The ability of certain antimutagenic agents to prevent development of antibiotic resistance.

Resistance to multiple antimicrobial agents has now become a prominent fact of contemporary life. It is believed that poor patient compliance, e.g. interrupted or premature cessation of therapy; and misuse or abuse of antibiotics, e.g. wrong antibiotic or insufficient dose, play important roles in resistance development. We present evidence that, this form of resistance often stems from spontaneous mutations accompanied by the positive selecting pressure of the doses of antibiotics being between the MIC and MBC levels. A number of antimutagenic agents, e.g. green tea catechins, and other antioxidants, etc. are able to suppress the emergence of resistance. In many cases, these agents are capable of exerting these effects at doses which by themselves produce no visible effect on growth. In a number of cases antimutagenic substances capable of preventing resistance emergence are present in normal food stuffs. These effects are exerted against resistance to tetracyclines, fluoroquinolones, macrolides, beta-lactams, aminoglycosides and the like. The implications of these laboratory findings for practical chemotherapy are discussed.

Role of antimutagens/anticarcinogens in cancer prevention.

Recently it has become increasingly clear that chemicals found in our foods and beverages can prevent the genetic damage that leads to cancer initiation. Such substances may also affect subsequent events in the pathways that lead to cancer, and may have the potential to inhibit the mutations that allow tumor cells to become resistant to antitumor agents. We describe here the antimutagenic potential of Glabrene analogs against EMS-induced mutations utilizing modified Ames tests in S. typhimurium TA 100 and E. coli JC 5088. Results of studies of the ability of well-known antioxidants such as EGCG and related compounds to prevent drug resistance mutations in microorganisms are described, and their possible significance in the prevention of chemotherapeutic drug-resistance in tumor cells is discussed.

Structure-antimutagenic activity relationship study of plicatin B.

A systematic structure-activity relationship study of plicatin B (1), an antimutagenic constituent of Psoralea juncea, was undertaken with a view toward elucidating its chemical mode of action and possibly optimizing its antimutagenic activity during the process. Compound 1 and its related analogues were examined for their antimutagenic activity against mutations induced by ethyl methanesulfonate, a direct acting mutagen and alkylating agent, in Salmonella typhimurium strain TA100, utilizing the modified Ames test protocol. The dihydro analogue 3 resulting from saturation of the conjugated alkene double bond of 1 was found to exhibit reduced cytotoxicity and enhanced efficacy relative to the parent compound. This result serves preliminarily to exclude a Michael acceptor role of the alpha,beta-unsaturated carbonyl moiety in connection with its antimutagenic activity.

Antimutagenic/antioxidant activity of green tea components and related compounds.

The ability of green tea components and other antioxidant compounds to function as antimutagens/antioxidants has been well established, and their role in cancer prevention is supported by numerous epidemiological studies. We have utilized modified Ames tests, superoxide scavenging assays, and assays for protection against DNA scissions to compare and contrast the protective effects of various teas and commercial and laboratory-isolated tea components to those produced by compounds such as resveratrol, selenium, curcumin, vitamins C and E, quercetin dihydrate, sulforaphane, ellagic acid dihydrate, glutathione reduced, trolox, butylated hydroxanisole (BHA), butylated hydroxytoluene (BHT), and N-acetyl-L-cysteine (NAC). In Ames tests, employing hydrogen peroxide as a mutagen, epigallocatechin gallate (EGCG) produced the highest level of protection of all antioxidants tested. Measurement of protection against DNA scissions produced results that again showed that EGCG produced the strongest protective effects. In scavenging assays using a xanthine-xanthine oxidase (enzymatic system), epicatechin gallate (ECG) showed the highest scavenging potential. In a nonenzymatic (phenazine methosulfate-NADH) oxidizing system, EGCG once again showed the strongest effects. The implications of these and similar results are discussed in relation to cancer prevention and prevention of drug/antibiotic resistance.

Tuberculosis: a search for novel therapy starting with natural products.

