Int. J. lmmunopharmac., Vol. 12, No. 4, pp. 4 2 7 - 4 3 4 , 1990. 0192-0561/90 $3.00 + .

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Printed in Great Britain. © 1990 International Society for Immunopharmacology.

EFFECTS OF LOW M O L E C U L A R C O N S T I T U E N T S FROM A L O E VERA

GEL ON O X I D A T I V E M E T A B O L I S M A N D CYTOTOXIC A N D
B A C T E R I C I D A L ACTIVITIES OF H U M A N N E U T R O P H I L S

L. A.'T HART, *t P. H. NIBBERING,~: M. TH. VAN DEN BARSELAAR,* H. VAN DIJK,~

A. J. J. VAN DEN BERG* and R. P. LABADIE*

*Department of Pharmacognosy, Faculty of Pharmacy of the University of Utrecht, Catharijnesingel 60,

3511 GH Utrecht; *Department of Infectious Diseases, University Hospital, Leiden;
~Immunology Section of the Eijkman-Winkler Laboratory of Medical Microbiology,
Faculty of Medicine of the University of Utrecht, The Netherlands

(Received 31 July 1989 and in final form 16 January 1990)

Abstract - - In traditional South-East Asian medicine the therapeutic value of the parenchymous leaf-gel of
Aloe vera for inflammatory-based diseases is well-reputed. The aim of this study is to investigate at which
level gel-constituents exert their activity. We show here that low -Mr constituents of an aqueous gel-extract
inhibit the release of reactive oxygen species (ROS) by PMA-stimulated human PMN. The compounds
inhibit the ROS-dependent extracellular effects of PMN such as lysis of red blood cells. The capacity of the
PMN to phagocytose and kill micro-organisms at the intracellular level is not affected. The inhibitory
activity of the low-Mr compounds is most pronounced in the PMA-induced ROS production, but is
significantly antagonized by the Ca-ionophore A23187. It is shown that the inhibitory effect of the low-Mr
compounds is the indirect result of the diminished availability of intracellular free Ca-ions.

In Third World countries and in many industrialized
countries medicines of plant-origin are commonly
used in basic health care. The preparation and use of
this type of medicines are largely based on empirism.
Despite the fact that a number of the preparations
have been used successfully for ages, only little is
known about the effects of plant constituents on the
processes that cause the illness.
A fairly well-documented preparation in tradition-
al medicine is the parenchymous leaf-gel o f A l o e vera
(Grindlay & Reynolds, 1986). In the crude form or as
aqueous extract the gel has been used to treat
different forms of inflammatory afflictions, ranging
from infected wounds (local) to rheumatoid arthritis
(systemic). In animal models a beneficial effect of
the gel has been demonstrated on infectious inflam-
mation (Solar, Zeller, Rasolofonirina, Coulanges,
Ralamboranto, Andriatsimahavandy, Rakotovao &
LeDeaut, 1982), rheumatoid arthritis (Saito,
Ishiguro, Imanishi & Suzuki, 1982) and X-radiation
dermatitis (Lusbaugh & Hale, 1953). The mechan-
isms underlying the therapeutic effects of the gel are
largely unknown. Recently, we have isolated poly-
saccharides from the gel which activate alternative
pathway complement activity in vitro and have an
adjuvants activity on specific antibody production
and T-cell activity in vivo ('t Hart, van den Berg,
Kuis, van Dijk & Labadie, 1989). Moreover, we
demonstrated that low-Mr substances from the gel
inhibit the release of ROS from stimulated
neutrophils ('t Hart, van Enckevort, van Dijk, Zaat,
de Silva & Labadie, 1988a),
The aim of this present study was to investigate the
effects of low-Mr gel-constituents on the oxidative
metabolism of neutrophils in more detail. Because
ROS are involved in the cytotoxic activity (McCord &
Wong 1978; Clark & Klebanoff, 1979; Weiss, 1980;
Vercelotti, van Asbeck & Jacob, 1985) of activated
neutrophils and in the intraphagosomal killing of
ingested micro-organisms (McCord & Wong, 1978;
Johnston, Keele, Misra, Lehmeijer, Webb, Baehner &
Rajagapolan, 1975) the effects of low-Mr gel-
constituents on these biological functions of the
neutrophils were investigated as well.

