J.Natn.Sci.Foundalion Sri Lanka 2007 35 (4): 2S5-23H 

K I . S K A k C H A R T I C L K 

Studies on organic non-covalent binders of p-sitosterol 
P. R . W i j c m a n n e 1 , P. M . J a y a w e e r a 2 a n d E . R . J a n s z 3 * 

' Department of Biochemistry, Faculty of Medical Sciences, University of Sri Jayewardenepura. Gangodawila. Nugegoda. 
' Department of Chemistry. Facult}' of Applied Sciences. University of Sri Jayewardenepura. Gangodawila. Nugegoda. 

1 29/1, Pietersz Place, Nugegoda. 

Revised: 13 October 2006 ; Accepted: 23 October 2007 

Abstract : The p-sitosterol moiety was known to bind acyclic 
carotenoids phytoene and phytofluene. Recently it was 
reported that impurities of p-sitosterol from plant sources 
ruled out isolation from those sources. These studies showed 
that common p-sitosterol contained many impurities which 
can be separated by using a chromatotron. Studies also showed 
that P-sitosterol on binding to phytofluene quenched the latter's 
fluorescence by forming a complex with log = 5.0. This 
complex does not significantly alter the fine structure of the 
phytofluene IR spectrum indicating no significant distortion in 
the condensed phase. 

Keywords: p-sitosterol, binders, phytofluene, quenching, 
fluorescence. 

I N T R O D U C T I O N 

p-sitosterol is one o f the most abundant phytosterols in 
plants , which a long wi th campesterol accounts for about 
9 5 % o f total s teroids in p lan t s ' . In addi t ion, P-sitosterol 
serves as the c o m m o n ag lycone of flabelliferins, a g roup 
o f steroidal saponins found in pa lmyrah fruit pulp ( P F P ) 2 . 
A n interest ing feature o f all flabelliferins is that they are 
a lways natural ly associated with a fluorescent b inder ' . In 
pa lmyrah fruit pulp this is an acycl ic fluorescent carotene 
phytoene or phytof luene 4 . The impor tance o f this 
fluorescent impuri ty is that it can affect the bioactivi ty 
of flabelliferins in P F P J , whi le the fluorescent complex 
of pa lmyrah flour s h o w s neurotoxic activity in ra t s s . 

C « H K 

Exact Mass: 542.49 
M o l . Wt.: 542.92 
C, 88.49; H. 11.51 

Figure 1: Phytofluene 

A recent report stated that p-sitosterol was difficult to 
purify due to the presence of other steroids' ' . Ou r hypot­
hesis was that the impurit ies were not only other steroids 
but a lso c o m p o u n d s that bind to the steroid nucleus . 
Compu te r model l ing studies have shown that phy toene 
and phytofluene could bind with P-si tosterol 7 . The 
objectives of this study were to determine; (i) whether 
p-sitosterol from a reputed manufacturer in USA had 
binding compounds , (ii) whether the binders were 
phytofluene or phytoene, (iii) whether phytofluene could 
bind to P-sitosterol in vitro and if so (iv) the association 
constant of the binding. 

METHODS AND MATERIALS 

P-sitosterol was obtained from Sigma, St. Louis , USA. 

Isolation of Phytofluene: Palmyrah fruit was collected 
randomly on site from Kalpit iya, extracted manually, and 
mixed well to g ive pulp (PFP). P F P ( 5 0 g ) w a s ground with 
an adequate amount o f celite and acetone (100 m L x 3) 
and- filtered under suction until the P F P exhibited no 
discernable colour. The final acetoneextract of carotenoids 
(300 m L ) was extracted into hexane in a separatory funnel 
and residual ace tone was washed using distilled water. 
The hexane extract was concentra ted under vacuum 
using a rotary evaporator and flushed with nitrogen gas to 
r emove oxygen . Open Column Chromatography ( O C C ) 
was performed with a column of M g O : celite (1:1) matrix 
(activated by heating at 110 °C for 4 h) t ightly packed 
under reduced pressure into a height of 12 cm in a glass 
co lumn. Anhydrous N a , S 0 4 was added on top to a height 
o f I cm. After equil ibrat ing the column with hexane and 
under applied pressure the carotenoid extract was added 
on to the N a 2 S 0 4 layer with a pipette and left to absorb 
into it. The extract was eluted with hexane and fractions 
were collected into dark bottles*. 

