SCREENING DATA FROM THE CANCER CHEMOTHERAPY NATIONAL SERVICE CENTER SCREENING LABORATORIES. XXXVU. PLANT EXTRACTS B. J. Abbott,1 J. L. Hartwell,1 J. Leiter3 R. E. Perdue, Jr.,3 and S. A. Schepartz1 SUMMARY Data are reported on 1197 plant extracts which were tested in the primary screens of the Cancer Chemotherapy National Service Center. Almost all the materials were tested in Sarcoma 180, Adeno- carcinoma 755, Leukemia 1210, and KB cells in culture; some tests were also carried out in Dunning leukemia ascites, Lewis lung carcinoma, Human sarcoma HS1, and Melanotic melanoma. Data are re ported only on materials which have not demonstrated sufficient activity in these systems to warrant fur ther investigation. The number of test systems in the screening program has recently been reduced; the reasons for the reduction are discussed. INTRODUCTION This report is a continuation of the series of publications of Cancer Chemotherapy National Service Center (CCNSC) screening data, and is the ninth report dealing with plant extracts. Data are included on systems incorporated into the primary screening spectrum a few years ago as well as those used ex clusively until that time. The presentation of data on these systems has been made possible by the re duction of all information to a unified line summary, which facilitates data storage and retrieval by an IBM 1410 computer. A substantial change in data presentation was made recently to permit the inclusion of the newer tumor systems. The following description of materials and methods, etc. , is similar to that of earlier publications, which first discussed preparation of plant extracts (18) and which first discussed the inclusion of the new test systems (19). MATERIALSANDMETHODS Service Screening Laboratories All the test data reported were obtained under contracts with the CCNSC. Test procedures are prescribed in a set of protocols followed by all contractors. Reports of the experiments are submitted to the CCNSC for review and statistical evaluation. The summary for each experiment gives the code identi fication of the screening laboratory; these codes are listed later. Source of Plant Extracts All plant materials were collected under the auspices of the New Crops Research Branch of the U. S. Department of Agriculture (USDA)under a transfer of funds from CCNSC. Botanists from the USDA named the plants and also submitted information on the location and date of the collection; a specimen of each collection is maintained by the USDA. Extraction was carried out by the Wisconsin Alumni Research Foundation under contract with the CCNSC. In Vivo Procedures Tumors and Host Animals. Almost all the materials reported in this publication have been screened in three transplanted mouse tumors: Sarcoma 180 (S180), Mammary adenocarcinoma 755 (Ca755), and an 1 National Cancer Institute, National Institutes of Health, USPHS, Bethesda, Maryland 20014. 2 National Library of Medicine, USPHS, Bethesda, Maryland 20014. 3 Crops Research Division, Agriculture Research Service, U. S. Department of Agriculture, Beltsville, Maryland. 1302 CANCER RESEARCH VOL. 26 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. Screening Data from the Cancer Chemotherapy National Service Center Screening Laboratories ascitic form of leukemia 1210 (L1210). These are well-known transplantable tumors and are adequately described elsewhere (5, 14, 17). The Screening Panel of the CCNSC selected these tumors on the basis of experience available in 1955; great weight was given to the ability of these screens to select almost all the materials then considered useful against cancer in man. Since it was also recognized that these test systems might not be the optimal ones to predict for clinical effectiveness, considerable effort was made to develop and evaluate a variety of additional sys tems. As a result of this work more systems in mice, rats, hamsters, and embryonated eggs were incor porated into the screen. A new material of unknown activity was tested in L1210 and 2 other test systems which were selected on a random basis. If quantities were limited, fewer test systems had to be used. Materials related to active agents were not tested in a random group of systems but rather in the system sensitive to the parent compounds, normally by multidose testing. In selecting additional test systems, an effort was made to find systems which were different from the original systems in their drug sensitivity and which could be used as reliable screens from the standpoint of reproducibility. After sufficient experience was gained in the evaluation of new agents against this random screen, a correlation study was undertaken to evaluate the predictability, if any, of these test systems against