticoagulant drug effects. Several studies have shown

COMPACT (Diagnostika Stago, France). Platelets that the TEG is able to detect hypercoagulable states in

count was performed using the Sysmex 3000 (Sysmex the clinical setting [7-11].

Kobe Japan). Thromboelastometry was performed The aim of this study is to evaluate the sensitivity,

using a ROTEM ® analyzer (Pentapharm GmbH, specificity, positive and negative predictive value of

Munich, Germany). The method, technique and the parameters of the rotation thromboelastography

variables of thromboelastometry were described (ROTEM ® Pentapharm Co., Munich, Germany) to

previously [12] (Fig. 1).

detect hypercoagulation state in patients with ® The following parameters of the ROTEM were thrombotic complication.

analyzed with measuring time to 20 min: CT-clotting time represents the initiation of coagulation,

CFT-clot formation time, α-angle, both are represent The study protocol for the present prospective cohort

2. Materials and Methods

the propagation of clot formation, reflecting thrombin

Table 1 Characteristics of the study population.

Patients w/

Patients w/

Patients with Patients w/

Pulmonary

thromboembolic

Patients w/ Patients w/

mesenteric Group

Thrombosis

artery (years)

Age Gender Myocardial of the prostetic embolism/

ischemic (F/M)

events due to the use of

infarction

heart valve

Deep venom cardiac assist device

thrombosis stroke

thrombosis

systems (INCOR)

(n) I-st group

(n) (n)

(n)

47 ± 20 18/16 0 0 0 0 II-nd group 55 ± 25 22/13

1 1 4 I-st group: healthy control: n = 21; II-nd group: group with thrombotic complication: n = 26.

30 Rotation Thromboelastography for Assessment of Hypercoagulation and

Thrombosis in Patients with Cardiovascular Diseases

factor, 300

μL blood. By the addition of cytochalasin D the platelets are blocked and clot formation is only depending on fibrin formation and fibrin

α angle (°)

polymerization. Chemicals and reagents were purchased from Pentapharm GmbH.

Maximum

Lysis ( %)

One-way ANOVA was used for statistical analysis.

M CF = Maximum Clot Firmness (mm)

All data are presented as mean and standard deviation (SD). Statistical comparisons of samples were

 Clot Quality

performed by student’s t-test. Differences were

CT = Clotting Time (se c)

considered significant when P < 0.05. The sensitivity, specificity, positive (PPV) and negative predictive

CFT = Clot Formation Time (Se c)

Fig. 1 Graphic expression of the thromboelastometry

value (NPV) of the CT, CFT, α-angle, MCF, TPI was

ROTEM ® .

calculated by using the receiver operator generation and early fibrin polymerization, characteristic (ROC) curves for two groups.

MCF-maximum clot firmness, which describes the

3. Results

final strength of the clot, is influenced by the fibrinogen concentration, the platelet count and fibrin-platelets

Thirty-five patients with thrombotic complications interaction, and TPI (thrombodynamic potential index),

were investigated. Nine patients (22%) were taking which is calculated from the curve and shows

vitamin K antagonists (acenocoumarol 4 mg) and five thrombodynamic potential of the temogram [13]

(14%) were taking antiplatelet agents (clopidogrel 75 (Fig. 2).

mg) at the time of admission to the hospital. The ROTEM ® device was tested regularly for

In the group of patients with thrombotic correct function using quality control serum complication the CT in EXTEM and INTEM assays (ROTROL ® ; Pentapharm GmbH).

was no significant differences between the two groups. We performed the following tests: EXTEM test: 20

The CFT in EXTEM and INTEM assays was μL CaCl 2 0.2 mol/L, 20 μL tissue factor, 300 μL blood,

significantly shorter, the MCF and the alpha angle in for monitoring the extrinsic system (factors VII, X, V,

EXTEM, INTEM and FIBTEM assays were

II, I and platelets); INTEM test: 20 μL CaCl 2 0.2 mol/L,

significantly higher when compared to the control

20 μL thromboplastin-phospholipid, 300 μL blood, for

group. (Figs. 3-5 and Tables 2-4.)

monitoring the intrinsic system (factors XII, XI, IX, The TPI in EXTEM and INTEM tests was

VIII, X, II, I and platelets), and FIBTEM test: 20 μL significantly higher in II-nd group (patients with CaCl 2 0.2 mol/L, 20 μL cytochalasin D, 20 μL tissue

thrombotic complication).

There were no significant differences between the two groups in routine clotting tests (Fig. 6 and Table 5).

In I-st group of healthy controls the sensitivity and NPV of ROTEM ® parameters were high, while the

specificity and PPV were very low. In II-nd group with TPI thrombotic complications the sensitivity, specificity,

PPV and NPV of the CFT and MCF showed a very high value, with the highest values of the TPI, while a

Fig. 2 TPI (thrombodynamic potential index).

α-angle showed a low specificity and PPV.

Rotation Thromboelastography for Assessment of Hypercoagulation and

Thrombosis in Patients with Cardiovascular Diseases

EXTEM

74.88 73.89 I-st N

60.5 62.26 6.55 II-nd TC

Fig. 3 Comparison of ROTEM parameters of healthy control and patients with thrombotic complication in EXTEM test.

INTEM

162.09 156.94 I-st controls

II-nd TC

Fig. 4 Comparison of ROTEM ® parameters of healthy control and patients with thrombotic complication in INTEM test.

FIBTEM

I-st controls 51.56

II-nd TC

Fig. 5 Comparison of ROTEM ® parameters of healthy control and patients with thrombotic complication in FIBTEM test.

32 Rotation Thromboelastography for Assessment of Hypercoagulation and

Thrombosis in Patients with Cardiovascular Diseases

Table 2 Comparison of ROTEM® parameters of healthy control and patients with thrombotic complication in EXTEM test. X

TPI EXTEM Group Mean SD Mean SD Mean SD Mean SD Mean SD

CT EXTEM

CFT EXTEM

α-angle EXTEM

MCF EXTEM

I-st healthy control 60.5 a 14 81.29 a 13.63 74.88 a 3.36 62.26 a 3.84 2.17 a 0.76 II-nd with thrombotic complication

81.8 a 48.62 47.82 b 9.18 80.4 b 1.96 73.89 b 4.18 6.55 b 2.67 Values with the same letter in a column are not significantly different at 95% level.

Table 3 Comparison of ROTEM ® parameters of healthy control and patients with thrombotic complication in INTEM test.

TPI INTEM Group Mean SD Mean SD Mean SD Mean SD Mean SD

CT INTEM

CFT INTEM

-angle INTEM

MCF INTEM

I-st healthy control 162.08 a 22.88 73.24 a 9.87 75.35 a 4.2 61.26 a 4.25 2.34 a 0.84 II-nd with thrombotic complication

156.94 a 33.36 45.82 b 9.75 80.68 b 2.29 73.54 b 3.96 6.73 b 2.85 Values with the same letter in a column are not significantly different at 95% level.

Table 4 Comparison of ROTEM ® parameters of healthy control and patients with thrombotic complication in FIBTEM test.

MCF FIBTEM Group

CT FIBTEM

-angle FIBTEM

Mean SD I-st healthy control

Mean SD Mean SD

51.56 a 12.1 73.53 a 4.70 18.29 a 5.38 II-nd with thrombotic complication

71.30 a 33 79.20 b 3.15 32.31 b 9.79 Values with the same letter in a column are not significantly different at 95% level.

ROUTINE CLOTTING TESTS

301.77 2.69 248.94 II-nd TC

Fig. 6 Comparison of routine clotting tests test of healthy control and patients with thrombotic complication.

Table 5 Comparison of routine clotting tests test of healthy control and patients with thrombotic complication.

РТ-INR APTT-R Fibrinogen-g/L Platelets X 109/ Group Mean SD

Mean SD I-st Healthy control

Mean SD Mean SD

1.21 a 0.25 1.32 a 0.72 3.54 a 89.4 248.9 a 79.5 II-nd with thrombotic

complication 2.69 a 2.5 1.36 a 0.52 4.20 a 94.7 301.8 a 194.74

Values with the same letter in a column are not significantly different at 95% level.

Very low values were for the CT, since 22% of patients greater than 85%, except APTT. In the second group received vitamin K antagonists at the time of the

with thrombotic complications only fibrinogen, PT and admission.

APTT were good specificity (> 85%), the other In the first group the sensitivity and NPV were

parameters showed very low sensitivity, specificity,

Rotation Thromboelastography for Assessment of Hypercoagulation and

Thrombosis in Patients with Cardiovascular Diseases

PPV and NPV. coagulation status assessment with thrombelastometry The “cut off”, sensitivity, specificity, PPV and NPV

is performed in whole blood containing all factors of the CT, CFT, α-angle, MCF, TPI and routine clotting

contributing to the clot formation, this method seems to tests are displayed in Tables 6 and 7, respectively.

mirror the overall coagulation more closely than classical clotting parameters or activation markers (e.g.

4. Discussion

thrombin-antithrombin complexes). Thrombelastometry The risk of a thrombotic event recurrence and its

is known to detect a hypercoagulable state in patients diagnosis is highly relevant to clinical practice.

who experienced venous or arterial thrombotic event Screening for thrombophilia in patients with increase

[11]. The new generation rotation thrombelastograph risk can guide the clinicians in selecting the aligns the naturality of classical TEG with the prophylactic strategy and duration of the anticoagulant

advantages of the modern technology. Activated treatment and therefore avoiding unnecessary ® ROTE М assays provide results much faster than

complications from the anticoagulant therapy. conventional assays, and due to the mechanical The conventional coagulation tests such as APTT,

modifications, described earlier, it can be used for fact PT, fibrinogen, platelets are now widely used in routine

screening and diagnosis of hypercoagulable state. clinical practice to assessment mainly the

The TEG has been used to evaluate both preexistent hypocoagulation. Hypercoagulation is not detected in

and acquired hypercoagulable states [7, 9, 11]. clinical practice with routinely performed blood

Compared to healthy reference subjects, patients with coagulation tests. Profiling whole blood coagulation

hyperhomocysteinaemia and heterozygosity for the with thrombelastography (TEG) can be used for

factor V Leiden polymorphism had a significantly indirect measure of thrombin generation. Because the

greater hypercoagulant whole blood coagulation clot

Table 6 The “cut off”, sensitivity, specificity, PPV and NPV of the CT, CFT, α-angle, MCF, TPI.