tTo whom correspondence should be addressed at: Department of Immunology, TNO Primate Centre, P.O. Box 5815,
2280 HV Rijswijk, The Netherlands.
427
428 L. A. 'T HARTet al.
EXPERIMENTAL PROCEDURES
Preparation of the low-Mr fraction from the gel
The leaves of Aloe vera plants (family Liliaceae)
were harvested in Sri Lanka. The mucilagenous
parenchymous tissue was excised from freshly cut
leaves, lyophillized and kept in sealed plastic bags
until use. To obtain an aqueous extract, 10 g of
lyophillized gel were swollen in 1 liter of saline at
room temperature under gentle stirring. The
yellowish extract was centrifuged for 15 min at
1000 × g. After decanting the supernatant the pellet
was suspended in 500 ml of saline, and the extraction
procedure was repeated. After a final extraction with
250 ml of saline the non-soluble residue (80 mg) was
discarded. The pooled supernatants were dialysed
three times against 10 liters of demineralized (demi)
water. The non-dialysable fraction, containing
polysaccharides not interfering with neutrophil
activity ('t Hart et al., 1988a), was also discarded.
The pooled dialysates were lyophillized and the
residue was extracted with excess ethylacetate to free
gel-compounds from NaC1. Attempts to purify the
active compounds from this fraction failed
repeatedly, because isolated compounds were highly
unstable under normal laboratory conditions and
therewith lost all activity. In semi-purified gel-
extracts the inhibitory constituents remain active.
Therefore the ethylacetate extract from the dialysate,
containing about 0.5 mg gel-extract per miUiter, was
used for the experiments. In this study two extracts
from the same charge of lyophillized gel were used.
Both extracts were compared by analytical thin layer
chromatography. The activity of the extracts was
determined by the concentration (v/v) giving 50%
inhibition of luminol-dependent chemiluminescence
(CL,u,0 by STZ- (serum-treated zymosan) stimulated
neutrophils 0cs0). The Ic50 of the extract used for
the experiments in Fig. 1 and Table 2 is 10% (v/v),
and of the extract used for the other experiments
20% (v/v).
Micro-organisms
Two different micro-organisms were used in this
study, which both are highly sensitive to ROS
toxicity: Staphylococcus (S.) aureus (type 42 D) was
grown overnight in Nutrient broth No. 2 (Oxoid Ltd,
Basingstoke, U.K.) at 37°C, and Candida albicans
(UC 820) was cultured for 24 h at 37°C in Sabouraud
dextrose broth (Difco Laboratories, Detroit, MI).
The suspensions of micro-organisms were centri-
fuged for 10 rain at 1500 × g, washed twice with
Hank's balanced salt solution (HBSS pH 7.4, Oxoid
Ltd) and diluted in HBSS, containing 0.1% gelatin
(HBSS-gel) to a concentration of 107 micro-
organisms/ml.
Neutrophils
Blood was obtained from healthy donors by
venapuncture. Neutrophils were collected by
sedimentation on a plasmasteril gradient for 25 rain
at 37°C. The PMN-rich layer was washed twice in
20 mM phosphate-buffered saline (PBS, pH 7.4).
Contaminating erythrocytes were then removed by
lysis with water for 40 s followed by the addition of
an equal volume of two-times concentrated PBS.
The cells were washed twice with PBS and finally
suspended to a concentration of 107 PMN/ml
HBSS-gel. In thus prepared suspensions more than
98% of the cells were viable neutrophils as
determined by trypan blue exclusion.
Serum
Serum was prepared from veneous blood of
healthy AB donors, and stored at - 2 0 ° C .
Measurement of Oz-consumption and the release of
0 2 and Hz02 by neutrophils
Oz-consumption by resting and PMA-stimulated
neutrophils (100 ng PMA/ml) (Sigma Chem. Co., St
Louis, MO) was measured polarographically (van
Dissel, Olivier, Leijh & van Furth, 1986). Briefly,
300 ~1 of a suspension of 5 × 106 PMN/ml
RPMI-1640 (Flow Lab. Ltd, Irvine, U.K.) were
added to a thermostated reaction vessel (37°C). The
Oz-tension in the medium was measured with a
miniature pOz-electrode and recorded on a BD 41
chart recorder (Kipp & Zonen, Delft, The Nether-
lands). The rate of Oz-consumption (nmol
02 × min ~') was calculated by measuring the slope
of the Oz-tension graph over a 10 to 15 rain period.
The results are expressed as nmol/(5 × 106 x 5 min).
The assay to measure Oz -release by neutrophils at
rest and after stimulation with 100 ng P M A / m l was
done as described (Babior, Kipnes & Curnutte,
1973). Neutrophils (106 cells/ml) were incubated
with 1 mM cytochrome c (Type IV, Sigma) at 37°C.
After a 15-min incubation the cells were centri-
fuged at 600 × g for 10 min (4°C) and the absorb-
ance at 550 nm was determined. In control incuba-
tions neutrophils were omitted or the incubates
contained 0.37 mg/ml superoxide dismutase (SOD,
