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Chapter 19

LIBS-Based Detection of Antioxidant Elements: A New Strategy

Geeta Watal, Bechan Sharma, Prashant Kumar Rai, Dolly Jaiswal, Devendra K. Rai, Nilesh K. Rai, and A.K. Rai

Abstract

The present study deals with the scientific evaluation of antioxidant potential of aqueous extract of Trichosanthes dioica fruits on diabetes-induced oxidative stress of diabetic rats. The most effective dose of mg/kg bw of fruit aqueous extract was given orally to diabetic rats for 30 days. Different oxidative stress parameters were analyzed in various tissues of control and treated diabetic rats. The observed elevated level of lipid peroxidation (LPO) comes down significantly (p < 0.05) and decreased activities of antioxi-dant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) got increased (p < 0.05) significantly of diabetic rats on extract treat-ment. Laser-Induced Breakdown Spectroscopy (LIBS) has been used as an analytical tool to detect major and minor elements like Mg, Fe, Na, K, Zn, Ca, H, O, C, and N present in the extract. The higher con-centration of Ca2+, Mg2+, and Fe2+, as reflected by their intensities are responsible for antioxidant potential of T. dioica.

Key words: Trichosanthes dioica, Diabetes, LIBS , Indian herbs

Laser-induced breakdown spectroscopy (LIBS) is a modern ana-lytical technique used for qualitative and quantitative analysis of trace elements present in any material. The principle of LIBS is based on the spectral analysis of radiation emanating from micro plasma generated by focusing a high power pulsed laser beam on surface of the sample. The characteristic emission from plasma is recorded as spectrum, which provides a fingerprint of constituents of target material. Thus, the spectrum contains qualitative and quantitative information, which can be correlated with sample identify or can be used to determine the amount of its constituents.

1. Introduction

D. Armstrong (ed.), Advanced Protocols in Oxidative Stress II, Methods in Molecular Biology, vol. 594 DOI 10.1007/978-1-60761-411-1_19, © Humana Press, a part of Springer Science + Business Media, LLC 2010

276 Watal et al.

It is a versatile, sensitive, real-time, and in-situ elemental analysis technique having microanalyses capability for any kind of material in any phase solid, liquid, or gas (1, 2). This technique is unique in the sense that it requires no sample preparation, as sample treat-ment is often prone to induced errors due to contamination and losses.

LIBS can scientifically unfold the mystery enclosed in medici-nal plants based on their inorganic constituents. Apart from hav-ing various organic molecules, plants are a rich source of inorganic mineral elements implicated in various health concerns. The crude material of these medicinal herbs is used in Ayurvedic medicines (3, 4). LIBS is a powerful and convenient method to detect the elements present in plant materials responsible for their medicinal value (see Note 1). It is potentially a useful method for rapidly analyzing multiple samples on site at low cost. A number of medicinal plants with antidiabetic activity have been screened and indexed (3–5) for their glycemic elements.

LIBS is a value-added efficient technique for preparing a high quality and most up-to-date elemental database of medicinal plants serving as a discovery tool to correlate a wide diversity in medicinal properties of herbs based on their elemental composi-tion. The world’s largest and most comprehensive collection of medicinal plants and their elements-related information can be prepared as a state-of-the-art information service with the help of this advanced analytical technique and can be explored further in the pharmaceutical industry.

The present study describes the LIBS-based detection of major and minor elements present in Trichosanthes dioica (pointed gourd) fruits responsible for their antioxidant potential, based on screening of the best set of oxidative elements involved. The assessment of antioxidant efficacy of T. dioica fruits has also been assessed in terms of their impact on various oxidative stress param-eters in various key tissues such as brain, liver kidney, pancreas, and spleen of normal as well as streptozotocin (STZ)-induced diabetic rats in order to validate the antioxidant potential of ele-ments present in T. dioica fruits. In brief, LIBS helps in assessing the impact of elemental composition changes on different medic-inal properties of plants such as antioxidant, antidiabetic, etc.

