effect of plant monofunctional pentacyclic triterpenes on the dynamic and structural properties of...

Chemistry and Physics of Lipids

89 (1997) 119–130

Effect of plant monofunctional pentacyclic triterpenes on thedynamic and structural properties of

dipalmitoylphosphatidylcholine bilayers

Sonia Rodrıguez a, Horacio A. Garda b,*, Horacio Heinzen a, Patrick Moyna a

a Farmacognosia y Productos Naturales, Facultad de Quımica, Gral. Flores 2124, Monte6ideo, Uruguayb Instituto de In6estigaciones Bioquımicas de La Plata (INIBIOLP), Facultad de Ciencias Medicas, Calles 60 y 120,

1900-La Plata, Argentina

Received 27 February 1997; received in revised form 14 July 1997; accepted 13 August 1997

Abstract

Fluorescence lifetime and rotational behaviour of 1,6-diphenyl-1,3-5-hexatriene (DPH) and 1-(4-trimethylammoni-umphenyl)-6-phenyl-1,3,5 hexatriene (TMA-DPH) were used to study the influence of several monofunctionalpentacyclic triterpenes on the structural and dynamic properties of dipalmitoylphosphatidylcholine (DPPC) bilayers.The plant monoalcohols a-amiryn, lupeol and taraxerol, the semisynthetic (H-18-a)-a-amiryn, and the ketonetaraxerone were studied in comparison with cholesterol. Among them, only a-amiryn is incorporated at as high levelsas cholesterol into DPPC bilayers. At 50 mol%, it results in the phase transition vanishing. Pentacyclic monoalcoholsare more potent than cholesterol to decrease the DPPC gel phase order, but less potent than it to increase theliquid–crystalline state order. They also produce more packing defects than cholesterol in DPPC bilayers. Theseeffects correlate with the absence of planarity in the pentacyclic skeleton, which would obstruct the ‘all trans ’ packingof acyl chains in the gel state and Van der Waals interactions in the liquid–crystalline state. Taraxerone has littleeffect on DPPC bilayers, probably due to its difficulty to form hydrogen bonds. © 1997 Elsevier Science Ireland Ltd.

Keywords: Pentacyclic triterpenes; Dipalmitoylphosphatidylcholine bilayers; 1,6-Diphenyl-1,3-5-hexatriene; 1-(4-Trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene; Fluorescence lifetime; Differential polarized phase fluores-cence; Bilayer ordering

Abbre6iations: DPH, 1,6-diphenyl-1,3-5-hexatriene; TMA-DPH, 1-(4-trimethyl-ammoniumphenyl)-6-phenyl-1,3,5-hexatriene;DPPC, dipalmitoylphosphatidyl-choline; t, fluorescence lifetime; rS, steady-state fluorescence anisotropy; r�, limiting anisotropy;PTs, pentacyclic triterpenes; Tt, phase transition temperature; DT, temperature range of the phase transition.

* Corresponding author. Tel.: +54 21 834833; fax: 54 21 258988; e-mail: [email protected], [email protected]

0009-3084/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.

PII S 0009 -3084 (97 )00068 -6

S. Rodrıguez et al. / Chemistry and Physics of Lipids 89 (1997) 119–130120

Fig. 1. Structure and molecular dimensions of cholesterol, a-amiryn, lupeol, taraxerol and [H-18-a ]-a-amiryn. Alchemy® was usedto calculate the molecular dimensions.

1. Introduction

Pentacyclic triterpenes (PTs) have a basic skele-ton of 30 C atoms (Fig. 1). They are biosynthetic

derivatives of isoprene, via squalene, and they arefound in relatively high proportions in plantwaxes (Moyna et al., 1983). Several PTs are toxicto insects (Cesio et al., unpublished results), and

S. Rodrıguez et al. / Chemistry and Physics of Lipids 89 (1997) 119–130 121

their presence in waxes was correlated with someevolutive advantages for the plant.

