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Industrial Crops and Products 45 (2013) 395– 400

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products

journa l h o me page: www.elsev ier .com/ locate / indcrop

solation of a unique dipyridodiazepinone metabolite nevirapine during largecale extraction of Cliv-92 from the seeds of Cleome viscosa

rnab Chatterjeea, Sunil K. Chattopadhyaya,∗, Sudeep Tandona, Ranjeet Kaura, Anil K. Guptab,rakas R. Maulikc, Ruchir Kantc

Process Chemistry and Technology Department, Central Institute of Medicinal and Aromatic Plants (CSIR), P.O.-CIMAP, Lucknow 226 015, IndiaGenetic Resource Management Department, Central Institute of Medicinal and Aromatic Plants (CSIR), P.O.-CIMAP, Lucknow 226 015, IndiaMolecular and Structural Biology Division, Central Drug Research Institute (CSIR), Chattar Manzil Palace, Post Box No. 173, Lucknow 226 001, India

r t i c l e i n f o

rticle history:eceived 22 August 2012eceived in revised form7 November 2012ccepted 1 December 2012

a b s t r a c t

Natural nevirapine (1), a dipyridodiazepinone metabolite has been isolated as a minor constituent witha yield of 0.00397% from the ethyl acetate extract during large scale extraction of Cliv-92 at a level of100 kg/batch from the seeds of Cleome viscosa. Column chromatographic purification of the ethyl acetateextract afforded the molecule which was characterized as nevirapine through spectral analysis and con-firmed by single crystal X-ray crystallography. The molecule also has been re-isolated from the same

eywords:leome viscosaevirapineipyridodiazepinone

RMS

batch of seeds to confirm its presence in the seeds of C. viscosa. The molecule nevirapine has been fullycharacterized by co-TLC, prep-TLC-MS/MS and chiral HPLC analysis. Isotope Ratio Mass Spectrometric(IRMS) analysis of natural nevirapine and the synthetic drug revealed it to be different. It is an interestingdiscovery to find out that the natural nevirapine exists as an endogenous metabolite of the plant. Thispaper also reports the isolation of salicylic acid from the same batch of seeds which afforded nevirapine

e to s

hiral-HPLC which may have been du

. Introduction

Cleome viscosa (Syn. Cleome icosandra) (Capparidaceae) is annnual wasteland weed with yellow flowers and strong penetrat-ng odour. This weed is very common in India. The leaves of thelant are rubefacient, vesicant and sudorific. The seeds are small,ark brown or black and granular. They are reported to have rube-acient, vesicant and anthelminthic properties. The seeds are usedccasionally as condiment in curries. (Anon, 1950)

Chemical investigation of the plant has disclosed the presencef several interesting classes of chemical compounds, macrocycliciterpene cleomaldic acid (Anon, 2001) glycoflavanones (Chauhant al., 1979), lipoflavanones (Srivastava and Srivastava, 1979),lucosinolates (Songsak and Lockwood, 2002) and cleomeolidediterpene lactone) (Mahato et al., 1979) which have anticancerroperties. Phytochemical investigation of the seeds of the plantlso has resulted in the isolation of a new class of chemical enti-ies known as coumarinolignoids in which a lignan (C6–C3) units linked with a coumarin moiety through a dioxane bridge. Four

oumarinolignoids cleomiscosins A, B, C and D have been isolatednd identified from the seeds of C. viscosa (Ray et al., 1985; Kumart al., 1988). Cleomiscosins A, B and C possess liver protective

∗ Corresponding author. Tel.: +91 5222718620; fax: +91 5222718620.E-mail address: chattsk@yahoo.com (S.K. Chattopadhyay).

926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.indcrop.2012.12.029

ome plant–pathogenic interaction.© 2012 Elsevier B.V. All rights reserved.

activity (Chattopadhyay et al., 1999a,b). The mixture of the threecoumarinolignoids cleomiscosins A, B and C is termed as Cliv-92which has hepatoprotective activity comparable to that of sily-marin the antihepatotoxic drug currently in use throughout theworld. Recent phytochemical investigations also have shown thepresence of terpenoids, saponins and alkaloids (Koche et al., 2010).The occurrence of nitrogenous compounds has been exemplifiedby the first isolation of lactam nonanic acid from the root exudatesof the plant (Jana and Biswas, 2011).

