South Pacific Study Vol. 19, No. 1-2, 1999

Mangrove Halophytes: A source of antiviral substances
Mariappan PREMANATHAN 1), Kandasamy KATHIRESAN 2)
and Hideki NAKASHIMA 1)

Abstract

Mangroves have been studied for their antiviral properties. The mangrove plants were sampled from Pichavaram
in the southeast coast of India. The extracts of different portions of mangroves were tested
against four
RNA viruses., Newcastle disease virus, encephalomyocarditis virus, Semliki forest virus and humanimmunodeficiency virus and two DNA viruses - vaccinia virus and hepatitis B virus. A broad spectrum antiviral
activity was exhibited in bark ofand leaves ofIn general, plants
belonging to the family - Rhizophoraceae are the source of potential antiviral substances.

Key words: mangrove, virus, antiviral, polysaccharides, lignin (not more than five)

Introduction
Mangrove plants are being used in folklore medicine for treatment of several diseases(KIRTIKAR and BASU, 1935; CHOPRA
1956; DATTA and DATTA, 1982). Althoughmangrove plants are known for medicinal use, specific reports on their antiviral activity are
only a few (KIRTIKAR and BASU, 1935). Bearing this in mind, we have conducted adetailed study about antiviral activity of mangrove plants (PREMANATHAN 1991;
PREMANATHAN
1992a,b; 1993b,c). This paper consolidates our previous reports on
antiviral properties of mangrove halophytes.

Materials and methods: The mangrove plant samples were collected from
Pichavaram mangrove forest (Lat. 11 27 N; Long. 79 47 E), Tamil Nadu, India. They
were washed, shade-dried, and powdered. The samples were macerated with 70％ aqueous
ethanol followed by an exhaustive percolation of the material with the same solvent (VAN
DEN BERGHE, 1986). The solvent from the extract was removed under reducedpressure at 40 C. The solid obtained was used in an antiviral assay after dissolving in
1) Department of Microbiology and Immunology, Kagoshima University Dental School, 8-35-1 Sakuragaoka,
Kagoshima 890-8544, Japan.
2) Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai 608 502, Tamil Nadu,
India.

50

South Pacific Study Vol. 19, No. 1-2, 1999

dimethyl sulphoxide (DMSO), taking into account that the maximum concentration of
DMSO in the text solution should not exceed 1％. The solution was further diluted withglucosol solution for Newcastle disease virus (NDV) or phosphate buffer saline for hepatitis B virus (HBV) or maintenance medium [minimum essential medium (MEM) ＋2％
fetal calf serum (FCS)] for vaccinia virus (VV), encephalomyocarditis virus (EMCV)and Semliki forest virus (SFV) or RPMI 1640 medium ＋10％ FCS for humanimmunodeficiency virus (HIV).
: Antiviral activity against NDV was carried out in chorioallantoic membrane (CAM) culture. For the assay, 1 ml of glucosol solution containing 75 μg of plant
extract and 0.064 haemagglutination unit of NDV was poured into each tube containing
CAM pieces and incubated horizontally at 37 C for 48 h. Then the medium was taken out
and a haemagglutination titre was carried out. Inhibition of the haemagglutination by eachextract was calculated as follows:
C−T
Percent inhibition ＝ ―――― × 100
C
where C - virus control; T - treated with extract
Antiviral assay for VV, EMCV, SFV were conducted in CEF (chick embryo fibroblast),
LM (mouse fibroblast) and Vero (monkey kidney fibroblast) cells, respectively, employing tissue culture microtiter plates. The cells in the growth medium (MEM ＋10％ FCS)
were seeded into microtiter wells of 100μl volume and allowed to grow to a confluent
monolayer. Once the monolayer was formed, the growth medium was changed to maintenance medium and challenged with VV (50 pock-forming units/ml) or EMCV and SFV(10 tissue culture infective dose 50/ml) and incubated at 37 C for 120 min (VV) or 90
min (EMCV and SFV) for adsorption of the viruses. Then the medium was changed to
fresh maintenance medium with plant extracts and incubated further at 37 C for 72h (VV)or 49h (EMCV and SFV); then the monolayer was stained with crystal violet. The antiviral
activity of the extracts was determined by the reduction of plaques formed by VV or
inhibition of cytopathic effects (CPE) for EMCV and SFV, compared with controls as
follows:
C−T
Reduction of plaques (％) or CPE inhibition (％) ＝ ―――― × 100
C
where C - virus control; T - treated with extract
The binding activity of HBsAg (hepatitis B virus surface antigen) was assayed following
VENKATESWARAN(1987). Plant extracts were mixed separately with an equal volumeof sera positive for HBsAg, and the mixture was incubated for one hour at 37 C. The
mixture was assayed directly for HBsAg using the Auzyme II ELISA Kit (Abbott, USA).
Binding activity was expressed as the percentage decrease in the adsorption of the test
sample compared to that of the controls composed of 1:1 (vol vol) mixture of HBsAg

