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Constituents of Tinospora sinensis andtheir antileishmanial activity againstLeishmania donovaniRakesh Maurya a , Prasoon Gupta a , Kailash Chand a , ManmeetKumar a , Preety Dixit a , Nasib Singh b & Anuradha Dube ba Division of Medicinal and Process Chemistry, Central DrugResearch Institute, Lucknow, Indiab Division of Parasitology, Central Drug Research Institute,Lucknow, India

Available online: 29 Oct 2009

To cite this article: Rakesh Maurya, Prasoon Gupta, Kailash Chand, Manmeet Kumar, Preety Dixit,Nasib Singh & Anuradha Dube (2009): Constituents of Tinospora sinensis and their antileishmanialactivity against Leishmania donovani , Natural Product Research, 23:12, 1134-1143

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Natural Product ResearchVol. 23, No. 12, 15 August 2009, 1134–1143

Constituents of Tinospora sinensis and their antileishmanial activity

against Leishmania donovaniy

Rakesh Mauryaa*, Prasoon Guptaa, Kailash Chanda, Manmeet Kumara, Preety Dixita,Nasib Singhb and Anuradha Dubeb

aDivision of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow, India;bDivision of Parasitology, Central Drug Research Institute, Lucknow, India

(Received 13 June 2008; final version received 21 November 2008)

Two new compounds 4-methyl-heptadec-6-enoic acid ethyl ester (2) and3-hydroxy-2,9,11-trimethoxy-5,6-dihydro isoquino[3,2-a]isoquinolinylium (7)were isolated from an ethanolic extract of the stems of Tinospora sinensis,along with six known compounds (1, 3–6 and 8). The structures of newcompounds were established on the basis of detailed spectroscopic studies.Compound 7 exhibited the highest in vitro antileishmanial activity againstLeishmania donovani promastigotes and intracellular amastigotes, whereascompounds 2, 4, 5 and 6 demonstrated moderate activity. Other compoundswere found to be inactive.

Keywords: Tinospora sinensis; Menispermaceae; alkaloids; lignans; Leishmaniadonovani; immunomodulation; antileishmanial

1. Introduction

Tinospora sinensis (syn: Tinospora malabarica) is commonly known as ‘Gurch’ and belongsto the family Menispermaceae. It is found almost throughout all of India and other SouthEast Asian countries. Traditionally, this plant has great therapeutic value in treatingdebility, dyspepsia, fever, inflammation, syphilis, ulcers, bronchitis, jaundice, urinarydisease, skin disease and liver disease (Anonymous, 1976). In addition, it is known to haveadaptogenic and immunomodulatory properties (Kirtikar & Basu, 1993). The aqueous andethanolic extracts of this species are reported to have various pharmacological potential,such as anti-inflammatory (Li, Lin, Myers, & Leach 2003), anti-diabetic (Yonemitsu,Fukuda, & Kimura, 1993), hepotoprotective (Khan, Gray, & Waterman, 1989), and haveimmunomodulatory and adaptogenic activity (Manjrakar, Jolly, & Narayanan, 2000).Phytochemical investigations have revealed that this species contains steroids, flavonoides,alkaloids and most importantly the furano diterpenoids class of compounds, such as10�-hydroxy columbin (Atta-ur-Rahman & Ahmad, 1988), menispermacide(Atta-ur-Rahman, Ahmad, & Choudhary, 1992), tinosporicide (Atta-ur-Rahman,Ahmad, Choudhary, & Malik, 1991), malaborolide (Atta-ur-Rahman & Ahmad, 1988),

*Corresponding author. Email: mauryarakesh@rediffmail.comyCDRI Communication No. 7405.