II-nd gr. TC Parameter

I-st gr. HC

“cut off”

Sensitivity Specificity PPV NPV Prcision “cut off”

Sensitivity Specificity PPV NPV Prcision

% % % CT EXTEM

< 50 & > 80 34 84 76 58 75 CFT EXTEM

89 94 94 89 100 α-angle EXTEM ≤ 67

97 74 79 96 97 MCF EXTEM ≤ 68

94 94 94 94 99 TPI EXTEM

93 93 100 99 CT INTEM

69 62 65 66 66 CFT INTEM

91 91 91 91 99 α-angle INTEM > 68

91 82 84 90 97 MCF INTEM

94 94 94 94 99 TPI INTEM

92 93 100 99 MCF FIBTEM > 12

Table 7 The “cut off”, sensitivity, specificity, PPV and NPV of the fibrinogen, PT, APTT, platelets.

II-nd gr. TC Parameter

I-st gr. HC

“cut off” Sensitivity Specificity PPV NPV Prcision “cut off” Sensitivity Specificity PPV NPV Prcision %

% % % Fibrinogen > 2.55

88 67 53 82 PT

92 86 63 87 APTT

88 69 54 69 Platelets

34 Rotation Thromboelastography for Assessment of Hypercoagulation and

Thrombosis in Patients with Cardiovascular Diseases

signature as defined by a shortened clotting time and an reference interval. Routine coagulation tests showed accelerated maximum velocity of clot propagation.

low sensitivity, specificity, PPV and NPV and they TEG can also serve as a useful adjunctive test for

were not available to detect hypercoagulable state. thrombophilia screening, particularly in patients in whom the regular thrombophilia screen tests were

5. Conclusion

normal [2]. The utility of TEG ® in the measurement of In conclusion, our study indicated that rotation

postoperative hypercoagulability is evident in many thrombelastography, was able to detect studies within a variety of noncardiac surgical specialty

hypercoagulability in patients with thrombotic fields including neurosurgery, orthopedic surgery [14],

complication. The value of TPI above 3.5 was defined abdominal surgery [9, 15], and vascular surgery [16 ].

as hypercoagulation with measuring time to 20 min. In our study, we compared 34 healthy controls with

35 patients with thrombotic complications. In the group

Reference

of patients with thrombosis the ROTEM parameters [1] J.A. Heit, M.A. Phelps, S.A. Ward, J.P. Slusser, T.M. CFT, α-angle and MCF were significant differences

Petterson, M. De Andrade, Familial segregation of venous compared with control group. The CT was no thromboembolism, J. Thromb. Haemost. 2 (2004)

731-736.

differences, because 22% of patients received vitamin [2] J.C. Souto, L. Almasy, M. Borrell, F. Blanco-Vaca, J. K antagonists at the time of the admission and the INR

Mateo, J.M. Soria, et al., Genetic susceptibility to was between 3.0 and 4.5. Clotting time is influenced

thrombosis and its relationship to physiological risk factors: The GAIT (genetic analysis of idiopathic

greatly by the deficit and inhibitors of coagulation thrombophilia) study, Am. J. Hum. Genet. 67 (2000)

factors and therefore did not change statistically.

1452-1459.

Nevertheless the other ROTEM parameters showed [3] T.B. Larsen, H.T. Sorensen, A. Skytthe, S.P. Johnsen, J.W. hypercoagulable state associated with clinically and

Vaupel, K. Christensen, Major genetic susceptibility for venous thromboembolism in men: A study of Danish

instrumental relevant thrombotic complications that twins, Epidemiology 14 (2003) 328-332.

was not detected with the standard coagulation

D. Wilson, E.A. Cooke, M.A. McNally, H.K. Wilson, A. laboratory monitoring. In this study we investigated the

Yeates, R.A. Mollan, Changes in coagulability as thrombodynamic potential index (TPI), which

measured by thrombelastography following surgery for proximal femoral fracture, Injury 32 (2001) 765-770.

describes the patient’s global coagulation. This [5] P.W. Collins, L.I. Macchiavello, S.J. Lewis, N.J. parameter was developed by Raby C with only one

Macartney, A.G. Saayman, R. Luddington, et al., Global activator and a small amount of samples for

tests of haemostasis in critically ill patients with severe Haemoscope [13]. Surprisingly, we observed the

sepsis syndrome compared to controls, Br. J. Haematol. 135 (2006) 220-227.

greatest differences in the TPI between the two groups [6] O. Toth, M. David, T. Habon, A. Nagy, Z. Keszthelyi, N.

with no differences in conventional clotting tests. Kovacs, et al., Type I antithrombin deficiency as a cause The “cut off”, sensitivity, specificity, PPV and NPV

of arterial and venous thrombosis in a family with severe of the CT, CFT, α-angle, MCF, TPI, PT, APTT,

thrombophilia, Orv. Hetil. 146 (2005) 2121-2125. [7] A.O. Spiel, F.B. Mayr, C. Firbas, P. Quehenberger, B.

fibrinogen and platelets were calculated by using the Jilma, Validation of rotation thrombelastography in a

ROC curves. The ROTEM parameters (CFT, MCF) model of systemic activation of fibrinolysis and demonstrated excellent value for sensitivity, specificity,

coagulation in humans, J. Thromb. Haemost. 4 (2006) PPV and NPV (> 88), with the highest values of the

411-416.

[8] TPI (> 90). TPI above 3.5 was defined as C.R. Bell, D.J. Cox, P.J. Murdock, M.E. Sullivan, K.J. Pasi, R.J. Morgan, Thrombelastographic evaluation of

hypercoagulation with measuring time to 20 min, coagulation in transurethral prostatectomy, Br. J. Urol. 78 independently of that, other parameters may be in the

(1996) 737-741

35

Rotation Thromboelastography for Assessment of Hypercoagulation and

Thrombosis in Patients with Cardiovascular Diseases

[9] E. Mahla, T. Lang, M.N. Vicenzi, G. Werkgartner, R. thromboelastometry, Clin. Lab. Haematol. 27 (2005) Maier, C. Probst, et al., Thromboelastography for

81-90.

monitoring prolonged hypercoagulability after major

C. Raby, Coagulation Intravascularies Disseininees et abdominal surgery, Anesth. Analg. 92 (2001) 572-577.

[13]

Locallisees, Paris Masson, Paris, 1974, p. 274. (in French) [10] J.M. Abrahams, M.B. Torchia, M. McGarvey, M. Putt, D.

[14] C.W. Whitten, P.E. Greilich, Thromboelastography: Past, Baranov, G.P. Sinson, Perioperative assessment of

present, and future, Anesthesiology 92 (2000) 1223-1225. coagulability in neurosurgical patients using [15] J.A. Caprini, J.I. Arcelus, M. Laubach, G. Size, K.N. thromboelastography, Surg. Neurol. 58 (2002) 5-11.

Hoffman, R.W. Coats, et al., Postoperative [11] L. Hvitfeldt Poulsen, K. Christiansen, B. Sørensen, J.

hypercoagulability and deep-vein thrombosis after Ingerslev, Whole blood thrombelastographic coagulation

laparoscopic cholecystectomy, Surg. Endosc. 9 (1995) profiles using minimal tissue factor activation can display

304-309.

hypercoagulation in thrombosis-prone patients, Scand. J. [16] M.J. Butler, Thrombelastography during and after elective Clin. Lab. Invest. 66 (2006) 329-336.

abdominal surgery, J. Thromb. Haemost. 39 (1978) [12] R.J. Luddington, Thrombelastography /

488-495.

Journal of Life Sciences 6 (2012) 36-40

New Silver Nanosensor for Nickel Traces. Part II: Urinary Nickel Determination Associated to Smoking Addiction

1 2 María Carolina Talio 1, 2 , Marta O. Luconi and Liliana P. Fernández 1. Chemical Institute of San Luis (INQUISAL-CONICET), Chacabuco y Pedernera, San Luis 5700, Argentine

2. Area of Analytical Chemistry, Faculty of Chemistry, Biochemistry and Pharmacy, Nacional University of San Luis, San Luis 5700, Argentine

Received: June 14, 2011 / Accepted: July 22, 2011 / Published: January 30, 2012.

“In memoriam” of Dr. Adriana Masi, prominent researcher, dear colleague and friend, who passed away prematurely, as a consequence of public insecurity, killed by a shot in the head at the door of her house.

Abstract:

A new fluorescence silver nanosensor assisted by surfactant has been recently synthesized and applied to ultra trace nickel determination. The methodology was validated by the standard addition method and satisfactorily applied to nickel determination in urine without previous treatment, coming from subjects with different smoking addiction levels and second hand smokers. Within-day precision was better than 0.011 CV. The reproducibility (between-days precision) was also evaluated over 3 days by performing six determinations each day with a CV of 0.025. The proposed methodology represents a promising approach in the area of biological monitoring due to its low operation cost, simplicity of instrumentation, high sampling speed and non-polluting solvents. Obtained results of urinary nickel concentration were successfully correlated with the tobacco addiction.

Key words: Fluorescence nanosensor, micellar silver nanoparticles, urinary nickel, smoker and non-smoker subjects, second hand smoke exposure.

1. Introduction Ni(II) in humans [2].

Plasma and urine nickel concentrations have shown The smoking habit represents the main preventable to be useful biomarkers of nickel inhalation exposure cause of human disease and death. Passive smoking or [3]. The development of new methodologies and exposure to second hand smoke (SHS) is an associated modern analytical techniques has allowed the use of problem to tobacco addiction and regulations are being other matrices that are less or non-invasive [4]. introduced to protect non-smokers in working and Because of the low concentration level of nickel in public places; however, 55% of young people are biological fluids, a preconcentration step must be exposed in their own homes [1]. introduced in analytical protocols when atomic Tobacco contains numerous harmful substances, spectroscopies are used [5], which involves among these are toxic metals, which can be inhaled contamination risks associated to sample manipulation. through both active and passive smoking. Although In a previous work a methodology was developed for nickel is an essential metal to human life, nickel urinary Ni(II) quantification [6]; a disadvantage of this compounds are human carcinogens by inhalation, and methodology is that the membranes must be there exists ample evidence for the carcinogenicity of conditioned with a dye and dried for retain by filtration

the analyte present in sample. This preparative step is Correpondence author: Liliana P. Fernández, chemical doctor, research fields: luminescent methods and time-consuming; added to this, each membrane can be

supramolecular systems. E-mail: lfernand@unsl.edu.ar.

