NATURAL MEDICINES FOR NAHINGA

edited by Prof. Nelson IMUNE

As our society develops more and more disdain for synthetic chemicals and synthetic chemo-phobia, there is also a growing appreciation for the natural medications. This is a basic right and freedom to choose natural medicine. There is more than substantial proof and strong evidence to support this choice. But, There are those from the synthetic chemical companies who do not want freedom of choice. They have lots of money and thus they can buy powerful friends. They can and do purchase influence and even bribe away human rights.

The Bible says that the healing of the nations will come from the leaves of the field. No nation has ever needed this more than the nations of Africa. An inexpensive easily accessible natural medicine is needed more than ever. With this in mind this set of studies is designed for scientific and intellectual defense of the natural medicines needed for saving people.

There is no profit motive hare as that this formula and science was developed as a gift to society, a gift to Africa, a gift to the world by Nahinga.

Phytolacca dodccandra: Continued interest in Endod (Phytolacca dodccandra) as the leading

plant-derived molluscicide and anti HIV retro-viral led both to raised hopes, even expectations, and to disappointment and frustration. Despite the many studies published on Endod toxicity--and the increasing data suggesting its relative safety as well as suitability--a standard reference point for the toxicological studies on Endod had not been established (a standard stock or done of the plant). Nor had standard procedures been followed on the Endod toxicity studies, although much useful data had been collected. In the increasingly vigilant and demanding arena of environmental pollution studies, other less standardised procedures no longer were acceptable. Endod workers were fully aware of the need for rigorous toxicity testing before it could be recommended for widespread use. But work was done in scattered facilities, some isolated and ill-equipped, following various standards and protocols.

The stimulus of an impartial, internationally recognised team of experts clearly was needed to set-gut the guidelines and firm up the standards for subsequent toxicologic study. No new molluscicide can be accepted as suitable for human and other non-target animal and plant expo-sure without adhering to such a standard. The availability--and acceptance--of such a standard is the only available path leading to credibility and acceptance of Endod as a valid test molluscicide. Just as the UNDP/World Bank/WHO meeting preceding the first workshop brought plant-derived molluscicides within scientific focus, so did the toxicology conference of experts set the stage for the current workshop. This will be reviewed and summarised in the present report.

One aspect of the two Endod workshops worthy of emphasis is that both meetings took place in Africa, far from the main research centers, in areas of high

disease endemicity--and great need. The plant involved is African (adaptable to Brazil as well), much of the interest is from scientists in endemic regions and the potential savings in bard currency will be to pressured economies in endemic areas. This combination of &cum-stances suggests local use, based on local need and responsibility, leading to locally formulated and tested applications. This is the key to the underlying appeal of Endod. If a suitable molluscicidal product could be locally produced and applied--and do the job--it might be possible to solve the great problem of continuous snail control: endemic long-term continuity of effort, based on local acceptability, production and application. In a word, self-help. in another word a gift to the Samgoma

The history of periodic foreign-funded and foreign-directed efforts using costly imported products and technology have been repeated time, and again. The results have been sadly consistent: rapid snail reduction! followed by equally rapid repopulation by these adaptable hermaphroditic' molluscs after the control team leaves. Reinfection of snails with schisto some parasites is assured within weeks to a few months after resurgence of the snail populations. No one can be justified in assuming that a product locally grown, prepared and applied will prove any more successful. But one can certainly point out possible advantages, establish some guidelines and test these possibilities. That is what this book is all about.

Brief Overview of Endod Studies Agronomic Studies

Over the last two years, selected new as well as known strains of the Endod plant

from Ethiopia, Phytolacca dodccandra, have been carefully introduced in Zambia and Swaziland by an international team of agronomists led by Prof. J. Lambert of Canada and Dr. Legesse Wolde-Yohannes of Ethiopia. In these new sites, the plants have proven to grow successfully, maintaining their high molluscicidal potencies and detergent properties. Mass cultivation of these strains for large-scale production of berries to be used for extraction and field trials are currently underway in all three countries.

These studies are being supported in part by small grants from the European Economic Community (EEC), the Canadian International Development Agency (CIDA), the University of Swaziland, the Zambian National Council for Scientific Research, the Ethiopian Ministry of Industry and Addis Ababa University.

There are successful large-scale Endod farms--in Holeta, 30 km north of Addis Ababa, systematically cultivated by Dr. Wolde-Yohannes; in Manzini, Swaziland at the College of Agriculture under the supervision of Dr. Lydia Makhubu, Acting Vice-Chancellor of the University of Swaziland; and in Lusaka under the direction of Dr. S.M. Silangwa, Secretary-General of the Zambian National Council for Scientific Re-search. These farms demonstrate the potential of mass production of Endod for local use and as a cash crop. In a parallel study being carried out by Professor Aluizio Prata at the University of Brasilia, the plant has also been successfully introduced and is growing well in Brazil, where schistosomiasis is a crucial public health problem.

Toxicological Studies

Along with the agronomic work, short- and long-term toxicologic studies are being undertaken by scientists in the United States, Brazil, Canada and Ethiopia. Early findings from these studies include the following: 1. Extensive mutagenic studies have been completed and published by Professor Norman It. Farnsworth of the World Health Organization (WHO) Collaborating Center for Traditional Medicine, College of Pharmacy, University of Illinois, Chicago. These studies have established that neither the water extracts nor any of the various alcohol extracts of Endod tested have mutagenic properties under the standard-ised test conditions in his laboratory. His findings significantly modify a 1983 UNDP/World Bank/WHO report (TDR/SCH.SWG(4)/83.3), which considered water extracts of Endod to be non-mutagenic but alcoholic extracts mutagenic. The work of Dr. Farnsworth established that neither are mutagenic. The full paper on this work has been published by J.M. Pezzuto, S.M. Swanson and N.R. Farnsworth in Toxicology Letters in 1983. No Mutagenic properties found. 2. Acute and chronic toxicologic studies of Endod in different animals and various aquatic biological systems have been underway since 1983 at the University of Brasilia. Professors Aluizio Prata and Fernando Ubatuba have been conducting this work with the financial support of the Brazilian Academy of Sciences. Preliminary results support earlier toxicologic findings from work done in Ethiopia and at the Stanford Research Institute in California, where a very low order of toxicity in test animals and biological systems was found. These and the agronomic studies on Endod in Brazil are continuing. 3. Acute and chronic toxicologic studies underway for several years at the Institute of Pathobiology in Addis Ababa by Dr. Ephraim Mamo, a veterinary pathologist, tested Endod in sheep, monkeys, dogs and other animals. These tests are also encouraging in terms of the low order of toxicity found. The tested animals showed high tolerance to doses of Endod extract, well above likely exposure levels when administered orally or even intravenously. These findings strongly counter earlier unverified statements that Endod is unsafe as a molluscicide and even poisonous to domestic animals. 4. Piscicidal (fish killing) properties of Endod remain of concern--a characteristic of all present-day moluscicides, which are comparably piscicidal at molluscicidal concentrations.

A proposal from Dr. Ronald Zweig, Director of Aquatic Studies, New Alchemy Institute, an environmental research center in Massachusetts, requested funds for a broad-scale comparative toxicologic study of the effects of Endod, Niclosamide and several commercially available pesticides on fish and other elements of the freshwater fauna as well as on aquatic plants and algae. So far, no funds have been found to support this work either from WHO or other sources.

Use at a potecy of 5x (one part per hundred thousand) has been shown perfectly safe for all. A 4x (one part per ten thousand) safe for most adults. More concentrated forms (3x) are somewhat Noxious not toxic. But 2x and 1x are not reccomneded. The 4x or 5x, will take a minimum use of every day for one month, two months recomended for successful anti-viral interference. Us of 5x imediantly after exposure to the virus have been shown to be quite effective (95%) in stopping the virus. so this Hemo A formula is a very effective morning after

remedy. This alone could save the nation if it's use was wide spread.

Detergent Studies

Based on encouraging results from preliminary studies under my direction at the Steifel Research Institute, a detergent formulation and testing laboratory in upstate New York, interest has been generated and has stimulated collaborative work in Ethiopia by scientists from Addis Ababa University, the Industrial Projects Services of the Ministry of Industry, as well as the National Chemical Corporation which operates the National Detergent and Soap Factories at Rappi (near Addis Ababa).

It is now well established that either the water or butanol (or any alcohol) extracts of Endod can be used as effective substitutes for dodecyl benzene sulfonic acid (DDBSA), a petrochemical by-product, as a surfactant in commercial detergent formulations that are particularly good for fine grades of cotton, linen and wool. The extraction process used for the detergent preparation is basically the same as that used for preparation of the molluscicide. The Endod extract prepared for detergent use can, therefore, also be used without any further processing for snail control purposes.

In two pilot-project runs conducted at the Rappi Soap Factory, Endod extract was substituted for DDBSA at 20 to 30 per cent of the total weight (consisting of "builder", wash-alkali, buffer, soil-suspending agent and brightener, the normal constituents of commercial detergent formulation). Two hundred kgs of Endod-containing detergent powder was prepared using this standard procedure with equipment already in use in the factory. The product was examined critically for its cleansing proper-ties in comparison with the DDBSA-based detergent (prepared at the same 20 to 30 per cent level). The Endod-based product proved quite comparable to the commercial one--a highly encouraging development!

Given these results together with the fact that Endod is more rapidly biodegraded than is DDBSA, the new Endod-based product may prove to have a commercial application and sales potential as a specialty detergent in both developed and developing countries. As a result of substituting a locally grown product for a costly imported material, Ethiopia could save up to US$25 million per year in foreign currency, which it now must spend to buy DDBSA. Comparable savings on other hard-pressed African economies could prove extremely important. Furthermore, a commercial incentive for upgrading the traditional use of Endod as a soap could make Endod an income-generating agricultural crop for farmers as well as a profitable locally manufactured product. This should make the material more readily available for its other important use: as a molluscicide for the control of schistosomiasis.

To protect this potential commercial use of Endod as a detergent, an application to patent the process has been filed in the United States, Europe and Japan. Returns from patent royalties, if any become available, will go to an "Endod Foundation", which in turn can support additional R&D on natural products in Africa.

Although these preliminary results are promising, many aspects of R&D, of product formulation and testing and of market research must be done on these Endod detergents and on other possible applications of Endod. These developmental studies, however, cannot be undertaken without additional financial support.

Studies on the Geographic Distribution and Chemical Characterisation of Endod

In another encouraging follow-up from the Lusaka Workshop, Dr. Robert A.

Adams (Hardin-Simmons University, Sandy, Utah) and Dr. Robert Parkhurst (Stanford Research Institute, Menlo Park, California), along with collaborating scientists in Ethiopia, Zambia, Swaziland and several other African countries, are commencing a study of the geographic distribution and the ecological and chemical characteristics of different genotypes of Phytolacca dodecandra to be collected from different parts of Africa. In a collaborative three-year study, funded by the United States National Science Foundation, plants will be collected from different countries and regions in Africa, their chemical structure and botanical identity determined and then tested for growth in different ecological areas.

Results should provide basic information from which useful new data on the ecological and chemical nature of different strains and species of the Endod plant can be derived. It will also provide an essential enlarged genetic pool for future crossbreeding experiments and the selection of improved strains of Endod to be used in different localities and for various other purposes. New tissue culture techniques, employed in Dr. Adam's laboratories, will both speed up the development of new strains and make possible comparative studies that would otherwise require many years and large acreages of specific soils for development of the various test crosses.

The World Health Organization

In January 1983, WHO looked into the possibility of plant molluscicides serving as a less costly substitute for Niclosamide. A special meeting of the UNDP/World Bank/ WHO Schistosomiasis Scientific Working Group (TDR/SCH.SWG(4)/83.3) was convened to review and evaluate studies on molluscicides of plant origin. These experts concluded that Endod was both the most studied and the most promising of the plant molluscicides they evaluated, and recommended that support should be given for further toxicologic, agrobotanic and other studies on Endod and on other plant molluscicides.

With respect to further toxicological studies, available data favour the probability that the compound is safe as a molluscicide and detergent for washing clothes (for which it has been traditionally used for centuries in many parts of Africa). However, I strongly concur, along with many others, that a thorough scientific study of its quantitative toxicity to humans, other animals and plants must be undertaken, and that all toxicologic studies done so far should be critically evaluated by competent toxi-cologists. Furthermore, in determining the toxicity of Endod to fish and other aquatic flora and fauna, I believe such studies should be done in parallel with Niclosamide and others, so Endod can be tested in direct comparison with approved and commercially available molluscicides and pesticides. All of them, for example, have some degree of toxicity to fish.

Support by Other United Nations Organisations

Other United Nations organisations, notably the United Nations Children's Fund (UNICEF), the United Nations Industrial Development Organization (UNIDO) and the United Nations Financing System for Science and Technology for Development (UNFSSTD), view the Endod project positively as a possible means of self-help for health and to stimulate local commercial development of other Endod products. UNICEF, for example, has officially expressed its interest in the development and wide use of Endod for the control of schistosomiasis on a community self-help basis in rural areas, where children are the primary targets of the disease. In 1982, the Executive Board of UNICEF approved a "noted" project of $2,000,000 for Endod studies in certain African countries. However, owing to lack of support and for other reasons (reservations by WHO), the project was not funded.

UNIDO's interest is in possible industrial production and processing of Endod for dual use as a molluscicicle and as a detergent, The UNIDO Investment Promotion Service in New York has attempted to identify potential investment partners from Canada and Norway. The initial search has been taken over, in a somewhat broadened form, by another United Nations organisation, UNFSSTD, which has shown an interest in turning the Endod project into a research, development and demonstration pilot project, involving the governments of Ethiopia, Zambia and Swaziland, as a follow-up to the Lusaka Workshop.

The UNFSSTD project, $1,750,000 for a four-year study in which UNIDO would play a major role, has been officially submitted for consideration by the governments of Norway, Canada and Belgium. The project is currently under review and evaluation by these governments.

