Baja California Plants Screened for Antitumor Activity

by

Richard Spjut and Richard Marin
World Botanical Associates
Laurel, Maryland and Temecula, California

Prepared August 2000

Presented in Spanish by Richard Marin at the Symposium IV on Botany Research of Baja California

Ensenada, Mexico (September 2000)

©The World Botanical Associates Web Page
Prepared by Richard W. Spjut
April 2003

Abstract

We reviewed plants that have been screened from Baja California, and list those species that are known to be active.  This includes an overview of plants screened for antitumor activity from the southwestern deserts.  Most samples from Baja California were screened in KB Cell Culture, P-388 Leukemia, and Astrocytoma. Additional collections have been obtained for many active species; however, we lack information on the active compounds.  The National Cancer Institute had terminated their screening program in 1981 just after most samples were collected and screened (1979-1980).  Fractionation and further screening has been undertaken for some species by chemists affiliated with drug discovery groups at universities.

 Introduction

The vegetation of Baja California is well known for its diversity of plant forms; thus, one might expect to find new chemical substances for treating cancer and other diseases.  We present a historical summary on plants that we know have been collected from North American Deserts for antitumor screening, and specifically mention those that have shown antitumor activity from samples we collected in Baja California. 

 Historical Overview

 The samples we have obtained for drug discovery programs generally follow three steps in screening:

Screening Plants for
Novel Active Chemicals:
Three Steps

1. Preliminary Screening—Crude Extracts—from Random or
    Select Samples: 100 g to 2 kg.

2. Isolation of Active Agents from Recollections: 5-100 kg,
    fractionation guided by bioassay results.

3. Clinical Evaluation of Active Agents: 50-20,000 kg
    Example: 8 g of maytansine from 8,000 kg of Maytenus
    buchananii stems for 200 patients.

Initial (general) samples of root, stems, twigs, leaves, or any combination of these, weighing 100 g to 2 kg dried, are collected for extraction in which crude extracts are tested in several or more bioassays.  During 1978-1980 general samples from ~ 400 species were collected from the southwestern deserts including Baja California.  These were extracted with aqueous/ethanol and chloroform by the NCI contract facility in Wisconsin (RALTECH).  Based on NCI reports of Cumulative Plant and Animal Materials considered active, the extracts of Baja California plants were tested in KB Cell Culture or Astrocytoma, and P-388 Leukemia.

A second stage in the evaluation requires recollections in amounts of 5-100 kg to isolate, identify and evaluate the active agents. Isolation of active agents is guided by bioassay results in which activity is determined by significant inhibitory effects on tumor or cell growth.  Not all recollections reconfirmed because activity may depend on environmental factors or stages in development of the plant.  For example, samples of Asclepias albicans collected by Spjut in the Chuckawalla Mountains of California showed activity in KB in an initial sample when plants were collected in flower bud, but failed to reconfirm when later collected in flower from the same locality.  No recollections of plants were made from Baja California for the NCI because the NCI had terminated the screening of natural products in October 1982; however, later collections were made of active plants that were screened in bioassays other than the ones originally employed.

A third stage in the evaluation requires massive samples—usually many tons—to obtain sufficient quantities of the active compound for clinical evaluation.  About one in every thousand species screened by the National Cancer Institute between 1960 and 1982 had reached this stage.  During this time the NCI screened ~35,000 species.  Examples of plants from the Sonoran and Chihuahuan deserts include Castela emoryi (holacanthone), Colubrina californica (colubrinol), and Bouvardia ternifolia (bouvardin).  None of these passed clinical studies, however. 

Thus far, the successful compounds are from plants collected elsewhere.  Taxol, currently the most successful drug from the NCI program, was discovered from stem-bark of Taxus brevifolia in Pacific Northwest America. Another is synthetic derivatives of camptothecin, which was isolated from Camptotheca acuminata (Nyssaceae), a species native to China that was collected in cultivation in southern California.  The need for more Camptotheca led to large-scale cultivation at a USDA plant introduction station in Chico, California. Despite the relatively few discoveries, the collection of 35,000 species of plants during the initial 22+ years (1960–1982) provided valuable pharmacological data on secondary metabolites in plants. 

 

The Baja Flora Screened
In Perspective to Other Collections Screened
From the Southwestern Deserts

Sonoran Desert plants screened for antitumor activity are summarized in the table below under four periods of time during which changes were made in the screen: (1) 1960–1964, (2) 1965–1969,  (3) 1970–1981, and (4) 1986–2000.  These are discussed in context with the major suppliers of samples.

