Table of Contents
Unander, D.W., P.S. Venkateswaran, I. Millman, H.H. Bryan, and B.S. Blumberg.
1990. Phyllanthus species: Sources of new antiviral compounds. p.
518-521. In: J. Janick and J.E. Simon (eds.), Advances in new crops. Timber
Press, Portland, OR.
Phyllanthus Species: Sources of New Antiviral Compounds
David W. Unander, P.S. Venkateswaran, Irving Millman, Herbert H. Bryan, and
Baruch S. Blumberg
- SOURCES OF ANTI-DNAP VARIABILITY
- Table 1
Phyllanthus has more than 700 species in at least 10 sub-genera
(Holm-Nielsen 1979, Webster 1956-58). A survey of 300 ethnobotanical
references of Phyllanthus spp. arranged taxonomically suggested some
uses were clustered by subgenus. A summary of the number of species per
sub-genus used traditionally in various places to treat possible symptoms of
liver disease (such as jaundice) is presented in Table 1. Among the herbs of
subgenus Phyllanthus used medicinally, we found that aqueous extracts of
P. amarus Schum. & Thom., P. debilis Klein, P. fraternus
Webster, P. niruri and P. urinaria L. all inhibited viral DNA
polymerase (DNAp) of hepadnaviruses in vitro. (Hepadnaviruses include human
hepatitis B virus and several animal hepatitis viruses.) Additionally,
extracts of less common species in the same sub-genus, such as P.
mimicus Webster or P. odontadenius Muell. Arg., also had such
anti-DNAp activity. Extracts of species from sub-genera not reported as being
used for symptoms of liver disease generally had much weaker inhibitory
activity (data unpublished).
The ultimate goal of our research is to develop a therapy for carriers of
hepatitis B virus (HBV) which would reduce the incidence of liver cancer.
Since its identification (Blumberg et al. 1967, Blumberg 1977), the virus has
been found to be associated with cirrhosis, chronic liver disease and primary
liver cancer as well as acute serum hepatitis. More than 200 million people
worldwide are estimated to be carriers, many of them asymptomatically (Ghendon
1987). A series of studies have shown that persistent HBV infection is
associated with a greatly elevated risk of developing liver cancer (Beasley et
al. 1981, Dodd and North 1987). Although a safe and effective vaccine exists,
and is being vigorously promoted, there is no effective therapy for carriers.
Aqueous extracts of P. amarus collected near Madras, India, (initially
published under the name P. niruri L.) inhibited viral DNAp in vitro.
In addition, they eliminated detectable virus from the sera of woodchucks
(Marmota monax) acutely or chronically infected with the woodchuck
hepatitis virus (WHV) (Venkateswaran et al. 1987). Endogenous DNAp is packaged
with viral DNA in hepadnaviruses like HBV or the closely related WHV. In a
small clinical trial in India, administration of powdered P. amarus
eliminated detectable antigen from the sera of 59% of the treated human
carriers as compared to 4% of controls (carriers receiving a placebo) who
cleared the virus naturally (Thyagarajan et al. 1988). Studies are in progress
to characterize the compound(s) responsible for these effects.
In Experiment 1, plants of P. urinaria and P. debilis were grown
in the greenhouse in a 2 x 2 factorial design comparing soils of differing
fertility (fertilized regularly with a 0.04N-0.06P-0.07K solution vs. not
fertilized) and different moisture and structure conditions (a commercial peat
mix regularly watered vs. sand infrequently watered). Six month old plants
were harvested, lyophilized and ground. Aqueous extracts were made using 5 g
ground plant in 20 ml water at 60°C for two hours, mesh-filtered,
centrifuged, and the supernatant passed through a 0.45 µm micropore filter
(Nalgene 125, Nalge Co., Rochester, NY). Dried whole and ground plants and
extracts were stored at -15°C. Extracts were made of two random samples of
ground plant per treatment combination. Activity was expressed as µg of
dry matter per ml of extract producing 50% inhibition of WHV DNAp, after
Venkateswaran et al. (1987), and analyzed after testing for homogeneity of
Various morphological variables (e.g., plant height weight, amount of
branching), were significantly affected by differing soil conditions (data not
shown). The only effects on inhibition of viral DNAp, however, were for gross
soil fertility on P. urinaria (84 and 38 µg/ml for fertilized and
unfertilized, respectively) and, in the pooled analysis, species (139 and 61
µg/ml for P. debilis and P. urinaria, respectively). Regression
analyses showed significant effects on P. urinaria of available N or P
or percent saturation of K. Genetic differences in viral DNAp inhibition were
greater than any effects from soil conditions.