The reemergence of tuberculosis and closely related diseases as significant public health problems is briefly reviewed with particular emphasis on the exacerbating role of AIDS and multiple drug resistance. Screening methods available for discovering new chemical entities active against resistant strains are discussed and their use in screening extracts and compounds for activity is illustrated with a number of newly discovered structures being presented. In particular, the properties of the potent and structurally novel indoloquinazolinone alkaloid, tryptanthrin, is described. Many analogs of this lead structure were synthesized by combinatorial and multiple parallel synthetic techniques and evaluated in vitro and in vivo for their potential in the chemotherapy of human infections.

Antitubercular natural products: berberine from the roots of commercial Hydrastis canadensis powder. Isolation of inactive 8-oxotetrahydrothalifendine, canadine, beta-hydrastine, and two new quinic acid esters, hycandinic acid esters-1 and -2.

Berberine (4) is responsible for the activity of an extract of a commercial root sample of Hydrastis canadensis against multiply drug resistant Mycobacterium tuberculosis. Two new quinic acid feruloyl esters, compounds 2 and 3, have been isolated from the same source along with canadine (1c), 8-oxotetrahydrothalifendine (1), and beta-hydrastine (5). These were found to be inactive. The structures of the new compounds were elucidated from spectral (1H, 13C, HMQC, HMBC, and H-H COSY) and chemical evidences.

A simple, inexpensive apparatus for performance of preparative scale solution phase multiple parallel synthesis of drug analogs. II. Biological evaluation of a retrospective library of quinolone antiinfective agents.

A series of pure fluoroquinolone antiinfective agents was prepared by multiple parallel synthesis using a simple new apparatus. These compounds were evaluated biologically against Gram-positive and Gram-negative microorganisms and against a BCG strain transfected with luciferase in a fluorescence-based antitubercular assay. Activity against relatively fast growing, acid-fast Mycobacterium smegmatis was determined in part by agar-dilution streak assays. Data obtained against Escherichia coli-derived DNA gyrase does not correlate well with whole cell assays against E. coli. These compounds were assayed by a convenient glass-fiber filter binding method modified for high throughput screening. In these analogs, the results with a N-1 cyclopropyl substituent were often inferior to those obtained with a N-1 2',4'-difluorophenyl substituent. None of the new compounds prepared was superior in its antimycobacterial potency to ciprofloxacin or temafloxacin.

Rapid screening of natural products for antimycobacterial activity by using luciferase-expressing strains of Mycobacterium bovis BCG and Mycobacterium intracellulare.

The object of this study was to investigate the ability of a rapid luciferase assay to detect antimycobacterial activity in plant extracts. Recombinant strains of Mycobacterium bovis BCG (rBCG) and Mycobacterium intracellulare expressing firefly luciferase were used as the test organisms. Assays were conducted in a 96-well minitube format under biosafety level 2 conditions. Control and test wells were sampled immediately after inoculation and after 3 (recombinant M. intracellulare) and 5 (rBCG) days of incubation to measure luminescence with a microplate luminometer, and the relative change in luminescence was calculated as a percentage of control values. As an alternative test method, Alamar blue was added after 12 days of incubation, and changes in color were read visually. A total of 480 extracts were tested. Sixteen extracts were active against rBCG, and of those, seven were also active against recombinant M. intracellulare. With activity defined as a relative change in luminescence of < or = 1% (i.e., > or = 99% inhibition) and a persistence of blue color after addition of Alamar blue, there was 99.0% agreement between the two methods. Our results suggest that the luciferase assay is rapid and accurate and has the potential to greatly accelerate the evaluation of antimycobacterial activity in plant extracts in vitro. With this method, it is possible to screen a large number of samples in a short period of time.

Natural antimutagenic agents.

Following a brief review of recent discoveries in the field of natural antimutagenic and tumor chemopreventive agents, contemporary findings in the author's laboratories employing the direct acting mutagen, ethyl methanesulfonate, in modified Ames tests and eukaryotic murine FM3A mammary tumor cells modified to be subject to thymidine-less death are described to illustrate the underlying principles. The EMS studies are illustrated with the isolation of the novel antimutagen, plicatin B, from the medicinal plants, Psoralea juncaea and P. plicata. The FM3A studies are carried out with extracts of Styrax asiatica, a plant previously studied extensively with the EMS system. The FM3A findings closely parallel the earlier work with EMS showing that the responsible agents, cinnamic acid, cinnamoyl ricinoleate and cinnamoyl cinnamate are effective both in prokaryotic and eukaryotic tests and that the new FM3A assay system has useful properties for screening and assay of novel antimutagenic agents.

