in vitro evaluation of surfactants with eucalyptus

8/14/2019 In Vitro Evaluation of Surfactants With Eucalyptus

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In vitro evaluation of surfactants with eucalyptus oil for

respiratory distress syndrome

R. Banerjee a,b,*, Jayesh R. Bellare c

a School of Medicine, Cardio6ascular Research Institute, Uni6ersity of California, 3333 California Street, Suite 150, San Francisc

CA 94118-1245, USAb School of Biomedical Engineering, Indian Institute of Technology, Bombay, India

c Department of Chemical Engineering, Indian Institute of Technology, Bombay, India

Abstract

The effects of low doses of eucalyptus oil (EO) were studied on the surface properties of phospholipid suspension

as exogenous surfactants, by in vitro analysis using a pulsating bubble surfactometer and a Wilhelmy balanc

Survanta, ALE C and Exosurf, commonly used surfactants in t herapy of respiratory d istress syndrome were used

controls for comparison. The test surfactants, in Ringers lactate at 1%, were pulsated at 40 cpm in the surfactomete

EO caused a significant improvement of adsorption of the surfactants. In the case of the binary mixture

dipalmitoylphosphatidylcholine and phosphatidylethanolamine (2:3), EO significantly improved the adsorptio

stability and minimum surface tension obtained. This combination performed better than ALEC and Exosurf and w

comparable to Survanta with respect to minimum surface tension attained. The re-spreading of a surface excess filof this mixture in a Wilhelmy balance was higher than that of ALEC and Exosurf. The ultrastructure of the E

enriched surfactants using cryogenic scanning electron microscopy showed easy facturability and formation of ope

membranous structures, which could have been associated with the improved surface activity.

Keywords: Mechanics of breathing; Alveolar surfactant; Methods; Wilhelmy balance; Surfactometer; Pharmacological agen

Eucalyptus oil; Surfactant; Phospholipid suspensions; Eucalyptus oil

1. Introduction

Pulmonary surfactant is a complex mixture of avariety of saturated and unsaturated phospho-

lipids, neutral lipids and surfactant specific

proteins SP-A, B, C and D (Wilkinson, 1993)

which lower the surface tension at the airalv

olar interface of the lungs. R espiratory distre

syndrome of neonates is most often due to developmental deficiency of pulmonary surfactan

along with an immature structure of the lun

(Avery, 1959). The predominant phospholipid

lung surfactant is dipalmitoyl phosphatidy

choline. It is capable of achieving near zero su

face tension during monolayer compression bu


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142

cannot function alone as an effective replace-

ment surfactant due to its poor adsorption and

re-spreading. Most surfactants available com-

mercially vary widely in their composition and

biophysical properties. Protein-free surfactants

have a relative disadvantage of poor re-spread-

ing when compared to their natural counter-

parts. The surfactant specific proteins SP-A, Band C are responsible for the rapid adsorption

and re-spreading of natural lung surfactants

(Creuwels et al., 1997). Ho wever, immunogenic

and cost considerations make the search of an

effective and affordable artificial protein-free

surfactant worthwhile in spite of their relative

lower effica cy. I n E xo su rf, t he a dd it io n o f

hexadecanol helps to partially overcome this

disadvantage o f protein-free surfactants by im-

proving the adsorption and re-spreading of the

base phospholipid namely dipalmitoyl phos-phatidylcholine (Yu and Possmayer, 1993). Cer-

t ain o th er n at ur al o ils a nd a lco ho ls co uld

perhaps be used instead of hexadecanol as re-

spreading agents and are the subject of the

present study.

One such natural oil is eucalyptus oil (EO)

which is commonly used as an expectorant in

chronic bronchitis (Gennaro, 1985). The efficacy

of EO in chronic bronchitis was documented by

Siurin (1997) with beneficial effects in a study of

150 patients with chronic bronchitis. Ho wever,

very few studies have described the performance

of this oil as a possible surfactant in respiratory

distress syndrome. Zanker et al. (1980) had eval-

uated the surfactant like effects of commonly

used remedies for colds including EO. They

found a decrease in surface tension at an air

water interface and improved lung compliance

in in vivo animal studies with EO. The present

study evaluates the in vitro performance of EO

as a component of phospholipid based surfac-tants.

The aims of the present study are to compare

the surface properties of different phospholipid

solutions with or without the addition of EO

and to approach the clinical issue of obtaining a

protein-free effective surfactant.