1. Q- switched Nd: YAG laser pulsed laser (Continuum Surellite III-10).

2. Multichannel spectrometer (Ocean Optics LIBS2000+) equipped with CCD.

2. Materials

2.1. Equipment

277 LIBS-Based Detection of Antioxidant Elements: A New Strategy

3. Software for analysis of LIB spectra, such as OOI LIBS software.

4. ANOVA Software for statistical analysis, such as SPSS com-puter software, version 7.5.

5. Centrifuge. 6. Lyophilizer. 7. ELICO VU-VIS Double beam spectrophotometer. 8. Vortex mixture. 9. Parafilm (American National Can, Greenwich CT). 10. Micro pipit. 11. Soxhlet.

1. Phosphate buffer (100 mM, pH 7.4) containing 150 mM KCl.

2. Isotonic ice-cold NaCl (0.9%, w/v) solution. 3. Pyrogallol. 4. H2O2 (10 mM). 5. Malondialdehyde (MDA)/Thiobarbituric acid reactive sub-

stances (TBARS). 6. Bovine serum albumin (BSA). 7. Tris Succinate (50 mM, pH 8.2).

Fresh fruits of T. dioica were cut into small pieces and shade dried. The dried pieces were mechanically crushed and extracted with distilled water using Soxhlet at 80–100°C up to 36 h. The extract was filtered and concentrated in rotatory evaporator at 35 ± 5°C under reduced pressure, to obtain semisolid material, which was then lyophilized to get the material in the form of powder (yield 14.9% w/w).

The sample was prepared by dissolving1 g of the extract powder of T. dioica fruits in 10 ml of distilled water at room temperature.

Diabetes was induced by a single intraperitonial injection of freshly prepared Streptozotocin (purchased from Sigma Aldrich Chem. Co. USA.) 55 mg/kg bw in 0.1 m citrate buffer (pH 4.5) to a group of rats fasted overnight. After 3 days of STZ adminis-tration, depending on their glucose levels (BGL), the animals were marked as a severely diabetic group. A thirty-day long

2.2. Reagents

3. Methods

3.1. Material Processing

3.2. Sample Preparation

3.3. Assessment of Antioxidant Potential

278 Watal et al.

treatment with the dose of 1000 mg/kg bw of T. dioica extract powder was given to normal and STZ-induced diabetic rats. After thirty days of treatment, the rats were fasted overnight and sacrificed by cervical dislocation causing minimal pain. The dif-ferent key organs were surgically removed, rinsed with isotonic ice-cold NaCl (0.9%, w/v) solution, blotted dry and weighed. A 10% (w/v) homogenate of each tissue was prepared in phosphate buffer (100 mM, pH 7.4) containing 150 mM KCl and centri-fuged at 9,000 × g for 30 min. The pellet was discarded and the cell-free supernatant was used for estimation of different oxida-tive stress parameters such as lipid peroxidation (LPO) and the activities of free radical scavenging enzymes such as catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and glutathione-S-transferase (GST) using standard pro-tocols (6–10). Data obtained was statistically evaluated using one-way ANOVA, followed by a post hoc Newman–Keuls Multiple Comparison Test. The values are considered significant at (p < 0.05).

A schematic diagram of the experimental setup for recording the LIBS spectra is shown in Fig. 19.1. The LIBS spectra of T. dioica fruits extract powder, dissolved in distilled water, was recorded for identifying the presence of best set of elements responsible for its antioxidant potential. The 4-channel spectrometer equipped with CCD (Ocean optics LIBS 2000+) consisting 4-grating was used to get the dispersed light from the plasma. A pulsed laser

3.4. Detection of Trace Elements

Laser power

unit

SpectrometerWith CCD

PC

Continum surelite III-10Wavelength Separator

prism

Lens Stand

Laserpower

unit

SpectrometerWith CCD

PC

Continum surelite III-10WavelengthSeparator

Fiber bundle

target

Collecting Lens

Fiber Stand

Fig. 19.1. LIBS experimental setup.