ISSN 1478–6419 print/ISSN 1029–2349 online

� 2009 Taylor & Francis

DOI: 10.1080/14786410802682239

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malaborolide B1 (Atta-ur-Rahman et al., 1994), magnoflorine, quercetin-3-O-glycoside,

kaempferol and its glycosides, palmatine (Atta-ur-Rahman, Ahmad, & Choudhary, 1992),

kokusaginine (Bowen &Motawe, 1985), N-formyl –anonaine (Atta-ur-Rahman & Ahmad,

1987), cyclo-euphordenol (Atta-ur-Rahman, Ahmad, Choudhary, & Malik, 1992),

tinosinen-I (Yonemitsu, Fukuda, & Kimura, 1993), di-O-methyl syringaresinol (Atta-ur-

Rahman & Ahmad, 1987), tinosporinone, dibenzoylethane and allyloxyflavone (Prakash &

Zaman, 1982; Prakash, Khan, & Zaman, 1983). �-Sitosterol, tetracosanoic acid and

tinosporine have also been characterised from stem of the plant (Banerji et al., 1981).

Recently, two new lignan glycosides, namely tinosposide A and tinosposide B, have beenreported from the stems of the plant (Li et al., 2004).

Leishmaniasis is a complex of disease syndromes, with a spectrum that has classically

divided into cutaneous, visceral and mucocutaneous forms, caused by protozoan parasites

of the genus Leishmania belonging to the family Trypanosomatidae. Leishmania donovani

is responsible for visceral leishmaniasis (VL) or ‘kala-azar’ in India. Pentavalent

antimonials and amphotericin B are the main drugs used for treatment but their irregular

effectiveness, emergence of resistance in parasites against them, especially for sodium

antimony gluconate in India, have necessasitated the use of other alternative drugs.

Toxicity and variable efficacy of existing drugs is also of serious concern. Despite the

development of new promising drugs, miltefosine and paromomycin, the search of new

active compounds, preferably from natural products, is greatly encouraged.Our preliminary studies revealed that ethanol extract and its n-butanol fraction

exhibited significant in vitro antileishmanial activity (Singh et al., 2008). This prompted usto perform activity-guided isolation of pure compounds from the bioactive fraction, and to

evaluate them for their antileishmanial activity. Our efforts resulted in the isolation of

eight compounds, 1–8. Out of these eight compounds we have subjected seven compounds,

1–7, to in vitro assays for determining their antileishmanial activity.

2. Results and discussion

Compound 2 was obtained as yellow coloured oil. The IR spectrum revealed absorption

bands at 1732 cm�1 for an ester group, 1660 cm�1 for C¼C stretching and 996 cm�1 for

C¼CH bending vibration. The FAB-MS showed a [MþH]þ molecular ion peak at m/z

311 corresponding to the molecular formula C20H38O2. This data, compiled with the 1H

NMR spectrum, suggested that the compound could be an unsaturated carboxylic acid

ester with a long hydrocarbon chain.The 1H and 13C NMR spectrum (Table 1) with the aid of 1H-1H COSY furnished a

signal at �H 4.08 (q, J¼ 7.2Hz; �C 60.4), assigned to oxygenated methylene, coupled with a

methyl group at �H 1.21 (brs; �C 14.5), a two proton triplet at �H 2.24 (2H, t, J¼ 7.5Hz;

�C 31.8) assigned to a methylene adjacent to a carbonyl group at (�C 174.0), which further

coupled with a methylene at �H 1.58 (2H, brt, J¼ 6.9Hz; �C 30.4), a four proton broad

doublet at �H 1.98 (4H, brd, J¼ 5.4Hz; �C 27.5, 34.7) was assigned to two methylene

protons adjacent to a double bond at �H 5.30 (2H, t, J¼ 3.6Hz; �C 130.0, 128.6), 0.85 (6H,

t, J¼ 6.3Hz; �C 18.0, 14.3) assigned to two terminal methyl groups and �H 1.72 (1H, m;�C 29.9) assigned to one methine showed coupling with the methyl group and two

methylenes adjacent to a double bond and � to carbonyl in COSY spectrum. Other

methylenes of the chain resonated in the range of �C 29.7–22.9. This NMR data confirmed

the conclusion drawn from IR and mass spectra; it also indicated an unsaturated

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long-chain carboxylic acid. The cis geometry of the double bond in the compound 2 wasindicated by the appearance of two methylene carbons adjacent to a double bond at �C27.5, 29.9. The HMBC spectrum (Table 1) gave the important correlation H-20/C-1; H-2/C-1, 4; H-3/C-1, 4, 5, 18; H-4/C-2, 6, 18, H-18/C-3, 5 to confirm the structure. Further,from the above data, the compound was elucidated as 4-methyl-heptadec-6-enoic acidethyl ester, which has not been previously reported.