Another country with a sustained interest and willingness to help in the development and use of Endod is the Netherlands. From 1976 through 1981, a dedicated, highly competent Dutch agronomist/natural product chemist, Dr. Ch. B. Lugt, in co-operation with Ethiopian scientists at Addis Ababa University, did most of the fundamental botanical/agronomic work that has been done on Phytolacca dodecandra, collecting over 67 different strains with high molluscicidal potency and improved growth characteristics.

Through Dr. Lugt, Professor C.FA. Bruijning, the well-known Dutch parasitologist, and Professor J. Koeman, an equally highly regarded toxicologist from Holland, also were involved in different aspects of Endod studies. In 1981, on the basis of a thorough evaluation of the work done to date on Endod, they joined in strongly recommending to their government support for the project at a multimillion-dollar level. The proposal, favourably considered by a Dutch evaluation team and subsequently officially offered by the Dutch government to Ethiopia, unfortunately did not materialise, owing to the political situation in Ethiopia at that time. The situation has now changed and the research support interest of the government and its institutions are now favourable to such a project. Dr. Lugt and other Dutch scientists have expressed continued interest in reconsidering this study, suggesting that it may also be possible to reactivate the Dutch government's interest and financial support.

Expert Group Meeting to Evaluate the Toxicity of Endod

As a consequence of the widely recognised need for independent standardised toxicologic evaluation of Endod, UNFSSTD and IDRC sponsored an "Expert Group Meeting to Evaluate the Toxicity of Ended". The meeting was held at the United Stations in New York, 27-28 February. A number of prominent toxicologists from different countries were invited. They were asked to review and assess toxicologic work done to date on Endod, establish criteria and protocols for further study, point out the chief gaps, and analyse the risks of immediate limited-scale use. The development and the rationale for a pre-registration "Harvard Evaluation" for Ended for its possible use as a molluscicide and a detergent were among the principal evaluation objectives.

This meeting proved to be highly successful. The full report will be presented by Dr. John Bruce, Director of the Center for Tropical Diseases of the University of Lowell and rapporteur of the meeting. On the basis of this, I believe we now have the opportunity to proceed with rigorous standardised tests and get the long-delayed development phase underway.

The Task Ahead

The primary task for the Second International Workshop on Ended is to review and implement the recommendations of the Expert Group Meeting in New York on Endod toxicology, assist in launching a national project on Endod in Swaziland, establish an "Endod network" of countries and institutions involved in Endod research and application, and review and critique current research proposals for research support from UNFSSTD and UMCEF.

PREVALENCE AND CONTROL OF

SCHISTOSOMIASIS IN SWAZILAND

Jean-Paul Chaine, Doctor of Public Health

Schistosomiasis, commonly called bilharzia in Swaziland, is a disease of man and animals caused by blood flukes called schistosomes. Three species of schistosomes commonly infect man: Schistosoma japonicum, limited to Asia; Schistosoma mansoni in Africa, South America and the Caribbean; and Schistosoma haematobium in Africa and the Middle East. In addition, several species are found with limited distribution, such as Schistosoma mekongi in Laos and Cambodia, Schistosoma intercalatum in Central Africa and Schistosoma mattheei in Southern Africa. It should be noted that schistosomes are not only found in man, but also in birds, rep-tiles and many mammalian species. Cercariae from these non-human schistosomes usually are immobilised in the skin of man, causing a dermatitis or "swimmer's itch', thereby reaching a dead end in their life cycle.

Life Cycle of Schistosomes

All schistosomes have a complicated life cycle with strict biological requirements. Each has a snail intermediate host, i.e., a part of the life cycle is spent developing in a suitable snail. Adult worms, male and female, live together in the blood vessels of man and produce eggs that are passed either in urine or faeces, depending on the species. The eggs hatch on contact with fresh water, releasing a ciliated miracidium that must penetrate an appropriate snail host within about six hours. The organism proliferates rapidly within the snail and a month later releases numerous fork-tailed swimming larvae--the cercariae, the form infective to man by direct invasion of the intact skin. Thus, a single egg produces a miracidium that infects a single snail, but this infected snail continues to shed thousands of cercariae for as long as it lives. To survive, these cer-cariae must find and penetrate a human host within one or two days. The cercariae migrate in the blood stream to the lungs, then the liver, where they develop into adults in the portal vein. Maturing adults, male and female, remain in copulation and migrate into the pelvic or mesenteric veins to complete the cycle by laying numerous eggs, which pass through the fine veins or capillaries. The eggs that work their way into the lumen of the gut or bladder pass to the outside in faeces or urine, hatch and seek a proper snail to start a new cycler-It is, incidentally, the eggs left behind, caught in human tissues, that induce the disease of schistosomiasis.

Prevalence Survey

There are three species of schistosomes that infect man in Swazi-land: Schistosoma haematobium, Schistosoma mansoni and Schistosoma mattheei. Each causes an epidemiologically distinct disease affecting different organs in the human host varying with the mode of passing eggs. Each also requires a different snail intermediate host and has a different geographic and demographic distribution. Recently, a National Schistosomiasis Prevalence Survey was conducted in Swaziland focussing on primary school children, the age group that throughout Africa as well as in Swaziland has the highest infection rates. A total of 2,035 students from 36 schools were included in this 1982 survey.

Schistosoma haematobium is responsible for urinary schistosomiasis and is the most common human schistosome in Swaziland. Very few cases (5 per cent) were seen in the Highveld and these are believed to have been imported from other parts of the country. The snail intermediate host was rarely found in the Highveld, but was common in the Middle and Lowvelds. During the survey, 34 per cent of school children tested in the Middleveld were found to be infected with S. haematobium, while the prevalence was 27 per cent in the Lowveld (Table 1).

In most areas, the population at greatest risk was the 10 to 14 year-olds, who had a 20 per cent prevalence. However, even the 1 to 4 year-olds of the highly endemic Lomati Basin had a 23 per cent prevalence. Surveys of adults showed that prevalence declined sharply in persons over 20 years old, even in highly endemic areas, presumably as a result of an acquired immunity and reduced water contact, although the relative role of each is controversial (see Table 2).

As shown in Table 3, provision of piped water and pit latrines substantially reduces prevalence of urinary schistosomiasis. Standpipes located well away from transmission sites reduce daily exposure to schistosomes of those who do the laundry or fetch water for household use. It is unlikely that pit latrines are used regularly for urination, but families with pit latrines probably have a higher awareness of sanitation and personal hygiene, which results in better health-related practices.

The Schistosoma mansoni distribution is confined for the most part to the Lowveld of Swaziland (Table 4). The three positive cases found in the Highveld were well scattered in three separate schools and are imported. In the Middleveld, prevalence was 2.3 per cent of the 511 stools examined. Only one school in the Middleveld had a prevalence more than 5 per cent. Intestinal schistosomiasis in the Lowveld showed an 18.3 per cent prevalence, although distribution within the Lowveld varied considerably (Table 5). This distribution is due to the presence of year-round surface water in the northern Lowveld and the relative absence of surface water in the central and southern Lowveld.

The age distribution of S. mansoni is basically the same as S. haematobium, except that adults have a more gradual decline in prevalence. Table 6 shows that the 10 to 14 year-old age group had the highest rates; thereafter, the rates declined slowly with age. In a separate study in the Lomati Basin, where 983 people of all age groups were examined, peak prevalence occurred in the 15 to 19 year-old group, with a 75 per cent infection rate, and adults over 30 years still had a 48.5 per cent infection rate.

Contrary to results expected, the highest prevalence of S. mansoni (15.8 per

cent) was seen in children from homes with access to piped water and with pit latrines (Table 7). The reason for this phenomenon is unclear, although the presence of both piped water and latrines in the National School Survey is heavily associated with worker housing compounds on the sugar estates. There the increased surface water, i.e., retendon ponds, canals and drainage ditches, create year-round snail habi-tats which contain snails that may become infected by a small number of individuals without latrines or not making use of those available. The multiplication factor in the snails and the higher density of the human population around surface water in these areas may be responsible for the skewed results. Exposure to cercariae is due to recreational use of the water habitats, such as swimming, bathing and fishing.

The snail intermediate host of S. mansoni in Swaziland is Biomphal aria pfeifferi, which is not found in the Highveld. In the Middleveld, this snail is confined to the larger impoundments such as Matsapha Dam and the hot springs areas, where the populations can be very high in the warm season. In the Lowveld, this snail can be collected throughout the year in a variety of habitat types, e.g., streams, dams, irrigation canals and drainage ditches. Schistosoma mattheei is common in cattle, sheep and goats in Swaziland and may occasionally be found in man. Although in the survey, S. mattheei was found only in the northern Lowveld, it has been seen in the wines of people presenting themselves at the Central Health Laboratory in Mancini. These cases are generally from the Middleveld, a function of the laboratory catchment area, rather than the true distinction of the parasite. To date, no one has been found shedding eggs of S. mattheei without also shedding eggs of S. haematobium. Both species of parasite share the same species of snail host--Bu/inns (Ph.) africanus--and the distribution of S. mattheei seems to follow that of S. haematobium. The prevalence of S. mattheei is probably less than 1 per cent in the Swazi population.

Control of Schistosomiasis in Swaziland

Schistosomiasis control in Swaziland is the responsibility of a small unit in the Ministry of Health consisting of ten people, one van and a very small budget. The activities of the Bilharzia Control Unit (BCU) are aimed at the school-aged children, the population group most at risk. The primary activity of the BCU is to screen for schistosomiasis eggs in the urine or stool and to treat all positive cases. First priority is given to schools in the northern Lowveld, where prevalence is highest.

Mollusciciding, the chemical control of snails, is not very practical in Swaziland, owing to the widely scattered transmission sites, and due to the small MOH budget for control. Snail control activities are most effective where transmission sites are limited, well-defined and utilised by a large proportion of the people. These conditions are rarely found in Swaziland, and mollusciciding is confined to worker camps and dams in the Lowveld. It should be noted that two large chemical companies (Bayer and Shell) conducted large evaluation programmes in Swaziland in the 1970s. Both programmes had resources far greater than the present resources of the Bilharzia Control Unit, but

the programmes were discontinued when no decrease in prevalence of schistosomiasis could be demonstrated. References Cawston, G.F. (1935). The Control of bilharzia infection in Swaziland. J. Trop. Med. & Hyg. 38 (24):305-306. Chaine, J.P. (1984). Schistosomiasis prevalence and control in the Kingdom of Swaziland. USAID Mission/Swaziland (Bureau for Africa) and American Public Health Association, Washington, D.C. Directions in Health Technology. (1982). PATH 2 (1). Eastman-Nagle. (1956). Schistosoma mansoni in Swaziland. Survey by rectal biopsy. S. Afr. Med. J. 30 (37):890-895. Gauldie, R.D. (1965). International report on bilharzia in Swaziland. The Health Office, Manzini, Swaziland. Green, E. (1982). A Knowledge, attitudes and practices survey of water and sanitation in Swaziland. Health Education Unit, Ministry of Health, Swaziland.

******* OUTLINE OF PRELIMINARY

DISCUSSIONS PRIOR TO SEPARATION OF PARTICIPANTS INTO WORKING GROUPS

April 7

Review of Endod Toxicology Meeting, 27-28 February 1986, at the United Nations in New York. Summary by Dr. Bruce, rapporteur of the meeting.

1. Key concern: standardisation of product, strain (done) methods of cultivation, of harvesting, drying, crushing, extraction and of pow-der preparation to ensure comparability of findings.

For example: Use of Ethiopian strain 44, picked at the appropriate immature stage, air-dried in dark, ground or crushed (comminuted), filtered/strained, sprayed or freeze-dried, assayed for LCgp (lethal concentration to kill 90 per cent of snails) level, following the WHO-prescribed protocol. Each step should follow the pre-scribed procedure and be accepted and adopted by other workers who are undertaking comparative tests. For the basic, definitive tests designed to determine and measure Endod toxicity on a rigorous comparable basis, the same batch of a standard Endod product should be used, one derived from a single harvest of about 1,000 kg, prepared in precise accordance with the accepted protocol. This material will then be made available to all labs for comparative toxi cologic study. It will be the comparison standard of Endod, to be called "Endod-S”.

2. Dr. Bruce also reviewed the conference recommendations for field molluscicide trials to follow WHO guidelines so that inter-country tests will be comparable.

3. There should be a risk-benefit evaluation and an environmental-impact study of Endod as a molluscicide On accordance with published FAO guidelines).

4. Earlier studies that were not in accordance with these standardised procedures should be repeated, using Endod-S as soon as it is available. Tests should emphasise acute oral toxicity. It should be possible to complete all of them within three to four years. Long-term chronic effects as well as full-scale mutagenicity, teratogenicity and other effects need not be undertaken so long as Enffdd is to be used as a molluscicide. If it is to be used internally in humans (as a spermicide, etc.) then a totally different set of far more rigorous requirements would apply.

5. Concurrent small-scale molluscicide trials can be undertaken during this period of standardised toxicity studies, at the same time that efforts are underway by various countries to expand the Endod agronomic and molluscicidal data base and fill gaps in our knowledge of the compound.

For example: The study of different methods and local variations in strain done selection, different methods of cultivation, harvesting, extraction, storing and field application procedures should proceed. These tests should be run wherever possible in comparison with a sample of Endod-S or, until the latter becomes available, its nearest equivalent, produced in accordance with the standardised protocol in order to achieve requisite quality and comparability. Other studies should also be undertaken to determine the long-term ecological effects as well as the efficacy of Endod against snails.

