Journul of Elhnophormucology. Elsevier Scientific
Publishers
33 ( I991 Ireland
63
) 63-66
Ltd.
Immunomodulatory
activity of three Sri-Lankan medicinal plants used in hepatic disorders
M. Ira Thabrewa,
de Silvab, R.P. Labadie’,
“Department
K.T.D.
of Biochemistry,
Faculty
of Medicine.
University
P.A.F. de Bie’ and B. van der Bergc
of Ruhuno. Guile (Sri-Lanka),
‘Department
of Chemistry,
of Sri-Jayawurdenupura. Nugegadu (Sri-Lunku) and ‘ Depurrmenr I$ Chemical Phurmacy. Sectian of Pharmucolagy. Luhorurary, Srute University of Utrechr, Cu~har~jnesingel60. 3511 GH Utrecht (The Netherlands) (Accepted
November
13, 1990)
The effects of aqueous extracts of Osheckiu acrundru whole plant. Melarhriu leaves on the human immune system were investigated. alternate pathways complement
of the human complement
pathway
human polymorphonuclear
Key words: Osheckiu
leukocytes
ocrundru:
system in vitro. The effects were dose-dependent a direct dose-dependent
upon stimulation
Melathriu
maderasputunu
whole plant and Phyllanthus dehelis
The extracts showed strong anticomplement
assay. The extracts also exhibited
Universi?
Phurmuceuticul
inhibition
effects on both the classical and
and most pronounced
of luminol-induced
in the classical
chemiluminescence
of
with zymosan.
muderuspatuna:
Phyllunthus
d&elis:
immune
system; a,lticomplement
effects: hepatic
disorders.
part be due to an influence on the immune system. Therefore, the immunomodulating capabilities of Osbeckia octandra, Melothria maderaspatana and Phyllanthus debelis were investigated with a view to obtaining a better understanding of their mechanisms of actions. The ability of these aqueous plant extracts to interfere with the immune systems involved in both humoral and cellular defence mechanisms were investigated by studying their effects on the classical and alternate pathways of the human complement (C) system in vitro and on luminol-induced chemiluminescence of human polymorphonuclear leucocytes (PMNs).
Introduction Over 40 different plants are at present thought by the traditional medical practitioners in SriLanka to be effective in the treatment of the various forms of liver disease (Attygalle, 1952; Jayaweera, 1982). The plants are usually administered in the form of fresh juice or as a gruel prepared with rice. Recent scientifically controlled trials (Syamasundar et al., 1985; Thabrew et al., 1987, 1988) have provided confirmatory evidence for the efficacy of Osbeckia octandra DC (Melastomaceae; local name: heenbovitiya), Melothria maderaspatana (L.) Cogn. (Cucurbitaceae; local name: heen kekiri) and Phyllanthus debilis Klein (Euphorbiaceae; local name: sudupitawakka) as antihepatotoxic agents. Results of recent studies with several plant extracts (Atal et ai., 1986; Van der Nat et al., 1987), including those exhibiting antihepatotoxic activity (Vallotton and Frei, 1981), suggest that the therapeutic effects of these plant extracts may in Correspondence Biochemistry,
to:
P.O. Box 70, Galle,
037%874l/SO3.50
Prof.
Faculty
M.I.
Thabrew.
of Medicine,
Department
University
Materials and Methods Plant material Whole plants of Phyllanthus debelis and Melothria maderaspatana and the leaves of Osbeckia octandra were used in the present study. The plants were collected from Galle in Southern Sri-Lanka. The botanical identity was determined by using the descriptions of Jayaweera (1982) and confirmed by direct comparison with authentic samples from the
of
of Ruhutla,
Sri-Lanka.
0
1991 Elsevier Scientific
Published and Printed in Ireland
Publishers
Ireland
Ltd.