Compound 7 was obtained as orange red needle shape crystals, m.p. 219�C. TheESI-MS spectrum exhibited a molecular ion at m/z 338 [M]þ, corresponding to themolecular formula C20H20NO4, confirmed by 1H, 13C NMR spectrum and pos. HR-ESIMS at m/z 338.123 (Calcd. 338.117). The UV spectrum (methanol) 243, 305, 377 nmwas characteristic of the protoberberine type alkaloids (Sangster & Stuart, 1965); thephenolic nature was evidenced by positive FeCl3 test. The infrared absorption spectrasupported the presence of a hydroxyl group by broad bands at 3418 and 3500 cm�1 and anaromatic system by band at 1522, 1495 cm�1. The 1H and 13C NMR data in combinationwith 1H-1H COSY and HSQC spectrum exhibited six aromatic protons at �H 6.84 (1H, s;114.7), 7.51 (1H, s; �C 108.6), 9.72 (1H, s; �C 144.4), 7.98 (1H, d, J¼ 1.2Hz; �C 126.7), 8.01(1H, d, J¼ 1.2Hz; �C 123.1), 8.71 (1H, s; �C 119.8), three methoxy signals centred at �H4.02 (3H, s; �C 55.9), 4.09 (3H, s; �C 56.6), 4.21 (3H, s; �C 61.6) and two mutually coupledmethylene at 3.32 (2H, t, J¼ 6.1Hz; �C 26.5) and 4.91 (2H, t, J¼ 6.1Hz; �C 56.7). The restof the quaternary carbons were deduced by DEPT 135 and assigned utilising HMBCspectra (Figure 1). These results were identical to compound 6, except the existence of twometa coupled aromatic protons instead of ortho coupled, as in compound 6, indicateddeference in both compounds. Further, using HMBC and NOESY correlation spectro-scopy (Table 2, Figure 2), compound 7 was elucidated as 3-hydroxy-2,9,11-trimethoxy-5,6-dihydro isoquino[3,2-a]isoquinolinylium, a new compound reported here for the first time.

The known compounds were characterised as lirioresino-�-dimethyl ether 1

(Kakisawa, Kusumi, Hsu, & Chen, 1970), �-sitosterol 3 (Greca, Monaco, & Previtera,1990), palmatine 4 (Atta-ur-Rahman, Ahmad, & Choudhary, 1992) palmatrubin 5

(Doskotch, Malik, & Beal, 1967), jatrorrhizine 6 (Cava, Reed, & Beal, 1965), and 8 (Fumioet al., 2005); (Fukuda, Yenemitsu, & Kimura, 1983), by direct comparison of NMR data

Table 1. NMR spectral data of compound 2.a

Position �H (J in Hz) �C HMBC

1 – 174.0 –2 2.24 (t, 7.5) 31.8 1, 43 1.58 (brt, 6.9) 30.4 1, 4, 5, 184 1.72 (m) 29.9 2, 6, 185, 8 1.98 (brd, 5.4) 27.5, 34.7 3, 18, 66, 7 5.30 (t, 3.6) 130.0, 128.6 4, 59–16 1.27–1.18 (m) 29.7–22.9 –17 0.85 (t, 6.3)b 14.3 –18 0.85 (t, 6.3)b 18.0 3, 4, 510 4.08 (q, 7.2) 60.4 120 1.21 (brs) 14.5 1

Notes: aRecorded in CDCl3:1H NMR, HMBC at 300MHz and

13C NMR at 200MHz (TMS as internal standard). bOverlappedsignal within the column.

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Table 2. NMR spectral data of compound 7 in DMSO-d6.