6. Comments by others: a) Dr. Lemma: Reviewed the background that led to the need for these

standardised guidelines, and the need for an independent evaluation by a team of experts, culminating in the recent February Endod Toxicology Meeting in New York.

b) Dr. Prato: Chronic studies, as well as acute toxicologic determinations, are needed to detect possible skin sensitisation after repeated exposure, especially among handlers and processors. Once these field and laboratory standards have been set up, truly international comparative tests can be run.

c) Dr. Parkhurst: Reviewed his paper on toxicologic effects of Endod on puppies and kittens (Appendix I-1). Conclusion: Although this was a small-scale effort made before procedural standards had been developed, the prompt emetic effect of Endod on dogs, cats and primates appeared to be the only demonstrable effects of the compound, unless massive doses are administered ("ingestion of 25 ppm [parts per million] in water would require the dog to drink four times its body weight to get an emetic effect").

d) Dr. Adams: It is essential that the clone of Endod to be used for testing be designated (now generally agreed that it will be Ethiopian 44). But we must also keep in mind the effects of environmentally induced differences (soil types, rainfall, local conditions). Also the effects of storage should be tested. Equally important is a "standard snail" for valid comparisons.

Therefore, we require a standard Endod source and production (Endod-S), with employment of standard laboratory procedures to follow, insofar as possible, GLP (good laboratory practice), although, as Dr. Parkhurst noted, this need not be carried to the extremes required for official GLP protocols.

e) Dr. Bruce: A "fingerprint" of the Endod saponins in terms of HPLC or

comparable chromatographic procedures should be made to determine the saponin portfolio and quantity that typifies each Endod strain, along with its bi-ological activity.

f) Dr. Lemma The real task of our Workshop has now been delineated: to agree upon applicable, practical and appropriate aspects of the New York Toxicology meeting resolutions that will provide the comparative Endod data base that is so clearly needed. The details of this selection will be an important responsibility of the Working Group on Toxicology.

g) Miscellaneous comments: It was noted by Dr. Parkhurst that grape husk oleonolic acids yield analgesics that have been tested for tumoricidal activity (after the sugars had been hydrolyzed off).

The neem tree is an extraordinary, multiply useful tree in India, which among many applications is a systemic insecticide when fed to cattle. The cashew nut has been shown in Brazil to produce a molluscicidal agent, tested in Mozambique.

April 8

II. Agrobotany – Taxonomy

1. Dr. Adams: Reviewed the long association of man with Phytolacca dodecandm. It frequently grows among cacti and along fences and with other forest plants in Ethiopia, where an acid pH and high N (nitrogen) levels occur. Wide variation is characteristic, even in the same plant. The three Ethiopian types (3, 17 and 44) are remarkably distinct in colour, size, form of plant and of fruits.

2. Dr. Wolde-Yohannes: Reviewed different growth characteristics of the three types on different soils and N concentrates.

Details of cultivation were then reviewed by Dr. Wolde-Yohannes. Harvesting must

be by hand, as the fruits mature from the top down and can consist of berries picked every second week.

Storage of seeds in water retains their potency for five years, possibly ten or more. Growth is optimal in loamy-sandy dry soil, but the plant is extremely hardy, as it has a high root-shrub ratio, and has shown excellent growth and potency at 750-metre elevation as well as the normal location at 1,600 metres. Still, P. dodecandra is widespread and has been discovered in a number of sites and soil types in many African countries. As Dr. Adams noted, many species are known with a worldwide distribution. As the plant is both hardy and adaptable, one can expect its successful cultivation in a number of harsh environments (as in the Fayoum oasis area of Egypt).

Yield: Higher after three years than with earlier crops. Third-year crops in Debre Zeit for:

Type 44 = 2,700 kg/ha/yr Type 3 = 1,300 kg/ha/yr Type 17 = 900 kg/ha/yr

It will be of interest to compare the production figures for Debre Zeit with

forthcoming data from Brazil, where the adaptation and growth of type 17 appears to be excellent.

Current studies at Carleton University with Dr. John Lambert have been productive. A study of extraction levels shows that a 25 to 30 per cent yield in dry powder can be obtained, i.e., at 900 kg/ha, the yield = 222 kg dried berries, giving a 5 ppm potency using either tap or distilled water extraction, after drying berries by solar energy.

Elevation of sites of P. dodecandm natural growth:

Zambia 1,000 metres Harare (eastern Ethiopia) 2,000 metres Nigeria 300 to500 metres Durban, South Africa sea level Ghana (near Accra) sea level

Treatment levels: If 5 kg are needed to treat 1 km streamshore, and four treatments are given per year, this would require 20 kg/km/yr. Seeds are viable and retain their potency for many years, so storage should not be a problem.

III. Country Reports

1. Swaziland - reviewed by Dr. Makhubtl(see Appendices 1-4 and 1-5) Experiments have been conducted on soil types, pruning intensity and form, and

fertilisation required at the major site (Middleveld at the Faculty of Agriculture campus). At the second site in the Highveld, cuttings and seedlings have been planted and are doing well, although the soil is poorer than at the Middleveld site.

At neither site is irrigation or watering required. Growth is extremely vigorous and healthy for all three types. Most encouraging is the high potency of these berries: 10 ppm of crude berries gives a 100 per cent kill in 24 hours. Levels of 5 ppm for the extracts have been achieved.

Dr. Chaine: Biomphalaria pfeiifen is the most abundant snail in Swaziland, but Butkus is common as well; Lymnaea is also found. Endod is applied as a spray at 35 ppm, giving a 100 per cent 24-hour kill, even in vegetation (as in the hotel ponds where it was tested).

An intensive programme to take advantage of this progress has been proposed in a new funding request (to be reviewed by this Workshop on April 10). Special study will be made of fertilisation variables, plot-potency levels and growth in low-yield, highly endemic areas of schistosomiasis.

Dr. Wolde-Johannes: The Swaziland results are better than those from Ethiopia! Since the leaves have very high levels of N, P, K (nitrogen, phosphorus, potassium), they should also be tried as fertiliser or animal feed after harvesting. The berry husks remaining after extraction should also be tested as a fertiliser and a cattle or chicken feed (after appropriate toxicity testing).

Dr. Lemma: Accolades for Dr. Malchubu! She has demonstrated that one scientist's skill, dedication and enthusiasm can make the difference. The kill with crude berries at 10 ppm is better than the Ethiopian experience. A 35 ppm kill for sprayed material is also excellent.

We need photographs of plants, harvesting, processing, application and results.

Regarding use of berry wastes as animal/chicken feed: this has been successfully tried in Ethiopia. It also has been employed with good results as a fertiliser. Yeast on the residue is also an excellent chicken feed supplement. It should be noted that the berry product after extraction is non-toxic and safe for animal or fowl food.

Discussion followed on local application of these findings, especially in regard to the proposed establishment of a Technical Cooperation in Developing Countries (TCDC) network:

• Can the Endod be tested locally as is? • Is it possible to utilise it as combined soap and molluscicide? • If tests are going to be conducted (at this stage) using crude materials, how are

we to standardise them? • Who establishes these standards? • Can we use the insoluble material? • Is it a good soap matrix? • Are the fish kills economically and socially tolerable in these areas of

application? • Should we do what was done at Adwa in 1964, simply pass water over the

berries to leach them and use the soluble toxins for snail killing, then the remaining leached product as a soap?

• If crude material can still kill snails at 10 ppm, do we need to process further than grinding? Or should we simply use whole-berry unground water extracts?

Dr. Makhubu: We must test both methods, using all variations in berry and processing type against the agreed-upon standard (or its equivalent if Endod-S is not yet prepared). The simple raw ground product would be most appropriate for village-level application; the more sophisticated standardised procedure should be used elsewhere in order to pro-vide scientific data and repeatable procedures to give credible results. If the product is to be registered in Canada as a pesticide, these procedures must be followed precisely.

Dr. Katz: Why has Bayluscide been favoured for general use? The testing procedure described will take five to six years. We should develop a single standard so that all areas can be measured against it. In Brazil, the regions of schistosomiasis endemicity are vast and scattered. Since each place may be markedly different, growth patterns may differ with the soils which may be reflected by different levels of Endod potency as well (although there is no confirmed evidence of this yet). The retesting of past studies will be costly and long. Therefore, we need to agree on one plant done to produce one harvest for one yield to follow one extraction process, which then is the sole source of bagged powder for all areas to be tested against, viz: Endod-S.

WORKING GROUP

REPORTS I. Introduction

There has been important progress in research on Endod by Africans since the Lusaka workshop in 1983. At the international level, encouraging progress has also taken place. Toxicologic studies by Professor N.H. Farnsworth and colleagues at the WHO Collaborating Laboratory at the University of Illinois in Chicago have shown that neither the water nor the butanol extracts of Endod have mutogenic activity under the widely accepted test conditions employed in his laboratory.

With financial support from the U.S. National Science Foundation, Dr. Robert Adams

of the Hardin-Simmons University laboratory in Utah and Dr. Robert Parkhurst of the Stanford Research Institute, Menlo Park, California, have begun a systematic collection of Phytolacca dodecandra plants from different parts of Africa for determination of their biochemical characteristics. These strains will be tested for cultivation characteristics and local adaptation in various countries in Africa for enrichment of the genetic bank of P. dodecandra.

In addition, both known and unknown strains of Endod have been successfully introduced from Ethiopia to Zambia, Swaziland and Zimbabwe. Search for local varieties in Zambia and Zimbabwe led to the discovery of plants with no molluscicidal potencies equal to or better than those in the parental strains from Ethiopia. In Swaziland, the cultivation of introduced strains appears to be doing better than the parent stocks because the insect pest that damages young shoots in Ethiopia is absent from Swaziland.

Since concern over possible toxicological effects of Endod was the major block to its acceptance for further development, the UNFSSTD and IDRC of Canada convened an independent international group of prominent toxicologists from the United States, Canada, Holland, Den-mark and Africa to meet at the United Nations in New York in February of 1986 (as was noted in the Foreword). The responsibility was to evaluate past studies on Endod toxicology, identify gaps and recommend a course of future action. The group, chaired by WHO, developed specific recommendations for standardising the done to be used for testing and for standard methods of extraction. They also recommended establishing a standing committee of experts to oversee toxicologic studies and advise on future design of Endod studies. These recommendations are being implemented.

In the light of these important developments and in view of the potential economic advantages in using Endod as a molluscicide in Africa, the Swaziland meeting found it timely and appropriate for interested re-searchers to form a network of individuals and institutions to facilitate, co-ordinate and standardise research and development of Endod for its possible use in integrated health care delivery systems in Africa. Further, research and development of Endod is seen as a model for development of other natural products by the participating institutions, in the spirit of the Technical Cooperation among Developing Countries (TCDC) pro-gramme.

In considering the potential role of Endod as a molluscicide to help control schistosomiasis in Africa, the workshop noted that of the 200 mil-lions estimated to be infected with schistosomiasis in the world, about half are in Africa. This number continues to increase as a result of well-intentioned but snail-spreading agricultural irrigation projects supported by various development assistance organisations, such as the World Bank, UNDP and numerous bilateral aid donor countries. The ensuing disease has a variably debilitating impact, especially on children, who are also most strongly associated with its transmission. Reduction of the productivity and general well-being of the labour force in irrigated agricultural development areas is an undocumented but probable effect of the continued spread of this chronic disease.

Ideally, schistosomiasis control is an integrated combination of chemotherapy, mollusciciding, dean water supply, improved sanitation and provision of appropriate health education for behaviour modification. However, apart from chemotherapy and mollusciciding, the other measures are not attainable in Africa at the present time. Chemotherapy and mollusciciding, separately or in combination, have reduced morbidity and disease transmission levels to significantly lower levels when these measures were applied on a systematic and continuous basis. However, the high cost of the best available drug for treatment (Praziquantel) and of the only molluscicide approved by WHO for wide use (Niclosamide) has prevented African governments from undertaking the sustained control efforts needed.

After 21 years of research and development, a pathway for final testing and future

application of Endod has emerged. The schistosomiasis Expert Group that met in Geneva to review plant molluscicides in 1983 concluded that of over 1,000 species of plants screened for molluscicidal potency, Endod was the most studied and is the most promising. The just-completed toxicologic review in New York recommended establish-ment of standards for future testing, as described earlier in this report. That assures appropriate rigour and standardisation of raw material to be studied, of procedures for Endod preparation and of comparability of results of testing.

The combined stimulus of these groups of experts has therefore given the go-ahead for further study of Endod as the most promising molluseicide, and has established the essential guidelines for reproducibility and confidence in the toxicological studies and in subsequent developmental testing. Participants in the Swaziland workshop based their considerations and recommendations for future action on these milestones of international evaluation and encouragement.

II Report of the Working Group on Agrobotany and Extraction

Leader & Rapporteur: Dr. Robert P. Adams

Members:

Dr. Robert M. Parkhurst Dr. J.P. Chaine Dr. Legesse Wolde-Yohannes Dr. Jerry Ndamba Dr. Ahmed K. Bashir Dr. E.G. Chimbelu Dr. Martin A. Odei

A. Assessment of Work to Date In addition to pioneering Endod research done in Ethiopia, new re-search is

underway in Swaziland, the United States, Zambia and Zimbabwe. The new research areas appear to reflect interactions stimulated by the First Workshop in Lusaka in 1983. In Swaziland and Zambia, test plots with types 3, 17 and 44 have been established to determine the range of adaptability of these types and cultural practices needed to maximise Endod productivity. In Zimbabwe, research has centered on exploration and evaluation of the Endod germplasm native to Zimbabwe. Research is well underway to examine P. dodecandra, collected throughout its natural range in Africa to determine patterns of regional variation in morphology and leaf chemistry, summarised in Appendix I-2 (Adams). In addition, germplasm will be collected from each region and disseminated to collaborators throughout Africa to establish gene banks, clonal seed orchards and replicated test plots. When implemented, these tests plots can serve as germbanks for further agronomic and breeding needs. Additional re-search has been done by Wolde-Yohannes and Lambert on extraction at Carleton University in Canada (see Appendix 1-3, Wolde-Yohannes) and at Lowell University in Massachusetts by Bruce and colleagues. Their preliminary data, supported in a memo to Bruce by N.W. Flood, suggest that water extraction from whole berries may be comparable in yield and potency to ground or crushed berries, in marked contrast with previous experience and

universal practice. This implies that a new research direction of exciting potential should be pursued,

perhaps in parallel with the studies undertaken with the Endod-S ground berries. The preparation of Endod-S will follow the preparation guidelines suggested at the 27-28 February New York Toxicology Meeting for preparation of the Endod standard. But if it subsequently proves that a simple water extract of whole berries provides as potent and large a yield as that from the crushed product, this would be of tremendous interest, procedural simplification, cost reduction and ease of standardisation. At present, however, this stands only as a challenge to be vigorously pursued without delay.