PREMANATHAN: Mangrove Halophytes: A source of antivial substances51

positive serum and PBS.
Antiviral assay for HIV was carried out in MT-4 cells using the MTT method. Briefly,
MT-4 cells were suspended at 3 × 105 cells per ml and infected with HIV at a multiplicity
of infection (MOI) of 0.01. The HIV-infected or mock-infected MT-4 cells were placed in
96 well microtiter plates (200 μl/well) and incubated at 37 C in a CO２ incubator in thepresence of the test substance. After 5 days, cell viability was quantified by the MTT assay,
as described previously (PAUWELS1987), from which 50％ cytotoxic concentration(CC50 ), 50％ effective concentration (EC50 ) and selectivity indices (SI ＝ CC50 /EC50 )were calculated.

Results and Discussion
Mangrove plant samples were extracted in aqueous ethanol and their antiviral activities
were evaluated. The results of the overall
screening are summarized in Table 1. The
extracts which showed more than 50％ of antiviral activity
are described in detail.
: Plant extracts were tested
against NDV in CAM culture andtheir gross antiviral activity was evaluated in terms of percent inhibition of
haemagglutination and is shown in Table 2. Of the 51 extracts, 10 were found effective
when treated along with the virus, by inhibiting the haemagglutination (HA) titer within arange from 60 to 100％. Bark and stilt root
showed minimum andmaximum HA inhibition, respectively.
: Plant extracts were tested
in CEF cell culture and their antiviral
activity was evaluated in terms of reduction in number of plaques by the extracts and the
50％ effective concentration (EC50 ) of each test sample was calculated. The results areshown in Table 3. Of the 51 extracts, only four showed anti-VV activity. The EC50 ranged
from 18.2/ ml in bark of
to 48.1/ ml in leaf of: Plant extracts were tested
against EMCV in LM cells andtheir antiviral activity was evaluated in terms of inhibition of cytopathic effect (CPE) andthe results are shown in Table 4. Of the 51 extracts, 13 showed anti-EMCV activity whenassayed along with the virus. The CC50 ranged from 32.3 /ml in hypocotyl of
to 326 /ml in leaf of
The EC50 ranged from 6.1 to 50 /ml
in bark and flower of, respectively. The SI ranged from 2.0 in leaf ofto 15.4 in bark of
The extracts which were found effective when treated along with the virus, were tested
further for their prophylactic activity against EMCV. The LM cells were first treated withplant extracts, washed after 24 h and then subjected to viral infection. The prophylactic
activity was evaluated in terms of inhibition of CPE and the results are shown in Table 5.
Of the 13 extracts, four were effective with the range of CPE inhibition. The EC50 ranged
from 7.5 /ml in bark of
to 21 /ml in leaf of
The extracts which were found effective when treated along with the virus, were tested

52South Pacific Study Vol. 19, No. 1-2, 1999

Table 1 . Results of antiviral screening of extracts of mangrove plants＊
Plant name

NDVa

Part

VVb

EMCVc

SFVc

HBVd

HIVc

Acanthaceae
calyx
corolla
fruit
leaf
root

＋＋
＋＋
＋＋＋＋

＋
＋

＋＋

NT
NT
NT
NT
＋＋

NT
＋＋＋
＋＋＋
NT
NT

＋
＋＋
＋

leaf

-

-

-

NT

＋＋＋

-

-

＋
-

＋
＋＋＋＋
＋
＋
-

NT
NT
＋
NT
NT

NT
NT
＋＋＋
＋＋＋
NT
NT
NT

＋
＋
NT
＋
＋
NT
NT

leaf
stem
leaf
leaf

＋
-

-

＋＋＋
＋

＋＋
NT
NT
NT

＋＋＋
NT
NT
NT

＋
NT
-

Willd.

bark
leaf
stem

＋＋＋＋ ＋＋＋
-

＋＋＋
＋＋
-

＋＋
＋
NT

NT
NT
NT

＋
NT

L.

flower
fruit
leaf

＋
＋＋＋
-

＋
＋＋
＋＋＋＋ ＋＋
-

NT
NT
-

NT
＋＋
＋＋＋＋

(L.) Blanco

fruit
leaf

＋＋＋＋ ＋＋＋(L.) Blume

bark
fruit
hypocotyl
leaf
bark
fruit
hypocotyl
leaf
barkflowerfruit
hypocotyl
leafstilt root
flowerleaf
stilt rootbark
flowerfruit
hypocotyl
leafstilt root

＋＋＋＋
＋＋＋
＋＋＋
＋＋
＋
＋
＋＋
＋
＋＋
＋
＋＋
＋
＋＋
＋＋＋
＋＋
＋
＋
＋＋＋＋

leaf

-

(L.)

