DOI: 10.

1007/s10967-007-1130-0

Journal of Radioanalytical and Nuclear Chemistry, Vol. 274, No.2 (2007) 411419

Thermal neutron activation analysis of essential and trace elements and organic constituents in Trikatu: An Ayurvedic formulation
R. Paul Choudhury,1 A. Kumar,1 A. V. R. Reddy,2 A. N. Garg1*
1 Department 2 Radiochemistry

of Chemistry, Indian Institute of Technology, Roorkee 247 667, India Division, Bhabha Atomic Research Centre, Mumbai400085, India (Received September 18, 2006)

Trikatu, an Ayurvedic formulation of three dried powder spices, ginger, black pepper and pipali in equal proportion is widely used to promote digestion, assimilation and bioavailibility of food. It works synergistically, and hence, is more effective than an equal amount of any of its three ingredients taken separately. Five different brands and its three constituents were analyzed for 31 elements by instrumental neutron activation analysis (INAA) using 5-minute and 6-hour thermal neutron irradiation followed by high-resolution J-ray spectrometry. Heavy toxic metals Cd, Ni and Pb determined by atomic absorption spectrometry (AAS) were found below permissible limits. Most elements in different brands vary in a narrow range. Ginger is particularly enriched in Ca, Fe, Mg and Mn whereas black pepper is enriched in Cr, Se, P and Zn. Cu/Zn shows linear relationship (r = 0.92) with Cu whereas Fe and Mn exhibit inverse correlation (r = 0.89) in different brands. Hydro distillation of pipali yielded an essential oil whereby 10 organic constituents were identified by GC-MS. Also barbituric and tannic acids were isolated from the aqueous methanolic extract of pipali.

Introduction Herbal medicines are the staple of medical treatment in many civilizations including those of Africa, China, Egypt, India, Latin America and others.1 According to the World Health Organization (WHO) estimates, 70% of the world population use herbal medicines and herbal products for primary health care.2 Herbal medicines are considered as natures pharmacy and form a major component in all indigenous traditional medicines. It is a common element in Ayurveda, Homoeopathy, naturopathy, Unani, traditional oriental and native American Indian medicines.2,3 The sophistication of herbal remedies used around the world varies with the technological advancement of the countries that produce and use them.3,4 These remedies range from medicinal tea and crude tablet to concentrated standardized extracts produced using modern pharmaceutical facilities and taken under the supervision of a physician. Trikatu is a Sanskrit word meaning three spices in powder form. This ancient formulation is made up of three herbal stimulants; black pepper (Piper nigrum), Indian long pepper (Piper longum) and the rhizomes of ginger (Zingiber officianilis) in equal amounts. The combination of three promotes the secretion of digestive juices, increases appetite and stimulates the formation of acid thus inhibiting gaseous distension. Its thermogenic effect, metabolic enhancement and nutrient bioavailability enhancing properties render it particularly beneficial in the support of respiratory health. It is used internally in the treatment of asthma, bronchitis, cough, dysentery, pyrexia and insomnia. It also acts as an aphrodisiac and strengthens reproductive functions and energizes the reproductive organs. Trikatu
* E-mail: agargfcy@yahoo.com 02365731/USD 20.00 2007 Akadmiai Kiad, Budapest