For one of our ongoing projects, we needed cleomiscoins A, Band C in large quantities. In order to get the compounds, we startedextracting 100 kg seeds of C. viscosa to get the molecules. In thatendeavor we isolated a molecule with a yield of 0.00397% whichwas characterized as nevirapine. In order to confirm the struc-ture unequivocally, the crystal structure of the molecule also wasstudied.

After its structure was fully characterized, we published ourfindings in Tetrahedron (Chattopadhyay et al., 2011a). After pub-lication of the paper in Tetrahedron, some criticisms have appearedon the natural occurrence of nevirapine as well as on its chiral prop-erty. Therefore, we had retracted the paper to verify the findingsbeyond doubt (Chattopadhyay et al., 2011b).

We have re-isolated the molecule from the same batch of seedsand confirmed that the natural nevirapine isolated from the seeds ofC. viscosa was different from synthetic nevirapine through isotoperatio mass spectrometric (IRMS) analysis. In addition to nevirapine,

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96 A. Chatterjee et al. / Industrial C

e also have isolated salicylic acid during isolation of nevirapine.n this paper we are reporting the occurrence of nevirapine in theeeds of C. viscosa and confirmation of its structure by detailedpectral analysis including X-ray crystallography.

. Materials and methods

.1. General

Melting points were uncorrected and were recorded on auchi – Melting point Apparatus. UV spectra were recorded on apectronic® GENESYSTM with a 10 mm quartz cell and IR spectraere recorded on a Perkin-Elmer Spectrum BX FT-IR spectrometer.

ample pellets were prepared in KBr using hydraulic pellet pressf Kimaya manufacturers. NMR spectra were recorded on a Bruker

Avance 300 MHz FT-NMR using CDCl3, a deuterated solvent, thehemical shift of which was used as an internal standard. DART-RMS data were obtained with a JEOL ACCU TOF DART JMS-T100LCass spectrometer. IRMS analysis for ∂13C and ∂15N was performedith Thermo Finnigan Flash1112 elemental analyzer linked with

Thermo Finnigan Delta V Plus isotope ratio mass spectrome-er. Sucrose with a ∂13C certified value of −10.8 and ammoniumulphate with a ∂15N certified value of +20, procured from IAEAInternational Atomic Energy Agency) were used as standard for13C and ∂15N. The overall analytical precision for replicates oftandard was 0.06‰ for ∂13C and 0.08 ‰ for ∂15N.

.2. Plant material

The seeds of C. viscosa were collected from the outskirts ofucknow during the month of October. The seed samples haveeen deposited in the “National Gene Bank of Medicinal and Aro-atic Plants” CIMAP, Lucknow (accession number CIMAP 3426).

he harvested plants were dried in diffused sunlight and seeds wereemoved by thrashing.

.3. Extraction and isolation of nevirapine from synthetic drugevimune

One tablet containing 200 mg API nevirapine was powdered in mortar and pestle. The powdered tablet was then allowed to stirith 1N aqueous HCl for about 1 h and was filtered. The aqueousltrate was extracted subsequently with hexane and chloroform.evirapine (120 mg) was isolated from the chloroform fraction.hen the acidic layer was basified with ammonia (liquor) andxtracted with chloroform. The chloroform layer gave an extrauantity (50 mg) of nevirapine.

.4. Chiral HPLC method development for the analysis ofevirapine

Several methods were used for chiral resolution of nevirapinend its acetate. It was found to be well resolved, with good repro-ucibility, in a NP-isocratic elution mode consisting of solvent An-hexane) and solvent B (ethyl acetate containing 0.1% diethy-amine) with 80:20 ratio and 0.5 mL/min flow in CHIRALPAK-IA250 mm× 4.6 mm i.d.) column procured from DAICEL Chemicalndustries, Tokyo, Japan. The solvents were procured from MERCK,ndia and were of analytical grade. Detections were performed at

max values of 283 nm for nevirapine and 340 nm for acetylatedevirapine in a Waters HPLC system (Waters Corporation, Milford,A, USA) consisting of a Waters 600E multi solvent delivery system

nd equipped with Waters Empower software for data acquisition.