2. Materials and methods

1,2 dipalmitoyl phosphatidylcholine (DPPC

abbreviated here as PC), palmitoyloleoyl pho

phatidylethanolamine (PE) and palmitoyloleo

phosphatidylglycerol (PG) were purchased (a

\99% purity) from the Sigma Chemical C

Calcium chloride was obtained as \98% puri

from Qualigens Fine Chemicals-Glaxo, IndiSterile Ringers lactate solution was obtaine

from Albert David Ltd., India. Exosurf N eon

tal, Survanta and ALEC were obtained as kin

gifts from Burroughs Wellcome Co., Ross Labo

ratories, and Britannia Pharmaceuticals, UK, r

spectively. Technical grade EO, having specifi

gravity 0.91590.005 at 25C, was purchase

from Gopaldas Visram and Co., India. Steri

distilled water and sterile normal saline we

used, as supplied, for reconstitution of Exosu

and ALEC. Other chemicals used in t he experments were HPLC grade chloroform and ac

tone (Qualigens Fine Chemicals-Glaxo, India

Chloroform was used as the spreading solvent.

We studied the effects of EO at low doses (

part oil to 9 parts of phospholipid v/v), on th

surface properties of phospholipid monolaye

by in vitro analysis using a pulsating bubb

surfactometer. This instrument tests the dynam

surface properties of surfactants at a spheric

interface and can be pulsated at high speed

This would be relevant to the lung surfacta

system as pulsation at a high speed of 40 cp

would correspond to the respiratory rate o

neonates (Schubring et al., 1976; Yu and Pos

mayer, 1993) and also to the frequency of vent

lator settings used for neonates (Koyabashi

al., 1990). The minimum surface tension, stat

surface tension at a bubble of maximum size

as a measure of the adsorption to an air liqu

interface, and stability index of liposomal su

pensions of the main phospholipids of pu

monary surfactant and their binary mixed film

in the ratio of 2 parts of PC with 3 parts of Por PG, forming the mixtures PCPE and PCPG

respectively, are studied both in the absence an

in the presence of eucalyptus. The surfactants i

t he p resen ce o f E O a re r efer red t o P CE O

P EE O, P GEO, P CP EE O, a nd P CP GEO. E

was added in the ratio of, 1 part of E


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(2 ml) to 9 parts of the phospholipid (single/

mixed). The performance of the test formulations

with EO was compared with that of three differ-

ent commercial surfactants Survanta, ALEC

and Exosurf. This was supplemented by the study

of the performance of E O enriched surfactants on

the re-spreading of phospholipid monolayers us-

ing a Wilhelmy balance. The ultrastructure of

liposomal suspensions of the surfactant formula-

tions with eucalyptus were also studied in an

attempt to analyze the structural changes, if any,

which could explain the effects of EO.

2.1. Pulsating bubble surfactometer experiments

All the test surfactant formulations were stud-

ied in the form of phospholipid suspensions in

Ringers lactate (a polyelectrolyte solution con-

taining 2 mM calcium and buffered to a pH of

7.4). Ringers lactate was used due to its similarity

to the chemical composition of the alveolar fluid

as studied by Nielson (1986). Thin films of 20 ml

of single or mixed phospholipid solution in chlo-

roform (concentration 50 mg/ml) were formed

and the solvent was allowed to evaporate under

nitrogen gas for half an hour. EO was added in

the ratio of, 1 part of EO (2 ml) to 9 parts of the

pho spholipid (single/mixed). The final concentra-

tion of the phospholipid-oil mixture in the liposo-

mal suspension was 1%. The thin film, after

evaporation of the solvent, was dispersed in 100ml of Ringers lactate, shaken by hand and then

left undisturbed at room temperature for 12 h to

allow formation of liposomes by hydration (Yu

and Possmayer, 1993). The therapeutic surfac-

tants were tested after reconstitution in the form

recommended for clinical use in a concentration

of 1 mg phospholipid per ml. Control experiments

were performed using EO suspended in Ringers

lactate in a concentration of 1%.

All samples were tested at 37C in a pulsating

bubble surfactometer (Surfactometer, E lectronet-ics Corp.). This instrument and its use in dynamic

surface pro perty evaluation were first described by

Enhorning (1977). Briefly, a disposable plastic

chamber is loaded with 25 ml of the test sample.