is ideally taken one hour before or immediately after each meal in a dose of 1/2 to 1 teaspoon or a capsule (0.5 to 1 g) twice a day or as directed by the physician for up to 1 month to have marked improvement. As trikatu has a bitter taste, it is better consumed with honey in a thick paste or else it may be taken with luke warm water. Each of three constituents of trikatu has wide uses in Indian household as listed in Table 1. QURESHI et al.5 analyzed the essential and toxic elements in black pepper from Islamabad/Rawalpindi (Pakistan) using instrumental neutron activation analysis (INAA). SHERIFF et al.6 employed INAA for the determination of Al, As, Au, Br, Ca, Cd, Co, Cr, Cu, Fe, La, Mn, Mo, Sb, Se, W and Zn in ginger and black pepper. In earlier reports from our group all three constituents were analyzed for up to 20 elements by INAA.7 JOHRI and ZUTSHI8 reviewed trikatu and suggested its use could be due to the bioavailability enhancing action on other medicaments. SIVAKUMAR and SIVAKUMAR9 ascertained its efficacy as a hypolipidaemic agent. ATAL et al.10 evaluated the scientific basis of the use of trikatu group of acrids in a large number of Ayurvedic prescriptions using 3Hlabeled vascine and sparteine as model drugs. KARAN et al.11 observed significant lowering of peak plasma concentration of rifampicin and isoniazid in rabbits treated with a single dose of trikatu. LALA et al.12 studied the effect of trikatu on the pharmokinetics and pharmodynamics of diclofenac sodium, a non steroidal anti-inflammatory drug. Most work carried out on trikatu deals with its pharmacological aspects. Nothing has been reported on the essential and trace element contents, which play a significant role in various physiological and metabolic processes of the body.13

Akadmiai Kiad, Budapest Springer, Dordrecht

R. P. CHOUDHURY et al.: THERMAL NEUTRON ACTIVATION ANALYSIS OF ESSENTIAL AND TRACE ELEMENTS

WEI et al.14 isolated several diaryl heptanoids and related compounds from ginger and studied its cytotoxic and apoptotic activities against promycocytic leukemia cells. BADMAEV et al.15 isolated piperin from black pepper and studied the mechanism of its gastrointestinal absorption enhancement. PARK et al.16 studied antifungal activity of piperoctadecalidene, a piperine alkaloid isolated from pipali. However, it is not known if any of the metals are bound with organic molecules. In the present study we have analyzed five brands of trikatu for seven minor (Al, Ca, K, Na, P, Mg and Cl) and 24 trace (As, Au, Ba, Br, Ce, Co, Cr, Cs, Cu, Eu, Fe, Hf, Hg, La, Mn, Rb, Sb, Sc, Se, Sm, Sr, Th, V and Zn) elements using thermal neutron activation analysis (TNAA). For one of the brands, both capsule and raw powdered form were analyzed. Also Cd, Ni and Pb were determined by AAS. Furthermore, individual components in duplicate were analyzed for their elemental contents. Two reference materials (RMs) obtained from the National Institute of Standards and Technology (NIST), USA and Institute of Nuclear Chemistry and Technology (INCT), Poland were also analyzed for internal quality control and data validation. Essential oil from long pepper, a relatively less investigated species, was extracted by hydro-distillation and 10 organic compounds were identified by GC-MS. Furthermore, barbituric and tannic acids were isolated from the aqueous methanolic extract of pipali. Experimental Sample collection and preparation Four different brands of trikatu were obtained from the Vyas Pharmacy (Indore, M.P.), Yogi Pharmacy (Haridwar, UA), Zandu (Mumbai) and Sushrut Pharmacies (Nagpur) both from Maharashtra. A powdered sample was also procured from Mumbai. The