Position �H (J in Hz) �C HMBC

1 7.51 (s) 108.6 –1a – 117.82 – 150.5 –3 – 148.3 –4 6.84 (s) 114.7 –4a – 128.45 3.32 (t, 6.1) 26.5 –6 4.91 (t, 6.1) 56.7 C-7, 8, 107 – – –8 9.72 (s) 144.4 C-6, 9, 108a – 121.79 – 144.1 –10 7.98 (d, 1.2) 126.7 –11 – 150.2 –12 8.01 (d, 1.2) 123.1 C-2, 10, 40, 60

12a – 133.913 8.71 (s) 119.8 C-10, 40, 50

13a – 138.82-OCH3 4.09 (s) 56.6 –3-OCH3 4.02 (s) 55.9 C-10, 30, 40

9-OCH3 – – C-2, 10, 20, 40

10-OCH3 4.21 (s) 61.6 C-40, 30 0, 500

O

O

OCH3

OCH3

OCH3H3CO

H3CO

H3CO 1

H H

HO

3

N

R1O

H3COOR2

OCH34: R1 =R2 = CH35: R1 = CH3, R2 = H6: R1 = H, R2 = CH3

2

OH3C

O

CH3

CH3

N

HO

H3COOCH3

OCH3

NH

O

7 8HO

OH

1 4

172′

18

11

13

Figure 1. Chemical structure of compounds 1–8.

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with those reported in the literature. All the known compounds were isolated for the firsttime from this plant (Figure 1).

2.1. In vitro antileishmanial activity

The activity of T. sinensis ethanol extract, its fractions and compounds isolated fromchloroform and n-butanol fraction were evaluated in vitro against GFP taggedL. donovani promastigotes (Pms) and intracellular amastigotes (Ams) by flow cytometry(FACS) as described previously (Dube, Singh, Sundar, & Singh, 2005; Mishra, Singh,Ahmad, Dube, & Maurya, 2005; Tewari et al., 2006). Briefly, log-phase GFP-tagged Pms(5� 105 cells permL) were incubated with 10, 25, 50 and 100mgmL�1 concentrations oftest samples for 78–96 h. FACS analysis was performed to quantify the fluorescencelevels in treated and untreated groups. Decrease in fluorescence intensity indicated thegrowth inhibition of parasites. J774 macrophages (5� 105 cells perwell) in 12-well cultureplates (Nunc) were infected with GFP-tagged Pms at a ratio of 10 : 1. The level ofinfection in infected macrophages before and after drug treatment was measured byFACS. The assays were performed in triplicate. Miltefosine was used as positive control.The activity of the samples was also assessed by Giemsa staining method.

Ethanol extract exhibited 91.0� 5.0 and 81.6� 7.3% inhibition of Pms and Amsrespectively at 100 mgmL�1 concentration (Figure 3). n-Butanol fractions showed96.3� 2.1 and 80.6� 3.2% inhibitions, respectively, at similar concentration. This fractionrevealed better efficacy than chloroform fractions as it was active (475%) even at25 mgmL�1 concentration against Pms, whereas chloroform fraction was toxic to themacrophages at 100 mgmL�1 concentration (Figure 3). Aqueous fractions were found tobe inactive at this concentration and therefore are not shown in Figure 3.

Among the compounds isolated and tested, compound 7 was the most active. At theconcentrations of 100, 50 and 25 mgmL�1 it resulted in 96.6, 93.7 and 81.0% inhibitions ofPms and 86.6, 81.3 and 49.7% inhibitions of Ams (Figures 4 and 5). Compound 2

exhibited 55.3� 3.8 and 54.0� 3.4% inhibition of Pms and Ams multiplication,respectively, at 100 mgmL�1 concentration. Compounds 4–6 showed moderate activitieswhereas compound 3 was found to be toxic to the macrophages at 100 mgmL�1.Compounds 2 and 4–7 were found to be non-cytotoxic at similar concentrations. The restof the compounds were inactive against both the stages of the parasite. Miltefosine, thereference drug, was highly active against both Pms (100% at 25 mgmL�1) and Ams (100%at 10 mgmL�1) (Figures 4 and 5).

N+

HO

H3CO

OCH3

OCH3

HMBC

NOESY

Figure 2. Selected HMBC and NOESY correlation of 7.

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In conclusion, activity-guided purification of ethanol extract of T. sinensis resulted ineight compounds 1–8; among them, two compounds, 2 and 7, are new. Compounds 2 and7 have demonstrated significant in vitro antileishmanial activity. Compounds 4–6 have alsoshown activity but lower than compound 7, whereas compounds 1 and 3 were foundinactive and showed certain levels of cytotoxicity.

