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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

  1. INTRODUCTION
  2. SOURCES OF ANTI-DNAP VARIABILITY
  3. CULTIVATION
  4. CONCLUSIONS
  5. REFERENCES
  6. Table 1

INTRODUCTION

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sub-genera not reported as being used for symptoms of liver disease generally had much weaker inhibitory activity (data unpublished).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.

SOURCES OF ANTI-DNAP VARIABILITY

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 variance.

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 significant

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.

CULTIVATION

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.

CONCLUSIONS

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.

REFERENCES

Table 1. Phyllanthus subgenera, number of representative species, regions where found, number of species used as liver disease remedies and references.z

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
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.
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 aw