sample from Sushrut was analyzed in capsule as well as in raw powdered form. Individual constituents of trikatu in powder form were procured in duplicate at different intervals. These were passed through a 100 mesh sieve and oven dried at 80 C for 2 hours before use. The samples were stored in precleaned polyethylene capped bottles and handled with extreme care in a glove box. Two reference materials (RMs) of biological origin; Peach Leaves (SRM-1547) procured from NIST, USA and Mixed Polish Herbs (MPH-2) from INCT, Poland were used as comparator standards. Irradiation and counting 3050 mg aliquots each of samples and RMs in dried form were packed in Alkathene and irradiated together in a batch of 5/10 at a thermal neutron flux of 1012 n.cm2.s1 for 5 min/6 hrs in the APSARA reactor at the Bhabha Atomic Research Centre (BARC), Mumbai. For short irradiation (5 min), counting was followed at the reactor site on an 80 cm3 coaxial HPGe detector (EG & G Ortec) with 4k MCA at the Radiochemistry Division of BARC, Mumbai. Peak to Compton ratio was 40 :1. Care has been taken to account for the counting losses keeping the dead time around 5% at the start of the measurements. Long irradiated samples (6 hrs) were air lifted to our laboratory in Roorkee and counted on an 80 cm3 coaxial HPGe detector with 8k MCA (Canberra, USA). 137Cs, 22Na and 152Eu sources were used for energy calibration and standardization of the detector at different distances (5 15 cm) from the detector. Counting schedule was followed up to 2 months as given in Table 2. The detector system had a resolution of 1.8 keV at 1332 keV of 60Co. Phosphorus was determined by measuring Eactivity of 32P by an end window GM counter, Alabsorber of 27 mg.cm2 thickness.17 In all cases, statistically significant counts (>104) were collected.

Table 1. Use of three constituents of Trikatu from Ayurvedic literature3,4 General name (English name) Botanical name Dried ginger (zinger) Zingiber officinale Black pepper (black pepper) Piper nigrum Pipali (long pepper) Piper longum Family Zingiberaceae Uses Analgesic, Blood purifier, Carminative, Expectorant, Appetizer, Digestive, Stimulant, Sciatica, Lumbago, Rheumatism, Slip disc, Gout, Chronic arthritis, Muscular trouble, Asthma, Cough, Chronic bronchitis. Stimulant, Carminative, Antacidic, Anti periodic, Stomachache, Digestive, Throat problem, Liver pain, Muscle pain, Piles, Spleen disorder, Leucoderma, Lumbago, Paralysis, Chronic fever, Vertigo, Arthritis, Urinary disorder, Flatulance, Indigestion. Tonic, Alterative, Rejuvenator, Digestive, Carminative Cough, Chronic bronchitis, Sedative, Antidote to snakebite, Anti-inflammatory, Anti-malarial, Dyspepsia, Throat disorder, Lumbago, Spleeno-megaly.

Piperaceae

Piperaceae

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Table 2. Irradiation, delay and counting schedule Irradiation 5 min Delay 5 min 30 min 1d 10 d 1520 d 3060 d Counting 60, 100 s, 5, 10 min, 1, 2 hrs 2, 4 hrs 4, 6 hrs 6, 12 hrs Nuclides identified (EJ , keV) (1778), 52V (1434), 49Ca (3084), 27Mg (1014), 38Cl (1643), 56Mn (846), 64Cu (511) 24Na (1369), 42K (1524), 82Br (554, 776), 198Au (411), 76As (559), 64Cu, 153Sm (103), 140La (1596)
27Al 51Cr

6 hrs

(320), 233Th (312), 203Hg (279), 131Ba (495) (1099, 1291), 141Ce (145), 203Hg, 75Se (264), 134Cs (605, 796), 124Sb (603), 46 Sc (889, 1120), 86Rb (1077) 32P (E max = 1710), 85Sr (514), 181Hf (482) 65Zn (1115), 141Ce, 203Hg, 75Se, 134Cs, 60Co (1173, 1332), 124Sb, 152Eu (122, 1408)
51Cr, 233 Th, 59Fe