Aizoaceae
L.
Avicenniaceae(Forssk.) Vierh. barkflowerfruit
leaf
Pneumatophore
bark
L.
leaf
Pneumatophore
stem
Chenopodiaceae
Roxb.
(L.) Dumort
Forssk.
Combretaceae

Euphorbiaceae＋

Myrsinaceae
-

＋＋＋
-

NT
＋＋＋

＋＋
＋＋＋＋

＋＋
＋
＋＋＋
＋＋
＋＋
＋＋
＋＋＋
＋
＋＋
＋
＋＋
＋
＋＋
＋＋
＋＋＋
＋＋

＋＋＋
＋＋＋
＋＋＋
＋
＋＋
＋＋＋
＋＋＋
＋
＋＋
＋＋＋＋
＋＋＋＋
＋＋＋
＋
＋＋
＋
＋＋＋＋

＋＋
＋＋
NT
＋＋＋＋
＋
＋＋＋＋
＋
＋＋
＋
＋
＋＋＋
＋＋
＋＋＋＋
＋＋
＋
＋
＋
＋＋＋

＋＋＋＋
＋＋＋
NT
＋＋＋＋
＋＋＋＋
＋＋＋＋
NT
＋＋＋＋
＋＋＋＋
＋＋＋＋
＋＋＋＋
＋＋＋
NT
NT
＋＋＋＋
＋＋＋＋
NT
＋＋＋＋
＋＋
＋＋＋
＋＋＋＋
＋＋＋
＋＋＋＋

＋
NT
NT
＋＋
＋＋
NT
NT
＋＋＋＋
＋＋
NT
NT
NT
＋＋＋＋
＋＋
NT
＋＋＋＋
＋＋
＋＋＋＋
NT
NT
NT
＋＋＋
＋

-

-

NT

NT

-

Rhizophoraceae(Griff.) Ding Hou

BlumeMontr.
Poir.

Sonneratiaceae
B. Ham.

No activity＋
1-24％ activity＋＋
25-49％ activity＋＋＋
50-74％ activity＋＋＋＋ 75-100％ activityNT
Not tested＊

data taken and modified from Premanathan

a％ Inhibition of haemagglutination
b％ Plaque reduction
c％ Inhibition of cytopathic effectd％ Binding inhibition

1992

PREMANATHAN: Mangrove Halophytes: A source of antivial substances

Table 2.

gross anti-NDV activity of mangrove plants＊

Plant name＊data taken from Premanathandata taken from Premanathan

Table 4.
Plant namePartdata taken from Premanathan

root
fruit
leaf
bark
flower
leaf
fruit
bark
bark
Stilt root

90
85
72
90
70
62
70
76
60
100

CC50 (

ml)

EC50 (

ml)

3.8
3.1
3.9
4.7

18.2
33.3
48.1
18.5

68.5
102.3
189.3
87.1

SI

1994a

anti-EMCV activity of mangrove plants＊
Part
leaf
bark
fruit
leaf
fruit
bark
flowerhypocotyl
leaf
barkflowerstilt rootleaf＊

HA inhibition (％)

anti-VV activity of mangrove plants＊

bark
leaf
leaf
bark
＊

Part

1993a

Table 3.
Plant name

53

1994b

CC50 (

/ml)

326.0
111.0
82.4
115.0
132.0
72.5
78.9
32.3
119.0
92.5
162.0
88.5
62.1

EC50 (

/ml)

46.7
14.6
25.0
23.1
16.7
8.4
28.6
10.0
30.0
6.1
50.0
18.7
30.8

SI
7.0
7.6
3.3
5.0
8.0
8.5
2.8
3.2
4.0
15.4
3.2
4.7
2.0

54

South Pacific Study Vol. 19, No. 1-2, 1999

Table 5. Prophylactic anti-EMCV activity of mangrove plants＊
Plant name

Part
leaf
bark
Stilt rootbark＊data taken from Premanathandata taken from Premanathan

115
93
88
76

EC50 (

/ml)

21.0
7.5
17.9
8.0

SI
5.5
12.3
4.9
9.4

anti-SFV activity of mangrove plants＊
Part
leaf
leaf
fruit
flowerbark
Stilt root

＊

/ml)

1994b

Table 6.
Plant name

CC50 (

CC50 (

/ml)

139
209
112
143
110
111

EC50 (
14
18
18
36
6
15

/ml)