cancer in humans (16). This retrospective analysis of 88 drugs which have been evaluated clinically showed that, of the 45 compounds with definite clinical activity, the activity of 33 was predicted by lymphoid leukemia L1210, 9 by Walker 256 (intramuscular), 1 by Dunning ascites leukemia and 2 by Ca755. As a result of this evaluation, many of the test systems previously used as primary screens have now been discontinued as unproductive. Emphasis is now placed on screening new agents in the L1210 and Walker 256 tumors. Data on drugs tested in the various tumors will be published in this and future issues even though these tumors are no longer used. In addition, the procedures described here are those in effect when the compounds were tested, not necessarily the procedures in effect at the present time. The test systems which are or have been used as primary screens are given below, along with the host used for propagation and test: Tumor Host for Propagation Host for Test Adenocarcinoma 755 C57BL/6 female mouse BDFXmouse (C57BL/6 female x DBA/2 male) Cloudman melanoma (S91) DBA/2 BDFi mouse Ehrlich ascites Randombred albino mouse Randombred albino mouse Friend virus leukemia DBA/2 mouse BT>71mouse Hepatoma 129 C3H/He mouse C3H/He (or hybrid) mouse Lewis lung carcinoma C57BL/6 mouse mouse Lymphoid leukemia 1210 DBA/2 mouse mouse Osteogenic sarcoma HE10734 C3Hf mouse C3H/He mouse Sarcoma 180 Randombred albino mouse Randombred albino mouse Dunning leukemia ascites Fischer/344 rat Fischer/344 rat Human sarcoma HS1 Suitable randombred rat Suitable randombred rat Murphy-Sturm lymphosarcoma Suitable randombred rat Suitable randombred rat Walker 256 carcinosarcoma Suitable randombred rat Suitable randombred rat (intramuscular) Adenocarcinoma of duodenum Hamster Hamster Adenocarcinoma of endometrium Hamster Hamster Adenocarcinoma of small bowel Hamster Hamster Melanotic melanoma Hamster Hamster Human sarcoma HS1 Embryonated egg Embryonated egg Preparation of Plant Materials. Currently, a single aqueous/ethanol extract is made for each plant sample by treating approximately 100 gm of finely ground dried plant with 300 ml of anhydrous ethanol and 300 ml of distilled water stirred for one hour at room temperature. The extract is then filtered through cheesecloth and evaporated to remove the alcohol. The alcohol-free filtrate is then freeze-dried to a NOVEMBER 1966 (Part 1303 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1966 American Association for Cancer Research. B. J. Abbott, J. L. Hartwell, J. Leiter, R. E. Perdue, Jr., and S. A. Schepartz powder. However, for some of the samples reported here, two extracts were prepared as follows: Aqueous extracts were made by stirring or shaking the finely ground plant sample with water (5-6 ml/gm) at room temperature for at least one hour. In some cases, such as fresh fruits and bulbs, the sample was ground and extracted simultaneously in a high-speed blender. The extract was decanted through a fast filter, concentrated under reduced pressure at 40°C or less, and lyophilized. The alcoholic extracts were made by combining a portion of the original dried plant material with 2-3 parts of 95% ethanol, and stirring, or by using a Soxhlet extractor. The material was filtered and the residue extracted a second time. The 2 filtrates were then combined, and concentrated to a semisolid consistency under reduced pressure at 40°C or less. The lyophilized aqueous extracts were dissolved in saline or suspended in 0. 5% methylcellulose (MC) or carboxymethylcellulose (CMC). Alcoholic extracts were dissolved in a small quantity of 95% ethanol and then suspended in MC or CMC. Occasionally alcoholic solutions were diluted with saline or with MC solution to a concentration not exceeding 2% alcohol. Because of the gummy nature of these extracts, satisfactory suspension of the material was not always possible. Also marked changes in toxicity occasionally occurred when there was a long interval between successive tests. These facts must be considered in evaluating the data. Test Procedures. The detailed protocols for each of the tumor systems given above have been published (23), and they will therefore not be repeated here. Copies of these protocols are available on request.4 These protocols include the methods in use at the time of testing the materials reported here. For a discussion of some of the earlier procedures, one may refer to a previous publication in this series (20). Tumors are transplanted under aseptic procedures including sterilized instruments and hooded areas. Fragments or suspensions are sampled for sterility in every experiment by using thioglycollate broth tubes. If bacterial growth occurs within 48 hours, the entire experiment is discarded.