New Silver Nanosensor for Nickel Traces. Part II: Urinary Nickel

Determination Associated to Smoking Addiction

used only once for a sample or standard, resulting in an

2.3 Biological Sample Collection additional cost per analysis.

First morning urine samples were collected from In this work, a fluorescence sensitive nanosensor is

eight nickel occupationally unexposed subjects. In presented as an advantageous alternative to traditional order to assure the obtaining of representative samples, instrumental methods. AgNPs are synthezised in SDS subjects previously received detailed information (Sodium Dodecyl Sulfonate) medium (SDS-AgNPs) about the collection protocol [6]. Biological samples and the obtained nanomaterials are applied to trace were collected in nickel-free polystyrene test tubes nickel quantification. between 8:00 and 10:00 a.m. to reduce possible

2. Experimental

circadian contributions. Studied subjects were asked to respond to a written questionnaire in order to obtain

2.1 Reagents information about smoking habits (frequency, length of

Urine samples were tested using Urine Strip-Wiener addiction), age, sex, occupation, etc.. Written informed Lab (Rosario, Argentina). Ni(II) stock solutions 1 ×

consents were obtained from all participants.

10 -9 mol·L -1 were prepared by dilution of 1,000 g·mL -1 Samples were centrifuged 10 min at 1,000 g and standard solution plasma-pure (Leeman Labs, Inc.).

processed immediately after arriving at the laboratory. Tris (Mallinckrodt Chemical Works, NY, USA)

No stabilizing agents were added to avoid the solution 1 × 10 -2 mol·L -1 was prepared by weighting

incorporation of analytes as impurity [7]. The obtained and subsequent dilution with ultrapure water and

samples (approximately 10 mL each) were centrifuged adjusted to the desired pH, with aqueous HClO 4 for 10 min. Supernants (approximately 5 mL) were

(Merck, Darmstadt, Germany) or NaOH (Mallinckrodt reserved for nickel quantification.

Chemical Works, NY, USA). AgNO 3 (Sigma-Aldrich,

2.4 Physical Characterization and Semi-Quantitative

St. Louis, USA) 1 × 10 -3 mol·L -1

was prepared by

Determination of Clinical Parameters in Biological dilution of 17 mg in 100 ml ultrapure water. Citric

Samples

acid (Hopkin and Williams, England), hexadecyl trimethyl ammonium bromide (J.T. Baker,

Biological samples were physically characterized Mallinckrodt Baker, Inc., NJ, USA) and sodium

(colour, odor and appearance, presence of sediment, dodecylsulfate (J.T. Baker, Mallinckrodt Baker, Inc.,

blood and mucus) in order to establish variables that NJ, USA) were used without further purification. All

could affect the obtained results. Urine samples were used reagents were of analytical grade.

tested using commercial reagent strips.

2.2 Apparatus

2.5 Dilution Test

1 mL of each biological sample was taken and Shimadzu RF-5301 PC spectrofluorometer equipped

Fluorescence measurements were made using a

dilutions were carried out to obtain dilution factors of with a 150 W xenon lamp and 1.00 cm quartz cells. A

1/2, 1/4, 1/8, 1/16 and 1/20.

combined glass electrode and a pH meter (Orion

2.6 Proposed methodology

Expandable Ion Analyzer, Orion Research, Cambridge, MA, USA) model EA 940 were used for pH

Appropriate aliquots of urine/standard solution Ni(II)

2 adjustments. A centrifuge was used in biological -1 (1.2 × 10 -2.93 × 10 ng·L ), 100 L buffer Tris

sample processing and AgNPs purification. All used -1 solution 1 × 10 mol·L (pH = 6.3) and 500 L of glass materials were previously washed with a 10% v/v

synthesized SDS-AgNPs, were placed in a 10 mL HNO 3 water solution and then with ultrapure water.

graduated centrifuge tube. The whole mixture was

38 New Silver Nanosensor for Nickel Traces. Part II: Urinary Nickel Determination Associated to Smoking Addiction

diluted to 3 mL with ultrapure water. Fluorescent blood and mucus) in order to establish variables that emission was measured at  em = 348 nm using  exc =

could interfere in the determinations. All processed 240 nm.

samples can be considered within the normal physical parameters. Samples were immediately centrifuged for

2.7 Accuracy Study

10 min at 1,000 g, and supernants were reserved for

1 mL of biological samples was spiked with

nickel examination.

increasing amounts of Ni(II) (1.2 × 10 -4 -2.93 × 10 2 The clinical parameters (pH, urobilinogen, glucose, ng·L -1 ). Nickel contents were determined by the

ketones, bilirubin, proteins, nitrite, blood, especific proposed methodology.

gravity and leucocytesin) of the urine samples, as determined by commercial urine strips, can be

2.8 Precision Study considered within normal values.

Repeatability (within-day precision) of the method

3.3 Dilution Test

was evaluated preparing urine replicate samples (n = 6) containing 5.81 ng·L -1 of nickel, and analyte contents

In order to establish the appropriate volume of each were determined by the proposed methodology.

sample for performing Ni(II) determination, several sample volumes were assayed. The adequate dilution

3. Results and discussion

for each sample was that whose signal fell into the Urine represents easily accessible body fluid using

linearity range of the developed methodology. Dilution non-invasive sampling procedures and it can reflect

test was of 25 μL for subjects with minor exposure and levels of biomarkers [4].

of 2.5 μL for the most exposed subjects. Dilution factors were adopted for the following studies.

3.1 Studied Subjects

3.4 Analytical Performance

Subjects with different degrees of tobacco addiction (Table 1) were selected to evaluate U-Ni (urinary

At optimal experimental conditions, a detection limit nickel) contents.

of 0.036 pg·L -1 and quantification limit 0.12 pg·L -1

3.2 Physical and Chemical Characterization of were obtained. The calibration sensitivity was 2 × 10

Biological Samples L·pg ·cm for the new methodology, with a range of linearity of six orders of magnitude between 0.12 and

Once in the laboratory, biological samples were

5 2.93 × 10 -1 pg·L (Table 2).

observed and characterized for physical appearance The accuracy of methodology was tested using (colour, odor and appearance, presence of sediment,

the standard addition method. The reproducibility

of the method was evaluated by performing 6 replicate

Table 1 Addiction levels for studied subjects.

Subjects Daily smoked cigarettes Exposure time (years)

Table 2 Quality parameters for nickel determination using 10 0 SDS-AgNPs sensor.

20 0 Parameters Regression Ni(II) (pg·L -1 ) 3 SHS*

55 (1) 8 Range of linearity

Range of linearity (2)

2,440-293,000 7 40

25 Range of linearity (3)

40 (1) Slit excitation: 5 nm; slit emission: 3 nm. (2) Slit excitation: 3 8 TChH**

40 nm; slit emission: 1.5 nm. (3) Slit excitation: 1.5 nm; slit * Second hand smoker; ** Tobacco chewing habit.

emission: 1.5 nm.

New Silver Nanosensor for Nickel Traces. Part II: Urinary Nickel

Determination Associated to Smoking Addiction

experiments. Ni(II) contents in each type of sample application of a surfactant assisted fluorescent based on the average of replicate measurements are

nanosensor for ultra trace nickel quantification, presented in Table 3. Although urine fluorescent

using the enhancement of AgNPs fluorescent signal emission constitutes a severe interference in in presence of Ni(II). The method was successfully fluorescence measures, the high sensitivity of applied to the determination of trace amounts of developed methodology permitted to realize urinary

nickel in urine without previous treatment, with good Ni(II) determinations using a very small volume of

tolerance to regular foreign constituents. The biological sample (0.0025-0.025 mL depending on

proposed methodology may constitute a promising exposure tobacco level), minimizing the spectral

approach in the area of biological monitoring with interference. The results showed that the proposed

low operation costs, simplicity of instrumentation, method was suitable for Ni(II) determination in urine

high sampling speed and non-polluting solvents. biological samples, and for all the range of studied

Results of urinary nickel were successfully concentrations.

correlated with the tobacco addiction. Considering The precision was better than 0.011 CV for U-Ni.

that high levels of this carcinogenic metal in the The reproducibility (between-days precision) was also

studied urine samples from smokers may contribute evaluated over 3 days by performing six to pathologic effects, efforts should be made by the determinations each day and was 0.025 CV. These

control agencies and health agents to discourage the results showed that the biological samples were stable

consumption of cigarettes and the tobacco chewing during this period of time.

habit.

4. Conclusions

Acknowledgments

In the present work we have described the bioanalytical The authors wish to thank Instituto de Química San

Table 3 Urinary nickel determination of subjects ordered

Luis-Consejo Nacional de Investigaciones Científicas

by increasing tobacco addiction level of recovery study.

y Tecnológicas (INQUISAL-CONICET), FONCYT Sample * Ni(II) added

Ni(II) found

Recovery

(ng·L -1 )

(Fondo Nacional de Ciencia y Tecnología), National -

CV (ng·L -1 )

(%, n = 6)

University of San Luis (Proyect 22/Q828) for the

financial support.

14.88 　 0.06 - 2 59 74.01 　 0.05 100.87

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99.95 cloud point extraction and preconcentration method

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for Cd and Ni prior to flame atomic absorption

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4129-4134.

Journal of Life Sciences 6 (2012) 41-47

Studies on the Antioxidant Potential of Extracts from Unripe Fruit of Carica papaya

1 2 Omotade Oloyede 2 , Daniel Roos and Joao Rocha 1. Biochemistry Department, University of Ado-Ekiti, Ekiti State 5363, Nigeria

2. Departmento de Quimica, Bioquimica Toxicologia (CCNE) Universidade Federal de Santa Maria, Santa Maria 97105-900, Brazil

Received: April 07, 2011 / Accepted: May 31, 2011 / Published: January 30, 2012.