Insects are unable to biosynthesize ‘de novo’the steroidal skeleton and thus they requiresteroids in their diet for their normal developmentand reproduction (Svoboda et al., 1982). This isthe only proved difference in the nutritional re-quirments between insects and mammals, and soit is an interesting base from which to search fornew natural pesticides (Svoboda, 1994; Svobodaand Feldlaufer, 1991). Insects require steroids tobiosynthesize hormones such as ecdysteroids, andit was proposed that the toxic action of some PTscan be due to enzymatic inhibition in this path-way (Svoboda, 1994; Smith, 1989). Like all ani-mals, insects also require steroids as fundamentalbiomembrane components. The possibility of abiological action of PTs on membranes could bebased on a certain character as ‘false steroid’.(Cesio et al., unpublished results).

The aim of this work was to compare theability of several PTs with that of cholesterol, fortheir incorporation into dipalmitoylphosphatidyl-choline (DPPC) liposomes and their influence onthe bilayer structure and dynamics. The PTs usedin this study were those on which measurementsof biological activity were made (Cesio et al.,unpublished results) and those which have a singlepolar group which allows the prediction of theirpositioning in the bilayer. They were the naturalmonoalcohols a-amiryn, lupeol and taraxerol, theketone taraxerone, and the semisynthetic isomerof a-amiryn, (H-18-a)-a-amiryn.

The influence of these PTs on the structure anddynamics of DPPC bilayers was studied by mea-suring the rotational behaviour of two probes: thehydrophobic 1,6-diphenyl-1,3-5-hexatriene(DPH), with a deep localization in the bilayer,and the amphipathic 1-(4-trimethyl-ammoni-umphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), that anchors to the bilayer hydrophilicinterface through its charged group locating itsfluorescent moiety more outwardly than DPH(Prendergast et al., 1981). The temperature depen-dence of the steady-state fluorescence anisotropy(rS) of these probes was used to study the influ-ence of the PTs on the DPPC phase transition.Lifetime (t) was measured by phase and modula-

tion fluorometry (Weber, 1981) and used as anindication of the extent of water penetration inthe bilayer. Differential polarized phase fluores-cence (Lakowicz et al., 1979; Lakowicz, 1983) wasused to calculate the limiting anisotropy (r�) inorder to obtain information on the acyl chainordering.

2. Materials and methods

2.1. Materials

Synthethic DPPC (99% pure) was obtainedfrom Sigma, DPH from Aldrich, and TMA-DPHfrom Molecular Probes. Pure cholesterol, m.p.148.5°C (CRC. Handbook of Chemistry andPhysics, 61st ed. CRC Press, Boca Raton, FL,1980), was obtained by two succesive recrystalliza-tions of commercial cholesterol (Sigma).

2.2. Natural pentacyclic triterpenes

a-Amiryn was obtained after ethanol recrystal-lization from a mixture of benzoates of a and b

amiryns isolated from Manila elemi resin. Afterbasic hydrolysis, a-amiryn was characterized spec-troscopically (IR, NMR, MS) according to theliterature. Lupeol and taraxerol were isolatedfrom Colletia paradoxa Spr. wax as previouslyreported (Moyna et al., 1983). Taraxerone, m.p.245°C (Heinzen, 1993), was isolated from the waxusing a silica gel 40–100 mesh (J.T. Baker) openchromatographic column eluting with a gradientof toluene/CHCl3.

2.3. Isomerization of a-amiryn

2.3.1. Allilyc oxidation of a-amirynN-bromosuccinimide (15 mg, 0.1 mM) was

added to a stirred solution of a-amiryn acetate(16 mg, 0.03 mM) in dioxane. The suspension wasirradiated at room temperature with a Tungstenlamp (60 W, Phillips) for 12 h. The reaction wasquenched by adding water (20 ml) and extractedwith ethyl ether (3×10 ml). The organic layerswere washed with brine and dried over sodiumsulfate. Flash chromatography (petrol ether:di-


chloromethane, 2:8) of the crude reaction mixtureseparated unreacted a-amiryn acetate (7 mg), 11-hydroxi-a-amiryn acetate and 11-oxo-a-amiryn(3.5 mg). Jones oxidation of 11-hydroxi-a-amirynacetate gave 11-oxo-a-amiryn (4.5 mg).

