Phytol and Glycerol Derivatives from the Marine Green Alga Codium iyengarii of the Karachi Coast (Arabian Sea) Muhammad Shaiq Ali*, Muhammad Saleem, Viqar Uddin Ahmad and Simin Shameel H. E. J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan * Reprint requests to Dr. M. S. Ali. E-mail: [email protected] Z. Naturforsch. 56b, 837-841 (2001); received March 3, 2001 Codium iyengarii , Chlorophyceae, Codiosides The ethyl acetate soluble part of a methanolic extract of the marine green alga Codium iyengarii, collected from the Karachi coast of the Arabian Sea, afforded four new derivatives of glycerol named : codioside A,B,C,D (1-4), a new derivative of frans-phytol named codio- ester (5) and rrans-phytol (6). The structures of 1-5 were elucidated with the aid of acid hydrolysis, methylation, GC-MS, and spectroscopy. The structure of 1 was confirmed through the HMBC NMR technique and the structures of compounds 2 -4 were based on the assign­ ments made for 1.

Introduction H- jC?—R* A number of fatty acids (saturated and unsatu­ rated), sterols, terpenoids, and sugars of various classes have been already detected from seaweeds [1-2]. A wide range of these marine natural pro­ ducts showed biological activities [3]. The mem­ bers of the genus Codium are known for their anti­ cancer activity [4]. Rl R2 R3 The marine alga Codium iyengarii ia a member of Chlorophyceae (green algae) found in bulk Codioside A 1 palmitate" OH H around Karachi and adjacent coastal areas of the Codioside B 2 oleate" OH H Arabian Sea. The results of a previous study pub­ Codioside C 3 oleate/palmitate" palrrdtate/oleate" " gals lished by Usmanghani and Shameel showed that Codioside D 4 palmitate" palmitate'" H water extract of C. iyengarii is capable to inhibit the growth of gram-positive bacteria [5]. The pre­ Fig. 1. Palmitic acid: CH 3(CH2)i4COOH; oleic acid: sent paper describes the results of an attempt CH3(CH2)7CH=CH(CH2)7COOH. made to investigate the chemical constituents of C. iyengarii. As a result of this study, five new com­ enough to allow some chemical reactions and pounds were isolated and characterized. They in- therefore, their structures were elucidated with the clulde: four glyco-fatty acid derivatives of glycerol aid of a combination of chemical reaction as well (1-4, codioside A to D) and a fatty acid derivative as physical data including the HMBC NMR tech­ of mms-phytol (5, codio-ester). nique together with the comparison of literature values. As the skeleta of 1 -4 are rather similar Results and Discussion they are discussed here together. The fatty acids The methanol soluble part of C. iyengarii af­ attached to the glycerol (1-4) and rrans-phytol (5) forded four new glycoglycerides named codiosides were identified by treating 1 -5 with sodium meth- A -D (1-4) (Fig. 1) and a new fatty ester of trans- oxide and the resulting methyl esters of fatty acids phytol named codioester (5). The quantities of iso­ were analyzed by GC-MS using known reference lated constituents 1 -5 (average 20 mg) were compounds. Similarly, after the detachment of the

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Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. 838 M. S. Ali et al. • Phytol and Glycerol Derivatives from Marine Green Alga Codium ivengarii