Research on storage of dry berries and of dry powdered extract has continued in Ethiopia with no loss of activity after five years. Fully expanded unripe (green) berries have been found to yield the most potent extracts. It is this stage that is to be selected for harvesting the Endod-S. Longevity and productivity studies are continuing in Ethiopia, which indicate that five-year-old type 44 plants may have reached a plateau in fruit production, but no decline in yields have been observed. Nor have pathologic declines been observed attributable to aging. B. Identification of Gap in Knowledge

1 .Ecological: Some new ecological data concerning soil types and pH has been gathered in Zimbabwe, but little ecological in-formation is available from most of the range of P. dodecandra in Africa.

2. Genetics and breeding data: No breeding studies have been undertaken to date. Saponin yields in natural populations are being examined in Zimbabwe, but extensive study of the compounds is needed. The data should be important for making inferences about the mode of inheritance of saponins. It is not known if one Endod genotype can be adapted throughout Africa, or if several cultivars are needed to cover a range of habitats. The genetic basis and mode of inheritance of secondary compounds have been shown to be under multigenic control with additive effects. Crossing studies, carried through the Fzz (tlourine) generation, will be needed to determine this for Endo? Since the Endod plant is apparently dioecious, it would be extremely valuable if markers could be found in male plants to indicate their genetic components directly affecting or linked to levels of saponin yields in their progeny. One index may be the level of saponins in the leaves. The breeding system is not known and needs to be understood before such studies can start. Propagation may be by seed, cuttings or tissue culture, but standard methods for rooting cuttings are now needed. Tissue culture has been successful in the propagation of Endod and two of these plants have produced seeds in Ethiopia.

3. Agronomic trials: A key unresolved question is: Can needed production be obtained from reasonably sited areas at costs competitive with synthetic molluscicides? Other areas of needed re-search include: yield trials, involving fertiliser rates, and irrigation effects and density trials in different environments. Finally, can yields be sustained (resources sustained), and if not, what additional inputs will be needed? The gaps here identified can be traced to lack of resources and failure to recognise the range of potential uses of Endod in various African countries and possibly elsewhere in the tropics.

C. Recommendations for Future Work on AgroBotany

1. Pruning: Training is needed to aid technicians in learning proper methods of pruning, branch training and time of season to prune. These training sessions could be expedited by the TCDC network.

2. Fertilisation: Each collaborating country should plan experiments with different kinds and rates of fertilisation, based on soil conditions. In addition to standard N, P, K treatments, research is needed on the effects of Ca, Na (calcium, sodium) and other ele-ments on fruit yields and potency. Research is needed to deter-mine fertiliser requirements conducive to root/crown production, vigorous growth and enhanced fruit production.

3. Adaptation trials: Trials are needed in additional areas to determine the specific influence and the relationship between soil types and climate on yields and potency. We recommend that all trials use clonal material cuttings from the Endod strain type 44, presently growing in a clonal (isogenic) plot at the Pathobiology Institute in Addis Ababa. These cuttings should be surface-sterilised, packaged and labelled "PHYTO-SANITARY" so they can be imported through plant quarantine in the recipient country. The TCDC network should co-ordinate this procedure. We cannot over-emphasise the importance for collaborators to distinguish clearly between plants derived from open-pollinated seed and those obtained from isogenic material. As far as practical, comparative studies among countries should use isogenic materials.

ity.

4. Agroforestry: Research is needed to determine if and how Endod can be grown intermixed with other crops, the effects of Endod on other crops and vice versa. Possible benefits of growing Endod as an intercrop are: windbreaks, reduced insect damage, reduced soil erosion and firewood supply from aged shrubs.

5. Weed control: Research is needed to determine the susceptibility of Endod to various herbicides, and new cultural practices need to be developed to minimise weeds. The effects of various kinds of weeds on growth and productivity of Endod should also be examined.

6. Plant culture: Optimum spacing, pruning and branch alignment need to be examined. This is currently being studied in Zambia and appears to be a little-researched but important factor with respect to productiv

7. Longevity: Research is needed to determine production yields as a plantation begins to mature, then goes into decline. We must be able to estimate crop rotational age and pathological rotation age. Economic analysis is needed to estimate the point when the profit/cost ratio is not acceptable and the plantation should be cleared and replanted. These are critical factors in the crucial matter of cost determination--the bottom line that underlies all agronomic studies.

8. Gene banks: Test plots of accession from throughout Africa should be established in several countries so that important agronomic characters may be conserved for future breeding work. The project currently being implemented, to grow seed sources from throughout Africa (Ethiopia, Kenya, Swaziland, Zimbabwe, Zambia, Ghana and Nigeria) in replicated test plots, needs to be sup-ported with funds for at least three years.

9. Variation in pericarp/seed mhos: New studies should examine variation in the ratio of pericarp to seed weight to select individuals that produce a greater amount of pericarp, where nearly all of the molluscicides are found.

10. Breeding and genetics: The genetics of the inheritance of saponins need to be determined so appropriate breeding programs can be started. A method for selecting (field screening) high saponin-yielding female plants should be developed. As previously noted, a marker needs to be found to permit male plants to be used for high saponin content (possibly based on leaf saponin content as a marker). Variations in saponin content within a natural population need to be examined, as these may lead to infer-ences on the mode of inheritance of these compounds.

11. Processing: Research on harvesting appears to be adequate, but additional study is needed on drying and storage throughout the expected range of utilisation. Year-round storage will be needed in many locations, and methods to prevent loss of molluscicidal activity should be developed for different environments.

12. General administration: Both centralised and decentralised germplasm depositories should be established to conserve selected germplasm and serve as a bank of genetic resources for future plant breeding. Co-operative agreements among developing countries would facilitate exchange of plant material and plant propagules, including seeds and/or in vitro tissue-cultured plantlets--another valuable advantage of the proposed TCDC Endod network (Working Group 3).

13. Reporting of yields: All yields of harvest and of molluscicides (or saponins) should be measured on a dry-weight basis (e.g., a small number of berries should be weighed, then placed in an oven at 100°C for 48 hours; the berries should then be weighed immediately, as they quickly begin to reabsorb moisture). The percentage of moisture can then be determined as:

100x(initial berry weight – dried berry weight)/initial berry weight

This determination must be made as a correction factor on each test involving Endod potency.

14. Phwmacognosrical monograph: A standard pharmacognostical monograph is needed to describe Endod and the identification of its macro- and microscopic characteristics, including the powdered form.

15. Grower acceptance: If Endod is grown by local farmers, economic and sociological research is needed to determine if there may be traditions or fears that could lead to resistance to growing Endod, and if Endod can compete on an economic basis with cash crops in the area.

16. Cost/benefit analysis: Determination of the real costs of molluscicide production and application is essential if a realistic comparison is to be made other than the crude $25,000 versus $500 respectively for the selling price of Baluscide and the cultivation + water extraction cost of Endod. Proper analytic methods are needed in order to make a valid comparison of the costs of different molluscicides as a function of the control/treatment package. These can then be offset against the medical and occupational cost savings or the social benefits gained after freedom from infection and disease has taken place. D. Recommendations for Future Work on Extraction Procedures

1. Beny Binding: The very nature of Endod as an indigenous plant in a given region that could be locally produced and processed, primarily to be used in self-help

projects, makes simple in-expensive extraction procedures and community acceptance essential. Since water as the extraction medium has yielded a dry product toxic to snails at 4 to 8 ppm, water appears to be the preferred solvent at this time. Furthermore, unverified recent data on water extraction of whole versus ground berries (Wolde-Yohannes, unpublished), as noted in the assessment portion of this report, should encourage new studies and possible challenge to general assumptions on extraction methods. Universal practice requires the use of ground berries for the obvious reason that the greater surface area results in greater yields. Yet a preliminary report from Lowell University indicates that a higher yield of saponins (measured as foaming action) was obtained from unground rather than ground berries. Time-course extractions of ground and whole berries (up to 24 hours) showed that the saponin (i.e., foaming ac-tion) was more concentrated in the extract of the whole berry at each time-interval of extraction compared with the crushed berry.

Not only are the yields from whole and ground berries comparable, but molluscicidal activities also appear to be similar against three snail species. Slightly lower activities reported for whole berries may be somewhat counterbalanced by larger yields obtained from the whole berries. Further study of these unverified findings should dearly receive a high priority.

2. Water extract of whole berries: simplified reference procedure for comparison studies with Endod-S (crushed beny standard) for TCDC studies: In order to facilitate comparisons between the re-search efforts of members of the TCDC network, the following procedure is proposed as a reference procedure 1.0 (RP1.0) as follows:

Place 1 gm (dry wt. basis) of whole, dry berries of type 44 (done 44E) into a standard graduated cylinder, containing 100 ml of distilled water at 25°C. Cap the cylinder and invert at 1, 2, 4, 19 and 21 hours. Allow to settle for three hours (24 hours total). The water extract then constitutes the Reference Procedure (RP1.0). This reference procedure can be used within the TCDC network to aid in making reproducible comparisons with the ground berry standard (Endod-S) and other kinds of extractions and protocols that may be developed (the above procedure being subject to verification of reproducibility).

3. Beny drying: Although drying berries in the shade has proven satisfactory in Ethiopia where there is very low humidity, significant problems have arisen in conditions-of high humidity. Re-search is needed to compare various methods for berry drying. Time-course experiments in which samples are extracted at regular intervals throughout the drying period should determine if degradation of the molluscicidal activity is occurring. Several drying methods should be utilised: a very harsh method (e g., oven drying 30, 40, 60 and 80°C), sun drying, drying in the shade or drying with forced hot air (such as that which might be obtained from a solar collector)

4. Stability of Endod saponins in water: The dry powder may be dissolved in water and carried for considerable distances before application. Thus research is needed to determine if the potency declines (and at what rate) after the dried saponins are reconstituted in water. Studies need to be conducted on the stability of the saponins in water with regard to temperature (i.e., effect of boiling).

5. Extract drying: The method used to dry the water extract could affect product potency. Research is urged to determine the effects of various drying methods on potency of the Endod saponins. These drying methods should include freeze drying, air drying at various temperatures and spray drying at various temperatures. Each of the

dried powder extracts should be redissolved in water and the reconstituted extracts compared to assess the changes in potency.

III. Report of the Working Group on Toxicology of Endod

Leader & Rapporteur: Dr. Naftale Katz

Members:

Dr. Aluizio R. Prata Dr. MA. Shehata Dr. John I. Bruce Dr. Donald Heyneman Dr. K.N.M. Mtera

a. Introduction

The group, having gone through the work and recommendations of the Committee in New York mooting to evaluate the toxicity of Endod (United Nations, 27-28 February 1986), has endorsed their recommendations.

At this meeting in New York, it was recognised that although Endod is a natural plant product, it cannot be considered as distinct from a synthetic molluscicide in terms of toxicologic testing since there is no proof that its mode of action is non-toxic or that it is target-species specific.

It is thus important to conduct toxicologic studies as a conventional molluscicide in the same way and to the same standards as with other chemical products. It is necessary to consider their potential hazards when molluscicidal preparations are applied to aquatic environments: its possible pollution of drinking water and potential accumulation of its products in the food chain, such as in fish and molluscs. If the chemical analysis for whole-berry and crushed-berry extracts prove to be identical, the toxicological findings for one product should apply equally to the other, without duplicating studies in the two equal products.

Although other biological activities have been recognised for Endod, in this context only its molluscicidal activities will be considered. b. Recommendations

1. Toxicological studies a. With respect to the toxicological evaluation recommended for Endod, it is

necessary to use a standard plant variety, following precisely standardised procedures, according to an acceptable protocol repeatable by others. A network of co-operating laboratories engaged in these studies and following the same guidelines is strongly encouraged.

b. For the preparation of an Endod standard (Endod-S), it is necessary to use berries from done type 44 from Ethiopia from which an aqueous extract will be prepared according to the recommendations of the New York Toxicological Meeting (see Appendix II).

Sufficient amounts (1,000 kg) of Endod-S should be prepared by the same laboratory to ensure that all toxicologic studies anticipated as well as effects on

non-targeted organisms are carried out from the same batch of extract.