Suppliers of Plant Samples and/or Extracts
 from the North American Deserts
for Antitumor Screening

Supplier                                                       # Species

 

University of Arizona:1960–1964                      1,043  fresh
            mostly Arizona and Sonora                     375  dried
University of Arizona: 1965–1969                        570  dried
USDA (Gentry & Barclay): 1965–1969                  ~500  dried
            Mexico and S California
WLRI (Marin): 1971 Baja California                       95 dried
USDA (Spjut): 1972 California                           ~200 dried
USDA (Spjut): 1978–1980                                  ~400 dried
            US (AZ, CA, NM, NV, TX, UT)
            Mexico (BCN, BCS)
University of Arizona: 1970–present                             ?
Morris Arboretum/NCI: 1997-2001                                ?
WBA (Spjut & Marin): 1986-2000                        ~400 dried
WBA (Spjut & Marin): 2001-

  1. 1960–1964. The University of Arizona. Many Sonoran Desert plants were screened by Jack Cole at the University of Arizona in Tucson during 1960-1964.  Most of their collections came from Arizona and Sonora (Mexico).  Aqueous, ethanol, and chloroform extracts were prepared and tested against Adenocarcinoma 755 (CA), Sarcoma 180 (SA), Melanotic Melanoma (MM), Lewis Long Carcinoma (LL), Lymphoid Leukemia (LE), Solid Friend Virus Leukemia (FV), Adenocarcinoma of the Duodenum (Dl), Walker 256 (WA), and KB (Eagle).  However, not all tumors were employed at the same time; SA, LE and KB were those most frequently used.   Collection data were summarized by Spjut in an accomplishment report submitted to Mexican authorities via the Agricultural Counselor at the American Embassy in Mexico City (Spjut 1981).  This report indicated that 1,043 species were screened.  Approximately three times as many species were extracted from fresh samples than dried samples; the dried samples were represented by 375 species.  Activity occurred more frequently in aqueous extracts of fresh samples, 1.9–2.6%, compared to dried samples, 0.8%–1.6%; most activity was in the KB assay.  Most active agents were later found to be tannins, sesquiterpene lactones, phytosterols, and cucurbitacins; however, one compound, a peptide, cesalin, that was isolated from Caesalpinia gilliesi, underwent clinical studies.

 2. 1965–1969. In 1964, the extraction procedure was modified to remove tannins and phytosterols, and extracts were then screened against WA, KB and LE.   From the desert regions ~570 species were collected by Dr. Cole’s group, and a similar number was also obtained by USDA explorers Howard Gentry and Arthur Barclay.  Significant discoveries from this procedure include holacanthone from Castela emoryi, colubrinol from Colubrina texensis, and bouvardin from Bouvaridia ternifolia.

 

Antitumor Active Compounds

 

     

                      

                 Colubrinol              Holacanthone               Bouvardin

   

Other compounds of interest were isolated from species of Aristolochia, Thevetia, Stemmadenia, and  Hunnemannia; however, these occur mostly outside the desert regions.

3. 1970–1981. In addition to these novel discoveries, the WA and KB assays were sensitive to sesquiterpene lactones, while activity in the LE 1210 bioassay was so infrequent that it was felt leads were being missed.  In 1969, the NCI substituted P-388 (PS) Leukemia for LE, and in 1980 replaced the KB assay with Astrocytoma (ASK).  They also modified the extraction procedure in 1976.  Further limitations were imposed on plant procurement in 1978; excluded from screening were many pantropical and pantemperate genera and many widely distributed species that were combined into a single listing know as SLOP for Species Low On Priority; collectively, these taxa amounted to ~60,000 species being precluded from further collecting.

In 1978 Spjut identified areas in the United States, Mexico and elsewhere where random collecting might be less impacted by SLOP and where many new plant genera might be encountered for collection.  From the North American deserts he obtained samples from ~ 400 species in 256 genera.  Represented from Baja California were 89 root samples, 129 stem or twig samples, and 32 samples of other plant parts.   Subsequently, he collected 74 samples during May 1986, and ~100  samples since then.  The active plants from Baja California are as follows:

 

Acalypha californica  (Euphorbiaceae)             stems-leaves and flower           KB

Acanthogilia gloriosa (Polemoniaceae)            root, twig-leaf                        1-5 tumors

Atamisquea emarginata (Capparaceae)            root                                       ASK

Bergerocactus emoryi (Cactaceae)                  root                                        ASK

Berginia virgata (Acanthaceae)                       stem-leaf                                 ASK

Bursera microphylla  (Burseraceae)                 root, stembark, twig-leaf          KB

Bursera sp.                                                    twig-leaf                                 KB

Camissonia crassifolia (Onagraceae)                 root-stem-leaf-flower              ASK

Castela peninsularis (Simaroubaceae)              twig-leaf                                 KB

Castela polyandra                                          root, twig                               KB, PS

Crossosoma bigelovii  (Crossosomataceeae)     twig-leaf                                 1-5 tumors

Dalea (Psorothamnus) juncea                         root, stem-leaf-flower              1-5 tumors

Dicraurus alternifolius (Amaranthaceae)          twig-leaf                                 ASK