In Experiment 2, plants of P. amarus were grown in the greenhouse in a 2
x 3 factorial design comparing soil moisture and structure conditions as in
experiment 1, and three levels of soil pH and in a 2 x 2 factorial comparing
soil moisture and structure conditions and high vs, low Ca levels. P.
amarus was commonly found in calcareous sites in Puerto Rico but much less
commonly in other soils, so a preference for high pH, high Ca or both was
hypothesized, and the effect of these variables on activity of plant extracts
against WHV DNAp was tested. Media of average pH 5.5, 7.5 and 10.5 were made
using aluminum sulphate, calcium sulphate and hydrated lime, respectively. The
treatments in sand had mean pH values about one log unit greater than those in
the peat mix (i.e., 11.5 vs. 10.5). To test the effect of Ca apart from pH,
pulverized limestone was added or not added to the sand or peat mix. In this
test, the pots with and without Ca had pH means of 8.0 and 7.0, respectively.
Water samples taken from the bottom of pots during watering were used to follow
pH. A more acidic treatment was also attempted, but P. amarus did not
survive more than two weeks at about pH 4.5. In this experiment individual
plants served as replicates with three replicates in the first test and two in
the second. Extracts, assays and analyses were done as described.
Soil conditions significantly affected plant growth. Optimum growth seemed to
be in soil slightly above neutral and with high Ca, as expected. For the
effects of plant extracts on viral DNAp, soil pH as a main effect was
non-significant. Using data from both tests, the linear regression of DNAp
inhibition on soil pH showed an effect at P = 0.09. The R2 value (0.11),
however, suggested relatively little variability in the inhibition of viral
DNAp was explained by pH. Soil moisture or structure significantly affected
DNAp inhibition of plant extracts in the pH test (158 and 263 µg/ml for the
sand and peat respectively). The interaction of moisture and pH had an effect
at P = 0.10. Means over pH treatments ranged from 174-246 µg/ml with greatest
activity at higher pH, over all six treatment combinations, means ranged from
85-332 µg/ml (greatest and least activity at high pH, sand and low pH, peat,
respectively). Plants from high pH in sand were significantly stunted relative
to other treatments, with average dry weight per plant of 1.7 g vs. an average
for the other five treatments of 6.6 g. Plants at high soil pH in peat had
mean inhibition values (262 µg/ml) similar to those at lower pH, illustrating
the interaction observed between pH level and peat vs. sand. No regressions of
DNAp inhibition on specific soil macronutrient data were significant. In the
test examining high Ca vs. no Ca and sand vs. peat, neither main effect was
In Experiment 3, genetic differences among accessions were tested. Plants of
P. urinaria from seed collected in India, the Ivory Coast Hawaii, Puerto
Rico, Trinidad, Venezuela or Vietnam were grown randomized on one greenhouse
bench using the same growing regime. Based on an analysis of extracts derived
from two plants/seed source, there were highly significant differences among
seed sources in inhibition, with means ranging from 70 to 532 µg/ml. This
suggests that genetic variability for inhibition of DNAp exists in P.
urinaria. Plants of P. amarus from seed collected from two sites in
India and three sites in Puerto Rico were grown in a similar experiment with
three plants/seed source but seed source was non-significant at P = 0.25, with
means for viral DNAp inhibition ranging from 192 to 409 µg/ml. A third test
using the same procedure examined different accessions of P. amarus from
sites in Florida, India and Puerto Rico. No significant differences were
found; means ranged from 122 to 163 µg/ml. These latter two tests were
respectively grown in the winter and the summer.
Although common in the tropics, the Phyllanthus species of interest are
generally small and often scattered in distribution. The only reference found
which discussed cultivation (Kangsu Medical Institute) recommended fertile,
well-drained soil for growing P. urinaria. To produce sufficient
quantities for large scale extraction, a system was developed at the University
of Florida Tropical Research and Education Center at Homestead, using black
plastic mulch and trickle irrigation Since plants with the strongest inhibition
of viral DNAp in our studies had consistently come from the warmest
environments, a tropical site was desired. Southern Florida was chosen as
being both tropical and efficient for shipment of harvested plant The soil at
Homestead, which is a crushed oolitic limestone, was also considered
well-suited to P. amarus. Wild plants of P. amarus were abundant
in fallow fields around Homestead following periods of rain.