64
Herbarium of the University of Peradeniya. Extraction procedure
The plant material (100 g) was refluxed for 8 h with 150 ml deionized water. After centrifugation and filtration, the aqueous extract was lyophilized. The respective w/w yields were: 0. octandra, 24.5% M. maderaspatana, 33.8%; P. debilis, 33.5% Complement tests
Alternate and classical C pathway activities were determined in human serum (Bloedbank Utrechet) using a microtitre assay (Klerx et al., 1983) with normal rabbit and sensitized sheep erythrocytes as the target cells, respectively. The alternative and classical C pathway activities were expressed in AP 50 and CH 50 units/ml (Van Der Nat et al., 1987). All tests were carried out in duplicate.
tration of 3.3 X 106/ml in Hank’s buffered salt solution (Gibco, Paisely, Scotland). PMNs were stimulated with zymosan-activated particles (ZAP, Packard Instrument Co., Downers Grove, U.S.A.) also containing the light enhancer luminol in a concentration of 10 mg/ml. The respiratory burst activity results in chemiluminescence (Allen and Loose, 1976) which was measured in a Packard Picolite 6500 luminometer for 16 min. The CPM values at every measurement were plotted against the reaction time. The area under the curve (AUC) is a measure for the total chemiluminescence in the test tube. The inhibitory effect of added samples on the chemiluminescence were expressed as follows: sample activity (‘%) = AUC (sample) x lOO/AUC (control). Each concentration of sample was tested in duplicate and all experiments were repeated at least once. Results and Discussion
Chemiluminescence assay for phagocytic activity
All three plant extracts under investigation showed strong anticomplement effects on both the classical and alternate pathways of the human complement (C) system in vitro (Figs. 1 and 2). As
PMNs were isolated from the blood of healthy volunteers using a density gradient centrifugation method (Boyum, 1968) and adjusted to a concen-
C Activity
C Activity
A
: (% controi)
I 0
10
30
Extract Fig.
I.
Human
added
90
270
810
2430
0
1
g/ml test mixture) /I
classical (A) and alternative
aqueous extracts of: A, 0. oc~untlru.
l
(B) complement
0.1 Extract
pathway
activity determined
. P. dehelis: o, M. n~rrderccspcrtunc.
0.5 added
0.25 (mg/ml
1.25
6.25
test mixture)
in the presence of graded amounts of the
65 TABLE I
Chemiluninescence (% of control)
SPECIFIC ACTIVITIES (CONCENTRATION CAUSING 501%~INHIBITION OF COMPLEMENT ACTIVITY) OF THE THREE PLANT EXTRACTS UNDER INVESTIGATION Plant extract
Specific activity @g/ml) Classical pathway
Os~e~kiu a~tandru Melothria
muderu.~put~na
Phy~l~n thus dehelis
I 0
500
100
Extract
added
1000
{bg/ml
test
1500
2000
mixture)
Fig. 2. Reduction of Iuminol-indu~ chemilumine~ence of human PMNs upon stimulation with zymosan by increasing doses of the aqueous extracts of: l , 0. ociunrlru, o , P. debelis; a
~M.
maderaspatana.
reported for some other medi~i~ai plants with antiinflammatory and antipyretic activities (Bamunuarachchi et al., 1984; Van der Nat et al., 198?), the effects of the plants under investigation were dose-dependent and most pronounced in the classical complement pathway assay. A comparison of the specific activities of the three plant extracts (Table 1) shows that ~sbeck~u oc~ff~~ru has the greatest inhibitory activity in both the CP and AP. Whether the inhibitory activities of the plant extracts on the C system is due to C consumption or to a more direct blockage of the C cascade (Obaseki et al., 1985; Van der Nat et al., 1987) has to be further investigated. Whatever the mechanism of complement inhibition, as a result of the actions of the plant extracts on the C system, the amounts of C components available for an interaction with other possible targets is strongly reduced. It is well known that decreased C activity
17.5 126.5 60.0
Alternative pathway 850.0 1000.0
1250.0
can lead to reduced C-elicited responses such as anaphylaxis, chemotaxis, opsonoization, stimuiation of macrophages and PMNs (Weir, 1983). As evident from Fig. 2, the extracts exhibited a direct dose-dependent inhibition of luminolinduced ~hemilumines~ence of human polymorphonuclear leukocytes upon stimufation with zymosan. Doses as low as 14 ,ug extract per test tube were found to be active. Luminol~induced chemilumine~ence reflects the myeloperoxidasemediated metabolic activity of PMNs (De Chatelat et al., 1982). This in turn is connected with the phago~ytic activity of the ceils (Ewetz et al., I98 1)~ The inhibition of complement activation and chemiluminescenc~ observed in the present investigation provides supportive evidence in favour of the view that the antihepatotoxic and other thera~uti~ effects of ~sbe~k~~ o~tundru, ~~lothr~u ~ad~ras~~i~~a and Ph~lla~~hu~~debelis extracts (~ayawee~, 1982; Thabrew et al., 1987, 1988) may, in part, be mediated through an influence on the immune system, References Allen. R.C. and Laose, L.D. (1976) phagocytic activation of a luminoi-de~ndent ChemiIumine~en~ in rabbit alveolar and peritonial macrophages. Biochemical and Biopi~ysicul Research ~ummuni~utian.~
59, 245-252.