Sigma). Oz--production was calculated from the
increase in A550.... using the extinction coefficient
of 2.9 × 10~ mM ~cm ~ (data from Sigma).
The results of these experiments are expressed as
 Effects of Low Molecular Constituents from Aloe vera Gel on PMN
nmol/(106 cells x 15 min. H202-release by resting
and PMA-stimulated neutrophils (100 ng PMA/ml)
was assessed by the peroxidase-dependent oxidation
of scopoletin (Root, Metcalf, Ostino & Chance,
1975). The results are expressed in nmol/(106 cells
x 5 min).
Chemiluminescence assay f o r R O S production
Procedures for measuring luminol-dependent
chemiluminescence (CLlum) of stimulated neutrophils
were described in detail previously ('t Hart et aL,
1988a). Stimulants in this assay were zymosan,
opsonized by incubation for 30 min at 37°C with
3 vol. pooled human AB serum (STZ), 10 ng/ml
PMA and 4 ~M of the calcium-ionophore A23187.
Luminol and A23187 were from Sigma Chemical
Company, St Louis MO, U.S.A.
Cytochemical staining o f granulocytes f o r
intracellular R O S
Intracellular 02 and H202 concentrations in
neutrophils were assessed semiquantatively after
specific cytochemical staining (Briggs, Karnovsky &
Karnovsky, 1975; Briggs, Robinson, Karnovsky &
Karnovsky, 1986).
Cytotoxicity assay
The 0 2 -dependent cytotoxicity of PMA-
stimulated neutrophils (20 ng PMA/ml) was
measured by determining the specific release of
radioactivity from 5~Cr_labelled rabbit red blood cells
as described in detail elsewhere ('t Hart, van
Enckevort, van Kessel, van Dijk & Labadie, 1988b).
Phagocytosis and intracellular killing o f
micro-organisms
The assay to measure phagocytosis and intra-
cellular killing of previously opsonized (in human
AB serum) S. aureus by human neutrophils has been
described in detail elsewhere (Leijh, van Zwet & van
Furth, 1986). Briefly, in a total volume of 1 ml
HBSS-gel supplemented with 5070 human AB serum
5 x 10 6 neutrophils and 5 x 10 6 S. aureus particles
were incubated at 37°C under rotation (4 rev./min).
At various time-intervals 50/al aliquots were taken
and mixed with 450/al ice-cold HBSS-gel to arrest
phagocytosis. Next, extracellular bacteria were
removed by differential centrifugation at 4°C for
429
4 min at 110 x g. The numbers of viable bacteria in
the supernates were determined microbiologically
using diagnostic-sensitivity-test agar plates (Oxoid
Ltd). Phagocytosis was expressed as the percentage
decrease of the number of viable extracellular
micro-organisms.
For assessment of the intracellular killing capacity
of neutrophils 500/al of 107 PMN/ml were mixed
with 500 tal of 107 opsonized S. aureus particles/ml
and incubated under rotation (4 rev./min) for 3 rain
at 37°C in HBSS-gel supplemented with 10°70 AB
serum. Non-ingested bacteria were removed by two
washes with ice-cold HBSS-gel using differential
centrifugation (4 min at 110 x g at 4°C). After this
procedure no decrease in the number of viable cells,
as determined by trypan blue exclusion, was found.
The neutrophils were suspended in HBSS-gel to a
concentration of 5 x 106/ml and again incubated
under rotation at 37°C in the presence of 10070 AB
serum. At various time-intervals 50/A samples were
taken and after lysing the neutrophils in water
containing 0.0107o bovine serum albumin (BSA,
Oxoid Ltd) the number of viable micro-organisms
was determined microbiologically as beyond.
Intracellular killing is expressed as the percentage
decrease in the number of viable ingested bacteria.
The assay for phagocytosis and intracellular
killing of C. albicans has been described in detail
elsewhere (Leijh, van den Barsselaar & van Furth,
1977). Yeast cells cannot be separated from neutro-
phils by differential centrifugation. Therefore, the
decrease in the number of free yeast cells in the
suspension was counted in a Bfirker haemocytometer
as a measure of phagocytosis. The decrease in the
total number of viable C. albicans in the incubation
mixture was taken as a measure of intracellular
killing.
Fluorometric assay f o r intracellular
Ca-concentration
The rise of intracellular free Ca 2- in stimulated
neutrophils was determined using the probe indo-1
AM (Molecular Probes Inc., Eugene, OR), which
upon binding of Ca-ions emits fluorescence pro-
portional to the amount of bound Ca-ions
(Grynkiewicz, Poenie & Tsien, 1985). Briefly,
107 neutrophils were incubated for 20 min with 4/al
Indo-I (1 mM) in a shaking waterbath at 37°C in a
total volume of 1 ml HBSS-gel. After addition of
4 ml warm RPMI-1640 (Gibco, Grand Island NY)
the incubation was continued for 40 rain. Next, the
cells were centrifuged, resuspended to a concentra-
430 L. A. 'T HART et al.
Table 1. Effect of the low-Mr fraction of Aloe vera gel-extract on neutrophil 02
metabolism.