The first plot served as an experiment to establish P. amarus as a row
crop and to test two fertility levels. Plots were fertilized with 560 or 1120
kg/ha 6N-5.2P-10K before mulching in a randomized block design with three
replicates. Seed of P. amarus were sown using 1.5% cellulose gel
(N-Gel, Hercules Co.) at 0.15 g seeds/liter (100 seedweight = 0.015 g).
Approximately 50 cc of gell/hill were applied. Hills were holes set 30 cm
apart in the mulch. Hills were in four rows spaced 30 cm apart on raised beds
1.7 m wide for a planting density of approximately 7,500 hills/ha.
Six months after planting, whole plants weighed an average of 7.5 g
(lyophilized dry weight). Aqueous extracts were made as described. Extracts
were made of shoots and roots by fertility treatment and replicate. The
average DNAp inhibitory activity over all samples was 142 µg/ml. The two
levels of fertility did not significantly affect viral DNAp inhibition. Roots
were significantly more active than shoots (66 and 151 µg/ml respectively), but
roots only accounted for about 10% of the total dry weight. This range of
activity was consistent with earlier results obtained with plants collected in
the wild (Venkateswaran et al. 1987). Cultivation, irrigation and
fertilization thus did not reduce DNAp inhibitory activity over the far more
laborious collection from the wild. Stand establishment measured as hills with
any plants six months after planting, was only about 70% in this first plot.
Variables affecting seed germination and longevity have not yet been
well-characterized. Total potential production was estimated to be at least 40
kg/ha (dry weight) but should be substantially greater once stand establishment
can be improved. At the rates used by Thyagarajan et al. (1988), 1.8 kg of
dried whole plant would have provided a course of treatment for 100 persons.
P. amarus and closely related species appear to contain activity against
the endogenous DNAp of hepadnaviruses. This may be the basis for traditional
uses of these species against disease symptoms such as jaundice, which,
retrospectively could, in at least some cases, have been caused by hepatitis B
virus. This DNAp inhibitory activity was relatively unaffected by soil
conditions, but differed among accessions, suggesting that genetic variability
does exist for this trait. Plants proved easy to grow and biological activity
of the cultivated material was equivalent to material collected from the wild.
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Table 1. Phyllanthus subgenera, number of representative species,
regions where found, number of species used as liver disease remedies and
zSummarized from a series of papers in preparation by D.W. Unander and G.L.
Webster examining 300 ethnobotanical references from a taxonomic perspective
and incorporating recent taxonomic changes by G.L. Webster.
| || ||Location|
|Subgenus ||Approx. no. of spp. ||North America ||South America ||West Indies ||Africa ||Asia ||Australia ||Oceania ||No. spp. used as liver disease remedies|
|Isocladus ||70 ||x ||x ||x ||x ||x ||x ||x ||3y|
|Kirganella ||50 ||x ||x ||x ||x ||x ||x ||x ||1x|
|Phyllanthus ||140 ||x ||x ||x ||x ||x ||x ||x ||9w|
|Gomphidium ||155 || ||x || || || ||x ||x ||0|
|Conami ||20 ||x ||x ||x ||x || || || ||0|
|Eriococcus ||30 || || || || ||x || || ||0|
|Phyllanthodendron ||10 || || || || ||x || || ||0|
|Botryanthus ||15 || ||x ||x || || || || ||0|
|Xylophylla ||60 || ||x ||x || || || || ||0|
yKangsu Medical Institute 1975, Kirtikar and Basu 1975, Stehlé 1986.
xAl-Kindi ca. 850, Antarkar et al. 1980, His Majesty's Government of Nepal
1982, Kirtikar and Basu 1975, Oliver 1950, Said 1969.
wAmadeo 1988, Bunyapraphatsara 1987, Cruz 1965, Freise 1934, Gooding et al.
1965, His Majesty's Government of Nepal 1984, Kangu Medical Institute 1975,
Kirtikar and Basu 1975, MacRae et al. 1988, Morton 1981, National Institute of
Health 1977, Oliver 1960, Petelot 1954, Purl 1970, Singh 1986, Stehlé
and Stehlé 1962, Watt 1892.
Last update March 31, 1997