nd Products 45 (2013) 395– 400

2.5. Extraction and isolation of nevirapine from the seeds

The process was aimed primarily at the extraction of coumarino-lignoids which involved the fixed oil removal from 100 kg of driedseeds by subjecting them to an oil expeller. The miscella (89 kg)was charged into pilot scale extractors of 100 kg/batch of a multiu-tility solid–liquid extraction plant where the initial defatting of thecrude material was done in hexane (200 L × 6). The marc left wasextracted with methanol (210 L × 6). The solvent free dark greencolored crude methanolic extract (12 kg) was then adsorbed ontocelite (6.4 kg) which served as a base for the solid-matrix parti-tion process. A nutsche type filtration unit of 50 kg capacity waspacked with celite (6.0 kg) in toluene and the adsorbed extractwas partitioned sequentially by toluene (25 L × 4), ethyl acetate(25 L × 4) and methanol (25 L × 4), respectively. The vacuum con-centrated toluene, ethyl acetate and methanolic extracts were of1.98%, 5.69%, 0.55% yields, respectively. The semi solid concentratedethyl acetate extract was adsorbed with 11.0 kg silica gel and wasloaded onto pilot scale stainless steel columns of 50 kg gel holdingcapacity. Silica gel (60–120 mesh) (23 kg) was packed with hexanealong with the adsorbed slurry and the column was eluted witha mixture of hexane and ethyl acetate in the ratio of 1:1, 1:3 andfinally with ethyl acetate. TLC analysis of the fractions of hexane:ethyl acetate (1:1) eluates in choloroform:acetone (93:7) showedalmost a major spot in UV at a Rf of 0.46 which was stained withDragendorff’s reagent. The fractions containing the above spot werepooled and concentrated. Recrystallization of the concentrate fromethyl acetate yielded colorless needles of nevirapine (1) (3.54 g,0.00397%); m.p. 241–243 ◦C; Rf (7% CH3COCH3/CHCl3) 0.46; IR �max

(KBr) 3047, 2921, 2868, 1744, 1656, 1586, 1463, 1417 cm−1; UV(CHCl3) �max (log ε) 325 nm (3.5), 275 nm (sh, 3.5) 240.4 nm (4.0);1H NMR and 13C NMR spectroscopic data see Table 1. DART-HRMSm/z 267.1231 [M+H]+ (calculated 267.1246).

2.6. Preparation of N-acetylated derivative 2 of 1

A mixture of 1 (50 mg, 0.019 mmol), acetic anhydride (4.0 mL,42.3 mmol) and triethylamine (2.0 mL, 14.35 mmol) was stirred onan oil bath at 80 ◦C for 4 h. Standard work up of the reaction mix-ture and subsequent silica gel column purification in chloroformgave a fraction which on crystallization from cold methanol yieldedthe acetate derivative 2 (30 mg,Yield = 53%): m.p. 174–176 ◦C (fromMeOH); UV (CHCl3) �max 343 nm, 262 nm (sh), 244 nm; IR (KBr)�max 1684, 1585, 1559, 1455, 1413,1297 cm−1; 1H and 13C NMRspectroscopic data see Table 1; ESI-MS m/z 309.2 [M+H]+; 331.3[M+Na]+.

2.7. Single crystal X-ray structure determination of nevirapine(1) at 293 (2) K

X-ray data were collected at 293 K with a Bruker Smart ApexCCD diffractometer with graphite monochromator and Mo K�radiation (� = 0.71073 A), using SMART32 (Bruker) and SAINT(Bruker) softwares. The structure was solved by direct meth-ods and refinements by full-matrix least-squares methods on F2

using SHELXTL-NT [Bruker AXS Inc.: Madison, Wisconsin, USA1997]. Crystal data: C15H14N4O, Empirical formula C17H14N4O2,Mr = 306.32, triclinic, space group P(−1), a = 7.767(3) Å, b = 8.420(4)Å, c = 12.466(5) Å, (deg) = 84.70, (deg) = 89.37, � (deg) = 68.39,V (Å3) = 754.5(5), Z = 2, �calc. = 1.348 Mg/m3, � (Mo K�) = 0.71073Å, � = 0.092 (mm−1). Data collection and reduction: crystalsize, 0.225 mm × 0.20 mm × 0.275 mm, range = 2.61–28.31, 4942

reflections collected, 3553 independent reflections (Rint = 0.0229),R indices (all data) = R1 = 0.1219 and wR2 = 0.3301 final R indices[I > 2(I)] R1 = 0.0931 and wR2 = 0.2450 for 233 variable parame-ters, GOF = 1.12. The X-ray crystallographic file of the synthetic

A. Chatterjee et al. / Industrial Crops and Products 45 (2013) 395– 400 397

Table 11H NMR Spectroscopic data of Nevirapine (1) and the N-acetylated derivative (2) in CDCl3.