2.3.2. (H-18-a)-11-oxo-a-amirynChlorotrimethylsilane (0.5 ml, 0.4 mM) was

injected under N2 to a stirred solution of 11-oxo-a-amiryn (8 mg, 0.02 mM) in triethylamine (7 ml).The solution was then refluxed for 16 h. The cooledreaction mixture was poured into a mixture of 3 lice and HCl 10% (40 ml), stirred for 10 min andextracted with diethylether. After working up, thecrude reaction mixture (6.4 mg) was 95% (H-18-a)-11-oxo-a-amiryn (controlled by capillary GC).

2.3.3. (H-18-a)-a-amirynA solution of (H-18-a)-11-oxo-a-amiryn (6 mg)

and hydrazine monohydrate (1 ml) in diethylengly-col (2 ml) and butanol (1 ml) was heated underreflux for 1 h. After cooling to 100°C, KOH (150mg) was added and the mixture slowly heatedwithout condenser to 200°C and maintained at215°C for 4 h with a condenser. After the usualworking up and flash chromatography of the crudereaction mixture, (H-18-a)-a-amiryn was crystal-lized (EtOH), characterized by spectroscopic meth-ods and differentiated from a-amiryn by GC.

2.4. Calculation of the triterpene:DPPC ratio

The final concentration of PT in liposomes wasmeasured by TLC scanning (Martınez and Moyna,1990). Half of the liposomal suspension was freezedried and the residue dissolved in 1 ml of CHCl3.Duplicates of the samples were exactly spotted ona 20×20 TLC plate. Five different concentrationsof a solution 1 mg/ml DPPC and five of a solutionof the PT to be quantified, were spotted. The platewas bi-dimensionally and monodirectionaly devel-oped with CHCl3-MeOH-NH4OH 2% (70:30:5) to8 cm (for DPPC), and then developed with CHCl3to 18 cm (for cholesterol and monotriterpenols).The plates were cut off at the site of the first solventrun end. DPPC was visualized using ammoniummolibdate in perchloric acid, and either PT orcholesterol were visualized with H3PO4–CuSO4

reagent (Rodrıguez et al., submitted). Each set ofspots was read in a densitometer (CS 9000, Shi-madzu, Tokio) at 454 nm, and the final concentra-tion calculated by means of linear regression. Thegiven data were obtained from regressions whosecorrelation coeficients were \0.9993.

2.5. Fluorescence measurements

Multilamellar liposomes (0.25 mM in DPPC)were prepared by mixing the correspondingamounts of DPPC and PT in chloroform. Thesolvent was evaporated, water was added andliposomes were formed by vortexing at 50°C. Forlabelling, samples were mixed with the same vol-ume of a suspension of 2.5 mM DPH or TMA-DPH in water, and incubated at 50°C for 30 min.Final concentration of DPPC in the samples was0.125 mM and the probe to DPPC molar ratio was1:100.

rS, t And differential polarized phase shift (D)were measured with a SLM 4800 C phase-modula-tion spectrofluorometer (SLM Instruments, Ur-bana, IL) according to Lakowicz et al. (Lakowiczet al., 1979; Lakowicz, 1983) with some modifica-tions (Tricerri et al., 1994; Garda et al., 1994a).Excitation wavelength was 361 nm, and the emit-ted light passed through a sharp cut-off filter(Schott KV 389). t Was measured with the excitinglight amplitude-modulated at 18 and 30 MHz by aDebye–Sears modulator. Glan–Thompson polar-izers oriented to 55° were used to eliminate effectsof Brownian motion (Spencer and Weber, 1970).The phase shift and demodulation of the emittedlight relative to a reference of known t weredetermined and used to compute the phase lifetime(tP) and the modulation lifetime (tM) of the sample(Lakowicz and Cherek, 1980). POPOP (1,4-bis(5-phenyloxazol-2-yl)benzene) in ethanol was used asreference, which has a t of 1.35 ns (Lakowicz,1983; Lakowicz et al., 1980). For all the samplesand both probes, differences between tP and tM

were within the experimental error and indepen-dent of the excitation frequency indicating a highdegree of homogeneity in the fluorophore popula-tion (Weber, 1981; Lakowicz, 1983). The reportedlifetimes are the average of tP and tM at bothexcitation frequencies.