AAS measurements For the analysis of Cd, Pb and Ni, AAS method was followed using atomic absorption spectrophotometer (GBC Avanta, Australia). About 2 g each of the sample was accurately weighed and dissolved in a (5 :1) mixture of nitric and perchloric acids18 and repeated heating. After a clear solution was obtained, a few drops of HCl were added and the solution was made up to 25 ml. Prior to the analysis, the instrument was calibrated using standard solutions of Ni, Cd and Pb salts of AR/HP grade. RMs were also analyzed for quality control. Isolation of the essential oil and GC-MS measurements Indian long pepper has been relatively less investigated species as far as its organic constituents are concerned. 50 g air-dried finely powdered sample was hydro-distilled for 4 hours using a Clevenger apparatus whence a pale yellow and odourless essential oil (yield 2.31% v/w) was obtained. It was dried over anhydrous sodium sulphate and after filtration, GC-MS was recorded using a Perkin-Elmer Clarus-500 gas chromatograph coupled with a mass spectrometer. The compound mixture was separated on a fused silica capillary column (HP-5MS) (5% phenyl methyl siloxane), 30 mmu0.32 mm, 0.25 Pm film thickness, in a temperature program from 50 (2 min hold) to 250 C (10 min hold) at a rate of 8 C/min. The injector temperature was 250 C, and the flow rate of He carrier gas was 1 ml/min. The interface which kept the capillary column end into the ion source block was at 280 C. Diluted samples (1/100 in acetone v/v) of 1.0 Pl were injected manually and in the split less mode. The components were identified based on the comparison of their relative retention time and mass spectra with those from the NIST spectral database.19

Extraction of organic constituents 100 g dried powder of pipali was extracted successively with 1 :1 aqueous methanol by using a Soxhlet extractor for 6 hours at a temperature not exceeding the boiling point of the solvent. The extract was filtered using Whatman filter paper and then concentrated. Column separation was carried out using a set of solvents with increasing polarity in the order: petroleum-ether < chloroform < ethyl acetate < ethanol < methanol < water. Methanol water fraction (25 :2) yielded two distinct spots at Rf = 0.74 and 0.59 which were then developed on a preparative TLC plate (20u20 cm2) in a mixture of methanol/water (50 :1). The two compounds, developed nicely were scrapped out, dissolved, filtered and distilled and finally recrystallized in acetone. Their C, H, N content (Elementar Vario-EL III, Germany) was determined and IR spectra (Thermo Nicolet, Nexus, USA) were recorded by KBr. Results and discussion Elemental concentrations in various trikatu brands and its 3 constituents (in duplicate) were calculated using RMs as comparators and listed in Table 3. Also included in the table are ranges of concentrations in 5 brands along with their mean rSD. Concentration data for samples were considered only if data for RMs matched within r510% of the certified values,. Elemental concentrations for Ni, Cd and Pb as obtained by AAS are also included. It is observed that SD values in most cases were <10% suggesting high order of accuracy and precision of our data. Therefore, it is assumed that elemental concentrations in different brands should be accurate within r10%. Typical J-ray spectrum for a short irradiated sample showing photo peaks due to short-lived nuclides such as 27Al, 52V, 24Na, 42K, 27Mg, 49Ca, 38Cl and 56Mn is shown in Fig. 1. Significance of elemental contents in different brands and constituents are discussed here.

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Elemental contents in trikatu The minor constituents of trikatu are Na, K, Ca, Cl, P and to some extent Mg and Fe whereas V, Mn, Cr, Cu, Co, Zn, Rb, and Se were in traces. Despite the fact that 5 brands of trikatu were derived from different parts of India where 3 constituents are likely to have been grown under different soil conditions, most elemental concentrations vary by a small factor of 23; Ca (3.02 4.80 mg/g), Cl (2.273.96 mg/g), Fe (0.310.90 mg/g), K (15.326.1 mg/g), P (2.713.84 mg/g), Zn (17.8 27.5 Pg/g), Se (0.080.21 Pg/g), Co (0.210.61 Pg/g) and V (0.852.39 Pg/g). However, Na (1721587 Pg/g) concentration varied by an order of magnitude. Such variations may be attributed to differential elemental uptake by the respective plants from the soil and other geo-environmental factors as suggested by ZAIDI et al.20 Higher Na concentration (>1000 Pg/g) in 2 brands incuding raw and capsule form from Sushrut Pharmacy, Nagpur may be due to deliberate addition of common salt to suppress its bitter taste. In general, trikatu exhibits higher concentrations of several nutritionally important elements such as Ca (3.90r0.60 mg/g), Fe (0.57r0.20 mg/g), Mn (138r42 Pg/g), Se (0.12r0.05 Pg/g) and Zn (21.2r3.7 Pg/g). High Fe content could explain the role of trikatu as a digestive healer.21 Two samples of Vyas Pharmacy collected at an interval of a year do not show widely different elemental contents exhibiting highest Ca and V contents. Similarly 2 samples from Sushrut Pharmacy, Nagpur (capsule and powder) do not differ significantly in their elemental concentrations. Out of the 5 brands analyzed in this

