During the course of chemical investigation of marine algae collected from Karachi coast of Arabian Sea, five sterols named as sarangosterol(1), 23-methyl cholesta-5, 25-dien-3ß-ol(2) from Endarachne binghamiae (brown alga), sargasterol(3) from Dictyota indica (brown alga), cholesterol(4) from Laurencia obtusa (red alga) and clerosterol(5) from Codium iyengarii (green alga) have been isolated. Their structures were elucidated with the help of spectroscopic means

Ahmad, V.U.

Perveen, S.

Rehman, A.

Shaiq Ali, M.

Shameel, M.

Uddin, S.

Aquatic Commons

P'lkistan Journal of Marine Sciences, Vol.1(1), 57-64, 1992 
STEROL COlVIPOSITION OF MARINE ALGAE FROM KARACHI 
COAST OF ARABIAN SEA 
Viquar Uddin Ahmad, Shaista Perveen, Mohammad Shaiq Ali, Shafi Uddin, 
Rehman Aliya and Mustafa Shameel. 
H.E.J. Research Institute of Chemistry, University of Karachi, Karachi-75270 
(VUA, SP, MSA, SU) and Department of Botany, University of Karachi, 
Karachi-75270, Pakistan (RA, MS) 
ABSTRACT: During the course ofchemical investigations of marine algae collected from Karachi coast of 
Arabian sea, five sterols named as sarangosterol (1), 23-methyl cholesta-5, 25-dien-38-ol (2) from En-
daraclme binghamiae (brown alga), sargasterol (3) from Dictyota indica (brown alga), cholesterol (4) from 
Laurencia obtusa (red alga) and clerosterol (5) from Codium iyengarii (green alga) have been isolated. 
Their structures were.elucidated with the help of spectroscopic means. 
KEY WORDS: Sterols - Marine algae - Endaraclme binghamiae - Dictyota indica - Law·enica obtusa -
Codittm iyengarii. 
INTRODUCTION 
Among the three major divisions of algae (Chlorophyta, Phaeophyta and 
Rhodophyta), the members of Phaeophyta (brown algae) have been studied moderat-
ly. Previously we reported hexadecanoic acid, 24-methylene cholesterol and D~man­
nitol from Endaraclme binghamiae (Bano et al., 1987) and secodolastane diterpenoids 
from Dictyota indica (Bano et al., 1990}. In this paper we report two sterols named as 
saran gosterol (1) and 24-methyl cholesta-5, 25-dien-313-ol (2) from E. binghaniae and 
a sterol, sargasterol (3) from D. indica. These three compounds have not been 
reported so far from their respective sources. 
The red algae of the genus Laurencia have been investigated extensively for their 
halogenated metabolities specially sesquiterpenoids. In our previous work we have 
isolated only halogenated sesquiterpenoids from L. pinnatifida (Bano et a!. 1988a, b, 
Ahmed and Ali, 1991). This time, during the search of sterol composition we found 
cholestrol ( 4) as a major metabolite isolated from L. obtusa. 
The members of Cholorphyta (green algae) are rarely studied and found complex 
with reference to their chemical composition, which might be due to the presence of 
:much chlorophyll. As a part of studies on marine natural products from Karachi coast 
of Arabian sea, we found a sterol named as clerosterol (5) from Codiwn iyengarii. 
MATERIALS AND METHODS 
EXTRACTION AND ISOLATION: 
E. binglzamiae (3 kg., wet wt.) was collected from Buleji, Karachi. It was extracted 
58 Pakistan Journal of Marine Sciences, Vol.l (1), 1992 
with methanol. The methanolic extract was evaporated under vacuum to dryness and 
partitioned between ethyl acetate and wateL The ethyl acetate fraction after evapora-
tion yielded 2.3g of crude extract which was chromatographed on silica gel column. 
Elution with hexane:ether (6.5:3.5) yielded compound 1 (18.25 mg). Further elution 
with hexane:ether (7:3) afforded compound 2(5.51 mg). 
D. indica (1kg) was extracted with acetone. The acetone extract was evaporated 
under vacuum and the residue obtained was subjected to column chromatography on 