- low-Mr + 20°7o low-Mr

n n P*
(a)
O2-consumption
(nmol/5 x 106 cells × 5min)
at rest 14 _+ 3 5 35 + 7 4 <0.001
PMA 102 _+ 43 5 110 _+ 22 4 >0.5
(b)
O2 -release
(nmol/106 cells x 15 min)
at rest 5+ 2 14 0 4 <0.001
PMA 44 + 16 14 0 6 <0.001
(c)
HzOz-release
(nmol/106 cells x 5 rain)
at rest 11 + 3 5 9_+6 5 >0.5
PMA 18_+ 2 4 4_+2 4 =0.014
The results are expressed as the mean +_ S.D. The level of significance (*) of the effect of
the low-Mr fraction was determined with Student's t-test. Final concentration phorbol
myristate acetate was 100 ng/ml.

tion of 4 x 106cells per ml R P M I and kept on ice till
further use. Immediately before the test, 0.5 ml of
the cell suspension was centrifuged. The pellet was
suspended in an equal volume of incubation buffer
(pH 7.4), consisting of 145 m M NaC1, 5 m M KC1,
1 m M NazHPO4, 1 m M CaClz, 0.5 m M MgSO4,
5 m M glucose and 10 m M N a - H e p e s and trans-
ferred to a 1 ml quartz cuvette. After adding 150/xg
Con A (Calbiochem, American Hoechst Corp.,
San Diego, CA) (Cohen, Chovaniec, Wilson &
Newburger, 1982) the increase in light emission
was recorded in a spectro-fluorometer (Aminco
Bowman) (excitation wavelength: 3 4 0 n m ; light
emission was recorded at 410 and 485 nm).
Statistical analysis
Statistical analysis of the effects of the l o w - M r
fraction was done with Student's t-test. Differences
were regarded as statistically significant when P was
<0.01.
RESULTS
Effect o f low-Mr compounds from the gel-extract on
the oxidative metabolism o f human neutrophils
The finding that l o w - M r compounds of Aloe vera
extract abolish the production of CL~umby stimulated
neutrophils ('t H a r t et al., 1988a), points at a
possible interference with the oxidative metabolism
of these cells. Therefore the effect of a
l o w - M r gel-extract on the O2-consumption and the
O2 and H202 release by resting and PMA-stimulated
neutrophils was investigated. It should be noted that
scavenging of Oz- by l o w - M r compounds is
negligible ('t Hart et al., 1988a). The results in
Table la show that the l o w - M r fraction had a
significant stimulatory effect on the consumption of
O2 by resting but not by PMA-stimulated
neutrophils. The stimulated 02 consumption could
not be attributed to the oxidation of l o w - M r
constituents. The decrease of the 02 tension in
RPMI-1640 was 0.45 in the absence and 0.50 nmol
O2/min in the presence of 20% l o w - M r fraction. The
l o w - M r fraction completely abolished the release of
02- by resting and PMA-stimulated neutropbils
(Table lb), while the H202-release by P M A -
stimulated, but not by resting neutrophils was
significantly reduced (Table lc).
In a separate experiment intracellular 02 and
H202-production in PMA-stimulated neutrophils
was visualized by cytochemical staining. F r o m the
intensity of the dye-precipitate, ROS concentrations
can be assessed semiquantitatively. The results show
no difference in the staining intensities when
stimulation occurred in the presence as compared to
in the absence of the l o w - M r fraction.
Effect o f the low-Mr fraction on the microbicidal
and cytotoxic function o f neutrophils
W h e n released into the pericellular space 02
mediates the lysis of 5~Cr-labelled R R B C , which is a
 Effects of Low Molecular Constituents from Aloe vera Gel on PMN 431

Table 2. Effect of the low-Mr fraction from Aloe vera gel-extract on

PMA-induced cytotoxicity

S~Cr release (disintegrations/min _+ S.D.)

Incubation time
Concentration 1 hour 2 hours 3 hours
Low M r
0 3451 + 470 5925 _+ 371 7327 _+ 20
0.3 3757 _+ 263 5633 _ 40 7246 _+ 0.7
1.6 3540 + 129 4839 _+ 315 5830 _+ 435
8.0 2499 _+ 10 3661 _+ 243 4594 _.+ 134
-PMA 176 _ 18 270 _+ 11 572 _+ 103
100% release 15707 _+ 549
105 neutrophils were incubated for 60 min at 37°C with 51Cr-loaded
rabbit red blood cells. The incubation medium contained various
concentrations of the low-Mr gel-extract. Neutrophils were stimulated
with 20 ng/ml PMA. The results are given as the mean of three
incubations.

Table 3. Effect of the low-Mr fraction from Aloe vera gel-extract on

the phagocytosis and intracellular killing of Staphylococcus aureus
and Candida albicans by neutrophils

-low-Mr + 20% low-Mr

n n P*
S, abiFeus
Phagocytosis 99% 4 99°7o 4 >0.5
Intracellular killing 92% _+ 2 4 81% _+ 4 4 >0.2
C. albicans
Phagocytosis 95°70 + 2 3 97% 3 =0.35
Intracellular killing 59% _+ 11 3 64°70 _+ 6 3 >0.1
The results are expressed as the mean of three or four experiments
(_+ 1 S.D.). Significance levels (.) of the difference between the
measured parameters in the presence or absence of the low-Mr
fraction were calculated with Student's t-test.