The differential polarized phase shift (D) wasdetermined according to Lakowicz (Lakowicz etal., 1979; Lakowicz, 1983) by exciting with lightmodulated at 18 and 30 MHz and vertically po-larized, and by measuring the phase differencebetween the parallel and perpendicular compo-nents of the emitted light. The measured valuesof rS, t, and D, and the fundamental anisotropy(rO) which had been previously estimated in0.390 (Garda et al., 1994b), were used to calcu-late the rotational rate and r� as previously de-scribed (Tricerri et al., 1994; Garda et al.,1994a) in accordance with the theory (Weber,1978). r� Values at both excitation frequencieswere similar within the experimental error andthe average values are reported.

3. Results

3.1. Incorporation of pentacyclic triterpenes inDPPC multilamellar liposomes

Table 1 shows that a-amiryn is incorporatedat levels as high as those of cholesterol (50mol%), but lupeol and taraxerol are incorpo-rated in relatively low levels, 12 and 5 mol%,respectively.

3.2. Effect of pentacyclic triterpenes on the gel toliquid crystalline phase transition of DPPCmultilamellar liposomes

The influence of PTs on the temperature de-pendence of rS for DPH and TMA-DPH inDPPC liposomes, is shown in Figs. 2 and 3.The middle point (Tt) and the temperaturerange (DT) of the phase transition were ob-

tained from the first derivative of these plotsand they are summarized in Table 2. The gel toliquid crystalline phase transition of DPPC lipo-somes at about 41°C abruptly decreases the rS

of both probes. Similar rS values are observedfor DPH and TMA-DPH in the gel state. In theliquid crystalline state, however, the rS valuesfor TMA-DPH are higher than those for DPH.This fact is due to both, lower mobility andshorter t of the interface anchored TMA-DPHprobe in comparison with DPH (Prendergast etal., 1981).

Increasing amounts of cholesterol produce asmall decrease in the rS of DPH and TMA-DPH for the gel state, and a large increase inthis parameter for the liquid crystalline state(Fig. 2). At 12 mol%, cholesterol increases theTt, as it is detected by DPH, but not by TMA-DPH. At 50 mol%, it results in the disappear-ance of the transition and the formation of anintermediate state.

As compared to cholesterol, a-amiryn evokesa larger decrease in the rS of the gel state, and asmaller increase in the rS of the liquid–crys-talline state. It broadens the transition tempera-ture range indicating a loss of cooperativity. At50 mol%, like cholesterol, a-amiryn results in anintermediate state without phase transition, butwith lower rS values.

The effects of lupeol, taraxerol and taraxe-rone, tested at their maximal incorporationlevel, on the phase transition of DPPC lipo-somes are shown in Fig. 3. Lupeol at 12 mol%,like a-amiryn, produces a decrease larger thanthat of cholesterol in the rS of DPH and TMA-DPH for the gel state; but it produces only asmall increase in the rS for the liquid–crystallinestate. Lupeol decreases the Tt as it is detectedby both probes. Taraxerol and taraxerone at 5mol% do not appreciably affect the temperaturedependence of DPH rS. However, they decreasethe TMA-DPH rS for the gel state and the Ttas detected by this probe, being the effect morenoticeable for taraxerol. Like a-amiryn, thesemisynthetic [H-18-a ]-a-amiryn at 12 mol%broadens DT, decreases TMA-DPH rS for thegel state and increases it for the liquid–crys-talline state.