Wallifoliol, a taxol congener with a novel carbon skeleton, from Himalayan Taxus wallichiana.

A new taxoid, wallifoliol [3], has been isolated, along with five known taxoids (taxol, cephalomannine, 10-deacetylbaccatin III, brevifoliol, 2-acetoxy brevifoliol = taxchinin A) from extracts of the needles of Himalayan Taxus wallichiana. The structure of wallifoliol has been assigned primarily from nmr studies. Wallifoliol [3] is assigned a structure in which rings A and B of the taxane system have undergone putative rearrangements producing a novel skeleton. Wallifoliol is the first diterpene to be found in nature with this particular 5/6/6/6/4 ring system.

Antimutagenic activity of extracts of leaves of four common edible vegetable plants in Nigeria (west Africa).

Organic solvent extracts of leaves of 4 common edible vegetable plants--Bryophyllum pinnatum, Dialium guincense, Ocimum gratissimum and Vernonia amygdalina--had inhibitory activity for His- to His+ reverse-mutations induced by ethyl methanesulfonate acting on Salmonella typhimurium TA100. The concentrated ethyl acetate, methanol and petroleum ether extracts were heat-stable when dissolved in dimethyl sulfoxide. The Bryophyllum ethyl acetate extract was fractionated into alkaloidal/water-soluble, acids, polar lipid and non-polar lipid fractions. The polar and non-polar lipid fractions inhibited reversion mutations induced by ethyl methanesulfonate acting on TA100 or TA102, and were also active against reversions induced by 4-nitro-O-phenylenediamine and 2-aminofluorene in TA98. The alkaloidal/water-soluble and the acid fractions had no appreciable antimutagenic activities.

Extracellular interception of mutagens.

Extracellular interception of mutagens by excreted enzymes or by chemical agents that react with or bind to formed mutagens provides an important means of defense against chemical mutagens/carcinogens. Kada and Shimoi have classified molecules that function in this manner as "desmutagens," and many of them are natural cellular metabolites. Among the specific mechanisms that such agents may employ are: prevention of the activation of "promutagens" to mutagens; stimulation of enzymes (e.g., glutathione-S-transferase) that catalyze the binding/inactivation of damaging electrophiles; direct binding and concomitant inactivation of promutagens or mutagens; interference with uptake of mutagens into cells; etc. De Flora and Ramel have provided an excellent discussion of the mechanisms of these agents and a proposed classification scheme. Drawing on work from our own laboratories and other recent examples in the literature, several examples of mechanistic approaches to these studies using natural plant-derived materials, e.g., humic acid, Glycyrrhiza glabra extract, glutathione, and bioflavonoids, are also described. Antioxidants and agents that conjugate electrophiles will be among the modes of action described for obtaining the goal of intercepting mutagens/carcinogens.

Glycyrrhiza glabra extract as an effector of interception in Escherichia coli K12+.

Glycyrrhiza glabra polar lipid extract contains a number of flavonoids and related chemical compounds. Studies on the effectiveness of Glycyrrhiza glabra polar lipid extract in intercepting reactive molecules generated from the illumination of the photosensitizers rose bengal and phenosafranin indicate that it is effective in preventing cytotoxicity against E. coli K12+ in a dose-related fashion using illuminated rose bengal. Since only a modest scavenging of singlet oxygen generated from phenosafranin is observed, the effects of the extracts are less related to singlet oxygen-mediated oxidation of substrate (type II reactions) than non-singlet oxygen-mediated oxidation of substrate (type I reactions). Elevated levels of glutathione observed in exponentially growing cells of E. coli K12 were also observed.

Antimutagenicity of secondary metabolites from higher plants.