silica gel. The sargasterol (3) (13.37 mg) was eluted with hexane:ether (8:2) and 
purified by thick layer chromatography developed in hexane:ether:acetic acid 
(7.5:2.0:0.5). 
L. obtusa (1.5kg, wet wt.) was collected from Manora coast of Karachi and ex-
tracted in a Soxhlet apparatus with chloroform and then with methanol. The 
chloroform extract was condensed under reduced pressure to afford gummy mass. 
This was subjected for column chromatography using solvent systems hexane, 
hexane:chloroform, chloroform, chloroform:methanol and finally with pure methanol. 
Elution with hexane:chloroform (1:1) yielded crystalline compound, identified as 
cholesterol ( 4). 
C. iyengmii was collected from Manora, Hawksbay and Buleji. The alga was 
washed with water and dried in shade. The air-dried alga (1.3 kg) was soaked in 
ethanol for one month. Ethanolic extract was evaporated under reduced pressure to 
obtain a gummy residue which was then partitioned with ethyl acetate and water. The 
ethyl acetate extract (8 g) after evaporation under reduced pressure was subjected to 
column chromatography on silica gel. The elution was carried out with mixtures of sol-
vents in the order of increasing polarity starting with hexane, ether, chloroform and 
methanol. The fraction eluted with hexane:ether (9:1) gave pure compound 5. 
RESULTS AND DISCUSSION 
The column chromatography of ethyl acetate extract of E. binghamiae yielded a 
fraction eluted with hexane:ether ( 6.5:3.5) was further purified on a silica gel column. 
The compound 1 was isolated during re-column chromatography with chloroform: 
methanol (9.8:0.2) in pure form. The HRMS of 1 showed a molecular ion peak at m/z 
428.36452 corresponding to the molecular formula C29H4s02. The presence of the 
peaks at m/z 255,231,213,145 and 119 which are common peaks in 115 sterols (Ikekawa 
eta!., 1966) indicated that the sterol has the same ring system as cholesterol. Further, 
the peak at m/z 314 showed the cleavage of the bond between C-22- C-23, together 
with a hydrogen transfer from the charge fetaining moiety and the intense peak at m/z 
271 appeared in the ElMS of 1, corresponding to the loss of side chain together with 
two hydrogen atoms from the steroid nucleus (Wyllie and Djerassi, 1968). 
The 13C-NMR (broad band) spectrum of 1, summarized in Table-1, which ex-
hibited 29 carbon resonances. The DEPT spectrum indicated the presence of five 
Ahmad et al: Sterols from Marine Algae 59 
methyls, eleven methylene and nine methine signals. The remaining 13C-NMR signals, 
in broad band spectrum, were due to the quaternary carbons. The methine signal at o 
71.84 was assigned to C-3 indicating the presence of a B-hydroxyl group at this carbon 
(Holland et a/. 1978). A multiplet at o 3.51 (W1h= 16.36 Hz), in the 1H-NMR 
spectrum, confirmed the presence of a B-hydroxyl group at C-3 of a sterol (Ikekawa et 
a!. 1966), while the carbon signals at o 140.85 ( -C =) and 121.75 ( = CH) showed the 
presence of an endocyclic double bond between C-5 - C-6 in a sterol (Holland et a!. 
1978). A distorted triplet at o 5.34 was correlated with C-6 methine signal at o 121.75 
in a hetero-Cosy (2D-NMR) spectrum. 
HO 
The 1H and 13C-NMR spectra suggested that 1 has the same ring system as 
cholesterol (Ikekawa et al., 1966; Holland et al., 1978). The methylene signal at o 
112.80 and methine signal at o 142.65, in 13C-NMR spectrum, which showed the 
presence of a vinyl group at C-24 (Guyot eta!., 1982). The vinyl protons give rise the 
three double-doublets at o 5.12 (J = 1.52, 10.84 Hz), 5.18 (J = 1.52, 17.36 Hz) and 5.79 
(J = 10.84, 17.36 Hz). The last signal correlated with the olefinic carbon signal ap-