measure o f the cytotoxic potential o f P M A - s t i m u -
lated neutrophils ('t H a r t et al., 1988b). The results
in Table 2 show that this activity o f P M A - s t i m u l a t e d
neutrophils was m a r k e d l y inhibited by the l o w - M r
fraction.
W h e n released in the p h a g o s o m a l c o m p a r t m e n t
ROS contribute to the killing o f ingested micro-
organisms. The results in Table 3 show that the
l o w - M r fraction h a d no effect on the capacity o f
neutrophils to p h a g o c y t o s e opsonized micro-
organisms. This result is in agreement with previous
findings on the phagocytosis o f radiolabelled and
opsonized S. aureus particles ('t H a r t et al., 1988a).
As is s h o w n in the same table the l o w - M r fraction
h a d no effect on the killing o f S. aureus or
C. albicans, a l t h o u g h b o t h m i c r o - o r g a n i s m s are
sensitive to ROS (Lehrer, 1970; Rodey, Park,
W i n d h o r s t & G o o d , 1969).
The possible site o f action o f l o w - M r constituents
In experimental conditions, ROS p r o d u c t i o n by
neutrophils can be induced with different stimulants,
such as opsonized particles, which bind to surface-
receptors, the p h o r b o l ester P M A , which directly
activates the regulatory cellular enzyme P K C , and
the C a - i o n o p h o r e A23187, which inserts an artificial
Ca-channel. In a next set o f experiments the
influence o f the l o w - M r fraction on CL~um) induced
with either o f these stimulants was tested. The
chosen l o w - M r c o n c e n t r a t i o n gave rise to a b o u t 50°7o
inhibition o f STZ-induced CLum (Fig. 1). As is
s h o w n in the same figure P M A - i n d u c e d CLlum was
432 L. A. 'T HARTet al.
100 -
verify this hypothesis the influence of the low-Mr
fraction on the rise of the intracellular free Ca 2÷
8O
concentration in stimulated neutrophils was
E determined. The lectin Con A was chosen for
d stimulation because in our hands this gave a strong
Z 60 and synchronous fluorescence signal. The results in
0
I---
Fig. 2 show that the low-Mr fraction abolished the
Con A-induced increase of the intracellular free Ca 2'
112 4 0
Z concentration.