J ff NaOMe ff H2C — O j-C — CH2— (CH2)13 CH3 ------► MeO— C— CH2— (CH2) i3 CT13

H2C — OH OC-MS with Standard fatty acid methyl «ster Hj:— OH

H2 C .OH *+ O— C

OH

Scheme 1. fatty moieties, the sugar units in 1 -4 were iden­ tified by acid hydrolysis to liberate the free glyco- units and then they were compared by TLC with the authentic sugar samples. The whole methodol­ ogy is explained for 1 in Scheme 1. Palmitic acid occurs very commonly in the green seaweed of the Karachi coast and is usually found in largest proportion, whereas oleic acid is the OH most commonly occurring unsaturated fatty acid Fig. 2. Selective HMBC connectivities in 1. [6 ]. Various phycochemical studies revealed that the main algal fatty acids are either saturated or ds-unsaturated with a 1 2 - 2 0 carbon chain and up sible to study the HMBC spectrum and establish to six double bonds [7]. Most of the fatty acids the structure of 1. However, the 13C NMR spectra are found as complex esters in marine algae or helped to establish the structures of 1-4. Based terrestrial plants. Glycerol can accommodate 1-3 on the information obtained from the HMBC molecules of fatty acids producing mono- to tri­ spectrum of 1, the structures of 2 -4 could easily glycerides. be established. The structures of 1 -4 were elucidated further The *H NMR spectrum of 1 displayed the anom­ by positive FAB-MS and NMR spectroscopy. The eric proton at <5 = 4.12 as a doublet (J = 7.5 Hz) positive FAB mass spectra of 1 -4 and their corre­ which showed HMBC interactions (Fig. 2) with sponding high resolution spectra gave the formu­ the methylene at d = 67.6 which in turn showed lae C2 5 H 4 9 O 9 , C2 7 H 5 1 O 9 , C4 9 H 5 1 O 1 5 and interactions with the anomeric carbon at d = 103.7. C4 1 H 7 9 O10, respectively. The IR spectra of all four Two more significant triplets resonating at (3 = 2.19 glycerides showed the absorptions of ester car­ (2 H, J = 7.1 Hz) and 0.81 (3H,/ = 7.0 Hz) were bonyl and hydroxyl function. Compound 3 attributed to 2"-H and 16"-H. A signal of two pro­ showed in addition an olefinic absorption at 1605 tons appeared at <5 = 4.32 as a doublet (/ = 6.2 Hz) cm-1. Most of the oxygenated methines and and was assigned to 1-H. The downfield chemical methylenes appeared in the XH NMR spectra as shift of this methylene group revealed that an het­ complex multiplets. Only anomeric and olefinic eroatom was attached to it which was proved methines, ester substituted methylenes, and through HMBC experiments (Fig. 2). Other oxy­ methyl signals could be detected in the XH NMR genated methines and methylenes were found to spectra. In addition, the methylene protons resonate at d = 3.80-3.22 as complex multiplets. (C3 H2) in 1 could also be traced in the spectrum The downfield carbon signals at (5 = 103.7 and by means of a selective decoupling experiment at 173.8 were assigned to anomeric and ester car­ d = 3.17 as a broad doublet and thus it was pos- bonyl carbons, respectively. The confirmation of M. S. Ali et al. ■ Phytol and Glycerol Derivatives from Marine Green Alga Codium ivengarii 839 palmitic acid [ 8 ] and glucose was performed as de­ An acyclic diterpenoidal fatty ester was also ob­ scribed in the experimental section and in tained from the same source and assigned as 5. The Scheme 1. Finally, the structure of 1 was elucidated molecular formula was depicted through HRMS as as 1-palmitate 3-gluco propanetriol and named C3 6 H 7 o 0 2 showing the presence of two double codioside A. bonds in the molecule. IR absorptions were found The *H NMR spectrum of 2 was highly similar to at 1725 (ester, C = 0) and 1610 cm “ 1 (C=C). The XH/ that of 1 except the presence of two olefinic signals 13C NMR signals and mass fragmentation pattern as doublet of triplets at <3 = 5.16 (J = 5.5,1.6 Hz) and provided strong evidence for the presence of a phy­ 5.04 (J = 5.6, 1.6 Hz) and their corresponding car­ tol type skeleton [11], A comparison of XH and 13C bons resonated in the 13C NMR spectrum at d - data of 5 with the data of cis- and rrans-phytols re­ 129.4 and 129.6 and were assigned to C-9" and C- vealed that 5 was the ester of fraws-phytol [12-13]. 