3. Experimental

3.1. General

Melting points (uncorr.) were recorded on a Complab melting point apparatus. IR spectrawere recorded on a Perkin-Elmer RX-1 spectrophotometer using either KBr pellets

10

25

50

75

10025

50100

Compounds

% P

aras

ite in

hibi

tion

2 3 4 5 6 7

Figure 4. Antileishmanial activity of isolated compounds of T. sinensis against L. donovani Pms.Data (means � SD) are representative of three independent experiments.

0

20

40

60

80

100

25 μgml−1

50 μgml−1

100 μgml−1

Ethanolextract

Chloroformfraction

n-Butanolfraction

Ethanolextract

n-Butanolfraction

Tox

ic

Promastigotes Amastigotes

% P

aras

ite in

hibi

tion

Chloroform fraction

Figure 3. Antileishmanial activity of ethanol extract and fractions of T. sinensis against L. donovaniPms and Ams. Data (means � SD) are representative of three independent experiments.

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or neat. UV spectra were obtained on a Perkin Elmer �-15 UV spectrophotometer, opticalrotations were measured on a Perkin-Elmer Model 241 digital polarimeter. 1H- and13C- NMR spectra were recorded on an Avance DPX-200 and Bruker DRX 300MHz

spectrometers and chemical shifts are expressed as � (ppm) values. The abbreviations of1H-NMR signal patterns are as follows: s, singlet; d, doublet; dd, double doublet; ddd,

doublet of doublet of doublet; t, triplet; m, multiplates. Proton-detected heteronuclearcorrelations were measured using HSQC and HMBC. The 2D spectra were recorded on a

Bruker DRX 300MHz spectrometer. FAB-MS were carried out on a Jeol SX 102/DA-6000 mass spectrometer using m-nitro benzyl alcohol as matrix. ESI-MS spectra were

obtained on an LCQ Advantage Max Thermo Finnigan. Preparative HPLC were

performed on a Shimadzu CLC-Octa decyl silane RP-18 (ODS) column with (20mmID� 25 cm length); 8mLmin�1 flow rate, PDA UV � 254 and 220 nm as detector. Column

chromatography was performed using silica gel (60–120 and 230–400 mesh); TLC: pre-coated silica gel plates 60 F254 or RP-18 F254 plates with 0.5 or 1mm film thickness

(Merck). Spots were visualised by UV light or by spraying with H2SO4–MeOH,anisaldehyde–H2SO4 reagents.

3.2. Plant materials

The plant was collected and identified by the Botany Division of the Central DrugResearch Institute, from Paschin Midnapur, West Bengal, India.

3.3. Extraction and isolation

The powdered stems of Tinospora sinensis (15.5 kg) were extracted with EtOH and yielded720 g extract. The EtOH extract was dissolved in water and fractionated successively with

CHCl3 and n-BuOH, which resulted in CHCl3 (270.1 g), n-BuOH (200.3 g) and aqueous(240.7 g) crude residues, respectively. A portion of chloroform fraction (80.0 g) was

1 20

25

50

75

100

2550

100

Inac

tive

Inac

tive

Compounds

% P

aras

ite in

hibi

tion

3 4 5 6 7

Figure 5. Antileishmanial activity of isolated compounds of T. sinensis against L. donovaniintracellular Ams. Data (means � SD) are representative of three independent experiments.