Abstract: The antioxidant activities of the ethyl acetate fraction and aqueous extract of unripe fruit of Carica papaya were compared and investigated in vitro using thiobarbituric reactive species (TBARS) assay, total phenol content, evaluation of reactive oxygen species (ROS) in liver mitochondria, Fe 2+ chelating and 2,2-diphenyl-1picrylhydrazyl (DPPH) scavenging activities. Both extracts had high antioxidant properties and could inhibit Fe 2+ and sodium nitroprusside lipid peroxidation in the liver. Total phenol content of ethyl acetate fraction and aqueous extract were 4.50 ± 2.26 and 1.21 ± 3.12 mg/g, respectively. Both extracts had a maximal effect at the lowest concentrations tested (15 µg/mL for ethyl acetate and 150 µg/mL for the aqueous). Ethyl acetate fraction showed that the highest values of antioxidant activity is probably associated with its high phenolic content, Fe 2+ chelating and DPPH scavenging activities. It is therefore concluded that ethyl acetate fraction and to a less extent the aqueous fractions are potent inhibitor of lipid peroxidation.

Key words: Carica papaya, thiobarbituric reactive species, antioxidant activity, lipid peroxidation.

1. Introduction  like cardiac glycosides alkaloids and saponins have been reported by Oloyede [7] and ripe fruit contains

The papaya, Carica papaya L. is a member of the glycine, phenylalanine and tryptophan [8, 9]. small family Caricaceae. It is a perennial, herbaceous Reactive oxygen species (ROS) such as free radicals plant and it originates from southern Mexico, central and peroxides represents a class of molecules that are America and northern South America. Papaya is derived from the metabolism of oxygen and exist actually cultivated in most countries with tropical inherently in all aerobic organisms [10]. Aerobic cells climate like Malaysia, Nigeria and west India where it possess antioxidant defenses that maintain ROS at is known as pawpaw [1]. levels compatible with normal cell physiology. In folkloric medicine, extracts of the fruits are used However, if there are overproductions in ROS or a for a variety of medicinal purposes ranging from decrease in the antioxidant defenses, oxidative stress treatment of ringworm, malaria, hypertension, (OS), which is a general term used to describe an inflammation, chronic skin ulcer, antifertility, jaundice excessive level of oxidative damage in a cell caused by and management of sickle cell anaemia [2], while the ROS, can take place in different cells types, tissues hypoglycemic effect have been reported by Fakeye et or organs. This damage can affect either a specific al. [1], Olagunju et al. [3], and Oloyede et al. [4]. molecule or different classes of biomolecules. Of Carica papaya fruit extract possesses antibacterial and particular importance for human health, oxidative antioxidant activities [5-6]. Presence of phytochemicals stress and damage to tissues can be common end points

Corresponding authors: Omotade Oloyede, Ph.D., senior of chronic diseases such as atherosclerosis, diabetes, lecturer, research fields: plant biochemistry and toxicology. E-mail: pjmoloyede@yahoo.com.

42 Studies on the Antioxidant Potential of Extracts from Unripe Fruit of Carica papaya

cancer, alzheimer , s, rheumatoid arthritis and Ibadan, Nigeria. The fruits were peeled, seeds removed inflammation [11-13].

and the pulp were cut into pieces, air dried and finely Antioxidants are molecules which can safely interact

powdered with an electric grinder. with free radicals and terminate the chain reaction

Aqueous extract was prepared by soaking the dry before vital molecules are damaged. Research on

powdered pulp in distilled water (5% w/v). Resulting relationships between antioxidants and prevention of

suspension was left overnight at room temperature and non-communicable disease such as cardiovascular

filtered. The filtrate was kept in the refrigerator (4 °C) disease has been increasing sharply in recent years.

prior to analysis. Also powdered pulp from Carica Epidemiological studies strongly suggest that food

papaya were extracted with cold 70% ethanol at room containing phytochemicals with antioxidants have

temperature (27 ± 2 °C) for 48 h. The filtrates (ethanol potential protective effect against many disease

extract) were evaporated to dryness using rotary including cancer and diabetes [14]. It is also

evaporator giving a percent yield of 35.33%. Obtained demonstrated that consumption of fruits and vegetables

residue was partitioned sequentially with light has contributed to the prevention of degenerative

petroleum ether (30-60 °C) and ethyl acetate. The ethyl process caused by oxidative stress [15]. In spite of this,

acetate fraction was evaporated to dryness and serial to the best of our knowledge, there is only limited

dilutions of this residue were made to obtain the literature data about the antioxidant properties of

desired concentration for the experiment. papaya fruits. Therefore, in view of the worldwide consumption of papaya fruit, the purpose of the present 2.3 Test Animals

investigation was to evaluate the antioxidant activities All animal procedures were in strict accordance with of Ethyl acetate fraction and aqueous extract from

the NIH guide for the care and use of Laboratory Carica papaya in an attempt to make systematic

Animals [16]. Two to three month old Wistar rats comparison and to determine whether the health

(200-250 g), from our own breeding colony, were used promoting properties of Carica papaya could be

for in vitro studies. For in vivo studies, male albino associated, at least in part, with its potential antioxidant

mice weighing 24-30 g were used. The animals were activity(ies).

kept in separate cages with free access to water and

food, in a room with controlled temperature (22 ± 3 °C) and in a 12 h light/dark cycle with lights on at 7:00 a.m.

2. Materials and Methods

2.1 Materials

2.4 Production of TBARS

Thiobarbituric acid (TBA), malonaldehyde-bis-dimethyl acetal (MDA) Production of TBARS was determined using a 2,2-diphenyl-1picrylhydrazyl (DPPH), were purchased

modified method of Ohkawa et al. [17] as described in from Sigma (St. Louis, MO, USA). Sodium Puntel et al. [18]. The rats were killed by nitroprusside (SNP) was obtained from Merck anaesthetizing them mildly in ether and the liver tissues (Darmstadt, Germany) and iron(II) sulphate from

were quickly removed and placed on ice. One gram of Reagen (Rio de Janeiro, RJ, Brazil).

tissues was homogenized in cold 100 mM Tris-buffer pH 7.4 (1:10 w/v) with ten up and down strokes at

2.2 Preparation of Plant Extracts approximately 1,200 rev/min in a Teflon glass

Fresh, unripe, mature fruits of Carica papaya homogenizer. The homogenates were centrifuged for (Variety Tsolo) were collected and authenticated in

10 min at 3,000 g to yield a pellet that was discarded National Horticultural Research Institute (NIHORT),

and the supernatant (S1) was used for the assay. The

43 supernatant (100 µL), with or without 50 µL of the

Studies on the Antioxidant Potential of Extracts from Unripe Fruit of Carica papaya

The reaction mixture was incubated at 45 °C for 40 min, various freshly prepared pro-oxidants (Iron and

and the absorbance was measured at 765 nm in the Sodium nitroprusside) different concentrations of the

spectrophotometer. Gallic acid was used as a standard plant extracts, and an appropriate volume of distilled

phenol [19]. The mean of three readings was used and water, which gives a total volume of 300 µL were

the total phenol content was expressed as milligrams of incubated at 37 °C for 1 h. The colour reaction was

gallic acid equivalents/g extract.

carried out by adding 200, 500 and 500 µL each of the

2.8 Evaluation of ROS Formation in Liver 8.1% sodium dodecyl sulphate (SDS), acetic acid (pH

Mitochondria

3.4) and 0.6% TBA, respectively. The reaction mixtures, including those of serial dilutions of 0.03

Formation of intracellular peroxides was detected mM standard MDA, were incubated at 97 °C for 1 h.

using an oxidant sensing fluorescent probe, The absorbance was read after cooling at a wavelength

2,7-dichlorofluorescin diacetate (DCFH 2 -DA) [20]. of 532 nm in a vis/uv spectrophotometer.

Fluorescence was determined at 488 nm for excitation and 525 nm for emission. The intensity of fluorescence

2.5 Iron Chelation Assay was standardized to protein concentrations [21] and The ability of the aqueous extract to chelate Fe(II)

expressed as percentage of untreated control values. was determined using a modified method [18]. Briefly,

2.9 Statistical Analysis

150 µL of freshly prepared 2 mM FeSO 4 were added to

a reaction mixture containing 168 µL of 0.1 M Data were analyzed statistically by one-way Tris-HCl (pH 7.4), 218 µL saline and the aqueous

ANOVA, followed by Duncan’s multiple range test extract of the plant (1.67-6.67 mg/mL). The reaction

when appropriate. Differences between groups were mixture was incubated for 5 min, before the addition of

considered to be significant when P < 0.05.