Position 1 2

ıHa, mult, intgt (J, Hz) ıC

b ıHa, mult, intgt (J, Hz) ıC

b

2 8.16, d,1H (4.8) 140.6 (CH) 8.30,d, 1H (4.8) 142.3 (CH)3 6.93, d, 1H (4.8) 120.7 (CH) 7.10, uns.q,1H (4.2, 8.1) 123.0 (CH)4 139.9 (C) 149.0 (C)

6 169.4 C O 168.8 C O7 8.11, dd,1H (2.1, 7.8) 144.7 (CH) 8.40, dd, 1H (1.8, 7.8) 148.0 (CH)8 7.06, dd, 1H (4.8, 7.5) 119.3 (CH) 7.10, uns. q, 1H (4.2, 8.1) 118.5 (CH)9 8.54, dd, 1H (2.1, 4.8) 152.4 (CH) 8.59, dd, 1H (1.8, 4.8) 153.4 (CH)

12 154.5 (C) 158.0 (C)13 125.3 (C) 117.1 (C)14 122.4 (C) 123.9 (C)15 161.0 (C) 159.7 (C)16 2.38, s,3H 18.1 (CH3) 2.28, s, 3H 18.1 (CH3)17 3.75, m, 1H 29.9 (CH) 3.74, m, 1H 30.0 (CH)18 0.45, 0.98, m, 2H 9.2 (CH2) 0.52, 1.08, m, 2H 9.0 (CH2)19 0.45, 0.98, m, 2H 9.4 (CH2) 0.52, 1.08, m, 2H 10.2 (CH2)

169.2 C O2.45, s, 3H 24.4 (CH3)

d, doublet; dd, double doublet; m, multiplet; uns. q, unsymmetrical quartet. 13C NMRm

ng

3

puaic

3

tmauts2o

N

NH

N

N

CH3

O

12

3

4

56

7

8

9

10 11

12

13

14

15

16

18

19

17

Key HMBC correl a�ons: H C

Fig. 2. Key HMBC correlations indicating the dipyridodiazepinone framework.

a Recorded at 300 MHz.b Recorded at 75 MHz. Multiplicity of the signals is given as follows: s, singlet;ultiplicities were obtained from DEPT-135 experiment.

evirapine has already been deposited in Cambridge Crystallo-raphic Data Centre, CCDC Nos. 649751-649756 (Caira et al., 2008).

. Results and discussion

As a part of our ongoing studies regarding optimization of batchrocess parameters for the large scale isolation of Cliv-92, we havendertaken an up scaling process from the dried seeds of this plantt a level of 100 kg/batch (Tandon et al., 2010). During the up scal-ng process, we have isolated a compound 1 (Fig. 1) which washaracterized as dipyridodiazepinone metabolite.

.1. Characterization of the isolated compound

Compound 1, obtained as transparent needles, was assignedhe molecular formula C15H14N4O from its DART-HRMS spectrum/z 267.1231, [M+H]+. The UV [�max 325, 275 (sh) and 240 nm]

nd IR spectra of the molecule indicated that it possessed a �,�nsaturated lactam system. The nature of 15C atoms present in

13

he molecule were revealed from C NMR of the compound whichhowed aromatic methyl (ıC 18.1), a cyclopropyl ring (ıC 9.4, 9.2,9.9) and five CH groups (ıC 119.3, 120.7, 140.6, 144.7 and 152.4),ne lactam carbonyl (ıC 169.4) and five quaternary carbons (ıC

N

NH

N

N

CH3

O

12

3

4

567

8

9

1011

12

13

14

15

16

17

18

19

Fig. 1. Structure of the isolated nevirapine (1) from the seeds of C. viscosa.