' Corresponding author 



236 P. R. Wijemanne et al. 

Phytofluene is a colourless carotenoid and only the 
colourless fractions which eluted first (fractions 1 and 2) 
were collected and analyzed by the UV-Visible double 
beam spectrophotometer (Shimadzu, UV-1601). The 
spectrum of each fraction was analyzed for the charac­
teristic shape of phytofluene given in the literature and 
the three ^ wavelengths of phytofluene at 331 nm, 
348 nm and 367 nm. The fractions that satisfied the above 
criteria were pooled together and the amount of phytofl­
uene was calculated using the equation given below 8 . 

X<ug)= A . y ( m L ) * 106 

A l % , * 100 

Where; X - Amount of the carotenoid 

A - Absorbance of the fraction 

y - Volume of the fraction 

A , %

k m - Absorption coefficient of the 
carotenoid in the solvent used 

Reaction of Phytofluene with ^-sitosterol: A P-sitosterol 
solution with a concentration of 1 mg in 5 mL of methanol 
(MeOH) was prepared. The phytofluene solution of 
concentration 100 ug in I mL MeOH was prepared by 
dissolving the isolated phytofluene (284 L t g ) from PFP 
in 2.84 mL of MeOH (Phytofluene and (3-sitosterol were 
mixed and placed in a dark environment overnight). 

The mixture was concentrated by gently flushing 
with nitrogen and reducing the volume to less than 0.5 mL 
in an Eppendorf tube. Using this solution Thin Layer 
Chromatography (TLC) was performed. Volumes of 5.10, 
20,30 and 50 p.L were spotted on to TLC paper (Whatman 
No.l) . The plate was developed using the solvent system 
of butanol: ethanol: ammonia (7:3:4)''. After drying the 
TLC paper, presence of fluorescence was ascertained by 
illuminating with 280 nm UV light. The presence of 
P-sitosterol was determined by spraying with a freshly 
prepared anisaldehyde / H 2 S 0 4 solution and heating at 
105 °C for 10 min for the development of dark spots. 

Chromatotron (Harrison Research, California, Model 
7924T): A rotor plate of 2 mm thickness was prepared 
by mixing of G w PF,M silica (50 g) in distilled water 
(110 mL). The plate was activated at 110 °C for 30 min 
just before it was used for the chromatotron run. For the 
eluent a methanol: propan-l-ol (1:1) mixture was used 9. 
P-sitosterol (0.5 mg) dissolved in 1 mL of methanol was 
eluted at a flow rate of 6 mL/min (FMI Lab Pump, Fluid 
Metering Inc, Model QD serial # 44 L 37672R), and 30 
fractions of 2 mL each were collected. 

Analysis of fractions: Fluorescence was detected by 
illuminating the fractions with 280 nm UV light. The 
presence of P-sitosterol was detected by spotting fractions 
on chromatography paper (Whatman No. 1), spraying with 
a freshly prepared anisaldehyde reagent and heating at 
105 °C for 10 min, to ascertain the presence of p-sitosterol. 
Rotor plate of the chromatotron was also treated in the 
same manner to detect any residual P-silosterol. 

Reverse Phase High Performance Liquid Chromatograph 
(RP-HPLC): Fluorescent fractions were pooled, 
concentrated and 20 uL of each was injected into the HPLC 
(Waters). An isocratic solvent system of acetonitrile: 
methanol: tetrahydrofuran (58:35:7) was used as mobile 
phase while a C18 column (250 mm x 4.6 mm) was used 
as stationary phase. A flow rate of 1 mL/min (Waters 
HPLC pump, Model code 515, serial no. D 515423 M) 
was maintained and detected at 286 nm and 330 nm 
(Waters 2487 Dual X. Absorbance detector). A mixture of 
phytoene and phytofluene was used as the standard. 