study, a sample from Mumbai seems most enriched in Ba, Cr, Co, Fe, Zn, Rb and Cs whereas the sample from Zandu exhibits highest Se content. Se as glutathione peroxidase inhibits the replication of tumor virus and prevents the malignant transformation of cells.22 It is observed that a toxic element contents such as that of As, Hg, Cd and Pb are well below the permissible level.23 Hence, the product seems safe for human consumption as a drug. Considering a daily dose of 2 tsp or 4 capsules for up to one month, total intake of essential elements is found to be Ca (250 mg), Mg (40 mg), Fe (35 mg), Mn (8 mg), Cu and Zn (12 mg), V (100 Pg), Cr (70 Pg) and Se (10 Pg). All the elements are well known for their definite role in various biochemical processes and in cure of ailments.13 Out of three constituents of trikatu, ginger also called saunth in dried form, is a household spice-cummedicine. It is particularly enriched in Ca (12.6r0.1 mg/g), Mg (1.88r0.06 mg/g), Fe (427r34 Pg/g) and Mn (266r45 Pg/g) whereas black pepper another widely used spice is enriched in P (3.53r0.35 mg/g), Cr (8.65r1.48 Pg/g), Se (0.093r0.02 Pg/g) and Zn (37.1r6.7 Pg/g) contents. Pipali, on the other hand has a higher Cu (28.1r2.6 Pg/g) and V (1.12r0.05 Pg/g) concentrations. A histographic comparison of means of minor, trace and toxic elements in five trikatu brands and its 3 constituents are shown in Figs 2, 3 and 4, respectively. A perusal of the figures shows that all the elemental contents in three constituents are comparable with the mean values in trikatu suggesting that it is an intimate mixture of three constituents.

Fig. 1. Typical J-ray spectrum for short-lived nuclides in trikatu

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Fig. 2. Concentration of minor elements in trikatu and its constituents

Fig. 3. Concentration of essential trace elements in trikatu and its constituents

In accordance with the observation of correlation of several elements in biological tissues and plant species24 we have observed an inverse correlation with r = 0.89 between Fe and Mn in trikatu and its constituents (Fig. 5). However, Cu/Zn ratio varies linearly (r = 0.92) with Cu (Fig. 6). ATAL et al.10 have suggested that trikatu group of drugs increases bioavailability either by

promoting rapid absorption or by protecting the drug from being metabolized. Organic constituents GC-MS of the essential oil (Fig. 7) showed 10 compounds; D-thiocyanato D-(4-nitrophenyl) methylene

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acetic acid, decane, 1-decyne, 3,4-dimethyl 1-decene, undecane, bis-(1-methylpropyl) disulfide, 2-nonynoic acid, 2,4-decadienal, tetradecanoic acid, nonanoic acid exhibiting retention times (base peak) at 2.93 (49), 5.14

(43), 5.45 (81), 5.64 (43), 7.41(43), 9.40 (57), 10.27 (81), 10.46 (81), 10.54 (73), 12.81 (60) minutes, respectively. Identification was done after comparing the spectra with the NIST spectral database.19

Fig. 4. Concentration of toxic elements in trikatu and its constituents

Fig. 5. Correlation of Fe to Mn in Trikatu

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Fig. 6. Correlation of Cu/Zn to Cu in Trikatu