10", respectively. The GC-MS experiments with 2 In case of fram-phytol, the oxygenated methylene indicated the presence of oleic acid [9-10] and thus group (1-H) appears at <5 = 4.1, whereas in c/s-phy- 2 was characterized as 1-oleate 3-gluco propanet­ tol it shifts downfield by 0.3-0.4ppm. Moreover, riol and named codioside B. the literature shows that in ?ram,-phytol, oxy-meth- The *H NMR spectrum of 3, which showed two ylenic carbon (C-l) appears at d - 59.4 and olefinic triplets at d = 2.19 (/ = 7.0 Hz) for four protons and carbons at <5 = 123.1(C-2) and 140.0 (C-3), [13] at (5 0.82 (J - 6 . 8 Hz) for six protons, suggested two whereas in the c/s-isomer they appear at (3 = 61.9, fatty acid units in the molecule. This was further 118.2 and 142.5, respectively [11]. The palmitate supported by the 13C NMR spectrum which dis­ moiety was confirmed through the GC-MS tech­ played two carbonyl resonances at <5 = 173.7 and nique. On the basis of obtained data, 5 was charac­ 173.5. Signals for two olefinic methines at d = 5.14 terized as rra/w-phytyl palmitate and named codio- and 5.06 as doublets of triplets indicated that one of ester. In addition to trans-phytyl palmitate, trans- the two fatty acid moieties was oleate. The phytol ( 6 ) was also isolated for the first time from spectrum exhibited also two anomeric resonances our investigated source. at d - 4.24 (7.6 Hz) and 4.20 (7.4 Hz). The anomeric carbons were found to resonate at d = 103.5 and 105.1. The sugar units were confirmed as glucose and galactose and their linkage was determined with the help of 13C NMR assignments which showed the downfield shift of C-4' from d = 78.0 to 84.2. Thus, it was concluded that the galactose is at­ tached at C-4 of glucose. Based on these results, the structure shown in Fig. 1 was given to compound 3 and it was named codioside C. However, the posi­ tions of the fatty acid moieties in the molecule may 5 : codio ester, R = palmitate be reversed. 6 : frans-phytyi, R = OH The XH NMR spectrum of 4 was different from that of 3 only by lacking two olefinic and one anom­ eric methines. The two triplets of four protons and Experimental six protons, respectively, resonating at <5 = 2.19 (/ = General 7.3 Hz) and 0.82 ( / = 7.0 Hz) were attributed to 2"- H, 2"'-H and 16"-H and, 16'"-H, respectively. The The *H and 13C NMR spectra were recorded at signals for quaternary carbons appearing in the 13C 500 and 125 MHz, respectively, on a Bruker AM- 500 instrument. The GC-MS of fatty acid methyl es­ NMR spectrum at <5 = 173.8 and 173.5 were due to ters was performed on a Hewlett Packard GC in­ the two ester carbonyl atoms. An anomeric proton strument with a 11/73 DEC computer system and a and the corresponding carbon gave signals at d - packed glass capillary column (1.2 m x 4 mm), 4.24 (7.6 Hz) and 98.5. Finally, the structure of 4 was coated with gas chrome Q (100-120 mesh, OV 101, assigned as 1,2-dipalmitate 3-gluco propanetriol 1%). The column temperature was programmed and named codioside D. from 170 °C to 250 °C with a rate of increase rate 840 M. S. Ali et al. • Phytol and Glycerol Derivatives from Marine Green Alga Codium ivengarii