Measurement of quenching by spectroflourimetry: 
Association constant for the reaction between phytofluene 
and p-sitosterol was determined by analyzing the 
quenching effect of P-sitosterol on the fluorescence 
of phytofluene. The solutions used for the test were, a 
solution of phytofluene (0.040 mg/mL) and a solution of 
P-sitosterol (0.025 mg/mL). The p-sitosterol used was 
those purified by separating the fluorescent impurity using 
the chromatotron. Fluorescence was determined by an 
Aminco-Bowman Series 2 Luminescence Spectrometer 
and analysed by AB2 software. The fluorescence of 
phytofluene (2.0 mL) was measured, exciting at 366 nm 
and scanning the emission spectrum, p-sitosterol was 
added to the phytofluene in the cell using a micropipette 
and emission spectrum was obtained each time. For the 
whole series of fluorescence measurement the exact 
parameters (i.e. excitation wavelength, excitation voltage 
and bandpass) observed for phytofluene alone was kept 
constant throughout. 

Calculation of the association constant using the 
Stem-Volmer Plot*0: The Stern-Volmer plot was used 
for calculating the association constant for the complex 
by plotting the concentration of p-sitosterol against the 
quantum yield ratio ( 0 ° , / <tf). Quantum yield ratio can be 
calculated using the equation, 

<D°f/<t>f= A 0 / A 

Where; O 0

f - quantum yield of phytofluene in the 
absence of p-sitosterol 

<t>(- quantum yield of phytofluene in the 
presence of p-sitosterol 

A 0 - peak area when no P-sitosterol is 
present in the phytofluene 

December 2007 Journal of the National Science Foundation of Sri Lanka 35 (4) 



Organic non-covalenl binders offl-sitosterol 237 

A - peak area at different P-sitosterol 
concentrations. 

The associat ion constant was calculated with the 
equat ion, 

0 > ° r / ( D = K [ Q l + 1 
f f ass L ^ J 

Where ; K - associat ion constant 
7 ass 

[Q] - concentration of the quencher (R-sitosterol) 

Infrared spectroscopic analysis of ft-sitosterol-
phytofluene complex: Solut ions of concentrat ion 0.025 
m g / m L in methanol were prepared using phytofluene 
and purified P-sitosterol. Equivalent vo lumes of the two 
solutions were mixed and kept overnight . Phytofluene 
solution (1 m L ) , p-sitosterol solution (1 mL) and a solution 
containing the mixture of phytofluene and P-sitosterol 
(1 m L ) were separately evaporated to dryness by flushing 
with nitrogen. The residue of each solution was mixed 
with KBr (Merck, art. 4097) in 1: 100 proport ions and 

pressed into a clear, thin KBr pellet in a steel pellet 
maker. Each pellet was scanned by a Fourier Trans­
formed Infrared (FT-IR) spectrometer. A T h e r m o Nicolet 
Avatar 320 FT-IR was used for scanning and E Z - O M -
N I C software was used for data process ing and analysis . 
KBr background scanning was performed beforehand to 
subtract any absorpt ion peaks due to KBr alone. 

Figure 1: Peaks given by impurities at 286 nm 

RESULTS 

Fluorescent compound of p-sitosterol standard 

P-sitosterol showed fluorescence on spott ing on 
chromatographic paper. The binders were separated 
by the chromatot ron and the binder was analyzed by 
R P - H P L C (Figure 1). 