Dyssodia anthemidifolia (Asteraceae)              root-stem-leaf-flower               ASK

Eriogonum preclarum (Polygonaceae)              root-stem-leaf-flower              1-5 tumors

Esenbeckia flava (Rutaceae)                            stem-bark                               KB

Forchhammeria watsonii (Koeberliniaceae)      root                                       ASK

Frankenia palmeri (Frankeniaceae)                  stem-leaf-flower                     1-5 tumors

Gochnatia arborescens (Asteraceae)                stem-bark                                KB

Hermannia palmeri (Sterculiaceae)                  root-stem-leaf-flower              KB

Hoffmannseggia intricata (Fabaceae)               root, stem-leaf-flower              ASK

Jatropha cinerea (Euphorbiaceae)                  stem-bark                                PS

Krameria erecta (Krameriaceae)                     stem-leaf                                 ASK

Marina parryi (Fabaceae)                               root                                        ASK
Mascagnia macroptera  (Malpighiaceae)           root, twig-leaf                         ASK

Merremia aurea (Convolvulaceae)                  stem-leaf                                 ASK

Nama cf. hispidum (Hydrophyllaceae)             root-stem-leaf-flower               1-5 tumors

Olneya tesota (Fabaceae)                               root                                        1-5 tumors

Orobanche cooperi (Orobanchaceae)              stem-leaf-flower                       ASK

Pachycormus discolor (Anacardiaceae)            root                                         1-5 tumors

Phaulothamnus spinescens (Achaptocarp.)      root                                         ASK

Rhus integrifolia (Anacardiaceae)                   root                                        1-5 tumors

Salvia cedrosensis (Lamiaceae)                       root, stem-leaf-flower              1-5 tumors
Salvia mellifera (Lamiaceae)                          inflorescence (flower)               1-5 tumors

Sphaeralcea axillaris (Malvaceae)                   root, stem-leaf-flower               ASK

Stegnosperma halimifolium (Stegnosperm.)    root                                         ASK

Stillingia linearifolia (Euphorbiaceae)            root                                         PS

Viguiera deltoidea (Asteraceae)                     root                                         KB

Xylonagra arborea (Onagraceae)                    tuber                                        PS

 

Total: 39 Active Species                                19 root
                                                                      4   stem-bark
                                                                    24 stem-leaf or twig-leaf

                                                                      1 inflorescence

Because many of these samples were obtained just prior to the NCI terminating their agreement with the USDA in 1982, we have little knowledge of the active agents. Based on other screening data, phorbol esters were isolated from many Euphorbiaceae, lignans from Burseraceae, alkaloids from Rutaceae, and sesquiterpene lactones from Asteraceae.  We know nothing about the active compounds that might be responsible for activity in Astrocytoma (ASK).  According to Dr. McCloud at the Drug Development and Therapeutic Branch in Frederick, Maryland, this assay may have become lost.

 (4) 1986–2000. Extracts from 74 samples representing 56 species were collected in Baja California plants during 1986 by Spjut, Marin, John Cassady (Dean, School of Pharmacy at the Ohio State University; http://ntpd.pharmacy.ohio-state.edu/Dean/) and Tom McCloud (former student of Cassady at Purdue University, now at the NCI extraction lab in Frederick, Maryland; http://spheroid.ncifcrf.gov/stb/staff/mccloud/mccloud.cfm).  These were later screened against 60 tumor cell lines.

Examples of Human Cancer Cell Lines
Currently Employed by the National Cancer Institute for
Screening Plant Extracts

 Panel/Cell Line

Leukemia CORF-CEM            Breast Cancer
      HC-60                            MCF7
                 K562                              MCF8ADR-RES
                       MOLT-4                          MDA-MB-23UATCC
         RPML-8226                      HS-578T
               SR                                  MDA-MB-435
Prostrate                               MDA-N
         PC3                               BT-549
      DC-145                          T 43D
           

The cell lines are divided into panels.   Each panel includes one or more assays pertaining to a particular organ such as lung, colon, ovarian, etc.  None of the Baja plant extracts showed specific cytotoxicity—that is activity that is selective to one panel, as opposed to general cytotoxicity in many tumor panels that is seen more often. Additionally, extracts of these samples were screened for anti-AIDS activity, and the Baja plants were being considered for screening of drug resistant antibiotics.  The lack of discoveries thus far should not be interpreted as negative as screening criteria by which plants are being selected for drug development have become more stringent.