Rodent:

All tests should be carried out according to the following OECD guidelines for testing of chemicals (OECD Guidelines for Testing of Chemicals. Paris: Tier 1, Organisation for Economic Co-operation and Development, 1981).

c. Tier I: Recommendations Acute Oral Toxicity (Mammals) ................. DIG 13 Acute Dermal Irritation/Corrosion ............. DIG 14 Acute Eye Irritation/Corrosion ................... DIG 15 Skin Sensitisation ..................................... DIG 16 Repeated Dose (Oral) Toxicity--

14- or 28-day Study ............................ DIG 17 Mutagenicity ............................................. DIG 18 Alga, Growth Inhibition ............................. DIG 19 Daphnia sp., 14-day Reproduction

(including Acute Immobilisation) ......... DIG 20 Fish, Acute Toxicity .................................... DIG 21 d. The immature fruit as recommended for use from the New York meeting should

be referred to as fully grown green (unripe) berries. e. It is recognised, nevertheless, that much data are available on physicochemical

properties and toxicity of crude extracts (i.e., water, butanol, etc.). These data should be used for comparison and evaluation.

f.Additional studies must use Endod-S in order to meet the precise regulations stated in the OECD basic toxicological guidelines for product chemistry, environmental data for pre-marketed chemicals and to appraise the safety of Endod-S for handlers and consumers.

g. Mutagenic studies must be completed, using a mammalian system. Based on the data obtained, it may, of course, be necessary to conduct additional toxicological tests with other animals or forms of exposure.

h. Until further studies demonstrate the extent and nature of hazard to workers who handle, store, grind and process En-doe!, suitable precautions are advised.

i. Information on possible hazards from inhalation of different particle sizes of Endod-S must be obtained, repeated exposures to Endod-S must be monitored and degree of exposure under different circumstances measured.

j. Until the nature of the hazards of exposure to Endod-S has been established, it is recommended that handlers be protected by the use of a mask to prevent inhalation. Goggles should be used by individuals grinding the dry materials as a routine protective precaution. Although berries have been used as soap for centuries, and no skin effects have been observed among long-time users, patch test and other dermatologic hypersensitivity tests should be conducted, particularly with children and easily sensitised individuals. All responses, positive or negative, should be documented and made avail-able.

k. Priority should be given to toxicologic testing for supplies of Endod-S in order to assure an adequate supply of its designed purpose.

2. Field Studies As a molluscicide, Endod-S is a weapon in the control of trematode-carrying snails.

Clearly, it is not a panacea, but can only be viewed as one of several interventions

possible in the multiple measures available for schistosomiasis control. a. The bioaquatic environment of the test site should be studied before and after

application of Endod-S in order to observe ecologic effects on non-target organisms. Persistence, diffusion, mobility, toxic spectrum and other ecologic and environmental effects must be documented over appropriate periods of time. The guidelines of the Working Group at the New York Toxicological Meeting must be adhered to.

b. To ensure replicability of molluscicide field trials, the guide-lines recommended by the World Health Organization (WHO) for molluscicidal application must be used for all trials of Endod-S.

c. After or in conjunction with the requisitive environmental and ecologic studies, small-scale pilot projects may be carried out using Endod-S as a molluscicide. It is advisable, however, that such studies be conducted in places where the water test site is not a primary source for human drinking water and where locally caught fish are not a major source of food for the community, at least until toxicologic studies have been completed.

d. Correlation of concentration of Endod-S with molluscicide activity should be evaluated by such widely used tests as calorimetric and hemolysis tests.

e. It is advised that comparative studies be conducted where possible with Niclosamide as a reference compound.

IV. Report of the Working Group on an Endod/TCDC Network

Leader & Rapporteur: Professor F.MA. Ukoll (Nigeria)

Members:

Dr. S.K. Chandiwana (Zimbabwe) Dr. MA. Odei (Ghana) Dr. Ahmed Bashir (Sudan) Dr. P.C. Mphande (Zambia)

A. Introduction

The Working Group on an Endod/TCDC Network supported intensified research and development of Endod on a collaborative basis, principally as the source of a locally produced molluscicide for schistosomiasis control, but also as an important additive in detergent formulations for laundry use (as well as other spin-offs of potential commercial value). To mobilise our limited manpower and institutional resources and galvanise them into action, the establishment of an Endod/TCDC network is pro-posed. It will serve to facilitate this essential collaboration among participating countries, institutions and individuals from endemic countries. The collaboration within such a TCDC network will operate on a two-way system. First, among scientists in Africa and other Third World countries with similar problems, such as Brazil. Secondly, between institutions and scientists in these countries and other recognised scientists from non-endemic areas

considered experts in various aspects of Endod and schistosomiasis research and related fields. B. Resource Mobilisation

Ten countries in Africa and South America endemic for schistosomiasis are to constitute the nucleus of this TCDC network:

Brazil Sudan Ethiopia Swaziland Ghana Tanzania Kenya Zambia

Nigeria Zimbabwe The international group from non-endemic areas will initially comprise centres in

the following countries: Canada, Denmark, The Nether-lands and the United States. The main purpose of setting up such a network will be three-fold: a. to establish machinery for exchange of information, ideas and staff; b. to develop capabilities in identification/taxonomy of the strains of P. dodecandm

and of related species, Endod ex-traction and storage techniques, molluscicidal screening and toxicologic and snail-rearing methods; and

c. to foster standardisation of procedures and equipment for re-search in these fields and to ensure adoption of these standards by all collaborating groups.

For this system to function effectively the institutions of individual scientists with the relevant knowledge and experience should be designated Reference Centres. Thereby, it is hoped to make available experience, expertise and resources of these scientists to help other scientists who wish to initiate similar programmes in their own countries. The following individuals, well-known for their contributions to this area, are recognised as directing the first such centres:

1. Identification and taxonomic studies a. Institute of Pathobiology (Dr. Legesse Wolde-Yohannes) Addis Ababa University

Addis Ababa, Ethiopia; b. Science Research Center (Dr. Robert P. Adams) Hardin-Simmons

University 8555 S. Escalante Drive Sandy, Utah 84092

*********

Project Analysis of Geographic Variation in Phytolacca dodecandra in Africa Using Morphology and Non-Polar

Leaf Chemical Components

Robert P. Adams and Robert M. Parkhurst

(Science Research Center, Hardin-Simons

University, Sandy, Utah and

Stanford Research Institute,' Menlo Park, California)

Funding Agency: National Science Foundation (USA)

Duration: 1 May 1985--30 April 1987

Principal Investigators: R.P. Adams and R.M. Parkhurst Project Summary: Plant specimens of Phytolacca dodecandra are being collected from its entire natural range in Africa. Morphological data will be collected from each specimen and analysed by statistical methods to determine patterns of infra-specific variation. In addition, a portion of the dried leaves will be extracted with hexane and the individual components used as chemical characters to elucidate further the pat-tern of geographic variation in P. dodecandra. Collections from natural populations include: Ethiopia (2), Kenya, Burundi, Zambia, Zimbabwe (2), South Africa, Madagascar, Zaire, Ghana and Nigeria. Seed collected from the aforementioned areas will be distributed to standardised test plots (complete random blocks with three replicates) in Ethiopia, Swazi-land, Zimbabwe, Zambia, Ghana and Nigeria. These standardised test plots will then serve as a repository of germplasm from which local countries can select superior forms of Endod for their own utilisation. In addition, follow-up studies can be performed on material in the test plots to estimate the magnitude of environmental and genetic factors that deter-mine yields of Endod molluscicide.

Report on the Introduction of Types

3, 17 and 44 of Phytolacca dodecandra in Swaziland

Lydia P. Makhubu

(Department of Chemistry, University of Swaziland, Kwaluseni, Swaziland)

Background and Description of the Endod

Endod is the Ethiopian name for the soapberry plant Phytolacca dodecandra (L'Herit)--synonyms: P. abyssinica (Hoffm); Pircunia abyssinica (Mog)--a member of the Phytolaccaceae family. The distribution of this plant is east, west, central and southern Africa and parts of South America and Asia (Dalziel, 1936).

Endod has small berries which when dried, powdered and mixed in water yield a foaming detergent solution that has been traditionally used in Ethiopia and elsewhere as soap for washing clothes (Lemma, 1964). In Ethiopia, Endod exists as two main types: arabe with pinkish, and ahiyo with greyish berries.

Arabe (possibly meaning "from Arabia") has more powerful proper-ties than ahiyo (meaning "donkef and implying that it is less active than the other type). The plant is a climber with hanging branches. It grows very rapidly, reaching a height of up to 10 metres, although the average height is two to three metres. Under favourable climatic conditions in Ethiopia, the plant bears fruit twice a year--December through February and June through July (Wolde-Yohannes, 1983).

Phytolacca dodecandra (L'Herit) and a closely related P. americana (commonly known in the U.S. as pokeweed) have long been recognised for their varied medicinal and other uses. Different parts of the plant, including the leaves, fruit and roots, are widely regarded for their emetic, purgative, abortifacient, detergent, antisyphilitic and other properties (Watt and Breyer-Brandwijk, 1962). Phytolacca is dioecious and therefore produces both male and female plants. The ratio of male to female is about 45:55 (Wolde-Yohannes, 1983). As the individual sexes cannot be distinguished until flowering, all plants must be cared for. Propagation may be by seed, cuttings or tissue culture. The effectiveness of these methods needs to be evaluated. Tissue culture has been successful in propagation of Endod, but the resulting plants flowered and bore fruit only after two years under Ethiopian climatic conditions. Introduction

Schistosomiasis and HIV continue to be the major obstacles to development among nations in South America, Asia and, most painfully, Africa. Be-cause both the snail hosts of schistosomiasis and human agriculture depend on abundant fresh water, the very act of bringing irrigation into new lands also provides an ideal environment in which transmission of the disease takes place.

In the past, there have been efforts to find snail killing substances which are under local production and control, easily applied, continuously available and low cost.

The molluscicidal property of the remarkable Ethiopian highland plant Phytolacca

dodecandra, locally known as Endod, is of relatively re-cent knowledge. But as a home remedy (as an emetic and abortifacient among other uses) and especially as an effective, non-sudsing soap with a strong bleaching action on the cotton "shamma", the national dress of Ethiopia, Endod has probably been used for centuries.

The development of Endod should be understood partly in economic, social and political terms. A great deal of chemical, toxicologic and agronomic work as been done on Endod during the past 20 years, especially on the berries, the primary molluscicidal product. Strain selection, growth characteristics, extraction and concentration procedures all have received several years of study. But the major reason for refocussed interest on Endod and other plant molluscicides is the growing disenchantment with imported synthetic molluscicides: their cost, the associated technology required, uncertainties over long-term environmental and toxic effects and the need to apply them regularly over an indefinite period of time. As with chemotherapeutics, Western manufacturers are loathe to commit significant research and development funds to a fluctuating market largely dependent on international funding. Only a few molluscicides remain in current use. Their snail killing importance is unquestioned, but their long-term market potential is uncertain. The need for a homegrown, reasonably effective, relatively non-toxic natural product, requiring minimal technologic processing and handling and which can be purchased locally, has grown increasingly dear (Makhubu, 1982).

Several plant product candidates have been revived and reconsidered from this point of view. Each has limitations and problems. But an effective molluscicide that can be locally produced has become an overriding necessity, in spite of a preference among most health workers and administrators for a synthetic product manufactured and delivered in accordance with a centrally administered program as part of a general control package. Such programs are a mainstay of public health planning and projections in many areas. Yet the problem over-reaches the capacities of planners, economists and administrators to deal with it in such neatly pre-scribed ways. Something else is also required: self-help. Local responsibility for an ongoing effort is needed as part of the solution (Heyneman, 1983).

The need for new molluscicidal approaches and additional tools has grown more compelling as the economies of developing nations have worsened in recent years. Because of these growing concerns, the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR Scientific Working Group on Schistosomiasis) held a workshop on plant molluscicides in 1983 to evaluate the problem and consider the feasibility of plant moiluscicide products. A number of candidate molluscicides were considered and guidelines for their evaluation were established. It was concluded by the workshop participants that Endod is the most promising of the plant molluscicides evaluated, and that toxicologic, agrobotanic and other studies should be encouraged for its establishment as a safe and regionally available molluscicide-producing plant.

Soon thereafter, an international workshop on P. dodecandm was organised and convened in Lusaka, Zambia, 7-11 March 1983. Both the Geneva workshop and preliminary discussions leading to the Lusaka workshop emphasised the promise of Endod, but equally the need for further research: definitive toxicologic studies, agrobotanic research to select the most potent and adaptable strains of the plant, determination of growth requirements and limitations, and the feasibility of raising the plant under a variety of soil conditions, particularly in lowland arid areas of Africa and in other regions where schistosomiasis is endemic (Wolde-Yohannes, 1983).

The activities and research reviewed in this paper strongly exemplify the spirit of technical co-operation among developing countries that characterises work on this

remarkable plant. The authors want to express their gratitude to Addis Ababa and Swaziland Universities for their valuable interest and assistance in carrying out this project, and to the European Economic Commission (EEC) for financing the project. Summary

1. Endod plant identification. Of plants previously planted at the Luyengo Campus Faculty of Agriculture, Swaziland University, the fol lowing types were identified: type 3 (11 plants), type 17 (11 plants) and type 44 (47 plants).

2. Harvesting. Of the 336 plants, 46 have produced berries. 3. Transplanting and cultivation. Rooted cuttings were transplanted on expansion

of the field in Luyengo Campus. The Endod cultivation was successful and all types (3, 17, 44) have become adapted. This applies to both propagation methods--seed and cuttings. The berries were harvested twice (May and October) in the first year of growth. Pruning was done after berry harvest.

4. Laboratory experiments. Identified types have been screened under laboratory conditions for potency against Biomphalana snails. Results of the screening indicate that the Swaziland-grown Endod was comparable in potency to the Ethiopian Endod.

5. Field application: methods and materials. Field testing has been carried out to observe molluscicidal effects based on the laboratory results.

a. Potency screening: A series of dilutions of Endod stock solution was prepared for all types of Endod.

i. one gramme crude Endod powder was placed in one litre of distilled water;

ii. dilutions to 10 to 50 ppm (parts per million) were made from the stock solution;

iii. test solution was placed in one-litre jars. Five snails for 24 hours were placed in each jar, three jars for each dilution (i.e., 15 snails per dilution). Snails used were similar-sized adult Biomphalaria pfeifferi; and

iv. pH of the solution: 7.21. Experiments were monitored 24 hours after exposure to the solution. After 24

hours, snails were washed and placed in Endodfree distilled water for an additional 24-hour recovery period. For each set of experiments there were three sets of controls in which the snails were placed in distilled water for the same periods. The pH of the distilled water was 7.22; room temperature was 18-22°C.

b.Field applications: Encouraged by the laboratory experiments field applications were undertaken in collaboration with the Bilharzia Unit of the Ministry of Health. A site had been previously identified by the Bilharzia Unit and pre-control arrangements were made jointly. The test site is located in the Ezulwini Valley farm of the Holiday Inn Complex. It is an artificial pond used for irrigation purposes, 33 x 23 x 1 metres. Around the pond are homes, and the residents use the pond water for washing and bathing.