20
DISCUSSION

The aim of this study was to investigate the

STZ PMA A23187
Fig. 1. Site of action of the low-Mr fraction of A l o e vera
gel-extract: neutrophils were stimulated in the absence
or presence of the low-Mr fraction (10%), with STZ
(1.2mg/ml), 10ng/ml PMA, 2/aM A23187 or the
combination of these stimulants. Results show the
percentage inhibition of CLtum (individual measurements
and mean _+ S.D.).
- IMr + 2 0 % IMr
Con A Con A Triton X-100
Fig. 2. Effect of the low-Mr fraction of A l o e vera gel-
extract on calcium mobilization in stimulated neutrophils:
Indo-l-loaded neutrophils were incubated at 37°C in a
fluorometer (excitation 340 nm, emission recorded at
410nm). After equilibration of the emission signal
150 tag/ml Con A was added. Incubation was performed in
the absence (a) or presence (b) of 20% of low-Mr (1-Mr)
fraction.
inhibited for about 80%, while a significantly lower
inhibition of about 300/o was found on CL~uminduced
with A23187. Furthermore, simultaneous
stimulation of neutrophils with P M A and A23187
reduced the inhibitory effect of the low-Mr fraction
to about 40o70.
The latter observation suggests that low-Mr
compounds might interfere with the availability of
intracellular free Ca 2~ in stimulated neutrophils. To
PMA +
A23187 influence of low-Mr compounds from A l o e v e r a gel
on different neutrophil functions. The gel is well-
known in the traditional medicine of South-East
Asia for its anti-inflammatory effects (review by
Grindlay & Reynolds, 1986). By investigating the
biological effects of this type of medicines we
attempt to find a rationale for their use in traditional
medicine on the one hand. On the other hand,
purification of compounds from such plants guided
by a selected biological activity might result in
potentially new medicines.
The main conclusion from the present results is
that low-Mr constituents from A l o e v e r a gel reduce
the release of Oz- and H202 by stimulated
neutrophils to the background level, while the
intracellular ROS concentrations, detected by
cytochemical staining were not markedly altered.
Despite the inhibition of 02- -release to the
background level, HzO2-release by resting
neutrophils was not influenced by the low-Mr
fraction. These observations seem to be contradict-
ory to the general assumption that N A D P H oxidase
catalyzes only the one electron reduction of 02 to 02
after which U 2 0 2 is formed in a dismutation reaction
(Babior, Curnutte & McMurrich, 1976; Gabig &
Babior, 1979; Wakeyama, Takeshige, Takayanagi &
Minakami, 1982). Recently, however, it has been
reported that H20: can be directly formed by
N A D P H oxidase in a two electron reduction step
(Green & Wu, 1986; Green & Pratt, 1987). Our
results suggest that the A l o e v e r a low-Mr fraction
inhibits only the one electron reduction step and that
this is independent from the two electron reduction.
For definitive proof of this assumption purification
of the active constituent(s) in the low-Mr fraction is
required.
Interestingly, the low-Mr fraction seems to have a
stimulatory effect on the 02 consumption by resting
neutrophils and leaves that by activated neutrophils
intact. The effect on resting neutrophils might be
 Effects of Low Molecular Constituents from Aloe vera Gel on PMN
explained by the fact that A l o e vera gel is a rich
source of quinones such as anthraquinones (Grindlay