279 LIBS-Based Detection of Antioxidant Elements: A New Strategy

beam from a Q- switched Nd: YAG laser (Continuum Surellite III-10) was focused on the sample using a converging lens (Quartz) of 30 cm focal length, the temperature of the locally heated region rose rapidly and resulted in plasma formation on sample surface. The emitted light from micro-plasma was col-lected using an optical fiber tip placed in the vertical plane at 45° with respect to the laser beam and finally fed into an entrance slit of the multichannel spectrometer (Ocean Optics LIBS2000+) equipped with CCD and 4 gratings. The spectra presented in each Figs. 19.2 and 19.3 are the average of 100 scans (100 shots). The initial three gratings had the resolution of 0.1 nm covering the wavelength range from 200 to 310 nm, 310 to 400 nm and 400 to 510 nm, respectively, while the fourth grating, called as broad band grating, covered the wavelength range from 200 to 1100 nm and had a resolution of 0.75 nm. All the four gratings

200 250 300 350 400 4500

200

400

600

800

Zn II

OII

NII Ca

CaII

CaII

CaIICaIIFeII

FeII

Mg

MgII

MgII

C

CIII

Inte

nsity

(a.

u.)

Wavelength (nm)

Fig. 19.2. Laser-induced breakdown spectra of T. dioica in the spectral range 200–450 nm. The LIBS spectra of the fruit extract of T. Dioica samples were recorded at 2 Hz repetition rate of and 175-mJ-laser energy using the experimental arrangement shown in Fig. 19.1. Nd:YAG (Continuum Surelite III-10) laser delivers maximum pulsed laser energy of 425 mJ at 532 nm, with pulse width 3–4 ns and repetition rate 10 Hz. The laser beam is focused by a plano-convex quartz lens of focal length 30 cm onto the sample surface, the temperature of the locally heated region rises rapidly and results in plasma formation. The light emission from microplasma is collected using optical fiber placed in the horizontal plane containing the laser beam (and at 45o with respect to laser beam) and fed to 4-channel spectrometer (Ocean Optics LIBS2000+) equipped with CCD. The spectrometer had initial three gratings with the resolution of 0.1 nm covering the wavelength range from 200 to 310 nm, 310–400 nm and 400–510 nm, respectively, while the fourth grating covered the wavelength range from 200 to 1100 nm and had a resolution of 0.75 nm. All the four gratings were used simultaneously to record the LIBS spectra. Each spectra presented in Figs. 19.2 and 19.3 is the average of 100 number of scans (100 shots). Figures 19.2 and 19.3 clearly revealed the presence of Mg, Fe, Na, K, Zn, Ca, H, O, C, and N elements in the spec-tral range l 200 nm-900 nm.

280 Watal et al.

were used simultaneously to record the LIBS spectra. LIBS spectra were recorded for aqueous extract of T. dioica at 2 Hz repetition rate of and 175-mJ-laser energy.

A significant increase was observed in the activities of free radical scavenging enzymes like SOD, CAT, GPx, and GST on long-term treatment with T dioica (Table 19.1). However, the diabe-tes-induced LPO recovered its normal values indicating thereby a high antidiabetic activity of T. dioica fruits.

The spectra of T. dioica fruit extract, shown in Figs. 19.2 and 19.3 were taken at optimized experimental conditions (see Notes 4–6). It clearly revealed the presence of Mg, Fe, Na, K, Zn, Ca, H, O, C, and N elements in the spectral range l 200–900 nm. According to the Boltzmann distribution law, intensity is directly related to con-centration (11) therefore, the intensity of observed spectral lines corresponding to major and minor elements present in the extract speaks about their concentrations and helped in defining their role in diabetes-induced oxidative stress management.

3.5. Results

3.5.1. Impact on Oxidative Stress Parameters

3.5.2. Analysis of Antioxidative Elements

600 650 700 750 800 850 900

0

1000

2000

3000

4000

NNa

O

O

KN

H

Inte

nsity

(a.

u.)