SI
10
11
6
4
18
8

1995a

further for their therapeutic activity against EMCV. The LM cells were treated with virus
for 6 h and then were treated with extracts. Of the 13 extracts, only the bark of
was found effective with 70％ inhibition of CPE and with the EC50 value of 8.57
/ml (PREMANATHAN
1994b).
The extracts which were effective in prophylactic condition were tested
in Swissalbino mice at the rate of 31.25 mg/kg animal/day (0.5 mg/mouse/day). Only the extract ofbark ofshowed 30％ protection and 3.2 days increase in average survival
time (AST) (PREMANATHAN
1994b).
: Plant extracts were tested
against SFV in Vero cells along withthe virus. The antiviral activity was evaluated in terms of inhibition of CPE and the results
are shown in Table 6. Of the 36 extracts, six showed anti-SFV activity. The EC50 ranged
from 6 /ml in bark of
to 36 /ml in flower of
The extracts which were effective
were tested
in Swiss albino mice at therate of 31.25 mg/kg animal/day (0.5 mg/mouse/day). Bark ofprotected themice to an extent of 40％ with 5.8 days increase in AST. The stilt root of
could protect only 10％ with 1.7 days increase in AST (PREMANATHAN
1995a).
: Plant extracts were mixed at a concentration of 3 mg/ml with surface
antigen of HBV (HBsAg) and then allowed to react with antibody to HBsAg (anti-HBs).
The binding activity of the extracts was determined in terms of percent decrease inabsorbance at 492 nm as compared to control. The results are shown in Table 7. Of the 28
extracts, 24 exhibited binding activity with HBsAg and inhibited the reaction of HBsAg
with anti-HBs. The maximum and minimum inhibition was observed in fruit ofand fruit of, respectively.
: Plant extracts were tested
against HIV in MT-4 cells and theirPREMANATHAN: Mangrove Halophytes: A source of antivial substances

55

Table 7. Effect of mangrove plant extracts at 3 mg/ml on the binding of HBsAg to＊anti-HBs
Plant namePart

Inhibition of binding (％)

corolla
fruit
leaf
fruit
leaf
bark
fruit
leaf
bark
fruit
leaf
bark
flowerfruit
hypocotyl
flower
leaf
bark
fruit
hypocotyl
leaf
Stilt root
leaf
leaf

61.2
56.8
58.5
56.1
63.8
91.7
53.4
83.3
92.2
93.0
67.7
84.3
89.2
87.5
60.6
89.0
84.3
85.4
70.8
91.3
54.0
91.0
68.0
68.4

＊

data taken from Premanatham, 1991

antiviral activity was evaluated in terms of inhibition of cytopathic effects (CPE) as measured by MTT assay and the results are shown in Table 8. Of the 34 extracts, seven showedanti-HIV activity. The EC50 ranged from 7.3 /ml in leaf ofto 492.3/ml in leaf of
The SI ranged from 1.6 in leaf ofto 16.2 inleaf of
The active plant samples were further extracted in different solventsand tested for anti-HIV activity. Alkaline extracts were found effective (PREMANATHAN
1995b).
The overall screening of the mangrove plants reveals that plants from the family
Rhizophoraceae were more effective than the plants from other family. Fourteen plant
extracts were effective against any one of the viruses. Eight were effective against any twoviruses. The most promising extracts were bark ofand leaf ofwhich were effective against six and five viruses, respectively. Leaf of
stiltroot ofwere effective against four viruses. Bark and fruit ofleaf ofbark ofand leaf of
were effective

56South Pacific Study Vol. 19, No. 1-2, 1999

anti-HIV activity of mangrove plants＊

Table 8.
Plant name

Part
leaf
leaf
leaf
leaf
leaf
bark
leaf

＊

data taken from Premanathan

CC50 (

/ml)

48.3
216.5
77.8
998.2
331.5
38.8
798.4

EC50 (

/ml)

29.3
13.4
7.3
108.5
139.2
7.6
492.3

SI
1.6
16.2
10.7
9.2
2.4
5.1
1.6

1996

against three viruses. In general, plants belonging to the family Rhizophoraceae are thesource of potential antiviral substances. To our knowledge, none of the aforementioned
extracts have yet been reported by others as antivirals in literature. Our work reports for the
first time that mangrove halophytes have antiviral activity.
We have partially purified the active substances from the bark ofand leafofand identified the active substances are acid polysaccharides(PREMANATHAN
1999; PREMANATHAN
unpublished data). These acid polysaccharides blocked the virus binding to cells as mechanism of action for its anti-HIV activity(PREMANATHAN
1999). Lignins were identified as active substance from the leaf offor its anti-HIV activity (PREMANATHAN
unpublished data).
Indigenous drugs for the treatment of HIV infection are urgently needed as currently
available synthetic anti-HIV drugs are very expensive and limited in supply. Natural substances from plants are widely used in medicine in India. It is, however, essential that the
mangrove halophytes as source of new antiviral leads should be explored further.

Acknowledgment
M. P. is Research Resident of Japanese Foundation for AIDS Prevention, Tokyo. Part of
the work was supported by the Kagoshima University Project: Grants for interactive studies
on food, heath and environmental Science in Kagoshima.

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