Fig. 3. ORTEP-diagram: stereoscopic view of the molecule (1) with atomic number-ing scheme at 30% probability.

3 rops a

18C1HC

FiAd

98 A. Chatterjee et al. / Industrial C

22.4, 125.3, 139.9, 154.5 and 161). In the HMBC spectrum H-2 (ıH.16) showed cross peaks with C-3 (ıC 120.7), C-4 (ıC 139.9) and

-12 (ıC 154.5) and H-3 (ıH 6.93) to C-2 (ıC 140.6), C-4 (ıC 139.9), C-6 (ıC 18.1) and to the quaternary carbon C-13 (ıC 125.3). Also, theMBC correlation of H-7 (ıH 8.11) to C-8 (ıC 119.3), C-9 (ıC 152.4),-14 (ıC 122.4), C-15 (ıC 161.0) and the characteristic correlation

N

N

N

N

CH3

O O

CH3

A

C

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Minutes

5.00 10.00 15.00 20.00

AU

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Minutes

5.00 10.00 15.00 20.00 25.00 30.00

Nevirapine acetate 2

ig. 4. Chiral-HPLC profile of the naturally isolated nevirapine (A), synthetic nevirapine (solated nevirapine (D) from the seeds of C. viscosa run on CHIRALPAK-IA column with a

(n-hexane) and B (ethyl acetate containing 0.1% diethylamine) in 60:40 (A:B) ratio forerivative.

nd Products 45 (2013) 395– 400

of H-7 to the carbonyl C (ıC 169.0) suggested the presence of adipyridodiazepinone moiety in the molecule. The key correlation

diagram for the 1H–13C HMBC is shown in Fig. 2.

The presence of a cyclopropyl group, two pyridine rings, lac-tam carbonyl and the diazepinone framework accounted for elevendegrees of unsaturation in the molecule. The molecular formula and

N

N

N

N

CH3

O

O

CH3

N

N

N

N

CH3

O

O

CH3

B

D

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Minutes

5.00 10.00 15.00 20.00

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00

B), mixture of synthetic and isolated nevirapine (C) and N-acetylated derivative ofNP-isocratic elution mode at a flow rate of 0.7 mL/min and solvent composition of

nevirapine and 80:20 (A:B) ratio with a flow rate of 0.5 mL/min for the acetylated

A. Chatterjee et al. / Industrial Crops an

Table 2IRMS analysis of natural and synthetic nevirapine.

Molecular formula Source ∂13C/∂12C (‰) ∂15N/∂14N (‰)

1

rcaoda2

maNmpwttsfynmas(rttt22f

2tmmwdat

aiwpflb2cd

4

wb

Coplen, T.B., Brand, W.A., Gehre, M., Gröning, M., Meijer, H.A.J., Toman, B., Verk-

C15H14N4O Natural −31.12 +3.21C15H14N4O Synthetic −28.04 −4.36

3C NMR of compound 1 showed a close resemblance with nevi-apine (1) (Hargrave et al., 1991; Anon, 2006). Compound 1 wasrystallized from ethyl acetate as needles. The detailed structurend stereochemistry of compound 1 was established unambigu-usly by single crystal X-ray crystallography as shown by the ORTEPiagram of the molecule (Fig. 3) and the corresponding crystal datanalysis (Pereira et al., 2007; Caira et al., 2008; Hannongbua et al.,001).

To co-establish its identity unequivocally, we purchased thearketed drug Nevimune (Cipla Ltd., Mumbai, Batch No.: G04633)

nd compared its identity with compound 1 by co-TLC, UV, IR,MR and were found to be identical. We have re-isolated theolecule nevirapine 1 from the seeds of C. viscosa to confirm its

resence in the seeds. Accordingly, 100 g seeds from 100 kg/batchere extracted with methanol and the methanolic extract was par-

itioned between water and ethyl acetate. The ethyl acetate extract,hus obtained, was subjected to preparative TLC using the solventystem 5% methanol in choloroform. Nevirapine, thus obtained,rom the preparative TLC was subjected to MS and MS/MS anal-sis and the spectra were compared with the spectra of syntheticevirapine. Major peaks in the MS/MS chromatogram of both theolecules (natural as well as synthetic) appeared at m/z 226, 161