The capillary of t his chamber serves as an airway

for bubble formation. A thermoregulated water

bath surrounds the chamber. The bubble size ca

be changed by manual control from minimum siz

having radius Rmin=400 mm (minimum surfac

a rea Amin=201.1104 mm2) to maximum si

having radius Rmax=550 mm (maximum surfac

area Amax=380.3104 mm2). The bubble can b

pulsated between these two sizes at frequenci

upto 100 cpm. There is a 47% reduction in arewhen the bubble changes size from Rmax

to Rmi

The pressure gradient across the bubble (DP)

measured with the help of a precision transduc

having a pressure range of 010 Torr and sens

tive to very low volume displacements (0.04 ml/c

water). The surface tension at the air liquid inte

face (g) is then calculated at minimum and max

mum bubble size of Rmin and Rmax, respectivel

in accordance with the law of Laplace as applie

to spheres (DPmin=2gmin/Rmin an d DPmax=2gma

Rmax). The bubble was cycled in automatic modbetween Rmin a n d Rmax at 40 cpm for 10 cycle

and the minimum and maximum surface tensio

were measured as surface tension at Rmin an

Rmax, respectively, during pulsation at 40 cpm

Cycling was stopped at Rmin and the bubble wa

expanded manually to Rmax

and held static for 1

sec. The static surface tension was measured. Th

gives an indication of the adsorption of the su

factant at the air liquid interface of the bubble.

correction factor was applied to the static surfac

tension at Rmax by multiplication with the facto0.055/0.040 (equal to the maximum radius/min

mum radius in accordance with Putz et al. (1994

This was done to compensate for t he error arisin

due to the assumption by the inbuilt program o

the pulsating bubble surfactometer of a minimu

radius during start of static measurements b

default. Stability index (SI) was calculated

SI=2(gmaxgmin)/(gmax+gmin) at 40 cp

(Clements et al., 1961). 40 cpm was chosen as th

frequency of oscillation as it corresponds to th

neonatal respiratory rate (Yu and Possmaye1993) and is the frequency which is used clinical

in ventilator settings for a neonate of respirator

distress syndrome (Koyabashi et al., 1990).

Statistical an alysis of the effects of the additiv

wa s d o n e b y t h e M a n n Whitney U test usin

PB0.05 as the cut off level for significance.


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2.2. Wilhelmy balance experiments

Dynamic surface pressure-area (p-A) isotherms

of solvent spread interfacial films were measured

with a Wilhelmy surface balance (LB 5000, KSV

Instruments, Finland). Highly pure triply de-ion-

ized water (Milli-Q UV Plus System, Millipore

Corp.) and acetone were used in all balance clean-

ing procedures. F or subphase formation, sterile

Ringers lactate solution, which contains in mEq/

L N a+ 131, K+ 5, Ca++ 4, bicarbonate as lactate

29 and Cl 111, was used after adjusting to a pH

of 7.4 by adding concentrated NaOH drop-wise.

Surfactants were dissolved in chloroform to a

concentration of 1 mg/ml and the solution was

spread drop-wise from a syringe at the air water

interface under surface excess conditions. This

refers to a high initial surface concentration where

the amount of surfactant spread was greater than

that required for a monolayer coverage (15 A, 2/

molecule). For studying the effect of EO, 1 part of

EO (Gopaldas Visram & Co., In dia) was added t o

9 parts of the chloroform solution of the phos-

pholipid before spreading. All experiments were

carried out at a body temperature of 3790.5C.

A time span of 10 min was allowed for solvent

evaporation before commencing dynamic cycling.

Surface pressure (the difference in the surface

tension of the interface without and with surfac-

tant) was monitored continuously from the force

on a sand-blasted platinum slide. Seven successivecycles of compression/expansion at the interface

were recorded between maximum and minimum

areas of 772.5 and 193.1 cm2 (compression ratio

4:1) at a constant speed of 1.2 cpm. The compres-

sion ratio of 4:1 is much higher than that found in

normal respiratory cycles. However, this ratio was

used in the present study in order to obtain

information regarding the re-spreading capability

of the surfactants. This was done by comparison

of collapse-plateau ratios, which could be ob-

tained only on maximum compression of the filmspast collapse. The speed of 1.2 cpm was the

highest possible by the Wilhelmy balance used.

D ynamic re-spreading was characterized b y col-

lapse plateau ratio criteria of Turcotte et al.