Atal. C.K., Sharma, M.L., Kaul, A. and Khajuria, A. (1986) ImmunomoduIating agents of plant origin. I : Preliminary screening. Journui @ ~t~~~~P~arrnu~f~/~~~~ 18, I 33- I4 1.
66
Attygalle, J. (1952) Sinhalese Materia Medica, 2nd. Edn. M.D. Gunasena and Co. Ltd., Colombo, Sri-Lanka. Bamunuarachchi, A., Abeysekera, A., de Silva, K.T.D. and Labadie, R.P. (1984) Evaluation of effects of SriLankan plants on human complement in vitro. Pharmaceurisch Weekblad
Boyum,
of leucocytes
Scandinavian
from
Journal of Clinical
blood
and
and Labora-
97). 394. De Chatelat, L.R.. Long, G.D., Shirley, P.S., Bass, D.A., Thomas, M.J., Henderson, F.W. and Cohen, M.S. (1982) Mechanism of the luminol-dependent chemiluminescence of tory Investigations
21 (Suppl.
human neutrophils. Journal of Immunology 129, I589- 1593. Ewetz. L., Palmbid, L. and Thore, A. (1981) The relationship between luminol chemihrminescence and killing of Staphylococcus aureus by neutrophil granulocytes. Blur 43. 373-381. Jayaweera, D.M.A. (1982) Medicinal Plants Used In Ceylon, Vols. 1-5, National Science Council of Sri-Lanka Publications, Colombo. Klerx, J.P.A.M., Beukelman, C.J. Van Dijk, H. and Willers. J.M.N. (1983) Microscopy for calorimetric estimation of complement activity in guinea pig, human and mouse serum. Journal of Immunological
indica
on membranes
in
SyCimSUndai’.
K.V.. Singh. B.. Thakur, R.S.. Husain. A., Kiso. Hikino. H. (1985) Antihepatotoxic principles of Phyllunthus niruri herbs. Journal (4 Etltnc)ppharnlacolog~ 14.
Y. and 414.
119, 901-902.
A. (1968) Separation
bone marrow.
aqueous extracts of Azadirachta vivo. Fitoterapia 56. I1 I-l 15.
Methods 63, 2 15-220.
Obaseki, A.O., Adeyi. 0. and Anyabuike, C. (1985) Some serum enzyme levels as marks of possible acute effects of the
Thabrew. M.I., Joice, P.D.T.M. and Rajatissa. W.C. (1987) A comparative study of the efftcacy of Osheckia octundro and Pavetta indicu in the treatment of liver dysfunction. Plunto Medica
53. 239-241.
Thabrew, M.I.. Jayatilaka. K.A.P.W. and Perera. D.J.P. (1988) Evaluation of the efftcacy of Melothria maderaspatana in the alleviation of carbon tetrachloride-induced liver dysfunction. Journal of Etlznoplturnluc(~logy 23. 305-3 12. Vallotton, J.J. and Frei. P.C. (1981) Influence of (+) cyaninadol-3 on the leucocyte migration inhibition test carried out in the presence of PPD and HBs Ag. Infection und Immunity
32. 432433.
Van der Nat, J.M., Klerx. J.P.A.M., Van Dijk. H., de Silva. K.T.D. and Labadie. R.P. (1987) lmmunomodulatory activity of an aqueous extracts of Azadirochtu indicct stem bark. Journul os Ethnophurmacology
Weir.
D.M.
(1983)
Immunology.
19, 125-l
Medicine and Biology, 5th edn.. Churchill
burgh,
pp. 18&218.
3 1.
An Outline
fiw Students q/
Livingtone.