Wavelength (nm)

Fig. 19.3. Laser-induced breakdown spectra of T. dioica in the spectral range 200–900 nm. The LIBS spectra of the fruit extract of T. dioica samples were recorded at 2 Hz repetition rate of and 175-mJ-laser energy using the experimental arrangement shown in Fig. 19.1. Nd:YAG (Continuum Surelite III-10) laser delivers maximum pulsed laser energy of 425 mJ at 532 nm, with pulse width 3–4 ns and repetition rate 10 Hz. The laser beam is focused by a plano-convex quartz lens of focal length 30 cm onto the sample surface, the temperature of the locally heated region rises rapidly and results in plasma formation. The light emission from microplasma is collected using optical fiber placed in the horizontal plane containing the laser beam (and at 45o with respect to laser beam) and fed to 4-channel spectrometer (Ocean Optics LIBS2000+) equipped with CCD. The spectrometer have initial three gratings had the resolution of 0.1 nm covering the wavelength range from 200 to 310 nm, 310–400 nm and 400–510 nm, respectively, while the fourth grating, covered the wavelength range from 200 to 1100 nm and had a resolution of 0.75 nm. All the four gratings were used simultane-ously to record the LIBS spectra. Each spectra presented in Figs. 19.2 and 19.3 is the average of 100 number of scans (100 shots). Figures 19.2 and 19.3 clearly revealed the presence of Mg, Fe, Na, K, Zn, Ca, H, O, C, and N elements in the spectral range l 200 nm-900 nm.

281Table 19.1 Effect of most effective dose of Trichosanthes dioica fruit aqueous extract on BGL and lipid profile of severely diabetic rats (mean ± SD)