(1977). This parameter cannot be easily judged by

a pulsating bubble surfactometer. Hence, experi-

ments were conducted on a Wilhelmy balance t

study the re-spreading of PCPEEO in compariso

with the commercial surfactants. On a given p-

compression curve, a vertical line is drawn at th

point of steepest slope and a horizontal line

drawn at the post-collapse region. The distan

from the intersection of these lines to the end o

the compression stroke is defined as the collap

plateau length. The re-spreading ratio is given b

the ratio of the lengths of these plateaus for th

seventh and first cycles. A ratio of 1 indicat

complete re-spreading and of 0 indicates no r

spreading. Fig. 1 schematically represents th

method of calculation of this ratio.

2.3. Cryogenic scanning electron microscopy

experiments

All samples were tested as liposomal suspe

sions of 1% concentration as used for the pulsaing bubble surfactometer study. The techniqu

used for sample preparation for cryogenic scan

ning electron microscopy was developed b

Bhadriraju and Bellare (1993). In this cryo-tec

nique, a thin layer of the sample was sandwiche

between two copper meshes that were fixed o

two copper plates. The copper plates used were o

0.1 mm commercial grade copper and the meshe

were squares of:33 mm2. One of the coppe

plates was L-shaped to facilitate fracture of th

sample inside the airlock chamber of the micro

scope. The sandwiched sample was then plunge

rapidly into liquid nitrogen kept at 196C

Samples were kept in liquid nitrogen for 30 mi

and then transferred onto the cryo-stage of th

scanning electron microscope (JEOL 6400 SEM

The samples were then stabilized in the airloc

chamber fracture-stage and then freeze fracture

by removing the L-shaped copper plate with th

microscopes fracture k nife. The fractured samp

together with cryo-stage was then transferred t

the specimen chamber of the microscope. Bo

the fracture-stage in the airlock chamber and thimaging stage in the specimen chamber of th

microscope were maintained at 130C . T h

sample was imaged at an accelerating voltage of

kV and a scanning probe current of 109 A

working distance of 2732 mm and magnific

tions between 1000 and 4000 .


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Fig. 1. Calculation of re-spreading. A schematic representation of isotherms of surface excess films compressed post-collapse in

Wilhelmy balance. The re-spreading ratio is obtained by dividing t he length of the collapse plateau of the seventh cycle to that

first cycle.

Fig. 2. Minimum surface tension on addition of EO. The minimum surface tension on pulsation at 40 cpm in a pulsating bubb

surfactometer is compared for surfactants with and without addition of EO. A lower value of minimum surface tension

physiologically beneficial. All values are means with standard errors with n=5 8.


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146

Fig. 3. Effect of EO on stability of surfactants. The stability index of the various surfactants in the presence and absence of EO

depicted here. A larger value of the parameter indicates that the film is more stable and capable of maintaining a low tension witho

collapse, for a longer period of time. All values are means with standard errors with n=58.

3. Results

Fig. 2 shows the minimum surface tension at 40

cpm for the test formulations, without and with

EO. The stability indices are compared in Fig. 3

and the values of static surface tension on adsorp-

tion are compared in Fig. 4. EO in buffer, withoutany phospholipids, adsorbed to a surface tension

of 3592 mN /m and on compression reached a

minimum tension of 3091 mN /m.

The attainment of low static surface tension

values indicates the formation of a monolayer film

of the surfactant at the interface and gives a

measure of the adsorption of the compound. The

addition of EO caused a significant improvement

in the adsorption at Rmax of all the surfactant

formulations i.e. caused significant decrease in the

value of static surface tension attained (PB0.05using Mann Whitney U test, n=8). Ho wever,

the addition of EO was detrimental to the stabil-

ity of most surfactants except PG and PCPE.

An improvement in stability and minimum sur-

face tension were also obtained in the case of

PCPEEO. Table 1 summarizes the beneficial ef-

fects of EO on the surface properties of the su

factant formulations.

On comparing the surfactant formulations wi

eucalyptus, and the commercial surfactants, it w

observed that PCPEEO performed particular

well with respect to surface tension reduction wit

significantly lower gmin values when compared tSurvanta. Its adsorption and stability were als

comparable to that of Survanta and it was f

superior to ALEC and Exosurf with respect to a

the parameters considered. We could not test th

r e-sp rea din g o f Su rva nt a d ue t o t ech nic

difficu lt ies in spr ea din g of t he a queou

suspension.