Edin-
Publishers
33 ( I991 Ireland
63
) 63-66
Ltd.
Immunomodulatory
activity of three Sri-Lankan medicinal plants used in hepatic disorders
M. Ira Thabrewa,
de Silvab, R.P. Labadie’,
“Department
K.T.D.
of Biochemistry,
Faculty
of Medicine.
University
P.A.F. de Bie’ and B. van der Bergc
of Ruhuno. Guile (Sri-Lanka),
‘Department
of Chemistry,
of Sri-Jayawurdenupura. Nugegadu (Sri-Lunku) and ‘ Depurrmenr I$ Chemical Phurmacy. Sectian of Pharmucolagy. Luhorurary, Srute University of Utrechr, Cu~har~jnesingel60. 3511 GH Utrecht (The Netherlands) (Accepted
November
13, 1990)
The effects of aqueous extracts of Osheckiu acrundru whole plant. Melarhriu leaves on the human immune system were investigated. alternate pathways complement
of the human complement
pathway
human polymorphonuclear
Key words: Osheckiu
leukocytes
ocrundru:
system in vitro. The effects were dose-dependent a direct dose-dependent
upon stimulation
Melathriu
maderasputunu
whole plant and Phyllanthus dehelis
The extracts showed strong anticomplement
assay. The extracts also exhibited
Universi?
Phurmuceuticul
inhibition
effects on both the classical and
and most pronounced
of luminol-induced
in the classical
chemiluminescence
of
with zymosan.
muderuspatuna:
Phyllunthus
d&elis:
immune
system; a,lticomplement
effects: hepatic
disorders.
part be due to an influence on the immune system. Therefore, the immunomodulating capabilities of Osbeckia octandra, Melothria maderaspatana and Phyllanthus debelis were investigated with a view to obtaining a better understanding of their mechanisms of actions. The ability of these aqueous plant extracts to interfere with the immune systems involved in both humoral and cellular defence mechanisms were investigated by studying their effects on the classical and alternate pathways of the human complement (C) system in vitro and on luminol-induced chemiluminescence of human polymorphonuclear leucocytes (PMNs).
Introduction Over 40 different plants are at present thought by the traditional medical practitioners in SriLanka to be effective in the treatment of the various forms of liver disease (Attygalle, 1952; Jayaweera, 1982). The plants are usually administered in the form of fresh juice or as a gruel prepared with rice. Recent scientifically controlled trials (Syamasundar et al., 1985; Thabrew et al., 1987, 1988) have provided confirmatory evidence for the efficacy of Osbeckia octandra DC (Melastomaceae; local name: heenbovitiya), Melothria maderaspatana (L.) Cogn. (Cucurbitaceae; local name: heen kekiri) and Phyllanthus debilis Klein (Euphorbiaceae; local name: sudupitawakka) as antihepatotoxic agents. Results of recent studies with several plant extracts (Atal et ai., 1986; Van der Nat et al., 1987), including those exhibiting antihepatotoxic activity (Vallotton and Frei, 1981), suggest that the therapeutic effects of these plant extracts may in Correspondence Biochemistry,
to:
P.O. Box 70, Galle,
037%874l/SO3.50
Prof.
Faculty
M.I.
Thabrew.
of Medicine,
Department
University
Materials and Methods Plant material Whole plants of Phyllanthus debelis and Melothria maderaspatana and the leaves of Osbeckia octandra were used in the present study. The plants were collected from Galle in Southern Sri-Lanka. The botanical identity was determined by using the descriptions of Jayaweera (1982) and confirmed by direct comparison with authentic samples from the
of
of Ruhutla,
Sri-Lanka.
0
1991 Elsevier Scientific
Published and Printed in Ireland
Publishers
Ireland
Ltd.