Pre-control observations and snails sampled by net scoopings showed a large number of snails, predominantly Biomphalmia pfeifferi. Water volumes were kept constant by stopping up the pond in-let. Snails were collected and placed in three cages, 50 healthy adult snails (Biomphalaria pfeii feri) to a cage. Cages were set about 10 metres apart in the water. Water temperature and pH were measured prior to each experiment, with water samples taken from the three snail cage areas for microflora and fauna determination.

Previously water-soaked (30 min) crude Endod powder was placed in water in a spray can and repeatedly sprayed along the edges of the pond. The concentration of the solution was estimated by volume at 30 to 35 ppm and spraying lasted about 30 minutes. Water samples were again taken after 24 and 48 hours.

6. Results. The laboratory screening for molluscicidal effects showed that the minimum concentration for a 100 per cent snail kill is 10 ppm for Endod types 3 and 17: for type 44, about 12.5 ppm is required. In the controls, there was 100 per cent survival.

These results are closely comparable with those obtained under Ethiopian conditions. Results under field conditions showed that about one hour after spraying Endod, small fishes showed restlessness but none were seen to die. After 24 and 48 hours, caged snails were checked and counted; all were dead. Scooped snails (Biomphalaria, Bulinus, Lymnaea and others) were also all killed. Small and medium-sized fishes had also died, but many larger fishes were still alive. Fresh water crabs were observed trying to escape, although they did not die.

7. Discussion and conclusions: From these results, adaptation to Swaziland soil and climatic conditions has succeeded for Ethiopian Endod types 3, 17 and 44 tested at altitude levels (about 1,000 metres). Attempts to grow at a higher altitude (1,200 m) are underway. In addition, field studies of the Swazi Endod type will be undertakep. So far, Phytolacca americana has been found by the authors in Ezulwini Valley. The National Herbarium also reports location of Phytolacca dioica in Swaziland.

Screening under laboratory conditions will be continued after each harvest to determine whether potency is maintained over time. These observations will also be made with respect to berry yields.

Field applications will be continued and, if possible, expanded to different sites, depending on the yields of berries harvested. Since Endod is not ovicidal, frequent and regular application is important. It is also important to note that the non-ovicidal property of Endod applies to fish eggs as well. This means that the population of fish will reappear as soon as the Endod biodegrades in the water, beginning 24 hours after

application.

References

Heyneman, Donald. (1984). Conclusions and recommendations of the workshop. In Phytolacca dodecandra (Endod). Final report of the international workshop, eds.A. Lemma, D. Heyneman and S.M. Silangwa. Dublin: Tycooly International Publishing.

(1984). Presidential address. Development and disease--a dual dilemma. J. Parasitol. 70:17. Rloos, H. and F.S. McCullough. (1981). Plant molluscicides: A review. WHO/Shisto/81:59. (Unpublished WHO Document). Lemma, A. (1965). A Preliminary report on the molluscicidal property of Endod (Phytolacca dodecandra). Ethiop. Med. J. 3:187-190. _ (1970). Laboratory and field evaluation of the molluscicidal properties of Phytolacca

dodecandra. Bull. WHO 42:597-617.

, D. Heyneman and H. Kloos. (1979). Studies on the molluscicidal and other properties of the Endod plant Phytolacca dodecandra. Special publication. Institute of Pathobiology, Addis Ababa University, and Department of Epidemiology and International Health, University of California, San Francisco. Lugt, Ch.B. (1981). Phytolacca dodecandra berries as a means of controlling bilharzia-transmitting snails. Institute of Pathobiology, Addis Ababa University. Addis Ababa: Litho Printers.

ork.

Makhubu, L. (1984). Research pre-proposal. Testing the potential uses of the plant Phytolacca dodecandra as a molluscicide, pesticide and deter-gent. UNIDO Investment Promotion Servicelwith the assistance of Dr. Aklilu Lemma). (May 1983). Endod project: towards industrial production and processing of the natural plant produce Endod for use as detergent and pesticide. Proposal for a small-scale pilot-project in Ethiopia, Zambia and Swaziland. United Nations, New YWHO. (1980). Epidemiology and control of schistosomiasis. Report of the expert committee. Series 643. Geneva. Wolde-Yohannes, L. (1981). A Proposal into the evaluation and development of the detergency of Endod and improvement of molluscicide de-livery systems, for use as a biodegradable natural product. Institute of Pathobiology, Addis Ababa University. ___ . (1982). Research proposal. Development of improved methods of Endod

application as a molluscicide. Institute of Pathobiology, Addis Ababa University. ___ . (1982). Synthetic and plant molluscicides in the control of schistosomiasis with

special reference to the agrobotanical studies of Endod (Phytolacca dodecandra) in Ethiopia. Presented to the Symposium on Human Schistosomiasis, 1-4 November 1982, Addis Ababa. (1983). Past and ongoing agrobotanical studies of Phytolacca dodecandra (Endod) in Ethiopia. Presented to international workshop on Phytolacca dodecandra, 7-11 March 1983, Lusaka.

A Report on the Geographical Variation in the Molluscicidal Potency of Phytolacca dodecandra Berries

in Zimbabwe* Jerold Ndamba and Stephen K Chandiwana

(Blair Research Laboratory,

Harare, Zimbabwe) Introduction

Due to the rising costs in synthetic molluscicides, Zimbabwe, like many other developing countries where schistosomiasis is endemic, has been exploring other means of obtaining cheaper molluscicides. As a result, there is a growing interest in plants with molluscicidal properties.

Money, working at this laboratory, screened several plants to assess their value as molluscicides. Fruits of the tree Swartzia Madagascariensis were used for snail control programmes for a considerable period of time mixed with copper and sawdust. This mixture was lethal to snails within 24 hours of application. The main disadvantage was the size of the tree which is difficult to propagate, taking years before the fruits can be harvested. Evans also screened infusions from several plant species south-east of Zimbabwe. Euphorbia ingens was found to be molluscicidal within 24 hours if infusions made from stem and from sap were used at concentrations of 2 ppm.

The most extensively studied plant molluscicide is one derived from the berries of Phytolacca dodecandra, a plant indigenous to eastern, central, western and southern Africa. Its molluscicidal properties were first reported in Ethiopia where the berries are traditionally used as soap and as a medicine. It has since been established that the active molluscicidal ingredients of the berries are several derivatives of oleonic acid of a triterpenoid saponin.

Toxicity studies have shown that P. dodecandra berries are lethal to small fishes at molluscicidal concentrations that are required for snail control purposes. However, unlike some chemical molluscicides, the berry extract rapidly biodegrades in water thus reducing the possible ac-cumulation of toxic residues in the environment. Considerable interest has been shown in the plant and recently an international workshop was held in the Zambian capital, Lusaka, to review the work done so far on the molluscicidal, agrobotanical, toxicological and other aspects of P. dodecandra and to assess possible areas of collaboration among interested African countries.

In Zimbabwe, P. dodecandm is a shrub or semi-climber often found growing on anthills and relatively widely distributed in central, northern, eastern and southern Zimbabwe. However, there have been very few studies done to evaluate the molluscicidal potency of the local varieties and data from only two plants have been published. These berries were shown to be lethal to snails within 24 hours of exposure at concentrations ranging from 10 to 25 ppm.

This paper reports the geographical variation in the potency of P. dodecandra berries in Zimbabwe, and the possible causes of such variations are also discussed.

Mater ials and Method

A search for P. dodecandra plants was conducted in recorded and suspdcted

localities by asking local people if they knew where a plant similar to the specimen shown could be found. Anyone who could identify the plant was asked to give a) the local name of the plant; b) its traditional uses (including medicinal), if any, and c) the exact location of the plant in the area.

About 1 kg of green berries was collected into pre-labelled transparent plastic bags from plants whose racemes had reached the unripe fruit stage of development. These were brought back to the laboratory where they were oven dried for three days at 30°C. The dried berries were ground into a fine powder using an electric blender. The powder was further refined (hence standardising the particle size) by passing it through a 0.5 mm mesh and was then stored in screw-topped bottles for at least two weeks when bioassays were performed.

1. Molluscicidal potency tests. Bioassays to screen for the molluscicidal activity of the berries were performed using Balinus (Physopsis) globulus with a minimum height of 12 mm. B. globosus is the most important snail intermediate host of schistosomiasis in Zimbabwe. A stock solution of 50 ppm was prepared by weighing 50 mg of P. dodecandra berry powder and mixing this in 1,000 ml of dechlorinated water. Wild caught snails were left to acclimatize to laboratory conditions for at least seven days before being exposed to the molluscicide. Four replicates of five snails each were exposed to molluscicide concentrations ranging from 40 to 5 ppm in a total volume of 200 ml for 24 hours. The snails were then washed, provided with food and allowed a recovery period of a further 24 hours after which mortality was assessed. This was done by pricking those snails that showed no sign of movement with a sharp needle at the back. If no withdrawal movements were evident, such snails were transferred into an exposure vial filled with clean dechlorinated water and left to stand for at least six hours, when mortality was again assessed as be-fore. If no movements were again evident these were pronounced dead. Results

The areas where P. dodecandra berries were collected are shown in Figure 1. A total of 103 plants were located in the eastern district of the country (E on Figure 1) of which 27 were berry producing. Berries were also collected from 15 out of 21 plants located in the central district, from 6 out of 13 plants in the northern district, from 10 out of 15 in the south-ern district, and from 16 out of 32 plants in the southeastern district (C, N, S and SE on Figure 1, respectively), making a total of 74 wild plants from which berries were collected and bioassay tests performed out of 154 plants that were found growing in various parts of the country.

Table 1 shows the mean percentage mortalities that were recorded when B. globosus were exposed to a molluscicide concentration of 15 ppm prepared from P. dodecandra berries collected in each of the five districts. This was the minimum concentration (15 ppm) at which at least some snail mortalities were recorded for most of the berries collected. The table also shows the mean annual temperature, mean annual rainfall and the mean altitude of the places from which P. dodecandra berries were collected in each district.

Of all the 74 plants from which berries were collected, only berries from eight plants were lethal to 50 per cent of the snails at a molluscicide concentration of 10 ppm. The snail mortalities that were recorded when B. globosus were exposed to different molluscicide concentrations pre-pared from berries collected from these plants are shown in Table 2.

Discussion

To the authors' knowledge no studies have been undertaken else-where to determine the geographical variation in the molluscicidal

potency of P. dodecandra berries. The bioassay results show that there is a marked variation in the molluscicidal potency of the berries collected from various parts of Zimbabwe (Tables 1 and 2). Berries collected from nearly all the plants in the eastern, northern, central, southern and south-eastern districts of Zimbabwe (Figure 1) resulted in at least some mortality of the snails at a molluscicide concentration of 15 ppm. However, berries from only eight plants resulted in snail percentage mortalities greater than 50 per cent at a molluscicide concentration of 10 ppm. Of these eight, seven which were collected from the Masvingo area (whose mean altitude is 989±94 metres, receiving a mean annual rainfall of 760±5 mm with a mean annual temperature of

19.30±0.40°C) were 100 per cent lethal to B. globosus at molluscicide concentrations of 15 ppm. Berries from three plants were also 100 per cent lethal to the same snail species at 10 ppm. The only other plant whose berries were of an equally high potency were those collected from a plant that Was found near Elinor Mission (eastern district), grid reference 32°50'E/17°37'S, whose height above sea level is 960 metres with an annual rainfall of 700 mm and an annual mean temperature of 19.50°C.

The most potent berries were those collected from near Namamwa Growth Point, grid reference 30°55'E/20°20'S, which resulted in an 80 per cent snail kill at 8 ppm and 5 per cent at 5 ppm. This compares well with the Ethiopian types 3, 17 and 44 which were shown to be the most potent.

From the general trend in the variation in the molluscicidal potency of P. dodecandra berries, altitude, rainfall and temperature may play an important part in determining the potency of the berries. Adaptation trials in Ethiopia have shown that the plants, which naturally grow at altitude between 1,600 and 3,000 metres, can be grown at lower altitudes (750 m) and bear fruits. However, no mollusdcide potency tests were done on these berries. Thus, this is the first time an attempt has been made to try and relate the geographical location of a plant and the molluscicidal potency of the berries.

The results of this work have shown a relationship between the geographical location of a plant and the molluscicidal potency of the berries it produces. This information could be useful in trying to define the regions of the country where P. dodecandra plants can be grown for snail control purposes. Efforts are underway to propagate and cultivate plants with high molluscicidal properties in different climatic regions of Zimbabwe with a view to determining the effects of soil types, altitude, rainfall and temperature on the molluscicidal potency of P. dodecandra berries and hence define the regions of the country where plants might be grown for snail control purposes.

References Chandiwana, S.K., S. Mavi and J. Ndamba. (1985). A Preliminary report on the

distribution of P. dodecandra in Zimbabwe. Zimbabwe Agnes J. , H. Murare and S. Mavi. (1984). The Potential of the berries controlling

schistosomiasis in Zimbabwe. Paper presented at the Re-search Day Meeting. University of Zimbabwe. Evans, A.C. (1981). Evaluation of molluscicidal properties of some parochual plants. Annual report 1981. Ministry of Health, Zimbabwe. Lemma, A. (1965). A Preliminary report on the molluscicide properties of Endod (Phytolacca dodecandra). Ethiop. Med. J. 3:187-190. ___ . (1970). Laboratory and field evaluation of the molluscicidal properties of

Phytolacca dodecandra'. Bull. WHO 42:597-617. , D. Heyneman and S.M. Silangwa, eds. (1984). Phytolacca dodecandra (Endod).