nd 107. The IRMS analysis of synthetic and natural nevirapinehowed different values for the ratios of ∂13C/∂12C and ∂15N/∂14NTable 2) which were found to be −28.04, −4.36 for synthetic nevi-apine and −31.12 and +3.21 for natural nevirapine isolated fromhe seeds of C. viscosa. Thus, the IRMS analysis clearly establishedhat nevirapine isolated from the seeds of C. viscosa is different fromhe synthetic nevirapine (Smith and Epstein, 1971; Coplen et al.,006; Santamaria-Fernandez et al., 2008; Schimmelmann et al.,009; Voznesenskaya et al., 2007). The re-isolation of nevirapinerom the seeds of C. viscosa confirmed its occurrence conclusively.

The chiral HPLC analysis (Fujita et al., 1999; Kitagawa et al.,000; Rao et al., 2006; Zhang et al., 2010) study of the natural andhe synthetic nevirapine showed a single peak in the HPLC chro-

atogram (Fig. 4). This was due to the rapid interconversion of theolecule at room temperature (Burke et al., 2012). Furthermore,e acetylated natural nevirapine with Ac2O in Et3N and the acetateerivative 2 thus obtained was analyzed by chiral HPLC column. Thecetate derivative 2 showed two peaks in the HPLC chromatogramhus confirming the racemic nature of nevirapine.

It is worthwhile to mention that we also have isolated salicyliccid by pooling the fractions containing nevirapine and subject-ng them to column chromatography. The identity of salicylic acid

as confirmed by NMR and comparison with an authentic sam-le. The presence of salicylic acid in the nevirapine containingractions, clearly indicates that the plant might have accumu-ated salicyclic acid (SA) as a defense signalling molecule againstiotrophic pathogenic attack (Marek et al., 2010; Durrant and Dong,004; Raskin, 1992; Malamy et al., 1990; Métraux et al., 1990). Thehiral HPLC analysis of nevirapine and its acetate derivative clearlyemonstrated that natural nevirapine is a racemic molecule.

. Conclusions

The X-ray and detailed spectral analysis of compound 1, alongith co-analysis with the marketed drug, proved the compound to

e nevirapine.

d Products 45 (2013) 395– 400 399

The chiral-HPLC analysis of isolated nevirapine and the syntheticone appeared as a single peak due to the rapid interconversion ofthe two enantiomers at room temperature. The rapidly intercon-verting molecule showed atropisomeric behavior when acetylatedthus increasing the barrier to the N inversion leading to the possi-bility of separating the two enantiomers. The IRMS analysis of theisotope ratios ∂13C/∂12C and ∂15N/∂14N for the natural and syn-thetic nevirapine were different which clearly substantiated thatboth the molecules were different.

The re-isolation of nevirapine from 100 kg batch seed samplesruled out any kind of contamination at the extract/extraction levelthus establishing the endogenous nature of the compound. Thepresence of salicylic acid itself suggested that the plant might havebiosynthesized nevirapine in response to either some abiotic stressor may be the compound has been triggered in response to somekind of endogenous plant–pathogenic interaction.

The discovery of nevirapine from natural source clearly demon-strates that nature still holds the key to the discovery of uniquebiologically active molecules.

Supplementary data

1H and 13C NMR and 2D-NMR spectra for the key compounds areavailable. Prep-TLC-MS/MS chromatograms of the synthetic drugand the re-isolated nevirapine from fresh batch along with HPLCanalysis of the extracts and chiral assessment of the molecule andits respective derivatives also are available.

Acknowledgements

We thank the Director, CSIR-CIMAP, Lucknow for his coopera-tion. Financial assistance from the CSIR networking project NWP-09is gratefully acknowledged. We also are very grateful to Prof. Geof-frey A. Cordell for critically going through the manuscript and forhis valuable comments. We are also thankful to Dr. S.R. Shetye,Director, CSIR-NIO, Goa and to Dr. V.K. Banakar, Anita Garg, Dr. A.Mazumdar for IRMS analysis. Authors also are thankful to Dr. K.P.Madhusudanan, ex-scientist, CSIR-CDRI, Lucknow for his valuablesuggestions on IRMS analysis of the samples.

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