64
Herbarium of the University of Peradeniya. Extraction procedure
The plant material (100 g) was refluxed for 8 h with 150 ml deionized water. After centrifugation and filtration, the aqueous extract was lyophilized. The respective w/w yields were: 0. octandra, 24.5% M. maderaspatana, 33.8%; P. debilis, 33.5% Complement tests
Alternate and classical C pathway activities were determined in human serum (Bloedbank Utrechet) using a microtitre assay (Klerx et al., 1983) with normal rabbit and sensitized sheep erythrocytes as the target cells, respectively. The alternative and classical C pathway activities were expressed in AP 50 and CH 50 units/ml (Van Der Nat et al., 1987). All tests were carried out in duplicate.
tration of 3.3 X 106/ml in Hank’s buffered salt solution (Gibco, Paisely, Scotland). PMNs were stimulated with zymosan-activated particles (ZAP, Packard Instrument Co., Downers Grove, U.S.A.) also containing the light enhancer luminol in a concentration of 10 mg/ml. The respiratory burst activity results in chemiluminescence (Allen and Loose, 1976) which was measured in a Packard Picolite 6500 luminometer for 16 min. The CPM values at every measurement were plotted against the reaction time. The area under the curve (AUC) is a measure for the total chemiluminescence in the test tube. The inhibitory effect of added samples on the chemiluminescence were expressed as follows: sample activity (‘%) = AUC (sample) x lOO/AUC (control). Each concentration of sample was tested in duplicate and all experiments were repeated at least once. Results and Discussion
Chemiluminescence assay for phagocytic activity
All three plant extracts under investigation showed strong anticomplement effects on both the classical and alternate pathways of the human complement (C) system in vitro (Figs. 1 and 2). As
PMNs were isolated from the blood of healthy volunteers using a density gradient centrifugation method (Boyum, 1968) and adjusted to a concen-
C Activity
C Activity
A
: (% controi)
I 0
10
30
Extract Fig.
I.
Human
added
90
270
810
2430
0
1
g/ml test mixture) /I
classical (A) and alternative
aqueous extracts of: A, 0. oc~untlru.
l
(B) complement
0.1 Extract
pathway
activity determined
. P. dehelis: o, M. n~rrderccspcrtunc.
0.5 added
0.25 (mg/ml
1.25
6.25
test mixture)
in the presence of graded amounts of the
65 TABLE I
Chemiluninescence (% of control)
SPECIFIC ACTIVITIES (CONCENTRATION CAUSING 501%~INHIBITION OF COMPLEMENT ACTIVITY) OF THE THREE PLANT EXTRACTS UNDER INVESTIGATION Plant extract
Specific activity @g/ml) Classical pathway
Os~e~kiu a~tandru Melothria
muderu.~put~na
Phy~l~n thus dehelis
I 0
500
100
Extract
added
1000
{bg/ml
test
1500
2000
mixture)
Fig. 2. Reduction of Iuminol-indu~ chemilumine~ence of human PMNs upon stimulation with zymosan by increasing doses of the aqueous extracts of: l , 0. ociunrlru, o , P. debelis; a
~M.
maderaspatana.
reported for some other medi~i~ai plants with antiinflammatory and antipyretic activities (Bamunuarachchi et al., 1984; Van der Nat et al., 198?), the effects of the plants under investigation were dose-dependent and most pronounced in the classical complement pathway assay. A comparison of the specific activities of the three plant extracts (Table 1) shows that ~sbeck~u oc~ff~~ru has the greatest inhibitory activity in both the CP and AP. Whether the inhibitory activities of the plant extracts on the C system is due to C consumption or to a more direct blockage of the C cascade (Obaseki et al., 1985; Van der Nat et al., 1987) has to be further investigated. Whatever the mechanism of complement inhibition, as a result of the actions of the plant extracts on the C system, the amounts of C components available for an interaction with other possible targets is strongly reduced. It is well known that decreased C activity
17.5 126.5 60.0
Alternative pathway 850.0 1000.0
1250.0
can lead to reduced C-elicited responses such as anaphylaxis, chemotaxis, opsonoization, stimuiation of macrophages and PMNs (Weir, 1983). As evident from Fig. 2, the extracts exhibited a direct dose-dependent inhibition of luminolinduced ~hemilumines~ence of human polymorphonuclear leukocytes upon stimufation with zymosan. Doses as low as 14 ,ug extract per test tube were found to be active. Luminol~induced chemilumine~ence reflects the myeloperoxidasemediated metabolic activity of PMNs (De Chatelat et al., 1982). This in turn is connected with the phago~ytic activity of the ceils (Ewetz et al., I98 1)~ The inhibition of complement activation and chemiluminescenc~ observed in the present investigation provides supportive evidence in favour of the view that the antihepatotoxic and other thera~uti~ effects of ~sbe~k~~ o~tundru, ~~lothr~u ~ad~ras~~i~~a and Ph~lla~~hu~~debelis extracts (~ayawee~, 1982; Thabrew et al., 1987, 1988) may, in part, be mediated through an influence on the immune system, References Allen. R.C. and Laose, L.D. (1976) phagocytic activation of a luminoi-de~ndent ChemiIumine~en~ in rabbit alveolar and peritonial macrophages. Biochemical and Biopi~ysicul Research ~ummuni~utian.~
59, 245-252.