Higher plants contain both mutagens and antimutagens and are susceptible to mutagenesis but screening programs for detection of antimutagenesis rarely employ higher plant systems. Short-term bacterial and mammalian tissue culture systems are the norm. Using modified screening tests for detecting antimutagenic agents, higher plants have been shown to contain a variety of structurally novel antimutagenic agents. Systematic bioassay-directed methodology resulted in the isolation in pure form and biological and chemical characterization of the responsible individual active components from various plants. The methodology in use is illustrated by the isolation of cinnamic acid, cinnamyl cinnamate and cinnamyl ricinoleate as the active constituents of the classic medicinal plant product, Styrax asiatica. The methods which may be used to reveal structure-activity relationships and to explore putative molecular modes of action are illustrated with excerpts from the same study.

A modern look at folkloric use of anti-infective agents.

Infectious diseases are of ancient origin, and mankind has a venerable history of use of higher plant extracts for the therapy of such infections. Some such agents survive in use from earlier times--quinine, emetine, and sanguinarine, for example--but the modern use of fermentation-based antibiotics has greatly overshadowed work on agents from other sources. After a brief review of the present status of the field of antibiotics, this review focuses upon the present status of antimicrobial agents from higher plants with particular reference to agents from plants with a folkloric reputation for treatment of infections. In particular, recent work on the tropical genus Erythrina is emphasized. The use of modern microbiological techniques demonstrates that higher plants frequently exhibit significant potency against human bacterial and fungal pathogens, that many genera are involved, that many folkloric uses can be rationalized on this basis, that the active constituents are readily isolated by bioassay-directed techniques, that their chemical structures are types uncommon amongst fermentation-based agents but are familiar to natural product chemists, that their antimicrobial spectra are comparatively narrow but that their potency is often reasonable, that they are comparatively easy to synthesize and the unnatural analogues so produced can possess enhanced therapeutic potential and, thus, it is concluded that such work generates a gratifying number of novel lead structures and that the possibility of finding additional agents for human or agricultural use based upon higher plant agents is realistic.

Total chemical synthesis and antitumor evaluation of the 9-aza analogue of N-(trifluoroacetyl)-4-demethoxydaunomycin.

The 9-aza analogue of N-(trifluoroacetyl)-4-demethoxydaunomycin has been synthesized from 2,5-dimethoxybenzaldehyde. Pomeranz-Fritsch condensation followed by borohydride reduction and acid-catalyzed cyclization led smoothly to 4-hydroxy-5,8-dimethoxy-1,2,3,4-tetrahydroisoquinoline. Selective N-acetylation and subsequent Friedel-Crafts acylation with phthalic anhydride produced 2-acetyl-5,12-dihydroxy-1,2-dihydro-2-azanaphthacene-6,11-dione, which was protected as its dimethyl ether and epoxidized to an acylated aza Brigl's anhydride. This was converted to (+/-)-2-acetyl-4-hydroxy-5,12-dimethoxy-1,2,3,4-tetrahydro-2- azanaphthacene-6,11-dione by dehydration to the 4-keto analogue followed by cyanoborohydride reduction either stepwise or in situ. The protecting groups were removed with boron trichloride and the resulting aglycone glycosidated with optically active N,O-bis(trifluoroacetyl)daunosamine bromide and silver trifluoromethanesulfonate. The resulting diastereoisomers were separated by column chromatography and their structures established by CD and NMR spectroscopy. Unexpectedly it was not possible to remove the N-trifluoroacetyl blocking group without aromatization to the azanaphthaquinone. Both (R)- and (S)-acetyl-4-O-[N-(trifluoroacetyl)daunosaminyl]-5,12-dihydroxy-2- azanaphthacene-6,11-dione were inactive ip in mice carrying the P388 tumor. Drugs were given at various concentrations on days 0, 5, and 9.

Total chemical synthesis and antitumor evaluation of 4-demethoxy-10,10-dimethyldaunomycin.

The novel anthracycline analogue 4-demethoxy-10,10-dimethyldaunomycin was prepared in nine chemical steps from 5,8-dimethoxy-2-tetralone. It proved to be inactive as an antitumor agent in the mouse P388 lymphocytic leukemia model.