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chromatographed over silica gel (60–120 mesh, 900.0 g) and eluted with a gradient ofhexane: CHCl3 (95 : 05) to CHCl3: MeOH (85 : 15) sequentially. Seventy-eight fractions (of1000mL each) were sampled and their composition monitored by TLC, with those showingsimilar TLC profiles grouped into nine fractions (F-1 to F-7). Compound 1 was crystallisedfrom F-5 at room temperature; it was then recrystallised in cold methanol, which afforded1 (400mg). Purification of F-3 (22.3 g) over silica gel (60–120 mesh, 400.0 g) with a gradientof hexane: benzene (90 : 10) to hexane: benzene (50 : 50) afforded four pooled fractions (F-8to F-11) on the basis of TLC profiles from a total of 14 fractions of 100mL each. Furtherpurification of F-10 (2.8 g) using similar conditions over silica gel (60–120 mesh, 45.0 g)yielded compound 2 (2.3 g) as yellow coloured oil. Fraction F-2 (8.0 g) was rechromato-graphed over silica gel (60–120 mesh, 130 g) and eluted with a gradient of hexane: CHCl3(100–50%), which afforded four fractions (F-12 to F-15) from a total of 68 fractions of150mL each on the basis of TLC profiles. Subsequently fraction F-13 (1.1 g) was purifiedover silica gel (60–120 mesh, 35.0 g) and eluted with hexane: acetone as isocratic eluent. Atotal of 13 two sub-fractions of 100mL were collected, sub-fractions 3–15 containing 3weredried, which afforded 3 (150mg) as white solid. A portion of n-BuOH soluble fraction(95.0 g) was subjected to column chromatography (CC) over silica gel (60–120 mesh, 2.1 kg)and eluted with a gradient of chloroform:methanol (98 : 02) to (50 : 50) sequentially. Seventyfractions (1000mL each) were sampled and their composition monitored by TLC, withthose showing similar TLC profiles grouped into seven fractions (F-16 to F-22). Furthercolumn chromatography of F-17 (14.2 g) over silica gel (60–120 mesh, 360.0 g) with agradient of CHCl3/MeOH (95 : 5) to (75 : 25), afforded five pooled fractions (F-23 to F-27)on the basis of TLC profiles from a total of 14 two fractions of 200mL each. Furtherpurification of F-26 (1.6 g) was over silica gel bed (230–400 mesh, 60.0 g) eluted withchloroform with increasing volume of methanol from 10% to 20%. A total of 14 five sub-fractions of 75mL each were sampled and reduced to four fractions 1–15 fr., F-28; 16–24 fr.,F-29; 25–34 fr., F–30 and 35–45 fr., F-31. Fraction F-29, containing 4, was then dried andcrystallised in methanol and afforded compound 4 (40mg) as pink coloured crystals.Fraction F-18 (5.2 g) was chromatographed over silica gel (230–400 mesh, 30.0 g) and elutedwith a gradient of chloroform and methanol from (90 : 10 to 50 : 50). A total of 51 fractionsof 100mL each were collected, fractions 11–21 eluted in chloroform and methanol (85 : 15)yielded 5, which then crystallised in methanol and afforded 5 (30mg) as orange red crystals.Further, fr. 30–43 containing amixture of two compounds upon preparative HPLC (RP-18)purification with gradient H2O/MeOH 100% to 50% resulted in sub-fractions 1–45, 10mLin volume, which afforded needle shape crystals of compound 6 (40mg), fr. 12–22 and 7

(45mg), fr. 24–32. Fraction F-23 (2.2 g) was purified over silica gel (60–120mesh) and elutedwith a mixture of chloroform and methanol 100–90%; a total of 24 fractions were sampled,100mL each, and fr.13–16 yielded compound 8 (48mg).

3.3.1. Compound 2

Yellow oil (2.3 g); IR �max (Neat) cm�1: 1732, 1660, 1595 and 996. 1H NMR (CDCl3,300MHz) and 13C NMR (CDCl3, 75MHz) see Table 1; FAB-MS (pos.): m/z 311 [MþH]þ;Positive HR-ESIMS: m/z [M]þ 310.2931 (Calcd 310.2872 for C20H38O2).

3.3.2. Compound 7

Orange red crystals (47mg); m.p. 219�C; UV (MeOH) �max nm: 243, 305, 377; IR �max

(KBr) cm�1: 3500, 3418, 1601, 1596, 1522, 1495 and 1354. 1H NMR (DMSO-d6, 300MHz)

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and 13C NMR (DMSO-d6, 75MHz) see Table 2; ESI-MS (pos.): m/z 338 [M]þ; PositiveHR-ESIMS: m/z [M]þ 338.1437 (Calcd 338.1392 for C20H20NO4).

Acknowledgements

P. Gupta is grateful to the Indian Council of Medicinal Research, New Delhi for the award of SeniorResearch Fellowship. N. Singh, P. Dixit and K. Chand are thankful to CSIR and UGC, respectively,for financial support. We are thankful to S.C. Tiwari for technical assistance.

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