Atal. C.K., Sharma, M.L., Kaul, A. and Khajuria, A. (1986) ImmunomoduIating agents of plant origin. I : Preliminary screening. Journui @ ~t~~~~P~arrnu~f~/~~~~ 18, I 33- I4 1.
66
Attygalle, J. (1952) Sinhalese Materia Medica, 2nd. Edn. M.D. Gunasena and Co. Ltd., Colombo, Sri-Lanka. Bamunuarachchi, A., Abeysekera, A., de Silva, K.T.D. and Labadie, R.P. (1984) Evaluation of effects of SriLankan plants on human complement in vitro. Pharmaceurisch Weekblad
Boyum,
of leucocytes
Scandinavian
from
Journal of Clinical
blood
and
and Labora-
97). 394. De Chatelat, L.R.. Long, G.D., Shirley, P.S., Bass, D.A., Thomas, M.J., Henderson, F.W. and Cohen, M.S. (1982) Mechanism of the luminol-dependent chemiluminescence of tory Investigations
21 (Suppl.
human neutrophils. Journal of Immunology 129, I589- 1593. Ewetz. L., Palmbid, L. and Thore, A. (1981) The relationship between luminol chemihrminescence and killing of Staphylococcus aureus by neutrophil granulocytes. Blur 43. 373-381. Jayaweera, D.M.A. (1982) Medicinal Plants Used In Ceylon, Vols. 1-5, National Science Council of Sri-Lanka Publications, Colombo. Klerx, J.P.A.M., Beukelman, C.J. Van Dijk, H. and Willers. J.M.N. (1983) Microscopy for calorimetric estimation of complement activity in guinea pig, human and mouse serum. Journal of Immunological
indica
on membranes
in
SyCimSUndai’.
K.V.. Singh. B.. Thakur, R.S.. Husain. A., Kiso. Hikino. H. (1985) Antihepatotoxic principles of Phyllunthus niruri herbs. Journal (4 Etltnc)ppharnlacolog~ 14.
Y. and 414.
119, 901-902.
A. (1968) Separation
bone marrow.
aqueous extracts of Azadirachta vivo. Fitoterapia 56. I1 I-l 15.
Methods 63, 2 15-220.
Obaseki, A.O., Adeyi. 0. and Anyabuike, C. (1985) Some serum enzyme levels as marks of possible acute effects of the
Thabrew. M.I., Joice, P.D.T.M. and Rajatissa. W.C. (1987) A comparative study of the efftcacy of Osheckia octundro and Pavetta indicu in the treatment of liver dysfunction. Plunto Medica
53. 239-241.
Thabrew, M.I.. Jayatilaka. K.A.P.W. and Perera. D.J.P. (1988) Evaluation of the efftcacy of Melothria maderaspatana in the alleviation of carbon tetrachloride-induced liver dysfunction. Journal of Etlznoplturnluc(~logy 23. 305-3 12. Vallotton, J.J. and Frei. P.C. (1981) Influence of (+) cyaninadol-3 on the leucocyte migration inhibition test carried out in the presence of PPD and HBs Ag. Infection und Immunity
32. 432433.
Van der Nat, J.M., Klerx. J.P.A.M., Van Dijk. H., de Silva. K.T.D. and Labadie. R.P. (1987) lmmunomodulatory activity of an aqueous extracts of Azadirochtu indicct stem bark. Journul os Ethnophurmacology
Weir.
D.M.
(1983)
Immunology.
19, 125-l
Medicine and Biology, 5th edn.. Churchill
burgh,
pp. 18&218.
3 1.
An Outline
fiw Students q/
Livingtone.
Edin-