Towards controlling transmission of schistosomiasis with the use of a natural product. Final report of the international work-shop, Lusaka, Zambia, March 1983. Dublin: Tycooly International Publishing. Lugt, Ch.B. (1981). Phytolacca dodecandra berries as a means of controlling bilharzia-transmitting snails. Institute of Pathobiology, Addis Ababa University. Addis Ababa: Litho Printers. McCullough, F.S., P. Guyral, J. Duncan and J. Christie. (1980). Molluscicides in schistosomiasis control. Bull. WHO 58:681-689. Motley, A. (1952). Molluscicides. London: Lewis. Parkhurst, R.M., D.W. Thomas, WA. Skinner and L.W. Carry. (1974). Molluscicidal

saponins of Phytolacca dodecandra: Lemmatoxin. Can. J. Chem. 52:702-705. Wolde-Yohannes, L. (1983). Past and ongoing agrobotanical studies of Phytolacca dodecandra (Endod) in Ethiopia. Presented to the international workshop on Phytolacca dodecandra, 7-11 March 1983, Lusaka.

********

Control of Schistosomiasis

Using Indigenous Plants

Ahmed K Bashir and Suaad M. Sulaiman

(Medicinal and Aromatic Plants Research Institute, National Council for Research;

and Bilharzia Research Unit, National Health Laboratories,

Khartoum, Sudan)

Introduction Schistosomiasis is a widespread parasitic disease in tropical and subtropical

countries of Africa, Asia and South America. In Sudan, the establishment of irrigation schemes resulted in the rapid spread of the disease. The prevalence of Schistosoma mansoni was as high as 80 per cent among school children in the Geizera area.

Plants with molluscicidal activity have received considerable attention after the encouraging results of Phytolacca dodecandra as a potential for the control of snails. In Sudan, more than 100 plant species were screened for their molluscicidal activity. The present paper was directed for the comparative study of molluscicidal, cercaricidal

and miracidicidal activity of nine Sudanese plants. Results and Discussion

The extracts of nine plants were tested for their effect on Biomphalaria pfeiffed, miracida and cercariae of Schistosoma mansoni. The results (Table 1) showed that cercariae are more resistant than miracidia to the extracts tested.

The aqueous extracts of B. aegypdaca, G. tufea and R. nilolica were found to be the most potent miracidicidal and cercaricidal extracts in the present study. The active molluscicidal compounds identified in the above-mentioned extracts were found to be saponin in nature. G. kranssiana, which was found to be the most promising molluscicide out of 51 plants investigated, demonstrated poor miracidicidal and cercaricidal activity.

The extracts tested were found to have no toxic effects on cercariae or miracidia at concentrations lethal to the snails. However, many plant samples need to be investigated before any general conclusion can be drawn. Materials and Methods

1. Molluscicidal test. Two adult snail species were used, Bulinus uuncatus and Biomphalana pfeifferi. Screening tests were carried out ac-cording to the method recommended by WHO. The concentration of the different solutions is expressed in ppm of the powdered plant material.

2. Eggs, miracidia and cercariae of S. mansoni. Stools collected from bilharzia patients were pooled and mixed with physiological saline. The eggs were concentrated through a double-layer filter sac. The sediment was washed with cold water, followed by warm water (30°C) and the flask was placed under a source of light until egg-hatching. The miracidia were then collected for toxicity testing.

The snails were infected with miracidia. After four weeks, the snails were screened for cercarial shedding.

3. Preparation of plant extracts. Ten grammes of the dried coarsely powdered

plant material was macerated with distilled water for 24 hours in a perculator. The volume of the perculate was adjusted to 1,000 ml with distilled water to give a concentration of 10,000 parts per million (ppm). This was considered as the stock solution. Further serial dilutions of 1,000, 500, 250 and 10 ppm were made from the stock solution.

4. Toxicity tests a. Ea-hatching: The eggs of S. mansoni were concentrated as mentioned before

and transferred to petri dishes containing the different concentrations of each plant extract. To each petri dish, containing 10 ml of the extract, three drops of the egg de-posits were added and mixed. This mixture was left under a source of light for hatching. The effect of the extracts on egg-hatching was noted under a dissecting micros

iod of three hours. The tim

hours under a dissecting microscope. The LCtm and time ta

when they stopped the r movement and the oral and ventral suckers were extended.

tries, environmental

uscicidal -195.

dal properties of .

HO Technical Series, Report No. 214 (1961).

Schistos rgeon's Point of View

(Mba ital, Mbabane, Swaziland)

gree with some of the

cope b. Miracidiw Five miracidia in a minimum volume of water were transferred to

microtitre plates containing 1 ml of the different concentrations of the extract. The miracidia were examined under a dissecting microscope over a per

e taken to kill all of the miracidia and the LCt® were recorded. c. Cercariae: 15-25 cercariae suspended in 0.1 ml distilled water were incubated

with 1 ml of the different concentrations of each extract in microtitre plates. They were examined over a period of three

ken for it were determined. Dead miracidia showed granular shape and were immobile. Cer cariae were

considered dead i

References Amin, MA. (1976). Arid lands and irrigation in developing counproblems and effects, eds., E. Barton and Worthington, pp. 407-411. Daffala, A.A. and MA. Amin. (1976). Laboratory and field evaluation of the mollproperties of Habatit-el-Mollok (Croton spp.) E. Aft I. Med. Res. 3(4):185El Hadi, MA., A.K Bashir and Y.M. El Kheir. (1984). Plana Medico 1:74. El Kheir, Y.M. and M.S. El Tohami. (1979). J. Trop. Med. & HA. 82:237.

Lemma, A. (1970). Laboratory and field evaluation of the mollusciciPhytolacca dodecandra. Bull. WHO 42:597-617W

********* omiasis: A Su

Dr. Lidia Baiocchi

bane Government Hosp

You are now going to hear the point of view of a surgeon working with bilharzia in this region. I can tell you from the beginning that you might disa

thin

ry tract supra-infections which lead to re

sy. The fresh specimen if examined right away is generally positive for a dual infe

ne. Again, the remarkable fact is that they were negative or had only one or two

ranulomatous reaction around the eggs that

r GI bleeding caused by s

gran

es of this meeting, I have some reservations to make on the ideal drug to wor

e disease. Praziquantel probably remains the drug of choice for out-

gs said because we look at them from a very different angle. I will begin by observing that the disease encountered in the hospital is acutely lethal

and disabling. I refer to massive bleeding from esophageal varices caused by portal hypertension, to carcinoma of the bladder which brings the patients to a miserable end, to incapacitating chronic cystitis, to ascending urina

nal failure. I have seen all of this in Swaziland. The most frequent appearance is rectal bleeding in the 30 to 40 age group. If these

patients are submitted to rectal examination, which is generally negative, I proceed to rectal biop '

ction. My colleagues and myself have seen six cases of upper GI bleeding caused by

schistosomiasis, especially in Pigg's Peak where there occurs a splenomegaly every two months in children 12 to 18 years. In addition, several older women diagnosed with anemia gave a history of haematemesis and melena over several occurrences. What is common to most of those patients is that they were negative for eggs in their stool or urine, but positive for liver biopsy. Some 52 patients, examined in Maputo with portal hypertension due to schistosomiasis and ruptured esophageal varices, had rectal biopsies do

eggs. From the data presented at the Workshop, positiveness for eggs does not mean that

the patient is necessarily going to acquire this disease. By the term disease, I do not mean presence of the infection but the gcauses the symptoms mentioned above. Schistosomiasis is now regarded as an immunological disease due to granulomatous hypersensitivity to the parasite eggs. The person producing a large amount of eggs (perhaps he does not retain them) is likely to be considered an asymptomatic carrier, at least in S. mansoni infections. Again in Maputo, we could detect patients admitted for hernias or hydro-cods who had large egg counts and were at ages from 20 to 40 completely asymptomatic. On the other hand, our patients with portal hypertension and in the same age group were almost dying because of severe uppe

chistosome portal hypertension, and had negative egg counts. So again on clinical grounds, the information about egg shedding capacity is not

useful. What we had in mind was to find a way to identify the person infected with bilharzia who eventually develops portal hypertension, carcinoma of the bladder or the

ulomatous reaction to the eggs which is the prime motor of schistosomal disease. The Ross Institute in London has developed the specific antigen of Schistosoma

mansoni and, as I learned from the Workshop, WHO is offering it on request. We are shortly going to be able to test the serum, patiently collected of patients affected by the disease with the Enzyme-Linked Immunosorbent Assay (ELISA). In order to have a tool to spot them, we hope to be able to see if patients who develop the disease have a different immunological profile from those who simply carry the worms. Coming back to the objectiv

k with. I quite agree with the task you have undertaken, to produce a local, cheap

molluscicide, and wish you the highest success. I plan to use Dr. Chaine's school packet that permits regular examinations of urine in the schools and would be inclined to treat with Praziquantel all those having haematuria. Regretfully, effective drugs are unavailable for treating patients who have the worst manifestations of the disease. Niridazole has the capacity of inhibiting granuloma formation and suppresses delayed hypersensitivity in experimental animals. Because of this important activity, I think it should remain in our armamentarium to treat those patients proven to have granulomas and therefore the true disease. Use should remain restricted to hospitals, linked to specific aspects of th

hospital patients.

Perhaps the presence of the Hon. Dr. Friedman at this Workshop will facilitate the adoption of future policies for a) dean-up of waterways; b) improvement in patient treatment, emphasising hospital care; and c) development of tools to identify those likely

come down with the disease.

********

DO-TOXIN STIMULATION OF THE IMMUNE SYSTEM

Bertok Jr., National Research Institute for Radiobiology and Radiohygiene

otic ria, el

l

l .

omeopathy,

ined

that igen-antibody combination. This led to the

to

EN Immune Stimulation as a Substitute for Anti-Biotics By: W.C. Nelson, Prof. Homeopathy College of Practical Homeopathy, London, England L. ABSTRACT Lately there has been much press about the massive problems of antibitherapy. Antibiotics have prompted the development of many new resistant bactecaused many iatrogenic disease, and prompted immunosuppression by disturbing bowflora. The anti-biotic attacks the bacteria directly and thus furthers the immunosuppression by preventing the natural immune system from working. The press has carried many reports of the continued problems of antibiotics. The medicacommunity is searching for a substitute for these antibiotics. Endotoxins are the lipopolysacharides in the outer levels of bacteria. These toxic compounds can stimulate the immune system to defeat bacteria. Homeopathy is a legal medical art that use this concept. In homeopathy a smaldilute amount of the bacteria is given to the patient to stimulate the immune systemThis is known as nosodal therapy. This article reviews the scientific and homeopathic literature around this therapy as a substitute for antibiotics. KEYWORDS Endotoxins, Lipopolysaccharides, Homeopathy, Initial Values, Nosode HImmune Stimulation BACKGROUND ON ENDOTOXINS Exotoxins are excreted from living microorganisms, whereas Endotoxins are retainside the cell. The Endotoxins are set free when the organism dies. These toxins have powerful stimulating capacities on the immune system. They stimulate antibody production, antitoxins, T-cell, B-cell, and immune cell formation, and immune cell efficiency. One mechanism is that these toxins are modified to inactivate the toxicophore group of the molecule, leaving the antigenic group unchanged.(ref. Tyler, Brady, Robbers 1988). These Exo and Endotoxins can be used to build immunityas in immunization. Certain of these endotoxins however produce general stimulation of the immune system. In other words they increase and fortify the entire immune system towards all intruders.(ref. Tyler, Brady & Robbers.1988) Over one hundred years ago Jules Bordet first detected the presence of factors could augment and stimulate ant

complement fixation test. The most important part of the complement activation involve the third complement component C3. The proposed classical pathway of activation and

s e

sential n

l activity and they are thus also known as 2 demonstrates this.

roduct stimulated

entire immune system. Research has shown LPS to be involved in metabolism, immunology, physiology,

s e

tities

n Budapest Hungary.

e icro

4,

e

ma cove

s that enhance the Endotoxin

ability and safe methods of administration have also been investigated. 1994 saw

OBLEMS WITH ANTIBIOTICS

eemed to offer so much for medicine. Many previously ubborn diseases responded to their touch. But was it just a short term result at then would have later complications. The problems of antibiotics came not

severe

conversion appears in the first figure. There are many other ways that Endotoxincan help the body. The Endotoxins are rich in Lipopolysaccharides (LPS) which arcontained in the cell wall of gram-negative bacteria. These bacteria are esfor life and must be part of the healthy bowel flora. In the bowl they help inutrient absorption, assimilation, detoxification, and systemic regulation of immunity. Endotoxins can also act to excite B-cellyclonal B-cell simulators. Figure po

FIGURE 1 *(Rapidly dissociates unless stabilization factors are present such as Endotoxins, some lgA or lgG)(ref. Reeves and Todd, 1990) **(end pimmunity) FIGURE 2 POLYCLONAL STIMULATION OF B-CELL BY T-CELL Thus the Endotoxins are nonspecific stimulators of the toxicity, and biosynthesis.(ref. Strain 1983, Munford 1981, Morrison 1981, Galano

induc1977, Kurtz 1982, Openheim 1986, Rick 1982, Skelly 1979) LPS re shown tosynthesis of interleukins and T independent antigens. However in large quanthey are pyrogenic. So how do we take advantage of these naturally immune stimulating compounds? One of the finest experts in Endotoxins is Dr. Lorand Bertok working at theNational Research Institute for Radiobiology and Radiohygiene iHis work started in the 1950’s and he has authored hundreds of articles on Endotoxicity, some with Dr. Hans Selye. Dr. Bertok writes in several articles andbooks about the stimulation of nonspecific resistance by Endotoxins. Thesrefined Endotoxins have wide range stimulation effects against all pathologic mintruders. The effects extend to viral and fungal defence to as well (ref. Bertok,1962, 1963, 1964, 1965, 1966, 1968, 1969, 1973, 1977, 1978, 1980, 1983, 1981985,1988, 1990, 1992, 1994) A review of this research will show the dramatic benefits and safe levels of use for these Endotoxins. The research has demonstrated positive effects of these Endotoxins on immunfunction, alcohol damaged livers, radiation defence, ACTH level, serum T4, lymphotropic sensitivity, peripheral lymphocyte number and effectiveness, traure ry, serum ribonuclease activity, catecholamine storage, digestion, and positive effects on bowel flora. The research has also demonstrated certain cofactoreffect. Stthe start of a specific journal for Endotoxins.(ref. Endotoxins,) PR The antibiotic revolution sstthonly from overuse but from a allopathic short fix philosophy. The most

problem of antibiotic use was the secondary immunosuppression they caused. defeating the bacteria directly they robbed the immune system of its livelihooand produced weakness. Antibiotics disrupted bowel flora balance and thus further disturbed ifunction as well as nutrient absorption and detox. Nature responded to the antibiotics by producing resistant strains which make current