Approximately 100 samples collected during 1986-1990 from Baja California were also screened by university chemists, Dr. Ching-jer Chang at Purdue University, and Dr. John Cassady at Ohio State University. They also employed the NCI human cancer cell lines, but on a more limited scale. Included were five cancer lines in four panels involving lung (1), breast (1), colon (1) and two of the skin (2). Species that were of interest in these screens are indicated as active in 1 to 5 cell tumor lines.  However, most recollections did not meet activity criteria to justify further pharmacological study.  Those that did are Dalea (Psorothamnus) junceus and Pachycormus discolor. A paper was reportedly in press by Dr. Chang and his associates on the active chemical compounds they isolated from samples of Dalea juncea.  Dr. Cassady reported only marginal activity in Pachycormus, presented by one of his students in a poster paper at the 1988 annual meeting of the American Institute of Biological Sciences.

Additionally, there are other active plants that were collected outside Baja California that occur in Baja California.  These include KB active Colubrina californica as mentioned earlier and Crossosoma bigelovii. Colubrinol is an ansamacrolid related to maytansine that underwent clinical trials but trials were discontinued due to cytotoxicity with a low therapeutic index.  Recollections of Crossosoma, on the other hand, were reportedly inconsistent in activity; nevertheless, one active compound, 5,7-Dihydroxy-8-methoxy-2-methylochromona, was isolated from samples collected in Arizona.

Two active compounds isolated early in the NCI program include synthetic derivatives that are either currently used in treating cancer, or are undergoing further clinical evaluation.  These are derivatives of camptothecin from Camptotheca acuminata (Nyssaceae) and quassinoids in Simaroubaceae.  Synthetic derivatives of holacanthone and other naturally occurring quassinoids are actively being studied by Paul Grieco at Montana State University who has found other novel compounds in Castela such as polyandrol in C. polyandra and peninsularinone in C. peninsularis ((http://www.chemistry.montana.edu/grieco.html) .

 

      

 

Polyandrol

It is interesting that 23 of the 39 species listed from Baja California are represented by root and stem-bark samples.  Twelve species showed activity only in their root part (or tuber), compared to eight species for aerial parts only.  These results are consistent with KB and P-388 data on plants screened from Kenya, Tanzania, Ghana, Peru, and Turkey as presented elsewhere (Spjut unpublished, see home page and click on World Botanical Associates).  Generally, active chemical agents in tropical species are most often found in stem-bark, whereas activity in plants from drier regions is more often found in extracts of root.  The importance of root samples is also evident from ethnobotanical studies. A case in point is root of octotillo (Fouquieria splendens) that has been used by the Apache Indians to treat painful swellings (Krochmal et al., Economic Botany, 1954). This species usually grows on rocky soils where it is not easy to dig, and the spreading thorny stems also makes access to the root even more difficult; thus, if the Apache Indians had to expend considerable effort to get to the root, we should make the same effort to collect root for screening.

In view of the preceding data on active plants in Baja California, the flora of Baja California probably contains many novel active chemicals yet to be discovered. Such discoveries are likely if plants are sampled systematically.  Our efforts in previous years have focused on perennial and woody plants, but we have not had the resources to compile a record as to what plants might best be collected in the future.  Currently, we draw mainly from our knowledge and experience as we travel in the field for collections.  Further information about our program can be found on our website, www.worldbotanical.com.

  Acknowledgments

 

Agricultural Attaché, American Embassy, Mexico, D.F.
Consejo Nacional de Ciencia y Tecnologia
Direccion General de Investigación y Capacitación Forestales
Aprovochamientos Forestales
General de Eonomía Agrícola
Universidad Autonoma de Baja California, Facultad de Ciencias
Herbario (BCMEX) y Farmaclogía Marina

  Memoranda, Permits, and Reports

American Embassy letter, 3 Jan. 1979, indicating permit status and where voucher specimens and accomplishment report are to be deposited.

1979 Mexico-US Travel Report

[USDA ARS] Accomplishment Report.  Procurement of Plant Samples from Mexico & U. S. for Antitumor Screening. August 1981.  17 pp.

1982 Correspondence: Travel Plans to recollect antitumor active plants in Mexico

1982 Plant Collecting Permit

 

Wall M. E., M. C. Wani, G. Manikumar, H. Taylor and R. McGivney. 1989.  Plant antimutagens, 6. Intricatin and intricatinol, new antimutagenic homoisoflavonoids from Hoffmanosseggia intricata. J Nat Prod. 52(4): 774–778.

Zhang H, X. Li, C. L. Ashendel and C. J. Chang.  2000. Bioactive compounds from Psorothamnus junceus. J. Nat. Prod. 2000 63(9): 1244–1228.  “During a search for bioactive compounds from Psorothamnus junceus, four heterocyclic compounds, psorothamnone A (1), psorothamnone B (2), dalrubone (3), and emorydone (4) were isolated from the ethanol extract of the stem bark. Psorothamnones A (1) and B (2) demonstrated inhibitory activity against protein kinase C (PKC), a key enzyme involved in the signal transduction of cell proliferation and differentiation. Dalrubone (3) and emorydone (4) showed cytotoxicity against several human tumor cell lines.”