The ability of the surfactants to re-spread to th

air liquid interface on dynamic cycling is also

physiologically important parameter. While com

paring PCPEEO with that of the therapeutic sufactants, only ALEC and Exosurf were used fo

comparison, as we were unable to spread th

aqueous suspension of Survanta as a chloroform

solution at the air liquid interface of the L

t ro ugh . F ig. 5 d ep ict s t he p er fo rm an ce

PCPEEO with respect to minimum surface te


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1

sion, stability index, adsorption and re-spreading

in comparison with ALEC, Exosurf and Sur-

vanta. PCPEEO had a high re-spreading ratio

of 0.7290.03 which was higher than that of

Exosurf and ALEC (0.1890.02 and 0.6590.03,

respectively).

We also compared the ultrastructure of the

surfactants in the absence and presence of euca-

lyptus in a cryogenic scanning electron micro-

scope. The structure of EO alone did not sho

any membrane formation and had a homog

nous stippled appearance of oil micro-drople

in buffer. The surfactants with eucalyptu

formed liposomes, which were easily fracture

to reveal their internal membranous organiz

tion. Wavy membranous folding was more com

monly formed in the case of liposomes co

taining EO.

Fig. 4. Effect of EO addition on adsorption. The static surface tension at Rmax is depicted as a measure of adsorption to t

air liquid interface. A lower value of this parameter indicates more efficient adsorption with more surface-active material reachin

the interface. All values are means with standard errors with n=5 8.

Table 1

Beneficial effects of EO on surface properties-PBS studya

Minimum surface tension (%) Stability index (%) Static surface tension: adsorption at Rmax

(%)Compound

PCEO 22b 50b 37c

PEEO d 37b 14c

33c18cPG EO 43c

PCPEEO 79c 149c 45c

41c 30cPCPG EO 27c

a All comparisons have been made between surfactant in the presence of EO vs surfactant without any additive. The percentag

of change have been calculated.b Indicates a detrimental effect.c Indicates a significant improvement (MannWhitney U test PB0.05).d Indicates absence of any significant change.


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Fig. 5. Comparison of surface properties of PCPEEO and

commercial surfactants. The performance of PCPEEO is com-

pared with that of ALEC, Exosurf and Survanta. The mini-

mum surface tension is measured after pulsation at 40 cpm for

30 sec in a pulsating bubble surfactometer. The static surface

tension is measured at Rmax in a pulsating bubble surfactome-ter as a measure of adsorption. The re-spreading ratio is

measured by comparison of the collapse plateau ratios in

surface excess films in a Wilhelmy balance. No data was

available for the re-spreading of Survanta.

8 mm size, which are fractured revealing the inte

nal wavy layers.

4. Discussion

The present study found an improvement in th

adsorption of surfactants at an air liquid inte

face on addition of EO. The effects of EO o

minimum surface tension and stability were no

consistent for all surfactant formulations. Th

addition of EO was beneficial consistently o n

for film adsorption. A beneficial effect on all th

parameters studied was observed in the case

PCPEEO when compared with PCPE. This w

associated with a decrease in the size of the lipo

somes on addition of EO (8 10 mm in the case

P CP EE O a nd 20 30 mm size in t he ca se

PCPE).

The addition of EO, which contains eucalypto

as one of its main components, could promo

dissolution of large micelle structures and cau

fusion of micellar contents. Such properties hav

been observed in the case of hydrophobic surfa

tant specific proteins causing more homogenou

behavior of phospholipid suspensions (Rana

al., 1993). A similar phenomenon may account fo

the improved adsorption of the phospholipids o

addition of EO. Microscopically, the surfactan

in the presence of EO formed open membranou

structures, which could represent this proces

This is in accordance with the finding of Sasa(1990) that open membranous structures are fun

tionally beneficial when compared to close

structures.

The small sized, easily fracturable liposomes

PCPEEO could perhaps account for its superio

in vitro performance. This is in partial agreeme

with the findings of Denizot et al. (1991) th

small sonicated liposomes show faster adsorptio

a t a n a i r liquid interface than the polydisper

larger liposomes. Though our study also foun

small liposomes of PCPEEO to be functionalbeneficial, it did not seem to be a necessity fo

adequate adsorption. Open membranous stru

tures, regardless of size, appeared to be favorab

for adsorption. However, the small size appeare

to be related to better surface tension reduction

stability and re-spreading.