Table 1Incorporation levels of cholesterol and pentacyclic triterpenesinto DPPC multilamellar liposomes

Maximal level of incorporation (mol%)Compound

50Cholesterola-Amiryn 50

12LupeolTaraxerol 5


Tab

le2

Eff

ect

ofpe

ntac

yclic

trit

erpe

nes

onth

eph

ase

tran

siti

onof

DP

PC

mul

tila

mel

lar

lipos

omes

,an

don

the

lifet

ime

and

limit

ing

anis

otro

pyof

DP

Han

dT

MA

-DP

Hflu

ores

cenc

e

Lim

itin

gan

isot

ropy

bF

luor

esce

nce

lifet

ime

(ns)

aSa

mpl

eP

hase

tran

siti

onpa

ram

eter

s(°

C)

TM

A-D

PH

DP

HT

MA

-DP

HD

PH

DP

HT

MA

-DP

H

50°C

30°C

50°C

30°C

50°C

30°C

50°C

Tt

DT

Tt

DT

30°C

8.3

6.1

3.4

0.33

10.

045

50.

314

41.0

0.11

810

.8D

PP

C40

.95

8.5

7.1

4.6

0.32

00.

120

DP

PC

+12

mol

%ch

oles

tero

l0.

306

42.1

0.16

46

41.3

69.

510

.08.

27.

70.

309

0.27

70.

301

10.9

0.27

6D

PP

C+

50m

ol%

chol

este

rol

n.o.

n.o.

n.o.

n.o.

c

811

.09.

67.

54.

90.

287

0.11

70.

288

0.16

7D

PP

C+

12m

ol%

a-a

mir

yn43

.110

44.0

9.8

6.6

4.8

0.27

90.

161

0.24

110

.10.

193

DP

PC

+50

mol

%a

-am

iryn

n.o.

n.o.

n.o.

n.o.

8.1

6.6

3.5

0.30

70.

057

0.26

8D

PP

C+

12m

ol%

lupe

ol0.

130

38.5

1038

.96

10.3

7.9

5.4

3.1

0.34

00.

045

0.29

410

.60.

130

DP

PC

+5

mol

%ta

raxe

rol

840

.26

41.3

69.

47.

86.

53.

40.

331

0.05

00.

310

0.11

940

.4D

PP

C+

5m

ol%

tara

xero

ne6

38.9

n.d.

5.8

3.4

n.d.

n.d.

0.28

0n.

d.0.

140

9D

PP

C+

12m

ol%

[H-1

8-a

]-a

-am

iryn

n.d.

dn.

d.40

.7

aT

hest

anda

rder

ror

ofth

ese

mea

sure

men

tsw

asab

out

0.1

ns.

bT

hest

anda

rder

ror

ofth

ese

mea

sure

men

tsw

asab

out

0.00

5.c

The

phas

etr

ansi

tion

isno

tob

serv

able

.d

Not

dete

rmin

ed.


Fig. 2. Temperature dependence of the steady-state anisotropy of DPH (A) and TMA-DPH (B) in liposomes of: pure DPPC (�),DPPC +12 mol% cholesterol (�), DPPC +50 mol% cholesterol (�), DPPC +12 mol% a-amiryn (), DPPC +50 mol%a-amiryn ( ).

3.3. Effect of pentacyclic triterpenes on thefluorescence lifetime (t) of DPH and TMA-DPHin DPPC multilamellar liposomes

DPH derivative probes have a low quantumyield and a very short t when exposed to water.Thus, their t is indicative of the extention ofwater penetration into the bilayer. The influenceof cholesterol and PTs on the t of DPH andTMA-DPH in DPPC liposomes, below (30°C)and above (50°C) the phase transition tempera-ture, is shown in Table 2. Shorter t are found forTMA-DPH in comparison with DPH, as a resultof the higher water content and polarity of theexternal region of the bilayer compared to thedeep region. Shorter t are found at 50°C ratherthan at 30°C, indicating more water penetrationin the liquid–crystalline than in the gel state.

Increasing amounts of cholesterol progressivelyincrease t for the externally anchored TMA-DPH,both at 30 and 50°C. At 50°C, DPH t is alsoprogressively increased by 12 and 50 mol% choles-terol. At 30°C, however, DPH t is decreased by12 mol% but it is not changed by 50 mol%cholesterol. This indicates that 12 mol% choles-terol causes packing defects, which increase waterpenetration in the deep interior of the bilayer.