Fig. 7. GC-MS of essential oil in pipali exhibiting 10 organic compounds; 1. D-thiocyanato D-(4-nitrophenyl) methylene acetic acid (149) 2. decane (43) 3. 1-decyne (81) 4. 3,4-dimethyl, 1-decene (43) 5. undecane (43) 6. bis- (1-methylpropyl) disulfide (57) 7. 2-nonynoic acid (81) 8. 2,4-decadienal (81) 9. tetradecanoic acid (73) 10. nonanoic acid (60)

Soxhlet extraction of pipali in aqueous methanol yielded two new compounds corresponding to Rf = 0.74 and 0.59. Elemental analysis and IR spectra25 suggested these to be barbituric acid and tannic acid. Surprisingly, tannic acid is not reported in pipali though it is a major constituent of black pepper.26 Both these compounds 418

may act as potential ligands to form complexes with various metals. Several workers have reported complexing ability of barbituric acid with transition metals and lanthanides.27,28 It has been reported that intracellular injection of cesium barbiturate alters neuron transmitter release by decreasing calcium entry.29 It is

R. P. CHOUDHURY et al.: THERMAL NEUTRON ACTIVATION ANALYSIS OF ESSENTIAL AND TRACE ELEMENTS

well known that barbiturates act as sedatives.30 Thus, use of pipali as sedative could be explained due to the presence of barbituric acid.31 It is assumed that essential elements may remain complexed with such organic compounds. Conclusions Trikatu, an Ayurvedic formulation of three spicesginger, black pepper and pipali in equal amounts used widely in Indian household to promote digestion, assimilation and bioavailability of food along with its three constituents were analyzed for 31 elements by instrumental neutron activation analysis using short and long thermal neutron irradiation followed by highresolution gamma-ray spectrometry. Cd, Ni and Pb were determined using AAS. K, Ca, Cl and P in mg/g and Fe, Mn, Cr, Cu, Zn, Rb, and V in Pg/g amounts are its main constituents. Most elements in various trikatu brands vary in a narrow range in spite of its origin from different parts of India. Inverse correlation was observed between Fe and Mn (r = 0.89) in trikatu and its constituents whereas Cu/Zn ratio varied linearly (r = 0.92) with Cu. Ten compounds were identified in the essential oil of pipali along with the isolation of tannic acid and barbituric acid in aqueous methanolic extract. The base line data may be helpful to the traditional Ayurvedic practitioners or pharmaceutical firms for developing new Ayurvedic drugs. The elemental analyses, including those of the organic constituents may help in understanding its pharmacological action. *
Financial assistance from BRNS (DAE) vide 2000/37/5 is gratefully acknowledged. This work is a tribute to Indias first nuclear reactor APSARA on its completion of 50 years. Sincere thanks are due to Drs. A. G. C. NAIR and R. N. ACHARYA, Radiochemistry Division, BARC for their active cooperation and to Dr. J. DEOPUJARI (Nagpur) for helpful discussions and literature support. MHRD assistantship to RPC is thankfully acknowledged.

References
1. R. P. STEINER (Ed.) Folk Medicine The Art and the Science, American Chemical Society, Washington DC, 1986, p. 223. 2. British Medical Association, Complementary Medicine: New Approaches to Good Practice, Oxford University Press, Oxford, 1993. 3. P. PARANJPE, Indian Medicinal Plants, Forgotten Healers: A Guide to Ayurvedic Herbal Medicine, Chaukhamba Sanskrit Pratisthan, Delhi, 2001, p. 316.