Fig. 6 (A) depicts a liposome of PCPE whereas

Fig. 6 (B), (C), and (D) depict structures in the

presence of EO. Fig. 6 (B) shows a PCEO lipo-

some, which is organized into wavy membranous

folds. Similarly, Fig. 6 (C) depicts the partially

fractured surface of a liposome of PCPE in the

presence of EO and the open membranous organ-isation of a totally fractured liposome of PCPG in

the presence of EO and 5 mM calcium is seen in

Fig. 6 (D). On comparing the ultrastructure of

PCPE and PCPEEO, it was observed that PCPE

formed large smooth liposomes 20 30 mm in size

whereas PCPEEO formed small liposomes barely


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14

PCPEEO was found to be comparable to Sur-

vanta in vitro with respect to surface tension

reduction and was superior to ALEC and Exosurf

with respect to all parameters compared mini-

mum surface tension, stability index and adsorp-

tion at Rmax. It also had a high re-spreading ratio,

as detected by the Wilhelmy balance study, com-

parable to that of ALEC and Exosurf. The im-proved adsorption and re-spreading, if extended

to in vivo conditions, could help in development

of a more ef ficient surfactant formulation which

could adsorb quickly to the air liquid interface of

the lungs and prevent collapse of the lungs by

lowering of surface tension. The improved re-

spreading would allow re-entry of surfactant ma-

terial into the interface with subsequent breaths

preventing a depletion of material from the inte

face due to losses. Further, the surface tension o

PC with EO and of PCPE with EO on adsorptio

at Rmax was B25 mN /m, which is similar to th

values observed for lung surfactant and its recon

stituted components (Ingenito et al., 2000). T h

presence of EO could perhaps help in the re-entr

of extra surfactant material from the surface assciated reservoir into the interface on expansion.

appears that PCPEEO may hold potential as

possible surfactant formulation.

However, we have not studied the compressibi

it y o f t he first compression monolayers in th

presence of EO, another important parameter fo

effective function. Furt her studies in a leak fr

captive bubble surfactometer would be inform

Fig. 6. Effect of EO on ultrastructure of surfactants. (A) depicts the ultrastructure of a liposome of PC:PE, 2:3. (B), (C) and (D

show the structures in the presence of EO. A liposome with wavy membranous folds is seen in (B) on addition of eucalytpus oil

DPPC. In (C), the structure of PCPE 2:3 in the presence of EO is seen. The liposomes are much smaller than the ones of PCP

The open membranous structure of PCPG with EO in the presence of 5 mM calcium is shown in (D).


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150

tive in this regard. Thus, the addition of EO (1%)

to PCPE (2:3) resulted in a favorable surfactant

formulation in vitro. It was found to be a promis-

ing candidate for possible exogenous surfactant

therapy in R DS based on its in vitro properties. It

is to be remembered that in vitro studies do not

necessarily provide an accurate determination of

in vivo exogenous surfactant efficacy due to in-

hibitory effects of plasma proteins, the in vivo

metabolism of the exogenously administered sur-

factant and inability to model the complex envi-

ronment of the lungs in vitro. Thus, one must be

cautious while extending these findings to in vivo

conditions. Further research in the form of animal

trials is required. Also, EO has never been used in

neonates and its safety remains to be established.

Though very low doses have been of EO have

been used in this study, far below the doses

described to have toxic effects 2 5 m l (Tibballs,

1995; Day et al., 1997) and Webb and Pitt (1993)

failed to find any toxic effects in children under 14

yr age even on oral ingestion of more than 30 ml

of the oil, this requires to be further con firmed.

In the event of its success in animal trials and

its passing the toxicity tests, PCPEEO could be a

promising surfactant formulation, which would be

inexpensive and could be beneficial to many ba-

bies in developing countries who are otherwise

unable to afford the present day surfactants.

Acknowledgements

We would like to thank Prof. S. Major for

extending the facility of the LB trough in the Thin

Films Laboratory, Department of Physics, Indian

Institute of Technology, Bombay, India. One of

the authors (R.B.) is very grateful to Dr. N.S.

Morton for having had the opportunity to con-

duct the surfactometer experiments in the Steroid

Laboratory of the Department of Child Health,

Royal Hospital for Sick Children, Glasgow, Scot-land, UK. Our sincere thanks to Prof. A. Wilkin-

son, Oxford University, UK , for his help and

encouragement which made this work possible.

We would also like to extend our deep gratitude

to Prof. R.R. Puniyani of the School of Biomedi-

cal Engineering, Indian Institute of Technology,

Bombay, India, for his fruitful suggestion

throughout the course of this study. The funds fo

the research trip to UK were partly met by th

J.N. Tata Endowment for The Higher Educatio

of Indians.

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