By d

mmune

use questionable. ups the

viral

nature is the best answer. By helping the natural process we are

CKGROUND ON HOMEOPATHY

medicine that today is legal in the United world. The art started with the concept

is best to use to cure. Jenner and the founding fathers immunization also agreed with this philosophy. Homeopathy concentrates

ng

de lp.

patient

ound e a weak immune system. In cases of extremely weak

alled

makes ss the

c

te for antibiotics.

ed our medical use. Complex homeopathy is an ever growing form of

dicine that can be easily learned. The College of Practical Homeopathy in London post graduate level via the internet

By etting the natural balance of microflora in the body and the environment antibiotics allowed for an increase in fungal and viral diseases. Also antibiotics are the most misprescribed medication, being given for colds and flu inappropriately. Much has been spoken about this in the press and medicine seeks new solutions.Encouragingparticipating in the most technologically advanced process in the world.(ref. Newsweek) BA Homeopathy is a hundred year old art ofates, Europe, India, and most of theSt

that what causes a disease ofon the minimal dosage or safest dosage and thus lost favor with an ever increasifast food culture. Antibiotics were developed for fast results and to attack the intruder directly. But immunosuppression, disease resistant strains, and sieffects resulted. Leaving modern medicine looking for help. Homeopathy can heHomeopathy also offers antifungal and antiviral capacities that make misprescribing much less likely. This can have a major impact on improving care. This is nosodal homeopathy. Since Endotoxins are toxic in large quantities, using the dilution principles of homeopathic succusion will offer a solution. By using a 7 x or 1 part per 10 million and combining the LPS derived from the bacterium we can engineer a combination homeopathic that could take the place of antibiotics. This compwill work to stimulatmuneim function other homeopathic stimulation will be desirable. This is c

sarcodal homeopathy. Homeopathy has been clinically shown effective for stimulating antibodies, treating infections, and stimulating the immune system.(ref. Nelson papers) The minimal dose philosophy of homeopathy along with its experienceit an ideal vehicle for helping medicine. To review these papers acceinternet at http:// usa > net / qmed. A patented Homeopathic process has blendeda combination homeopathic that captures the Endotoxins with homeopathistabilizers and enhancers to achieve a refined substitu The product type has been clinically tested and produced in an FDA registerlaboratory for ymeEngland even offers Continuing Education at the address.

CLINICAL SUGGESTIONS In our medical clinic in Budapest we almost exclusively use the BAC for all the bacterial conditions weways also recommend s

see. Our Clinical experience has been excellent. We tress reduction , good nutrition, exercise and avoidance

all immunosuppressants. The product comes as a liquid and is designed to be taken day. If the infection is in an acute state the take 4 drops e condition starts to abate. Then let the natural immune

en

reme

registered o e

circumvented the natural process there were disturbing long term side effects. rhaps the homeopathic-Endotoxin revolution will fare better.

medical,

Bertok L. Kemenes F. The effects of various experimental pathophysiological uction Z. IMMUN forsch 1962 124; 280-285 1962

Toth B ,Studies on the pathogenesis of the edema disease of swine LPS Vet Acad Sci Hung 13 421’-428 1963

Bertok L. Simon Gy. Winter M. Morava E. The pathophysioogy of LPS Endotoxins

ed.

oxins on the serum T4 Immunopharacology

st ihi K.N. Lange W.Immunological and nonspecific defence mechanism

amon

dman H. Klien ss 1990

ors in affecting plasma ACTH and cortcosterone levels J. endocrine

alof7 to ten drops twice a ery 30 min. till thev

system take over while continuing the twice daily administration for ten to fourtedays. This formula can also be used as a preventive taken during cold and flu season. It is safe for children and the elderly but half dosage is recommenced for toddlers and infants. No contraindications with other remedies other than extalcohol sensitivity are reported. DISCUSSION So after years of research and work we can now bring to you a refined NDC substitute for antibiotics. Complex homeopathy is safe, easy, and effective tuse. The antibiotic revolution was successful with short term results. But becausitPe REFERENCES 1. Barron D. N. The Essentials of Clinical Biochemistry. Elsevier BioAmsterdam, 1982. 2.conditions on the antibody prod3. Bertok L.effects Acta4.Acta Physio Hung. 1965 26-7 5. Bertok L. Conference on Bacterial endotoxins, 1968 Ann. Immunol. 6. Bertok L. Conference on immunological and pathological effects of bacterial Endotoxins Ann Immun Hung 1973 7. Bertok L. Immunological properties of detox LPS Immunology 1978 Separtum pp.463-470 8. Bertok L. Role of Bile in detoxification of LPS in Schelessinger D Microbiology 1980 pp. 91-93 9. Bertok L. U. Nagy Zs. The effect of Endot1984 8, 143-146 10. Bertok L. Lead-acetate induced Endotoxin hypersensitivity Experienta 1985 41; 575-576 11. Bertok L. Radiodetoxified Endotoxin a potent stimulator of nonspecific hodefence in Masagainst microbial infections Advances in the Biosciences Vol68, 365-371 PergPress Oxford 1988 12. Bertok L. Stimulation of non specific resistance by Endotoxin. FrieT.W. Endotoxin pp 677-680 Plenum Pre13. Elenkov I.J. Kovacs J. Kiss L. Bertok L. LPS Is able to bypass corticotrophinreleasing fact1992 23; 154-156 14. Essential Medicine. Edited by Ried Jones. Churchill Livingston, 1993.

15. Fust Gy. Bertok L. Interactions of the radio detox E-coli Endotoxin with thecomplement system. Infec. Immun 1

gton, DC June 1993. e

ty

and Dome Research

bach J. A. (1981)in contemporary Topics in Molecular

. Press, 1992. r Homeopathy II. cad. Press,

Acupuncturists, Chiropractors and Naturopaths.

troacupuncture.

0 P. D. et al J of Bacteriology 150 447(1982)

ase.

lymphocytes with Endotoxin

m. 5, 83 (1965)

Yerba Santa as an American Indian herb was used for asthma. It is an indigenous ant to America, with relatives indigen , India, and South America. The plan part of the waterleaf family and it’s parts are used for rope or broom making and it is lled devil’s broom in parts of Africa. It was first discovered as a remedy for Viral plication inte

DESCRIP ush or single

977 16;26-31 16. Galanos C. et al (1 977) International Review of Biochemistry of Lipids III vol. 14 pp. 2309-335 University Park Press Baltimore17. Harvey and Bordley. Differential Diagnosis. Saunders, 1976. 18. Homeopathic Pharmacopeia of the United States. Homeopathic Pharmacopeia Convention of the United States, Washin19. Kristina G. Bertok L. Effect of radiodetox Endotoxin and trace elements on threticulo-endothelial system damaged by ethanol in rats Acta Microbio Immun Hung1994 41;465-471 20. Kutas V. Bertok L. Effects of Endotoxins on the serum ribonuclease activiJ. Bact. 1969 100; 550-551 21. Kurtz H. J. et al Amer. J. Vet Res. 43, 262 (1982) 22. Lewis. Medical Botany. Wiley, 1977. 23. Merck Manual (15th ed.). Edited by Berkow, MD Merck, SharpLaboratories, 1987. 24. Munford R. S. et al J. Clin Invest 68, 1503 (1981) 25. Morrison DC and RudImmunology Vol. 8 pp. 187-218 Plenum Press 26. Nelson W.C. Quantum Quality Control. Acad. Press, 1993. 27. Nelson W.C. Experimental Evidence for Homeopathy. Acad28. Nelson W.C. Experimental Evidence fo1992. 29. Nelson W.C. Homeopathy for Acad. Press, 1993. 30. Nelson W.C. Homeopathy for Nutritionists. Acad. Press, 1993. 31. Nelson W.C. A Legal Outline of the Medical Practice of ElecAcad. Press, 1993. 32. Openhiem J.J. et al Immunol Today 7, 45, (1986) 33. Reeves G. Todd I .Lecture Notes on immunology. Blackwell Sci Publications 19934. Rick35. Pharmacognosy (9th ed.). Tyler, Brady & Robbers. Lea Febiger, 1988. 36. Skelly R. et al Infect. Immun. 23, 287 (1979) 37. Smith and Their Pathophysiology: The Biological Principle of DiseSaunders, 1981. 38. Strain S.M. et al J> Biol. Chem. 258, 2906 (1983)39. Temesi A. Bertok L. Stimulation of human peripheral Acta Microbio Acad Sci Hungary 1982 30; 1 3-1 40. Westphal O. an Jann K. Methods in Carbohydrate Che

*********

Brief Overview of Yerba Santa and Devil Broom Studies

Agronomic Studies

pl ous to Africaisac

re rference in the studies of Dr. Nelson in Hungary. TION- The Yerba S sinous evergreen banta is an Aromatic, re

plant. Sometimes forming thickets to 10 ft. Stems hairless and sticky. Leaves leathery and

d asthma remedy for tube

into the Kenyan game pres They tried to g

root and the Soap berry Phytolacca. The

lly, maintaining their

y Profes r extracts nor any of the various alcohol extracts of Yerba Santa tested have mutagenic properties under the standardised test conditions in his labora ificantly modify a 1994 IJMSH report which consid

sticky. lance shaped lower leaf with wooly hairs, margins coarsely toothed. May - Aug Flowers purple to white, tubular, in clusters, petals 5-lobed.

WHERE FOUND: dry arid rocky slopes and ridges, chaparral, roadsides, Yerba Santa is known as the "Saintly Herb" and has sweet spicy leaves. the leaves

were boiled, strained and added sugar to make a cold anrculosis, heating the leaves and inhaling the vapors used for dizziness, hayfever,

coughs, TB, pneumonia, and immune stimulation. The legend of it’s use in Kenya was from many years ago, when 5 AIDS patients in

a jail in Nairobi escaped. They made a suicide pact and went outerve. They traveled by night and slept during the day to escape detection. et the lions to eat them but the lions did not agree to the plan. The lions walked

away. So the resorted to trying to eat the most noxious plant possible. Many medicines that help us offer a noxious taste to the sick person. It is an instinct. They got sick when they ate the Yerba Santa, the Yucca Devil's broom

y got disentery, vomit, but they did not die. Instead they felt gradually better. After some months, they came out of the game preserve in Mombassa.

They were arrested and returned to jail in Nairobi. To the surprise of the doctors they were indeed better. In fact when tested there was no sign of AIDS. When this was reconfirmed in the research in Hungary.

Over the last ten years, selected new as well as known strains of the Yerba Santa plant from Kenya, have been carefully introduced in Zimbabwe and Mozambique by an international team of agronomists led by Prof. W. Nelson . In these new sites, the plants have proven to grow successfuhigh HIV anti-retro-viral potencies and immunological properties.

These studies are being supported in part by small grants from Nahinga. Toxicological Studies Along with the agronomic work, short- and long-term toxicologic studies

are being undertaken by scientists in Hungary, Mozambique, and Ethiopia. Early findings from these studies include the following:

1. Extensive mutagenic studies have been completed and published bor Nelson. These studies have established that neither the wates

tory. His findings signered water extracts of Yerba Santa to be non-mutagenic but alcoholic

extracts mutagenic. The work of Dr. Nelson established that neither are mutagenic. No Mutagenic properties found.

2. Acute and chronic toxicologic studies of Yerba Santa in different animals and various aquatic biological systems have been underway since 1993 at the IMUNE Professors Nelson and Sirbu have been conducting this work with the financial support of the IMUNE. Preliminary results support earlier toxicologic findings from work done in Ethiopia, where a very low order of toxicity in test animals and biological systems was found. These and the agronomic studies on Yerba Santa in Kenya are continuing.

3. Acute and chronic toxicologic studies underway for several years at the Institute of Pathobiology in Addis Ababa by Dr. Ephraim Mamo, a veterinary pathologist, tested Yerba Santa in sheep, monkeys, rabbits and other animals.

These tests are also encouraging in terms of the low order of toxicity found. The tested animals showed high tolerance to doses of Yerba Santa extract, well above likely exposure levels when administered orally or even intravenously. These findings strongly verify earlier unverified statements that Yerba Santa is safe as a HIV anti-retroviral.

So far, no funds have been found to support this work either from WHO or

other sources other than Nahinga. a potecy of 5x (one part per hundred thousand) has been shown perfectly r all. A 4x (one part per ten thousand) safe for most adults. More

ntrated forms (3x) are somewhat Noxious not toxic. But 2x and 1x are not neded. The 4x or 5x, w

Use atsafe foconcereccom ill take a minimum use of every day for one month, two months recomafter e

ended for successful anti-viral interference. Us of 5x imediantly xposure to the virus have been shown to be quite effective (95%) in stopping

the virus. so this Hemo A formula is a very effective morning after remedy. This alone could save the nation if it's use was wide spread.