At 50°C, a-amiryn increases DPH and TMA-DPH t at 12 mol%, with no additional changes at50 mol%. At 30°C, DPH t is not changed at 12mol% but it is decreased at 50 mol%. At the sametemperature, TMA-DPH t is increased more ap-preciably by 12 mol% than by 50 mol% a-amiryn.

No change in t of DPH and TMA-DPH isproduced by 12 mol% lupeol at 50°C. At 30°C,


Fig. 3. Temperature dependence of the steady-state anisotropy of DPH (A) and TMA-DPH (B) in liposomes of: pure DPPC (�),DPPC +12 mol% lupeol (�), DPPC +5 mol% taraxerol (), DPPC +5 mol% taraxerone ("), DPPC +12 mol% [H-18-a ]-a-amiryn (�).

however, 12 mol% lupeol evokes a small de-crease in DPH t and a small increase in TMA-DPH t. On the contrary, taraxerol decreasesTMA-DPH t mainly at 30°C, without a signifi-cant effect on DPH t. Taraxerone at 5 mol%has a similar behaviour to lupeol, with a higherability to decrease DPH t at 30°C. [H-18-a-]-a-amiryn at 12 mol%, opposite to a-amiryn,evokes a small decrease in TMA-DPH t at30°C, without changing it at 50°C.

3.4. Effect of pentacyclic triterpenes on theordering of DPPC bilayers

No marked effect of PTs was found on therotational rate of DPH and TMA-DPH (notshown). On the other hand, the PTs have differ-

ent effects on the r� of these probes (Table 2).r� Is related to the hindrance of the wobblingdepolarizing rotation of the probes, and it de-pends on the lipid bilayer order (Kinosita et al.,1977). The r� values are higher at 30°C than at50°C; fact that shows the large decrease in orderoccurring in the gel to liquid–crystalline phasetransition. Comparable high r� values are ob-tained for DPH and TMA-DPH at 30°C, indi-cating that in the gel state, the lipid order issimilarly high in the deep and the external bi-layer regions. At 50°C, however, the r� valuesfor DPH are smaller than those for TMA-DPH.This indicates that in the liquid–crystalline state,DPH rotates in the deep region with more free-dom than TMA-DPH in the external region ofthe bilayer.


Increasing amounts of cholesterol slightly de-crease r� of DPH and TMA-DPH at 30°C, butthey largely increase r� at 50°C. A high orderedintermediate state, relatively temperature indepen-dent, is produced by 50 mol% cholesterol. At 12mol%, a-amiryn produces the same increase ascholesterol in r� of both probes at 50°C. At 30°C,however, 12 mol% a-amiryn decreases r� of bothprobes to an extension greater than that producedby 12 mol% cholesterol. At 50 mol%, a-amirynhas a higher ability than cholesterol to decreaser� of DPH and TMA-DPH at 30°C, but a lowerability than cholesterol to increase r� of theseprobes at 50°C. Thus, 50 mol% a-amiryn resultsin an intermediate state, relatively more disor-dered than that resulting from 50 mol% choles-terol. The ordering of the intermediate state at 50mol% a-amiryn in DPPC has a larger temperaturedependence than that at 50 mol% cholesterol,specially in the deep bilayer region probed byDPH.

Lupeol and taraxerol at 12 and 5 mol%, respec-tively, do not have any effect on the ordering ofthe liquid–crystalline state as sensed by bothprobes. In the gel state, 12 mol% lupeol decreasesr� of both probes, but 5 mol% taraxerol decreasesr� of TMA-DPH and slightly increases that ofDPH. Taraxerone at 5 mol% has no effect on thebilayer ordering at both phase states as sensed byboth probes. [H-18-a ]-a-amiryn at 12 mol% has asimilar ability as a-amiryn to decrease TMA-DPH r� in the gel state, but a lower ability thana-amiryn to increase r� of this probe in theliquid–crystalline state.