13 µL of 0.25% 1,10-phenanthroline (w/v). The

3. Result

absorbance was subsequently measured at 510 nm in the spectrophotometer. 3.1 In vitro Assays

2.6 DPPH Radical-Scavenging Effects of aqueous and ethyl acetate fractions

obtained from unripe pulp of Carica papaya was Scavenging of the stable radical, DPPH was assayed

monitored on thiobarbituric acid reactive species in vitro [18]. The extract (1.67-6.67 mg/mL) was

(TBARS) production induced by different pro-oxidant added to a 0.5 mL solution of DPPH (0.25 mM in 95%

(10 µM of iron(II) sulphate and 5 µM sodium ethanol). The mixture was shaken and allowed to stand

nitroprusside) in rat liver homogenate. Values were at room temperature for 30 min and the absorbance was

compared with control containing pro-oxidants only. measured at 517 nm in a spectrophotometer. Percent

TBARS increased significantly in iron(II) inhibition was calculated from the control. Vitamin C

sulphate-induced oxidative stress as compared to the was used as a standard compound in the DPPH assay.

basal (Table 1). The extracts studied showed inhibition against the production of TBARS, though the

2.7 Phenolics Content inhibitory potency of the extracts varied. The lowest

The total phenol content was determined by adding concentration of ethyl acetate extracts from papaya

0.5 mL of the aqueous extract and ethyl acetate (0.13 mg/mL) caused a strong inhibition in respectively to 2.5 mL of 10% Folin-Ciocalteau’s

hepatic TBARS production (Table 1). However, reagent (v/v) and 2.0 mL of 7.5% sodium carbonate.

higher concentrations of ethyl acetate extract caused

44 Studies on the Antioxidant Potential of Extracts from Unripe Fruit of Carica papaya

Table 1 The inhibitory effect of ethyl acetate fraction on

obtained with aqueous extract was lower (1.21 ± 3.12

iron sulphate and sodium nitroprusside induced lipid peroxidation in rat liver homogenate.

mg/g). The two extracts had antioxidant activity and Pro-oxidants (nmol of MDA/g tissue)

most importantly, ethyl acetate fraction had a dual

Iron SNP effect (i.e., at low concentrations it decreased hepatic Basal

132.58 ± 26.56 a 124.99 ±16.28 a TBARS production, whereas it increased the TBARS Control

570.98 ± 40.46 b 498.80 ± 44.51 b production in a concentration dependent manner as 0.13 135.67 ± 12.36

concentrations rise from 0.13 to 2.08 mg/mL). 0.52 265.09 ± 7.81

DPPH radical scavenging and iron chelation of ethyl 1.04 315.34 ± 12.36

acetate fraction and aqueous extract were compared in 2.08 326.56 ± 34.32

order to provide explanation for the inhibitory potency Values represent means ± SE (n = 3). The values with different

demonstrated by the extracts (Figs. 1 and 2). Ethyl superscript are significantly different (P < 0.05) by DMR test

compared to control. acetate fraction had higher Fe chelating (56.10%

chelation) at low concentrations (65-260 µg/mL) than concentration-dependent increase in hepatic TBARS

aqueous extract that chelate iron with maximum production, indicating a biphasic effect of this extract.

percentage of 52.04%. Both extracts inhibit iron in a In contrast, aqueous extract caused significant decrease

concentration dependent manner. From the analysis, it (P < 0.05) in liver MDA levels in iron(II)

sulphate-induced lipid peroxidation at all tested Table 2 The inhibitory effect of aqueous extract on iron

sulphate and sodium nitroprusside induced lipid

concentrations (1.0-3.0 mg/mL). In fact, the lowest

peroxidation rat liver homogenate.

concentration tested (1 mg/mL) caused a maximal Pro-oxidants (nmol of MDA/g tissue)

Concentration(mg/ml)

inhibition and values were not significantly different Iron SNP

132.58 ± 26.56 a 124.99 ± 16.28 from basal (Table 2). It is interesting to know that a

Basal

570.98 ± 40.47 b 498.80 ± 44.51 b aqueous extract at all tested concentrations showed

Control

135.86 ± 27.37 similar inhibitory effect (71.37%-74.37%).

138.59 ± 23.25 The effect of extracts of papaya fruit on

128.48 ± 15.61 SNP-induced lipid peroxidation in liver was examined.

126.83 ± 30.39 Sodium nitroprusside (SNP) produced a statistically Values represent means ± SE (n = 3). The values with different significant increase in the formation of TBARS as

superscript are significantly different (P < 0.05) by DMR test compared to the basal (P < 0.05). Both extracts

compared to control.

produced a significant decrease in the formation of TBARS compared to control values (Tables 1 and 2).

Aqueous extract caused a maximal inhibitory effect at the lowest concentration (Table 2), but higher concentrations caused a similar effect to that observed with the lowest concentration and TBARS production determined in the presence of all concentrations of aqueous extract was similar to that obtained in basal conditions (no pro-oxidant added).

The results given in Table 3 showed that the highest

content of total phenolic compound was found in ethyl

Fig. 1 Radical scavenging and iron chelating ability of

acetate fraction (4.5 ± 2.26 mg/g) whereas the content

ethyl acetate fraction.

Studies on the Antioxidant Potential of Extracts from Unripe Fruit of Carica papaya

Fig. 3 Effects of ethyl acetate fraction on DCFH oxidation in liver mitochondria.

Fig. 2 Radical scavenging and iron chelating ability of

Aqueous extract.

could be concluded that there was a steady increase in the percentage inhibition of DPPH radicals by the ethyl acetate fraction and aqueous extracts with maximum inhibition of 75.89% (260 µg/mL) and 76.28% (6.67 mg/mL), respectively.

Effects of ethyl acetate fraction and aqueous extract on Basal reactive oxygen species (ROS) in mitochondria is as shown in Figs. 3 and 4. Both

extracts had a maximal effect at the lowest Fig. 4 Effects of Aqueous extract on DCFH Oxidation in

liver mitochondria.

concentrations tested (15 µg/mL for ethyl acetate and 150 µg/mL for aqueous) and after that there was a

[26] and the beginning of lipid peroxidation gradual increase in DFCH oxidation by both extracts.

determined by TBARS is highly dependent on the presence of even small amount of free iron. Increase in

the formation of TBARS in iron(II) sulphate-induced The involvement of free radicals in the pathogenesis

4. Discussion

oxidative stress, as compared to normal suggest of diseases like liver and heart disease, cancer, diabetes,

possible damage of tissue with overload of iron, which hormonal and immune system abnormalities has been

can cause considerable oxidative damage by increasing reported. And studies have shown that medicinal plants

superoxide production. The increased lipid with antioxidative properties might act as free radical 2+ peroxidation in the presence of Fe could be attributed

scavenger, reducing agents, chelating agents for 2+ to the fact that Fe can catalyze one electron transfer transition metals, quenching of singlet oxygen reactions that generate reactive oxygen species such as

molecules and activators of antioxidative defence the reactive OH radical which is formed from H 2 O 2 enzymes to suppress free radical damage in biological

through Fenton reaction. Iron also decomposes lipid system [22-25].

peroxides, which favours the propagation of oxidation There is a strong correlation between thiobarbituric

of lipids.

acid-reactive substance (TBARS) as a marker of lipid The protective action of aqueous and ethyl acetate peroxidation and products that reflect oxidative

fraction could be attributed to the presence of damage to DNA. Transition metals such as iron can

antioxidants especially ascorbic acid and phenols. participate in the generation of reactive oxygen species

Several studies have reported a significant correlation

46 Studies on the Antioxidant Potential of Extracts from Unripe Fruit of Carica papaya

between antioxidant activity present in some tropical In conclusion, the biochemical alterations induced vegetables and their phenolic content, which suggest

in liver homogenates following in vitro experiments that plants containing high phenolics can be a good

with aqueous and ethyl acetate fractions of unripe source of antioxidant [27]. Low concentrations of ethyl

pulp of Carica papaya provide evidence for the acetate fractions were effective at all in vitro assays.

potential use of unripe fruit of Carica papaya in Sodium nitroprusside has been reported to cause

folkloric medicine for the treatment of various cytotoxicity through release of cyanide and nitric oxide.

human diseases that has been reported to be Nitric oxide is able to cause neurodegenerating disease

associated with oxidative stress and emphasizes the like other reactive oxygen species [28]. The ability of

potential of Carica papaya fruit as an important the extracts to reduce nitric oxide induced TBARS

functional food.

back to basal levels could be related to a potential ability to scavenge free radicals due to the presence of

Acknowledgments

phenols. The Authors wish to acknowledge Conselho Nacional In this study, DPPH radicals were utilized to assess

de Desenvolvimento Científico e Tecnológico (CNPq), radical scavenging abilities of ethyl acetate fraction

Brazil and the Academic of Science for Developing and aqueous extract using ethanolic solution of the

Countries (TWAS), Italy; for granting Dr (Mrs) O.I stable free radical, DPPH. DPPH has the advantage of

Oloyede, a post-doctoral fellowship tenable at being unaffected by certain side reactions, such as

Biochemical Toxicology Unit, Department of metal on chelation and enzyme inhibition [29]. High

Chemistry, Federal University of Santa Maria, Brazil. radical scavenging activity (RSA) values exhibited by

FINEP research Grant: Rede Instituto Brasileiro de ethyl acetate fraction and aqueous extract suggest their

Neurociência, INCT-CNPq for excitotoxicity and antioxidant ability, due to their phenolic content. One

neuroprotection, and PRONEX-Rede Gaucha de of the most important mechanisms of action of

Estresse Oxidativo is greatly appreciated. antioxidant is by scavenging of reactive oxygen and

The authors disclosure statement: No competing nitrogen radicals and phenolics have shown to exert

financial interest.

their effect via this mechanism [28, 29]. Mitochondria ROS production inevitably produces

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Effect of Abscisic Acid on NaCl Stressed Callus Proliferation and Plant Regeneration in Rice

2 3 Ikram-ul-Haq 1 , Ghulam Yasin , Mumtaz Hussain and Ali Mohammad Dahri 1. Institute of Biotechnology and Genetic Engineering (IBGE), University of Sindh, Jamshoro 76080, Pakistan

2. Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan 3. Department of Botany, Universuity of Agriculture, Faisalabad 38040, Pakistan

Received: April 22, 2011 / Accepted: May 13, 2011 / Published: January 30, 2012.

Abstract: In rice, 2,4-Dichlorphenoxyacetic acid (MS 2 ) has been considered best for callus induction and combination of NAA, IAA, ABA (MS 3 ) for somatic embryogenesis. Efficient plant regeneration was observed when MS basal salts supplied with 3% sorbitol and 3% maltose as carbon sources without hormones (MS 4c ). During this experiment, effect of sodium chloride (NaCl) and abscisic acid (ABA) was assessed on callus growth, plant regeneration and root induction efficiency of rice (Oryza sativa L.) varieties IR-6 and Basmati-370. Callus proliferation rate was highly decreased in both varieties on MS 2b (100 mol·m -3 NaCl + 0.5 mg·L -1 ABA) than MS 2a (100 mol·m -3 NaCl) cultures significantly. Proline, glycine-betaine and reducing sugars were increased significantly in MS 2a and MS 2b

callusing cultures. However, total proteins were decreased in MS 2a , while slightly increased in MS 2b . Maximum plant regeneration (9.42 ± 0.54 and 10.67 ± 0.50 plantlets·callus -1 ) from somatic embryos was observed on MS 4c in IR-6 and Basmati-370, while 1.56 ± 0.06 (IR-6) and 0.95 ± 0.05 (Basmati-370) plantlets callus -1 were observed on MS 4b (100 mol·m -3 NaCl). No plant regeneration was observed on MS 4b (100 mol·m -3 NaCl + 0.5 mg·L -1 ABA) medium in both varieties. Inhibition of root induction efficiency was high in MS 5b (100 mol·m -3 NaCl + 0.5 mg·L -1 ABA) than MS 5a (100 mol·m -3 NaCl) in the stressed cultures (P < 0.05). In this experiment, it was concluded that ABA involved in somatic embryogenesis and elevation of NaCl stress, while it causes inhibition of cell’s growth as well as its regeneration into plantlets from somatic embryos.