peared at o 142.65 in the hetero-Cosy spectrum. The appearance of double doublet at 
o 5.79, in 1H-NMR spectrum, showed the absence of a proton at C-24. This informa-
tion allowed us to place a hydroxyl group at C- 24 (Ikekawa eta/., 1966). This was fur-
ther confirmed by a downfield quaternary carbon signal at o 77.73 in 13C-NMR 
spectrum which showed the presence of a quaternary hydroxyl group. Hence, on the 
bas~ of foregoing evidences the structure of 1 was elucidated as 24-hydroxy, 24-vinyl 
cholesterol. Some spectroscopic vlaues can also be matched with its 24-hydroperoxy 
isomer (Guyot eta!., 1982). This compound was previously reported as sarangosterol 
from brown alga Sargassum ringgoldiamun (Ikekawa eta!., 1966). 
The fraction eluted with hexane:ether (7:3) from the same column was further 
chromatographed on a silica gel column. The compound 2 was eluted with 
hexane: chloroform (7.5:2.5). The 1H-NMR spectrum of 2 showed three tertiary methyl 
signals at o 0.68 (s), 1.00 (s) and 1.43 (s), and two secondary methyl signals at o 0.88 
( d, J = 7.11 Hz) and 0.90 ( d, J 6.60 Hz). A multiplet at o 3.50 and a distorted triplet 
at o 5.34 were characteristic signals for 38-hydroxy-5-ene sterols (Ikekawa et a/., 1966; 
60 Pakistan Journal of Marine Sciences, Vol.l (1), 1992 
Guyot et al., 1982). The signals at o 4.65 (s) and 4.70 (s) indicated the presence of a 
methylene group in side chain of sterol. The downfield methyl signal at o 1.43 and 
methylene signals at o 4.65 and 4.70 allowed the placement of a double bond between 
C-25 and C-26 in a 24-methyl cholesterol skeleton. 
HO 
The ElMS of 2 exhibited a molecular ion peak at m/z 398 corresponding to the 
molecular formula CzsH460. The mass fragments at m/z 271, 255, 213, 145 and 119 
were characteristics of 115 sterols (lkekawa et al., 1966; Wyllie and Djerassi, 1968) 
which showed that the ring system of sterol was same as cholesterol. The other mass 
fragments at m/z 356 and 314 showed the cleavage of bonds between C-24- C-25, and 
C-22- C-23, respectively (Wyllie and Djerassi, 1968). These spectral results suggested 
that the structure of 2 was 24-methyl cholesta-5, 25-dien-3B-ol. 
The compound 3 was obtained from the column chromatography of acetone ex-
tract of D. indica. The FDMS and ElMS of 3 showed the molecular ion peak at m/z 
412 corresponding to the molecular formula C29H4sO. The mass fragments appeared 
in ElMS of m/z 314 and 271 showed the cleavage of the bonds between C-22- C-23, 
and C-17 - C-20, respectively (Wyllie and Djerassi, 1968). The remaining mass frag-
ments indicated that the sterol 3 has also same ring system as cholesterol. This fact 
was confirmed by the assignments of H-3 (o 3.51), H-6 (o5.34), H-18 (o0.68), H-19 (o 
1.00) and its 13C-NMR spectrum (see Table-1). 
The 1H-NMR spectrum of 3 indicated the presence of four secondary methyl sig-
nals. The downfield doublet at o1.56 (J =6.7 Hz) indicated that a methyl group is at-
tached to a double bond while the downfield quartet at o 5.14 allowed us to place the 
double bond between C-24 and C-28. These results indicate that 3 has same structure 
as reported for sargasterol. The 13C-NMR and hetero-Cosy (2D-NMR) spectra 
Ahmad et al: Sterols from Marine Algae 61 
showed the complete agreement with the structure elucidated by 1H and mass spectra. 
The compound 4 was eluted with hexane chloroform when the chloroform extract 
of L. obtusa was loaded on silica gel column. The EI mass spectrum of 4 exhibited the 
molecular ion peak at m/z 386 corresponding to the molecular formula C27H460. The 
other mass fragments observed at m/z 353, 273, 255, 213, 145 and 119 were same as 
cholesterol (Zaretskii et al., 1967). 