The fluorescent compound gave peaks at 2 . 1 , 2 . 7 , 4 . 9 
retention t ime (RT) at 330 nm and peaks at 2.1 (4.4 % ) , 2.5 
(1.3 % ) , 2.7 (4 % ) , 3.2 (1.5 % ) , 3.4 (2.0 % ) , 3.8 (0.5 % ) . 
4.1 (1.1 % ) , 4.2 (0.6 % ) , 4.4 (1.1 % ) , 4.9 (0.9 % ) , 5.2 
(0.3 % ) , 6.2 (0.1 % ) , 17.2 (79.7 % ) , 23.4 (0.8 % ) RT. 
Phytoene and phytofluene s tandards showed peaks at 
42.1 and 42.5 respectively at 330 nm. 

Reaction of p-sitosterol with phytofluene 

P-sitosterol (80 ug) was reacted with phytofluene 
(37 ug) in 870 uL. The spot containing fluorescence 
and anisaldehyde posit ive spot were same on the T L C 
showing that the complex was formed. It was noted that 
the spots of phytofluene was much larger than P-sitosterol 
due to higher sensitivity of fluorescence. 

sitosterol added 

Fluorescence 
Intensity ( I r ) 

Wavelength (nm) 

Figure 2: Fluorescence quenching of phytofluene by P-sitosterol 

Journal of the National Science Foundation of Sri Lanka 35 (4) December 2007 



238 P. R. Wijemanne el al. 

Quenching studies 

Addition of p-sitosterol to phytofluene showed gradual 
reduction of fluorescence emitted by phytofluene 
(Figure 2). For the Stern-Volmer plot, peak area of 
fluorescence was chosen. From the plotted graph an 
equation of y = 108674x + 1.0059 was given with a line­
ar regression of R2 = 0.9817. From the slope of the graph 
K = log 1.1 x 10 s, hence log K = 5.0. 

ass ° ' 1 0 ass 

FT-IR studies 

The infrared spectroscopic differences between the 
complex and phytofluene are minimal compared to those 
between complex and P-sitosterol. The characteristics 
of P-sitosterol are notable at peaks of 1460 cm 1 , 1380 
cm 1 , 1050 cm -' and 960 cm'. Phytofluene showed peaks 
at 1750 cm 1 , 1460 cm"', 1380 cm"1 and 1160 cm 1 , while 
the complex showed significant peaks at 1750 cm 1 , 
1460 cm', 1380 cm"' and 1160 cm'. 

DISCUSSION 

The peaks at 330 nm by the fluorescent compound (2.1, 
2.7, and 4.9) showed no correlation with the retention 
times of either phytoene (42.1) or phytofluene (42.5). 
Hence phytoene and phytofluene are not the compounds 
responsible for fluorescence. Furthermore, at 286 nm the 
fluorescent compound yielded a wide range of peaks, 
which leads to conclude that the impurity may not be 
just one compound but an array of compounds. However 
as it is possible to separate impurities from p-sitosterol 
by using a chromatotron as described, there is no need 
to go to lengths of developing a synthetic method for 
P-sitosterol production6. 

Quenching studies confirmed the binding of 
phytofluene to P-sitosterol took place with a logarithmic 
association constant of 5.0. The FT-IR spectra of 
p-sitosterol and the complex showed slight variations at 
1050 cm"' and 960 cm"' indicating that there was some 
distortion of P-sitosterol on binding but the distortion is 
not significant. 

It is interesting to note that flabelliferin-II (F-II), a 
tetraglycoside of P-sitosterol did not bind with phytofluene 
in vitro" and the binding of F-II to phytofluene was not 
favourable by computer modelling studies". Yet the 
F-II- phytofluene complex is found naturally in palmyrah 
fruit pulp2. It had been speculated" that P-sitosterol binds 

phytofluene first and thereafter the rest of the ca rbohydra te 
moiety is synthesized. The binding of P-sitosterol moie ty 
has significance as these affect antimicrobial and AT Pase 
inhibition activity of flabelliferins3 and has also been 
suggested to be the basis of the pro-toxin producing the 
neurotoxic effect of palmyrah flour45. 

Acknowledgement 

The authors thank IPICS SRI: 07 for funds. 

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December 2007 Journal of the National Science Foundation of Sri Lanka 35 (4)