Key words: Oryza sativa L., NaCl, ABA, embryogenic callus, somatic embryogenesis, bio-contents.

Abbreviations: ABA–Abscisic acid; IAA–Indole acetic acid; NAA–Naphthleinacetic acid; MS–Murashige and Skoog; 2,4-D–2,4 dichlorophenoxy acetic acid.

1. Introduction  During growth, both physiological and bio-chemical aspects are very crucial for the growth of

Agriculture production and its sustainability have plant tissue, while salinity causes their inhibitions. been affected significantly by salinity in arid and Abscisic acid (ABA) is only a plant hormone to be semi-arid areas of the world. It has lost both value and able to modulate the significant induced physiological yield rate of the crop. Such infertility conditions of the effects by salinity. Meanwhile, ABA has shown soil are because of the presence of enormous quantity contradiction in its biological functions like as of various salts. Identifications of the survival of inhibitory effects on plant biomass production of plants under that sever condition is worth of interest growing seedlings but in excised roots of soybean acts [1]. Each of osmotic stress, ion toxicity, lack of cell as growth promoter agent [3]. Its foliar application wall extension and cell-expansion causes growth

enhances overall growth cessation [4]. Largely Na + cessation in plants [2].

and Cl - transportation is inhibited after ABA supply in

Corresponding author : Ikram-ul-Haq, Ph.D., research field: intact tissue, while causes strong inhibition of plant biotechnology. E-mail: rao.ikram@yahoo.com.

Effect of Abscisic Acid on NaCl Stressed Callus Proliferation and Plant Regeneration in Rice

plasmalema influx and xylem transport [5-6]. In level [16, 17]. Such studies could constitute a addition, exogenous ABA induces accumulation of

convenient way for very close study the applied proline and leads to saline stress adaptation in cells in

growth retard stresses on plant growth and tissue culture [7-9]. The accumulation of this amino

morphogenesis. At the same time, NaCl stress could acid and soluble solutes like carbohydrates actively

bring specific in-valuable information for the cultures involved in osmotic adjustment and causes an assured

supplemented with ABA on developing tissues. These exertion of beneficial effects in plant tissues as well as

aseptic cultures may be helpful to measure the effect their regeneration from a single cell or tissues [10-11].

of salinity on plant regeneration.

Similarly, a number of organics have been acting as

2. Materials and Methods

plant fuel as well as osmo-protectant to alleviate saline effects through osmotic adjustment, which helps to

Mature and healthy seeds of rice (Oryza sativa L.) maintain turger pressure and detoxification of cell’s

cv., IR-6 and Basmati-370 were selected. They were organelles. Synthesis of such osmolytes under saline

dehusked and sterilized with 50% (1:1, v/v) stress has gained attentions recently. In particular,

commercial bleach (5.25% NaClO) through proline is playing an important role to enable a plant

sterilization with magnetic stirrer for one hour. They for salt tolerance [12-13]. Various genotypes have

were washed with sterile distilled water for three times been identified as salt tolerant for the synthesis of

(3-5 min).

higher levels of proline than salt sensitive genotypes. Surface sterilized seeds were cultured on callus Glycinbetaine and reducing sugars are also well

induction medium (MS 2 ). This medium was comprised pronounced agents in developing salt tolerance among -1 on MS [18] basal salts, B

5 vitamins [19] and 2.0 mg·L plant species [14-15].

2,4-D (Table 1). It was solidified with 1% (w/v) Plant tissue culture system is a useful tool to study

purified granulated agar (Difco). This culture was the effects of salts on plant development. However,

maintained for its proliferation and to get aged calluses salt tolerance mechanisms involved at the whole plant

(almost 8-week old).

level that could be quite different from those at cell Eight weeks old, well proliferating embryogenic

Table 1 Composition of different plant nutrient cultures used for plant regeneration of Indica rice through somatic embryogenesis and supplements of NaCl and ABA stresses.

Medium Cultures

Medium composition

Culture time

MS 2 MS salts, B5 Vit, 3% sucrose, 2.0 mg·L -1 2,4-D

-1 6-week MS

MS 2a Callusing -1 MS salts, B5 Vit, 3% sucrose, 2.0 mg·L 2,4-D, 100 mol·m -3

NaCl

MS salts, B5 Vit, 3% sucrose, 2.0 mg·L -1 , 2,4-D, 100 mol·m -3

2b ABA

NaCl, 0.5 mg·L

MS salts, B5 Vit, 3% maltose, 3% sorbitol, 2.0 mg·L -1 NAA, 2.0 mg·L MS -1 3 Somatic embryogenesis

IAA, 3.0

mg·L -1 ABA

10-day

MS 4 MS salts, B5 Vit, 3% sucrose MS 4a MS salts, B5 Vit, 3% sucrose, 100 mol·m -3 NaCl MS 4b MS salts, B5 Vit, 3% sucrose, 100 mol·m -3 NaCl, 0.5 mg·L -1 ABA

MS Plant regeneration

4c MS salts, B5 Vit, 3% maltose, 3% sorbitol MS 4d MS salts, B5 Vit, 3% maltose, 3% sorbitol, 100 mol·m -3 NaCl

6-week MS 4e MS salts, B5 Vit, 100 mol·m -3 NaCl, 3% maltose, 3% sorbitol, 0.5 mg·L -1 ABA MS 5 1/2 MS salts, B5 Vit, 0.75% sucrose

MS 5a Root induction

1/2 MS salts, B5 Vit, 0.75% sucrose, 100 mol·m -3 NaCl

2-week MS 5b 1/2 MS salts, B5 Vit, 0.75% sucrose, 100 mol·m -3 NaCl, 0.5 mg·L -1 ABA

50 Effect of Abscisic Acid on NaCl Stressed Callus Proliferation and Plant Regeneration in Rice

calluses were sub-cultured on MS 3 medium (IAA,NAA

Plant rooting (%) =

# of plantlets rooted

x 100

and ABA) from callus induction medium (MS 2 ) for 10

Total # of plantlets cultured

days under dark conditions (Table 1). These calluses After 6-week of treatment, different organic contents were placed on two sterile whatman -1 filter papers for

were determined in callus proliferation cultures. dehydration treatment in petridish. They were sealed

Reducing sugar contents were analyzed by Miller [20] with parafilm and incubated at 25 ± 2 °C in dark for

and total protein contents by Lowery et al. [21]

72 h [11]. Dehydrated callus cultures were methods. Proline and glycinebetaine contents were also

determined according to Bates et al. [22] and Bessieres Culture was incubated in light conditions for 6-week.

sub-cultured on plant regeneration medium (MS 4c ).

et al. [23], respectively.

The regenerated plantlets were harvested from The pH of cultures was adjusted between 5.7-5.8

before sterilization. These cultures were incubated at MS) medium in Magenta boxes for 2-week. Rooted

un-regenerated calluses and transferred to MS 5 (1/2

25 ± 2 ºC, while light conditions that culture required plantlets were transferred to soil in earthen pots

were 2 h in the daytime and night light conditions (light -2 covered with polythene bags. They gradually hardened -1 intensity 15 µmol·m ·s ) provided through white

for a week. The plants were finally shifted to the fluorescent tubes (36 W/54, 6500 K) in plant growth green house.

room. All cultures were comprised on 7 replicates per Three levels of treatments were maintained, i.e.,

treatment. Data significance among the treated and control (without NaCl or ABA), saline -3 (100 mol·m

non-treated cultures was computed with COSTAT NaCl) and salinity with ABA (100 mol·m -3 NaCl + 0.5

computer package (CoHort software, Berkeley, USA). mg·L -1 ABA). These were applied at three different

3. Results

plant growth stages, such as (a): callus proliferation stage; (b): plant regeneration stage; (c): root induction

Among the callus induction cultures of two rice stage (Table 1).

cultivars IR-6 and Basmati-370, callus induction in Almost four weeks old calluses of both varieties

Basmati-370 was observed first than IR-6. Calluses were fragmented into small pieces, weighed and

were multiplied for 4-week than well proliferating transferred ( 100 mg per petridish) on the same

calluses sub-cultured on same callusing medium (MS 2 ), callusing medium with different treatments for 6-week -3 while supplemented with 100 mol·m NaCl (MS

2a ) and

(Table 1). The callus proliferation was calculated by -1 100 mol·m NaCl + 0.5 ABA mg·L (MS 2b ) including applying formula as given below;

control (MS 2 ) culture as shown in Table 1. These Callus proliferation (%) = Callus Final Wt  Callus Initial Wt x

100 cultures were incubated under dark conditions for

6-week. Maximum callus proliferation was observed After somatic embryogenesis and dehydration

Callus Final Wt

on MS 2 medium (Basmati-370  IR-6), while diverse treatment, calluses were sub-cultured on plant effect of salinity (MS 2a ) was observed on callus growth. regeneration medium arranged with specific treatments

It was reduced to 41.08% from 56.66% in IR-6, (Table 1). After 6-week, numbers of the regenerated

while 33.46% from 58.22 in Basmati-370 (P < 0.05). plantlets per callus were counted.

By the addition of ABA in saline cultures (MS 2b ), there Regenerated plantlets were rooted on the rooting

slightly less decrease in callus multiplication was medium. The cultures were arranged in accordance

observed (Table 2).

with applied treatments (Table 1). After 2-week of Each callus cultures of saline (NaCl), saline culture, rooting rate was calculated by applying

with ABA stressed including control (MS 2 ) were formula as given below;

subjected for some bio-chemical analysis. Differential

Effect of Abscisic Acid on NaCl Stressed Callus Proliferation and Plant Regeneration in Rice

Table 2 Efficiency of different stages of plant regeneration through somatic embryogenesis in Indica rice (Oryza sativa L.) under NaCl and ABA stresses.