& Reynolds, 1986), which are also in the low-Mr
fraction, and which can function in an electron
transport chain (Woodbury, Parson, Gunner, Prince
& Dutton, 1982). Data from Gallin, Seligmann,
Cramer, Schiffman & Fletcher (1982) show that O2
consumption by resting human neutrophils can be
stimulated with menadione (vitamin K3; 2-methyl-
1,4-naphthoquinone), which is also an inhibitor of
02 production. Similar findings were described by
Nisimoto, Tamura and Lambeth (1988) in bovine
neutrophils. Taken that the low-Mr fraction is a
mixture of compounds, it cannot be excluded that
this effect is mediated by another constituent than
the inhibition of 02- release.
The inhibition of 02-- and H202-release in
stimulated neutrophils has clear effects on the ROS-
dependent cytotoxicity of stimulated neutrophils,
assayed by the O2 -dependent lysis of radiolabelled
rabbit red blood cells. Interestingly, the phagocytosis
and killing of ingested micro-organisms which is a
measure for the intraphagosomal activity of ROS
was unaffected. The mechanisms underlying the
different effects of the low-Mr fraction of the gel-
extract oi1 the oxidative metabolism of neutrophils in
the two compartments are now known.
In an experimental situation the molecular
processes initiated by receptor-ligand interaction
and leading to NADPH-oxidase activation
(Hamilton & Adams, 1987) can be mimicked with
PMA, binding directly with PKC, and with the
ionophore A23187 to enhance the intracellular Ca-
concentration. Our results show that PMA-induced
ROS production was strongly reduced by the low-Mr
fraction, but that the inhibitory activity was
433
significantly lower when neutrophils were simul-
taneously stimulated with the Ca-ionophore. These
findings imply that low-Mr constituents of the gel
might affect the availability of intracellular free Ca 2+
in stimulated cells. This assumption was supported
by the observation that the low-Mr fraction
prevented the Con A-induced rise of intracellular
free Ca 2+ in Con A-activated neutrophils. These
effects cannot be attributed to chelation of Ca 2+-ions
because classical pathway complement activity,
which is a highly Ca2+-dependent process, was
insensitive to the low-&lr fraction ('t Hart et al.,
1988a).
Taken together, low-Mr constituents from A l o e
vera gel reduce the harmful effects of ROS from
neutrophils at the site of inflammation, such as
toxicity towards tissue cells and inactivation of
protease inhibitors. The beneficial effects of
neutrophils, however, such as phagocytosis and
intracellular killing of infectious micro-organisms,
remain intact. This might explain the therapeutic
effect of the gel in concrete situations, e.g. when
applied to infected wounds. Low-Mr compounds
leaking from the gel might prevent oxidant-mediated
tissue-injury.
A c k n o w l e d g e m e n t s - - The authors thank colleagues from
the Section of Experimental Microbiology (Eijkman-
Winkler Lab. of Medical Microbiology, University of
Utrecht) for providing neutrophils, to Dr C. P. M. van
Kessel (same institute), Dr J . G . M . Scharenberg
(Department of Immunology, University Children's
Hospital Utrecht) and Mrs J. S. van de Gevel (Department
of Infectious Diseases, University Hospital Leiden) for
their valuable theoretical and technical support. Mr P. van
Dorp van Vliet and Mrs C. Luteijn are acknowledged for
making the figures.

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