4. Discussion

Cholesterol is inserted in lipid bilayers with itsOH group towards the external environment andits hydrocarbon skeleton lies inside it. The 3-b-OH is hydrogen bonded to the carbonyl group ofsn-2 fatty acid (Finean, 1990). The monoalcoholicPTs are possibly orientated like cholesterol in thebilayer, with their 3-b-OH towards the exterior.a-Amiryn incorporates into DPPC liposomes atlevels as high as those of cholesterol, but lupeoland taraxerol are incorporated in lower propor-

tions indicating that they do not interact fa-vourably with the bilayer structure. Amphipathicmolecules as phospholipids that aggregate in lipidbilayers, are approximately cylindric with a simi-lar cross sectional area in the hydrophobic andhydrophilic regions (Eibl, 1984; Marsh, 1991).Cholesterol and a-amiryn present this approxi-mately cylindric geometry (Fig. 1), but lupeol andtaraxerol have geometric characteristics that canmake the bilayer structure unstable. In lupeol, theisopropenyl group attached to C-19a increases themolecular dimensions in the E ring. The arrange-ment of the D and E rings in taraxerol increasesthe cross sectional area of this region which inter-acts with the bilayer interior. These structuralfactors could explain the low incorporation levelsof lupeol and taraxerol in DPPC bilayers.

When lipid bilayers are in the liquid–crystallinestate, the rigid tetracyclic skeleton and the lateralchain in C-17b of cholesterol interact throughVan der Waals forces with the phospholipid acylchains, restricting the interconversion among dif-ferent conformers. Thus, cholesterol decreases theacyl chain mobility reducing the area required perphospholipid molecule and increasing the bilayerorder. This is referred to as ‘condensing effect’(Houslay and Stanley, 1983; Mouristen and Jor-gensen, 1994). In the gel phase, however, thetetracyclic skeleton of cholesterol precludes the‘all trans ’ packing of the phospholipid acyl chainsinducing a more disordered state. Due to theopposite effects of cholesterol on the gel andliquid–crystalline states, the phase transition be-comes undetectable at 50 mol% cholesterol. Asobserved by Raman spectroscopy the transitiontakes place, but the cooperativity is lost and thereis no associated enthalpy change (Baret, 1981).

Among the PTs tested here, a-amiryn is themost powerful modulator of the DPPC phasetransition. Like cholesterol, a-amiryn at 50 mol%results in the DPPC phase transition vanishing asdetected by DPH and TMA-DPH. However, itsstructural differences with cholesterol result in adifferent influence on the acyl chain order. Theabsence of lateral chain, the molecular dimensionsand the arrangement of the E ring of a-amiryn(Fig. 1) introduce empty spaces in the bilayer deepinterior, endowing the phospholipids acyl chains


with more conformational freedom. These factorscan account for the lower potency of a-amirynthan that of cholesterol in the condensing effecton the liquid–crystalline state. On the other hand,the lack of planarity of the rigid pentacyclic skele-ton, and the absence of the lateral chain in a-amiryn can be responsible for its higher potency,compared to cholesterol, to preclude the phospho-lipid acyl chain packing and to increase disorderin the gel state. This is evident mainly in theexternal region of the bilayer sensed by TMA-DPH. Lower t values of DPH and TMA-DPH inDPPC bilayer with 50 mol% a-amiryn comparedto with 50 mol% cholesterol indicate a highernumber of packing defects, allowing more waterpenetration in the lipid bilayer. Studies with an-drostan-3b-ol have shown the importance of thecholesterol lateral chain for its condensing effecton liquid–crystalline bilayers (Houslay and Stan-ley, 1983). The planarity of the cholesterol tetra-cyclic nucleous is also important for its ability tomodulate the bilayer lipid order. Coprostanol,which has a non-planar steroidal nucleous, in-creases the area per molecule in phospholipidmonolayers (Demel et al., 1972). Lanosterol, abiosynthetic precursor of cholesterol, has a disor-dering effect on phospholipid bilayers as shownby NMR, and this fact was attributed to thepresence of the C-14a methyl group (Bloch, 1983;Nes et al., 1982). This group lies perpendicular tothe plane determined for the axial hydrogenatoms and it distorts the planarity of the a face.