Root growth Rice cultivars

Callus growth

Plant regeneration

Callus proliferation

# of plantlets/callus

Medium (%) 3% maltose + Medium Rooting Medium 3% sucrose (%) Medium

3% sorbitol

MS 5 86.20 ± 1.45 IR-6

MS 2 56.66 ± 1.56

MS 4 1.79 ± 0.60

MS 4c 9.42 ± 0.54

MS 2a 41.08 ± 1.75

MS 4a -

MS 4d 1.56 ± 0.06

MS 5a 22.32 ± 1.60

MS 2b 38.41 ± 0.98

MS 4b -

MS 4e -

MS 5b 14.64 ± 1.65

MS 2 58.22 ± 0.97

MS 4 0.90 ± 0.09

MS 4c 10.67 ± 0.50

MS 5 94.20 ± 2.14

Basmati-370 MS 2a 33.46 ± 1.45

MS 4a -

MS 4d 0.95 ± 0.05

MS 5a 16.82 ± 1.45

MS 5b 9.05 ± 1.24 Significance ***

MS 2b 34.23 ± 0.76

accumulation of various organic bio-components was observed in stressed callus cultures of both varieties (Fig. 2). Maximum proline contents were observed in

the cultures supplied with NaCl and ABA (MS 2b ) in

both salt sensitive (Basmati-370) and salt tolerant (IR-6) varieties. Similarly, various organic bio-components such as glycinebetaine and reducing sugars were also increased significantly under NaCl stress while total protein contents were decreased (P <

0.05) in NaCl cultures (MS 2a ) but slightly increased

among the MS -1 2b (100 mol·m NaCl + 0.5 mg·L ABA) cultures (Fig. 2). These cultures specific alterations in

concentrations of various organic contents were

Fig. 1 Representation of in vitro nutrient cultures

occurred in the multiplying tissues reflect the mode of

developed for plant regeneration through somatic

development of the cultured tissues.

embryogenesis of Indica rice (Oryza sativa L.) under NaCl

Somatic embryogenesis was carried by culturing and ABA stresses. a, b and c: 6-week old proliferating

calluses of rice on MS 2 MS 2a and MS 2b plant nutrient

8-week old embryogenic calluses from callusing

callusing medium; d: 10 days old well proliferating callus on

medium (MS 2 ) to MS 3 medium for 10 days and

somatic embryo induction medium (MS 3 ); e and f: dehydration desiccation for 72 h under dark conditions. Regenerated plantlets from somatic embryos on MS 4c and

MS 4d Large numbers of plantlets were regenerated when plant nutrient cultures after 6th weeks of culture.

cultured on plant regeneration medium under light ± 0.06 plantlets per callus were regenerated in IR-6, conditions (Table 1). First of all, plant regeneration was

while 0.95 ± 0.05 plantlets per callus in Basmati-370 started in Basmati-370 on MS -3

4c medium (Fig. 1) within

on 100 mol·m NaCl stressed cultures. With the

supplement of ABA in saline plant regeneration almost after 4th week of culture. Maximum 10.67 ±

2-week compared to MS 4 (MS, 3% sucrose) medium

medium (MS 4e ), no plant regeneration was observed in

0.50 and 9.42 ± 0.54 plantlets per callus were both IR-6 and Basmati-370. While in case of plant regenerated in Basmati-370 and IR-6 respectively.

regeneration medium supplemented with sucrose only Salinity (MS 4d ) strongly reduced the plant (represented as MS 4 ) the lowest plant regeneration was regeneration capacity in both rice varieties. Almost 1.56

observed. Complete plant regeneration inhibition (Table 2)

52 Effect of Abscisic Acid on NaCl Stressed Callus Proliferation and Plant Regeneration in Rice

lead to reduce the efficiency of plant developmental stages or causes plant regeneration inhibition ultimately. At the same time, the application of a specific salt like as NaCl which also affects the growth of cultured tissues. Meanwhile, supplement of ABA in the NaCl stressed cultures causes to elevate its stress on growing tissues. During this experiment, rice responses for NaCl and/or ABA stresses under aseptic conditions were observed. Salinity causes decrease in callusing and rooting abilities in rice cultivars (Basmati-370 > IR-6).

Callus induction as well as root induction rate is not remained consistent under saline stressed and saline with ABA in both varieties (Table 2). Variant

Fig. 2 Effect of NaCl and ABA stresses on different organic

morphological patterns appeared among the stressed

bio-components in 6-week old callus cultures of Indica rice varieties.

cultures in comparison to control. Development of specific traits in multiplied plant tissues is reflected

was observed on MS -3 4a (100 mol·m NaCl) and MS 4b because of presence of specific synthesized

(100 mol·m -1 NaCl + 0.5 ABA mg·L ) media which bio-components or internal physiology of tissues. The were supplemented with 3% sucrose as carbon source

regenerating capacity of multiplied embryogenic callus (Table 2).

is genotype dependent as Basmati-370 and IR-6 have Best root induction in the regenerated plantlets was

showed differential callus growth behaviors as well as observed on MS 5 , while decreased in MS 5a and MS 5b variant internal bio-components as represented in Figs.

media (Table 2).

1 and 2 [24]. Each biological trait is polygenic Root induction efficiency was observed higher

character or controlled by a group of variant genes (92.20 ± 2.11%) in Basmati-370 than IR-6 variety in

either stage of plant development like as callus

induction, its multiplications and then regeneration into induction was higher in Basmati-370 than IR-6 on

the control cultures (MS 5 ). The rate of decrease in root

plantlets. Each of these phenomena is justified when MS 5a medium. With the supplement of ABA in NaCl

plant regeneration system is optimized. Any addition or

removal of salts in plant nutrient medium causes to effect of both was observed on root induction rate

stressed cultures as in MS 5b medium, a detrimental

alter plant culture conditions. This changed form also (Table 2). Presence of ABA in saline stressed cultures

causes specific internal or external changes of the causes further decrease in root induction efficiency of

cultured tissues that are involved to hinder the plant the regenerated rice plantlets.

regeneration or induction of specific mode of plant development.

4. Discussion

Salinity (NaCl) stress in callus development, plant Plant regeneration under in vitro comprised on more

regeneration and root induction cultures had induced a than one nutrient culture based developmental stages.

detrimental effect that causes to decrease callus growth When each stage/step of plant regeneration is as well as plantlet regeneration [25]. Applied stresses optimized that huge mass production within a very

in the cultures of different plant developmental stages short time is possible. Under optimized conditions,

causes accumulation of specific bio-components in the addition as well as removal of aseptic components may

cells, which induce specific mode of plant growth.

Effect of Abscisic Acid on NaCl Stressed Callus Proliferation and Plant Regeneration in Rice

Organic substances in cell sap are acting as osmotica experiment, exogenous supplement of ABA causes that contributes up to 50% osmotic potential of cell

accumulation of various organics, while inhibit plant under saline conditions to protect cell membranes and

regeneration from somatic embryos as well as root reduces aggregations of denatured proteins [26].

induction in regenerated plantlets. Its supply in saline Meanwhile, ABA-dependent expression of genes like

cultures, elevation of salinity stress was observed in as ABA responsive element binding proteins (AREB1),

callus proliferation and root induction stages of rice drought responsive element binding protein (DREB2)

under aseptic conditions slightly. ABA is for somatic and AtNAC2, CBF2, OsDREB1 are activated because

embryo induction as well as its maturation that may be of dehydration and drought or salt stresses [27-29].

because of synthesis of LEA (late embryogenesis These genes or transcriptional factors show variant

abundant) proteins. However exogenous application of gene activation under different stresses, while

ABA linked with antioxidant enzymes or generation of over-expression directly or indirectly has been

H 2 O 2 , while molecular mechanism remained observed in salt tolerant plant lines [30]. incomplete. Today ABA has got much importance for However total protein contents have been decreased its involvement in various biotic and abiotic stress and accumulation of various amino acids has also been resistances. It is also useful for the selection or reported under saline stresses like glycinebetain and development of environmental stress tolerant lines proline contents [17, 31]. They are considered as a

through tissue culture in future.

good indicator of salt tolerance. Higher concentrations of free amino acids in IR-6 under saline stress or with

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Journal of Life Sciences 6 (2012) 55-60

Physiological Response of Hydrilla verticillata (l.f.) Royle Exposed to Cadmium Stress

1 Sibanarayan Mohapatra 2 and Surjendu Kumar Dey 1. Department of Botany, Laxminarayan Mahavidyalay, Jamsuli, Balasore 756081, India

2. Post-Graduate Department of Environmental Science, Fakir Mohan University, Balasore 756020, India

Received: May 26, 2011 / Accepted: June 29, 2011 / Published: January 30, 2012.

Abstract: Hydrilla verticillata (l.f.) Royle twigs were exposed to CdCl 2 (50, 100, 200 and 500 µM) under continuous light for 48 hrs and the physiological parameters like photosynthetic pigment (chlorophylls a, b and carotenoids) contents, activities of antioxidative enzymes like superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) along with lipid peroxidation level were determined. With respect to increase in Cd concentration in the medium and exposure duration, decrease in pigment contents, and decrease in the activities of SOD, CAT and POX were found. The increased levels of lipid peroxidation indicated the prevalence of oxidative stress situation in the tissues which might be one of the reasons behind Cd induced toxicity in Hydrilla verticillata. Since there was significant decrease in the activities of key antioxidative ezymes, the study suggests that Hydrilla verticillata may not be effective for phytoremediation of cadmium in these concentration ranges.

Key words: Photosynthetic pigments, antioxidative enzymes, lipid peroxidation, cadmium stress.