8 °C/min. The injector temperature was 250 °C, the Sodium metal (0.5 g) was added to freshly dis­ carrier gas (He) flow was 32 ml / min. tilled and dry methanol (25 ml) with stirring in an ice bath. When all the sodium metal was dissolved, Collection and identification 5 (10 mg) was added and left overnight at r.t. under nitrogen. Methanol was removed and the yellow The alga was collected in March 1999, from the gum thus obtained, was diluted with water, acidi­ Karachi cost (Bulejii) of the Arabian Sea as a drift fied with dilute HC1 and the organic material was material. It was then identified by Ms. Shaista extracted with ethyl acetate. The organic layer was Hameed, Center of Excellence in Marine Biology, washed several times till neutral pH. The resulting University of Karachi, where the voucher specimen methyl ester of the fatty acid was analyzed along is deposited in the herbarium (No. MBG-22). with a reference fatty acid methyl ester by GC-MS and was found to be methyl palmitate. Extraction and isolation Another fraction from the same column eluted with 1 0 % methanol in chloroform showed many The collected alga (wet weight 11.5 kg) was spots on TLC which was then subjected to repeated washed with tap-water in order to remove the sea- column chromatography for final purification using salts and dried under shade for a period of one the various combinations of chloroform and metha­ week. The dried material (2.0 kg) was then soaked nol as mobile phase. By elution with 5% methanol in methanol (2x5 1) for ten days at r.t. The extract in chloroform, compounds 1 and 4 were obtained as was concentrated (60.0 g) at low temperature jelly-like masses (1, 27.0 mg and 4 ,19.0 mg). (28° C) to avoid the thermal decomposition and Repeated column chromatography (eluent: 7% was diluted with water. The organic material was methanol in chloroform) yielded 2 as an oily mass recovered in ethyl acetate. The crude ethyl acetate (22.5 mg). soluble part was then chromatographed on a silica Compound 3 was eluted with 10% methanol in gel column using hexane, hexane-chloroform, chlo­ chloroform from the same column as a light yellow roform, chloroform-methanol and finally, pure colored oil (26.3.0 mg). methanol as mobile phase. The fatty acid(s) in 1 -4 and sugar unit(s) in 2 -4 The fraction obtained with 5% chloroform in were confirmed as described in 5 and 1, respec­ hexane yielded 5 (23 mg, yellow jelly) and 6 (17 mg, tively. The prime numbering in 1 -4 is explained in mobile oil) which were separated by repeated col­ Fig. 1. umn chromatography. Compound 6 was identified Codioside A (1): [a]D: +16.3° (MeOH, c = as ?rans-phytol [ 1 2 ]. 0.73 mg/ml). - IR (KBr): 3450 (OH), 1730 (ester, Codio-ester (5): [ö]d: +63.6° (CHCI 3 , c = 0.33 mg/ C=0), 1150 (C-O) cm“1. - :H NMR (500 MHz, ml). - IR (CHCI3 ): 1725 (C =0), 1610 (C=C), 1430, CDCI3 + CD 3 OD): <5 = 0.81 (t, / = 7.0 Hz, 3 H, 16"- 3 1150 (C-O) cm "1. - XH NMR (500 MHz, CDC1 ): H), 1.41-1.22 (br.s, CH 2 chain), 2.19 (t, J = 7.1 Hz, (5 = 0.87-0.85 (d, J = 6.5 Hz, 12 H, 7a-H, 11a, 15a, 2 H, 2"-H), 3.17 (br.d, 2 H, 3-H), 3.80-3.22 (m, 7 H, 16), 0.88 (t, J = 7.5 Hz, 3 H, 16'-H), 1.24 (br.s, CH 2 2-H, and sugar), 4.12 (d, J - 7.5 Hz, 1 H, 1 '-H), 4.32 chain), 1.65 (s, 3 H, 3a-H), 2.30 (t, J = 7.5 Hz, 2 H, (d, J = 6.2 Hz, 2 H, 1-H). - ^C ^H ] NMR (125 MHz, J = J 2'-H), 4.12 (d, 7.0 Hz, 2 H, 1-H), 5.31 (t, = 7.0 CDCI3 + CD 3 OD): (5 = 13.85 (C-16"), 29.51-28.24 Hz, 1 H, 2-H). - ^C^H] NMR (125 MHz, CDC13): (C-3"-14"), 31.73 (C-15"), 33.82 (C-2"), 61.22 (C- (5 = 14.11 (C-16'), 16.10 (C-3a), 19.79 and 19.77 (C- 6 '), 67.61 (C-3), 68.60 (C-l), 70.22 (C-2), 74.84, 7a, 11a), 22.63 (C-16), 22.72 (C-15a), 24.54 (C-9), 73.24, 71.03, 70.22 (C-2', 3', 4', 5'), 103.73 (C -l'), 24.81 (C-13), 25.11 (C-5), 28.03 (C-15), 29.72-29.13 173.84 (C-l"). - FAB MS (+ ve mode): m /z = 493 (C -3'-1 3 '), 32.73 (C-7), 32.82 (C -ll), 34.43 (C-2'), (M+H)+. - HR FAB(+ ve mode): m /z 493.33659 36.76 (C-6 ), 37.22 (C-12), 37.35 (C-8 ), 37.43 (C-10), (calcd. m /z = 493.33762 for C25H49O 9). 39.44 (C-14), 39.90 (C-4), 59.46 (C-l), 123.00 (C-2), 140.52 (C-3), 176.51 (C -l'). - EIMS: m/z 534 [M]+, 323, 279 (M-palmitate)+, 255 (palmitate)+, 95 and Acid hydrolysis o f 1 83. - HRMS: m/z 534.53672 (calcd. m /z 534.53754 The reaction vessel was charged with 1 (10 mg) for C 3 6 H 7 0 O2), 255.3218 (calcd. m /z 255.3228 and 2M HC1 in aqueous methanol (3.5 ml). It was Ci6 H 3 1 0 2), 279.2990 (calcd. m /z 279.30515 C 2 0 H39). then refluxed for 3 h, methanol was removed and The fatty acid moiety was identified by GC-MS. the remaining material was diluted with water. The water layer was extracted with ethyl acetate, then Detachment of the fatty acid as methyl ester from 5 neutralized with dilute NaOH, and concentrated. and confirmation by GC-MS The obtained material was then compared with M. S. Ali et al. • Phytol and Glycerol Derivatives from Marine Green Alga Codium ivengarii 841