4. V. V. SIVARAJAN, I. BALACHANDRAN, Ayurvedic Drugs and their Plant Sources, Oxford & IBH Publishing Co. Pvt. Ltd., New Delhi, 1994, p. 570. 5. I. H. QURESHI, J. H. ZAIDI, M. ARIF, I. FATIMA, Intern. J. Environ. Anal. Chem., 43 (1991) 25. 6. M. K. SHERIFF, R. M. AWADALLAH, A. H. AMRALLAH, J. Radioanal. Nucl. Chem., 57 (1980) 53. 7. V. SINGH, A. N. GARG, Appl. Radiation Isotopes, 48 (1996) 97; Food Chem., 94 (2006) 81. 8. R. K. JOHRI, U. ZUTSHI, J. Ethnopharmacol., 37 (1992) 85. 9. V. SIVAKUMAR, S. SIVAKUMAR, Phytother. Res., 18 (2004) 976. 10. C. K. ATAL, U. ZUTSI, P. G. RAO, J. Ethnopharmacol., 4 (1981) 229. 11. R. S. KARAN, V. K. BHARGAVA, S. K. GARG, J. Ethnopharmacol., 64 (1999) 259; Indian J. Pharm., 30 (1998) 254. 12. L. G. LALA, P. M. DMELLO, S. R. NAIK, J. Ethnopharmacol., 91 (2004) 277. 13. B. L. ODELL, R. A. SUNDE (Eds), Handbook of Nutritionally Essential Mineral Elements, Marcel Dekker Inc., 1997. 14. Q. Y. WEI, J. P. MA, Y. J. CAI, L. YANG, Z. L. LIU, J. Ethnopharmacol., 102 (2005) 177. 15. V. BADMAEV, M. MAJEED, L. PRAKASH, J. Nutr. Biochem., 11 (2000) 109. 16. B. S. PARK, W. S. CHOI, S. E. LEE, Agric. Chem. Biotech., 46 (2003) 73. 17a.) R. G. WEGINWAR, D. L. SAMUDRALWAR, A. N. GARG, J. Radioanal Nucl. Chem., 133 (1989) 317. b.) A. N. GARG, A. KUMAR, R. P. CHOUDHURY, J. Radioanal. Nucl. Chem., 271 (2007) 481. 18. J. ZHANG, Fenxi Kexue, 16 (2000) 1. 19. NIST/EPA/NIH Mass Spectra Library with Search Program: (Data version: NIST, Software version 2.0), NIST Standard Reference Database, 2002, No. 76442. 20. J. H. ZAIDI, I. FATIMA, I. H. QURESHI, M. S. SUBHANI, Radiochim. Acta, 92 (2004) 363. 21. C. A. STEWART, B. TERMANINI, V. E. SUTLIFF, J. SERRANO, F. YU, F. GIBRIL, R. T. JENSEN, Aliment. Pharmacol. Ther., 12 (1998) 83. 22. L. A. DANIELS, Biol. Trace Elem. Res., 54 (1996) 185. 23. WHO, Environmental Health Criteria, Mercury, Arsenic, Lead, World Health Organization, Geneva, 1989. 24. A. S. PRASAD, Essential and Toxic Trace Elements in Human Health and Disease, Alan R. Liss Inc., New York, 1986. 25. The Aldrich Library of FT-IR Spectra, 2nd ed., Vol. 2, Sigma Aldrich Co., USA, 1997, p. 2173. 26. H. WRBA, M. M. EL-MOFTY, M. H. SCHWAIREB, A. DUTTER, Exp. Toxicol. Pathol., 44 (1992) 61. 27. M. S. MASOUD, A. M. HEIBA, E. M. SOLIMAN, Transition Metal Chem., 14 (1989) 175. 28. O. V. KOVALCHUKOVA, A. K. MOLODKIN, R. K. GRIDASOVA, V. A. VYCHUZHANIN, Z. Neorg. Khim., 30 (1985) 3134 . 29. M. A. WERZ, R. L. MACDONALD, Mol. Pharmacol., 28 (1985) 269. 30. Wikipedia, The free Encyclopedia, http://en.wikipedia.org/wiki/barbiturate 31. Medicinal Plants Species Prioritized by Andaman & Nicobar Medicinal Plant Board Society, http://agri.and.nic.in/medicinal_species_priorit.html

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