1. Introduction  phyto-accumulator in aquatic bodies, studies on the physiological response of this plant to Cd exposure are

Among the different abiotic stresses that plants very essential. Reports on this aspect of study on encounter in the environment, stress due to exposure of Hydrilla verticillata are rare. Therefore, in this study, heavy metals is a matter of serious concern. The heavy effects of Cd on certain physiological parameters in metals like As, Hg, Sb, Pb, Cd are known to have no Hydrilla verticillata have been assessed in laboratory nutritional functions in biological systems and are condition. The parameters studied are the contents of more or less toxic [1-4]. Cadmium has many photosynthetic pigments; activities of antioxidative anthropogenic uses. To the aquatic environment, Cd enzymes like superoxide dismutase (SOD), catalase enters mainly from industries like electroplating, (CAT) and peroxidase (POX) along with the lipid batteries and chemicals. This is a nonessential metal for

peroxidation level in the tissues.

plants and is known to have many toxic effects on normal plant metabolic processes [5-9]. Among the

2. Experiments

different aquatic macrophytes, Hydrilla verticillata is

2.1 Plant Material, Growth Conditions and Stress found to grow luxuriantly in various polluted water bodies. Several workers have found this plant to have Imposition

potential to accumulate and remove toxic metals from Fresh twigs of Hydrilla verticillata (l.f.) Royle were aquatic ecosystems [10, 11]. To use this plant as a Cd

collected from local pond and were allowed to grow in the laboratory condition in half strength Hoagland

Corresponding author: Surjendu Kumar Dey, Ph.D., research fields: plant physiology, environmental solution for seven days with 8 h/16 h light/dark cycle. biotechnology and oxidative stress physiology. E-mail:

Then the twigs were exposed to different surjendufmu@rediffmail.com.

56 Physiological Response of Hydrilla verticillata (l.f.) Royle Exposed to Cadmium Stress

the control (without enzyme) and V is the absorbance 500 µM), prepared in half strength Hoagland solution.

concentrations of CdCl 2 solutions (50, 100, 200 and

of the sample (with enzyme). One unit of SOD is

10 mL from each concentration was taken separately in defined as the amount that inhibits the superoxide test tubes and one healthy twig was inserted in each

driven nitrite formation from hydroxylamine tube for Cd exposure. In one test tube, 10 mL of

hydrochloride by 50% under the assay conditions. Hoagland solution was taken as control. All the test

Catalase (CAT, EC 1.11.1.6) was assayed by tubes were exposed under continuous light intensity of

measuring the rate of decreasing concentration of H 2 O 2

90 µmol·m -2 s -1 for 48 h at 30 ± 1 °C. The source of light due to enzyme activity at 240 nm as described by Aebi was a bank of fluorescent white cool tube lights along

[14]. The activity of CAT was calculated using the

with incandescent bulb. At 24 h interval, the twigs were -1 extinction coefficient of 40 mM cm of H

2 O 2 at 240 collected, thoroughly washed and used for the study of

nm. Peroxidase (POX, EC 1.11.1.7) activity was various physiological parameters.

assayed taking H 2 O 2 as the substrate and guaiacol as the reduced co-substrate following the method of Kar

2.2 Extraction and Estimation of Photosynthetic and Feierabend [15]. The increase in the colour Pigments intensity due to tetraguaiacol formation was measured

For photosynthetic pigment analysis, the twigs were at 470 nm in a spectrophotometer and POX activity homogenized with cold 80% acetone. The was calculated using the extinction coefficient of 26.6 homogenates were centrifuged at 5,000 rpm in

mM -1 cm -1 of tetraguaiacol at 470 nm. The activities of centrifuge machine and the supernatants were collected.

CAT and POX were expressed in katal (kat), i.e., moles The absorbance of the supernatants were read at 470

of substrate used up or product formed due to enzyme nm, 646 nm and 663 nm in a spectrophotometer and the

activity per second.

contents of chlorophyll a, chlorophyll b and carotenoids were calculated following the method of 2.4 Extraction and Estimation of Malondialdehyde

Arnon [12]. The level of lipid peroxidation was determined by measuring the content of malondialdehyde (MDA), a

2.3 Extraction and Assay of Antioxidative Enzymes decomposition product of peroxidized polyunsaturated

For extraction of enzymes, the twigs were fatty acid components of membrane lipid taking homogenized under ice-cold condition in mortar and

thiobarbituric acid (TBA) as the reactive material, pestle with extraction buffer. The buffers used were

following the method of Heath and Packer [16]. sodium phosphate buffer, 50 mM, pH 7.4 for

superoxide dismutase and sodium phosphate buffer, 50 2.5 Data Presentation mM, pH 7.5 for catalase and peroxidase. The

All the experiments were performed for three times homogenates were homogenized at 10,000 rpm at

with three replicates in each time. The mean values -4 °C for 10 min and the supernatants were collected

along with the standard deviations are represented in separately for enzyme assay, after suitable dilutions.

figures.

Superoxide dismutase (SOD, EC 1.15.1.1) was assayed

3. Results and Discussion

following the method of Das et al. [13] in which the inhibition of superoxide driven nitrite formation from

In this study, the Hydrilla verticillata twigs were hydroxylamine hydrochloride was measured. The

exposed to CdCl 2 for 48 h under light and in 24 h enzyme activity was calculated from the value of

interval the twigs were collected for estimation of

V 0 /V -1 , where V 0 is the absorbance (read at 543 nm) of photosynthetic pigment contents. The contents of

57 chlorophyll a, chlorophyll b and carotenoids were

Physiological Response of Hydrilla verticillata (l.f.) Royle Exposed to Cadmium Stress

the photosynthetic process of the plant. The loss of assessed and the results are presented in Fig. 1. It has

chlorophyll is also an indication of the prevalence of been observed that with increase in Cd concentration in

oxidative stress situation in plants [20]. Thus, in this the medium, there was decline in the contents of all

case also imposition of oxidative stress can be three types of pigments in comparison to the control.

presumed.

Chlorophyll a content was always more than Superoxide dismutase is an important antioxidative

enzyme that scavenges the superoxide radicals to H 2 O 2 100 µM, the chlorophylls a, b and carotenoids contents

chlorophyll b content. Up to CdCl 2 concentrations of

and O 2 and thereby reduces its accumulation [21]. In of samples exposed for 48 h were higher than the 24 h

this study, the SOD activity declined significantly exposed samples of respective concentration. This

when compared to the control one both with respect to might be due to some additional synthesis of the

concentrations of Cd and duration of exposure (Fig. 2). pigments under longer exposure to continuous light.

The decline in SOD activity increases the chances of However, at all the concentrations of the metal, there

superoxide accumulation in the tissues. Decrease in was significant decrease in the pigment contents in

SOD activity has also been reported under exposure to comparison to the control and at 500 µM; the decrease

Cd in wheat seedlings [6] and in Lemna [7] (under in chlorophyll a was 70% (after 24 h exposure) and

prolonged exposure). Catalase is another antioxidative 81% (after 48 h exposure). Loss of photosynthetic

enzyme that detoxifies H 2 O 2 to O 2 and H 2 O and pigments in Hydrilla verticillata has also been reported

thereby reduces its accumulation [22]. In this study, a earlier under exposure to Hg [17], Cu [18] and Ni [19].

sharp decline in CAT activity was found with increase The loss of pigments due to exposure to Cd, which has

in Cd concentration in the medium (Fig. 2). The been found in this study, could have negative impact on

increase in the exposure duration had also significant

Chlorophyll a (after 24 h exposure) Chlorophyll a (after 48 h exposure)

1.2 Chlorophyll b (after 24 h exposure) Chlorophyll b (after 48 h exposure)

ght ei

Carotenoids (after 24 h exposure)

w sh

Carotenoids (after 48 h exposure)

CdCl 2 , µM

Fig. 1 Changes in the photosynthetic pigment contents in Hydrilla verticillata twigs exposed to CdCl 2 for 48 h under light.

58 Physiological Response of Hydrilla verticillata (l.f.) Royle Exposed to Cadmium Stress

this study, like SOD and CAT, the POX activity was

24 h exposure

A also decreased in response to increase in Cd

gh ei

48 h exposure

concentration and exposure duration (Fig. 2). Thus the

hw es

efficacy of the tissues to scavenge H 2 O 2 was weakened

fr g

due to Cd toxicity. In aerobic cells, superoxide radicals

150 nit.

and H

2 O 2 are known to interact in presence of

,u

ity t

transition metal ions via Haber-Weiss reaction forming

tiv

ac . 75 hydroxyl radicals ( OH), the most potential reactive

oxygen species (ROS) [21]. The unsaturated fatty acid

0 components of membrane lipid are highly susceptible to hydroxyl radical attack and are peroxidized in its

presence. Therefore, lipid peroxidation is the

consequence of free radical mediated reactions and is

ght

ei w

considered as the indicator of the prevalence of

sh 8

oxidative stress [23]. In this study, the lipid

fre

peroxidation level was determined by measuring the

kat.

contents of MDA in the tissues and was found to

,n ity

increase with increase in Cd concentration in the

tiv ac T

medium (Fig. 3). Thus, there was increase in lipid

peroxidation which indicated the imposition of

0 oxidative stress situation in the tissues.

4. Conclusions

In the present study, with increase in the Cd

ght ei w

concentration there was decrease in pigment contents,

sh 400 fre

SOD, CAT and POX activities and also increases in lipid

24 h exposure

200 ctiv

48 h exposure

CdCl 2 , µM

,n

Fig. 2 Changes in superoxide dismutase (SOD) (A),

MD 20

catalase (CAT) (B) and peroxidase (POX) (C) activities in

Hydrilla verticillata twigs exposed to CdCl 2 for 48 h under

light.

effect on decreasing the enzyme activity. Peroxidases 500 CdCl 2 are also H , µM

2 O 2 scavenging enzymes, similar to CAT, but

Fig. 3 Changes in the level of lipid peroxidation in Hydrilla

they do so by co-oxidation of reduced co-substrates. In

verticillata twigs exposed to CdCl 2 for 48 h under light.

59 peroxidation in the Hydrilla verticillata twigs. The

Physiological Response of Hydrilla verticillata (l.f.) Royle Exposed to Cadmium Stress

[6] S.K. Dey, J. Dey, S. Patra, D. Pothal, Changes in the antioxidative enzyme activities and lipid peroxidation in

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