Standard sugars by TLC [silica gel, development in 1"). - FAB MS (+ ve mode): m /z 519 (M + H )\ - ethyl acetate acetic acid water methanol ( 6 :1 :1 :2 )] HR FAB (+ ve mode): m /z 519.35674 (cacld. m/z and found comparable with glucose. 519.35327 for C 2 7 H 5 1 0 9). Codioside D (4): [a]D: +23.0° (MeOH, c = Codioside C (3)*: [a]D: +06.9° (MeOH, c = 0.29 mg/ml). - IR (KBr): 3450 (OH), 1725 (ester, 0.97 mg/ml). - IR (KBr): 3450 (OH), 1730-1740 C = 0), 1150 (C-O) c m 1. - *H NMR (500 MHz, (br., C=0, ester), 1605 (C=C), 1150 (C-O) cm“1. - CDC13 + CD 3 OD): 6 = 0.82 (t, J = 7.0 Hz, 6 H, 16"- *H NMR (500 MHz, CD3OD + few drops CDC13): H , 16'"), 1.38-1.20 (br.s, CH 2 chain), 2.19 (t, / = 7.3 Ö = 0.82 (t, J = 6 . 8 Hz, 6 H, 18"'-H, 16'"'), 1.52-1.21 Hz, 4 H, 2 "-H, 2'"), 3.36-3.25 (m, 8 H, 3-H and (CH 2 chain), 2.19 (t, J = 7.0 Hz, 4 H, 2"'-H, 2""), 3.63 sugar), 4.24, (d, J = 7.6 Hz, 1 H, l'-H ), 4.35 (d, J = (m, 1 H, 4'-H), 4.01-3.25 (m, 13 H, 3-H and two 6 . 6 Hz, 2 H, 1-H), 4.81 (m, 1 H, 2-H). - 1 3 C{1 H} sugars), 4.20 (d, J = 7.4 Hz, 1 H, 1"-H), 4.24 (d, J = NMR (125 MHz, CDC13 + CD 3 OD): (5 = 13.51 (C- 7.6 Hz, 1 H, 1 '-H), 4.35 (d, J = 6 . 6 Hz, 2 H, 1-H), 4.80 16", 16'"), 29.33-28.82 (C-3"-14" and C-3'"-14'"), (m, 1 H, 2-H), 5.06 (dt, J = 5.7,1.5 Hz, 1 H, 10"'-H), 31.54 (C-15", 15'"), 33.93 (C-2", C-2'"), 62.71 (C- 6 '), 5.14 (dt, / = 5.7,1.4 Hz, 1 H, 9"'-H). - ^CpH} NMR 65.75 (C-3), 66.9 (C-l), 69.90 (C-2), 72.92, 72.32, (125 MHz, CD 3 OD + CDCI3 ): <3 = 13.83 (C-18'", 16'" 71.40, 70.44 (C-2', 3', 4', 5'), 98.51 (C -l'), 173.53 '), 29.52-29.02 (CH 2 chain), 33.92 (C-2""), 34.11 (*C-1'"), 173.83 (*C-1"). - FAB MS (+ ve mode): m / (C-2'"), 62.61 (C-6 '), 63.44 (C-6 "), 66.01 (C-3), 67.82 z 731 (M+H)+; HR FAB (+ ve mode): m /z = (C-l), 69.93 (C-2), 72.00 (C-4"), 74.01 (C-2"), 74.93 731.56897 (calcd. m/z 731.56722 for C 4 1 H 7 9 O 10). (C-3"), 75.42 (C-2'), 77.91 (C-5"), 78.02 (C-3'), * Assignments may be reversed. 78.11(C-5'), 84.21 (C-4'), 105.10 (C-l"), 103.52 (C- Codioside B (2): [a]D: +79.9° (MeOH, c = 1'), 129.22 (C-9'"), 129.31 (C-10'"), 173.53 (C-l""), 0.11 mg/ml). - IR (KBr): 3450 (OH), 1725 ( C = 0, 173.74 (C-l'"). - FAB MS (+ ve mode): m /z 919 ester), 1610 (C=C), 1150 (C-O). - XH NMR (500 (M+H)+ . - HR FAB (+ ve mode): m /z = MHz, C D C I 3 + C D 3 O D ) : d = 0.83 (t, J = 7.2 Hz, 3 919.634218 (calcd. m /z 919.63574 for C 4 9 H 9 1 0 15). H, 18'-H), 1.56-1.21 (br.s, CH 2 chain), 2.19 (t, / = * Assignments of H'" / C'" and H"" / C"" may be 6.9 Hz, 2 H, 2"-H), 3.80-3.19 (m, 9 H, 2-H, 3 and reversed. sugar), 4.15 (d, J = 7.6 Hz, 1 H, l'-H ), 4.34 (d, J = 6.3 Hz, 2 H, 1-H), 5.04 (dt, J = 5.6,1.6 Hz, 1 H, 10"-H), Acknowledgements 5.16 (dt, J = 5.5,1.6 Hz, 1 H, 9"-H). - ^CpH} NMR One of us (M. S. A.) is very much thankful to the