Oxidative stress parameters in different tissues of rats

Treatment groups Treatment Liver Kidney Brain Pancreas Spleen

Activity of superoxide dismutase (SOD), IU/mg protein

NC DW 12.3 ± 2.5 11.4 ± 1.8 9.5 ± 2.6 4.3 ± 2.8 3.2 ± 1.9

NT TE 12.9 ± 3.1 10.9 ± 2.7 10.4 ± 2.5 4.5 ± 1.1 3.4 ± 0.9

DC DW 4.2 ± 0.7 4.7 ± 0.9 3.5 ± 1.2 1.7 ± 1.0 2.2 ± 0.8

DT TE 6.8 ± 1.5 5.9 ± 2.6* 4.7 ± 1.5** 2.5 ± 0.6 2.4 ± 0.5

Activity of catalase (CAT), IU/mg protein

NC DW 7.32 ± 2.2 6.41 ± 1.7 5.29 ± 1.9 3.42 ± 1.1 2.56 ± 0.6

NT TE 8.2 ± 2.6 7.12 ± 2.3 5.32 ± 1.4 3.67 ± 1.1 2.49 ± 0.7

DC DW 4.2 ± 1.8 4.7 ± 1.3 3.5 ± 1.0 1.7 ± 0.5 2.2 ± 0.8

DT TE 6.84 ± 2.5 5.98 ± 2.2* 4.72 ± 1.2* 2.51 ± 0.4* 2.43 ± 1.2**

Activity of glutathione S transferase (GST), IU/mg protein

NC DW 1.52 ± 0.4 1.30 ± 0.8 0.91 ± 0.9 0.72 ± 0.4 0.59 ± 0.2

NT TE 1.42 ± 0.3 1.51 ± 0.4 1.25 ± 0.2 0.68 ± 0.1 0.62 ± 0.3

DC DW 0.92 ± 0.5 0.41 ± 0.2 0.35 ± 0.2 0.50 ± 0.3 0.24 ± 0.1

DT TE 1.40 ± 0.8 0.63 ± 0.6* 0.56 ± 0.3* 0.62 ± 0.2* 0.31 ± 0.2***

Activity of glutathione peroxides (GPx), IU/mg protein

NC DW 9.31 ± 2.2 7.96 ± 1.5 6.53 ± 1.7 4.27 ± 1.9 3.52 ± 1.1

NT TE 9.9 ± 2.4 7.62 ± 2.1 6.84 ± 1.9 4.31 ± 1.3 3.47 ± 1.1

DC DW 5.4 ± 2.8 5.2 ± 2.2 3.6 ± 2.6 2.3 ± 0.9 2.1 ± 0.6

DT TE 8.84 ± 2.5 6.72 ± 1.9* 6.01 ± 1.7* 3.67 ± 1.6*** 2.95 ± 0.8**

Lipid peroxidation (LPO) in terms of nM of malondialdehyde (MDA) released/mg protein

NC DW 1.43 ± 1.1 1.35 ± 0.4 0.89 ± 0.3 1.17 ± 0.6 1.20 ± 0.3

NT TE 1.46 ± 0.5 1.24 ± 0.7 0.81 ± 0.6 1.02 ± 0.5 1.10 ± 0.4

DC DW 1.98 ± 0.4 1.85 ± 0.8 1.62 ± 0.7 1.42 ± 0.9 1.68 ± 0.2

DT TE 1.32 ± 0.2 1.14 ± 0.4* 1.12 ± 0.2* 1.13 ± 0.7** 1.21 ± 0.5***

Effects of T. dioica fruit extract treatment on the oxidative stress parameters in different tissues of rats. The treatment groups were normal control (NC), normal treated (NT), Diabetic control (DC), and diabetic treated (DT). The treatment regimen and doses have been discussed in materials and method section. DW and TE denote distilled water and T. dioica fruit extract respectively. The oxida-tive stress parameters were estimated in terms of activities of SOD, CAT, GST, GPX, and lipid peroxidation were determined. One international unit (IU) of enzyme activity has been defined as fifty percent inhibition of pyrogallol auto oxidation per min. One inter-national unit (IU) of catalase activity was defined as micromoles of H2O2 decomposed per minute. One international unit (IU) of GST activity was defined as expressed as nanomoles of GSH–CDNB conjugate formed per minute whereas, glutathione peroxidase activity was expressed as µg of GSH consumed/min/mg protein. Specific activity of enzymes is expressed as activity (IU)/mg protein. Lipid peroxidation (LPO) has been expressed in terms of nM of malondialdehyde (MDA) produced/mg protein. Values are expressed as mean ± SD; n = 6, where n = number of determinations. ***Indicates values significantly different from DC at p £ 0.001, **means values are significantly different from DC at p  0.01 and *indicates value significantly different from DC at p £ 0.05.

282 Watal et al.

The ratio of intensities of detected elements (Mg, Fe, Na, K, Zn, Ca, C, H, O, and N) to the intensity of reference lines C and O, which were the essential constituents of plant materials, was estimated to evaluate their proportional concentration. Since

Table 19.2 Intensity ratio of different elements with respect to Carbon (247.8 nm)