All the PTs studied are methylated on the a-face, but they have differences in the arrangementof their rings and in their planarity. In spite of itsC-14a methyl group, a-amiryn presents, the con-densing effect on the liquid–crystalline state, al-though in a lesser extension than cholesterol. Byanalizing its structure it can be observed that thearrangement of the E ring prevents the C-14a

methyl group from emerging out of the a-face.This methyl group lies on the plane determined bythe axial hydrogens in the a-face and the E ring.Then, although the a-face is not planar, it is acontinuous surface which would permit more Vander Waals interactions with the phospholipid acylchains; and this fact could explain its behaviourwhich is more similar to cholesterol than to lanos-

terol. Reports on cycloartenol (Bloch, 1983),which presents a condensing effect on lipid bilay-ers, would support this hypothesis. Cycloartenolis methylated in C-14a, but its conformationmakes the a-face continuous. The influence of theC-14a methyl group depending on the arrange-ment of the E ring, is confirmed by the resultsobtained with [H-18-a ]-a-amiryn. This compoundat 12 mol%, opposite to a-amiryn, has no con-densing effect on the DPPC liquid–crystallinestate. The only difference between both com-pounds is the arrangement of the E ring. Theabsence of bending in [H-18-a ]-a-amiryn deter-mines that the C-14a methyl group emerges out ofthe plane of the pentacyclic skeleton, which wouldinhibit van der Waals interactions with the phos-pholipid hydrocarbon chains.

The other monoalcoholic PTs, lupeol andtaraxerol, have no large effects on the liquid–crystalline state of DPPC. In lupeol, the C-14a

methyl and C-19a isopropenyl groups come out ofthe plane of the pentacyclic skeleton (Fig. 1),obstructing van der Waals interactions with acylchains. Taraxerol has a trans A/B/C structure,and a cis D/E union. The C ring, due to thepresence of the C13a methyl group, has a boatconformation. D ring is under torsion and E ringhas chair conformation. Thus, rings A, B and Care approximately in the same plane, but E ring isperpendicular to it. This fact determines, togetherwith the C-13a methyl group, a torsion in the a

face, which would inhibit van der Waals interac-tions with phospholipid acyl chains. Decreasedvan der Waals interactions would result not onlyin a decreased order but also in an increasedwater penetration in the bilayer in the liquidcrystalline state. This effect is particularly notori-ous in the external bilayer region sensed by TMA-DPH, whose t is increased by cholesterol and in alesser extension by a-amiryn, but it is not changedby lupeol and decreased by taraxerol.

The non planar and irregular structure of thepentacyclic skeleton of lupeol and taraxerol couldalso explain the increased disordering effect onthe gel state observed for these compounds incomparison with cholesterol. A non planar andirregular structure would be more effective than aplanar and regular one to preclude the ‘all trans ’


packing of the phospholipid acyl chains. In addi-tion, these compounds would produce more pack-ing defects than cholesterol and than a-amiryn inthe gel state, increasing water penetration in thelipid bilayer, as indicated by the shortening ofDPH and TMA-DPH t. The increased disorderand water penetration in the bilayer in the gelstate is mainly observed in the external regionsensed by TMA-DPH, owing to the lateral chainabsence which makes these molecules shorter thancholesterol.

Bio-assays with Machroziphum euphorbiae haveshown that lupeol and taraxerol, but not a-amiryn, are toxic to insects (Cesio et al., unpub-lished results). Moreover, lupeol and taraxerolinduce electrolytic leakage in Hordeum 6ulgare L.(sp.) (Heinzen and Moyna, 1993). The presentresults suggest that these effects of lupeol andtaraxerol, including their toxicity, could be due totheir ability to inhibit acyl chain packing in lipidbilayers.

Acknowledgements

Support from CONICYT (Uruguay) throughproject 122/94, CEE through project JR 1097 andIFS through grant F1080, is gratefully acknowl-edged. H.A. Garda thanks the Asociacion de Uni-versidades Grupo Montevideo for support. S.Rodrıguez thanks scholarships from PEDECIBAand CONICYT. H.A. Garda is a member of theCarrera del Investigador Cientıfico, CONICET(Argentina).

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