The identification of 4 as cholesterol was supported by the 1 H-NMR spectrum 
which showed two tertiary methyl signals, at o 0.67 (s) and 1.00 (s) for H-18 and H-19, 
respectively and three secondary methyl signals, appeared at o 0.91 (3H, d,J = 6.56 Hz) 
and 0.86 (6H, d,J=6.64 Hz) for H-21, H-26 and H-27, respectively. A multiplet at o 
3.51 (1H, W1h=15.20 Hz) and a distorted triplet appeared at o 5.35 were charac 
teristic signals of H-3 and H-6 in cholesterol (Ikekawa et al., 1966; Rubinstein et al., 
1976). 
HO 
The 13C-NMR (broad band) spectrum of 4 showed 27 carbon signals. The DEPT 
spectrum indicated the presence of five methyls, eleven methylene and eight methine 
carbon signals. The remaining three carbon signals, in broad band spectrum were due 
to the quaternary carbons. All the carbon signals, appeared in 13C-NMR spectrum, 
showed complete agreement with the structure of 4 as cholesterol. 
Compound 5 was obtained in pure form, through the silica gel column which was 
loaded by ehtyl acetate extract of C. iyengarii. The 13C-NMR spectrum of 5 showed the 
presence of twenty nine carbon signals. The DEPT spectrum showed five methyls, 
twelve methylene and eight methine signals, the remaining four signals in the broad 
band spectrum were due to quaternary carbon atoms. The 13C-NMR spectrum ex-
hibited two sets of olefinic carbon signals at o 147.6, 111.4 and 140.8, 121.7 due to a 
terminal double bond ( C = CH2) and trisubtituted double bond ( C = CH-) fun-
tionalities. The 13C- NMR showed the methyl group resonances at o 11.85, 12.06, 
17.82, 18.67 and 19.40.1t further showed the methine signal at o 11.80. Comparison of 
the 13C-NMR data with other sterols showed the presence of cholestane skeleton 
(Johnson and Jankowski, 1978). This was further supported by 1H-NMR spectrum 
62 Pakistan Journal of Marine Sciences, Vol.l (1), 1992 
Table-1: 13C-NMR data of compounds 1,3 and 5 
Carbon no. 1 3 5 
1 37.30 37.29 37.29 
2 31.71 31.70 31.61 
3 71.84 71.85 71.80 
4 42.35 42.34 42.26 
5 140.85 140.85 140.83 
6 121.75 121.76 121.74 
7 31.94 31.94 33.70 
8 31.95 31.95 31.93 
9 50.18 50.18 50.17 
10 36.53 36.54 36.52 
11 21.10 21.11 21.11 
12 39.80 39.80 39.82 
13 42.40 42.38 42.34 
14 56.80 56.80 56.80 
15 24.29 24.34 24.30 
16 28.23 28.26 28.18 
17 55.88 55.84 56.10 
18 11.87 11.86 11.85 
19 19.40 19.41 19.40 
20 36.17 36.45 35.35 
21 18.81 18.87 18.67 
22 29.13 25.74 33.70 
23 34.61 35.25 29.43 
24 77.73 147.80 49.55 
25 35.95 34.80 147.60 
26 16.47 22.14 17.82 
27 17.55 22.25 111.43 
28 142.65 115.62 26.53 
29 112.89 13.18 12.06 
Ahmad et al: Sterols from Marine Algae 63 
HO 
which showed the methylene group resonances at o 4.71 and 4.62 as broad singlets. A 
multiplet at o 3.50 and a distorted triplet at o 5.34 is the characteristic pattern of the 
1:::.5 - steroids (Findlay and Petil, 1985). The 1H-NMR spectrum further showed three 
tertiary methyl group signals at o 0.66, 0.99 and 1.55, a secondary methyl group 
resonance as doublet at o 0.89 (J:::: 6.6 Hz) and a primary methyl group signal as triplet 
at o 0.79. The mass spectrum of 5 displayed the molecular ion peak at m/z 412 and 
other fragment peaks at 394 (M-H20) + ,314 (M-part of side chain C7H14) + ,271 (M-
side chain + 2H) +. The presence of the common cholesterol nucleus was attested by 
the diagonostic ions at m/z 371, 314, 255 and 213. All these results suggest the struc-
ture of 5 as clerosterol. 
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Sterol composition of marine algae from Karachi coast of Arabian Sea

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Sterol composition of marine algae from Karachi coast of Arabian Sea

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