Elements Ratio

Zn (202.5 nm)/C (247.8 nm) 0.02511

Zn (206.2 nm)/C (247.8 nm) 0.01868

C III (229.62 nm)/C (247.8 nm) 1.38261

C (247.8 nm)/C (247.8 nm) 1

Fe II (234.3 nm)/C (247.8 nm) 0.04655

Fe II (238.2 nm)/C (247.8 nm) 0.10883

Fe II (239.5 nm)/C (247.8 nm) 0.08793

Fe II (240.4 nm)/C (247.8 nm) 0.03884

Fe II (249.3 nm)/C (247.8 nm) 0.02752

Fe II (258.5 nm)/C (247.8 nm) 0.0392

Fe II (259.8 nm)/C (247.8 nm) 0.18889

Fe II (260.7 nm)/C (247.8 nm) 0.05208

Fe II (261.1 nm)/C (247.8 nm) 0.29323

Fe II (273.9 nm)/C (247.8 nm) 0.04585

Fe II (274.9 nm)/C (247.8 nm) 0.04928

Fe II (275.5 nm)/C (247.8 nm) 0.05908

Mg II (279.5 nm)/C (247.8 nm) 1.48591

Mg II (280.2 nm)/C (247.8 nm) 1.13031

Ca II (315.8 nm)/C (247.8 nm) 0.10521

Ca II (317.9 nm)/C (247.8 nm) 0.34345

Ca II (393.3 nm)/C (247.8 nm) 3.38716

Ca II (396.8 nm)/C (247.8 nm) 2.02546

Ca II (422.6 nm)/C (247.8 nm) 0.55465

Mg (285.2 nm)/C (247.8 nm) 0.16581

K (766.4 nm)/C (247.8 nm) 0.02045

K (769.9 nm)/C (247.8 nm) 0.01339

283 LIBS-Based Detection of Antioxidant Elements: A New Strategy

gratings of different resolutions were used, the whole spectra was divided in to two parts: the first, covered the wavelength range from 200 to 510 nm with 0.1 nm resolution and the second lied in the wavelength range 510–1100 nm with 0.75 nm resolution. To find the intensity ratios of spectral lines, the C line (247.88 nm) as reference line for the spectral range of l 200–510 nm and O line (844.10 nm) as the reference line for spectral wavelength range of 500–1100 nm had been selected. Thus, the intensity ratios of Zn/C, Fe/C, Mg/C, Ca/C, K/O, Na/O, H/O, and N/O were calculated and are given in Tables 19.2 and 19.3. The higher concentration of Ca2+, Mg2+, and Fe2+ as reflected by their intensities are responsible for antioxidant potential of T. dioica. The salient features of the protocol are illustrated in Notes.

1. It has been observed that the elemental composition of medic-inal plants changes with change in their biological activities (5), therefore, changes in spectral characteristics of herbal samples can be correlated with their medicinal properties.

2. In liquid phase, repetition rate also becomes an important param-eter due to shock wave generation and splashing phenomenon.

3. In order to enhance the signal to background and signal to noise ratio and also to get reproducibility, average spectra should be preferred instead of single shot spectra.

4. Notes

Table 19.3 Intensity ratio of different elements with respect to Oxygen (777.2 nm)

Elements Ratio

O (777.2 nm)/O (777.2 nm) 1

O (844.6 nm)/O (777.2 nm) 0.07293

H (656.2 nm)/O (777.2 nm) 7.05396

Na (818.3 nm)/O (777.2 nm) 0.0419

Na (589.5 nm)/O (777.2 nm) 0.00796

N (744.2 nm)/O (777.2 nm) 0.05028

N (746.8 nm)/O (777.2 nm) 0.45277

N (868.3 nm)/O (777.2 nm) 0.06613

284 Watal et al.

4. LIBS spectra is sensitive to experimental protocols such as laser power, lens to sample distance, and position of emission collection optics with respect to plasma plume, which have to be optimized before recording.

5. The most suitable experimental protocols found for this study were laser energy – 175 mJ, lens to sample distance of 30 cm and tip of the fiber bundle at 45° with respect of laser beam.

6. Depending upon the sample the laser energy and laser fre-quency can be changed from 1 to 425 mJ and 0.001 to10 Hz, respectively.

7. Association of oxidative stress with deficiency of Ca2+, Fe2+, and Mg2+ has been validated by the present report, which in its turn confirms the antioxidant property of T. dioica fruits (12–15) (see Note 7).

Acknowledgements

The authors are grateful to DRDO, National Medicinal Plants board (NMPB) New Delhi, India, for providing the financial assis-tance. PKR and DKR are thankful to Indian Council of Medical Research (ICMR) and Council of Scientific and Industrial Research (CSIR) respectively, for the award of SRF to them. The drawing of the experimental setup by Mr. Vivek Kumar Singh, Department of Physics, University of Allahabad is acknowledged.

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