(125 MHz, CDCI 3 + CD 3 O D ) : <5 = 13.64 (C-18"), Office of Naval Research (U. S. A.) for financial 29.22-28.62 (CH 2 chain), 31.84 (C-17"), 34.11 (C- support (Project NP-13) and we are also thankful to 2"), 61.60 (C-6 '), 66.24 (C-3), 66.91 (C-l), 70.43 (C- Ms. Shaista Hameed, Center of Excellence in Ma­ 2), 73.52, 72.82, 71.75, 70.23 (C-2', 3', 4', 5'), 103.22 rine Biology, University of Karachi for identifying (C -l'), 129.41 (C-9 "), 129.61 (C-10"), 173.65 (C- the algal material.

[1] Atta-ur-Rahman, V. U. Ahmad, S. Bano, S. A. Ab­ [8] A. H. Memon, M. Shameel, K. Usmaghani, M. bas, K. A. Alvi, M. S. Ali, H. M. Lu, J. Clardy, Phy­ Ahmed, V. U. Ahmed, J. Bot. 23 (1), 33 (1991). tochemistry 27, 3879 (1988). [9] R. Aliya, M. Shameel, K. Usmanghani, V. U. [2] K. Usmanghani, M. Shameel, M. Sualeh, K. H. Ahmed, Pak. J. Pharm. Sei. 4 (2), 103 (1991). Khan, Z. A. Mehmood, Fitoterapia LV, 73 (1984). [10] K. Usmaghani, M. Shameel, M. Alam, Studies on [3] S. Bano, M. S. Ali, V. U. Ahmad, Planta Medica 53, fatty acids of a green alga Ulva fasciata from Kar­ 508 (1987). achi coast. Prospects for Biosaline Res., US-Pak. Bi­ [4] Jhy-Horng Sheu, C. C. Liaw, Chang-yih Duh, J. Nat. osaline Res. Workshop (Ahmad, R. and Pietro, Prod. 58, 1521 (1995). A. S., eds.), Botany Dept., Karachi University, Paki­ [5] K. Usmanghani, M. Shameel, in: Studies on the anti­ stan (1986). microbial activity of certain seaweeds from Karachi [11] N. Rasool, V. U. Ahmed, A. Malik, Phytochemistry coast. (R. Ahmad and A. S. Pietro, eds.). Prospects 30, 1331 (1991). for Biosaline Res., US-Pak. Biosaline Res. Work­ [12] J. J. Sims, J. A. Pettus, Phytochemistry 15, 1076 shop, Botany Dept., Karachi University, Pakistan (1976). (1986). [13] R. A. Goodman, E. Oldfield, A. Allerhand, J. Am. [6] R.Qasim, J. Chem. Soc. Pak. 8 , 223 (1986). Chem. Soc. 95, 7553 (1973). [7] K. Usmaghani, M. Shameel, Pak. J. Pharm. Sei. 9 (2), 53 (1996).