The use of and search for drugs and dietary supplements derived from plants have accelerated in recent years. Ethnopharmacologists, botanists, microbiologists, and natural-products chemists are combing the Earth for phytochemicals and “leads” which could be developed for treatment of infectious diseases. While 25 to 50% of current pharmaceuticals are derived from plants, none are used as antimicrobials. Traditional healers have long used plants to prevent or cure infectious conditions; Western medicine is trying to duplicate their successes. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimicrobial properties. This review attempts to summarize the current status of botanical screening efforts, as well as in vivo studies of their effectiveness and toxicity. The structure and antimicrobial properties of phytochemicals are also addressed. Since many of these compounds are currently available as unregulated botanical preparations and their use by the public is increasing rapidly, clinicians need to consider the consequences of patients self-medicating with these preparations.

Plant Products as Antimicrobial Agents

Antimicrobial host defense peptides are produced by all complex organisms as well as some microbes and have diverse and complex antimicrobial activities. Collectively these peptides demonstrate a broad range of antiviral and antibacterial activities and modes of action, and it is important to distinguish between direct microbicidal and indirect activities against such pathogens. The structural requirements of peptides for antiviral and antibacterial activities are evaluated in light of the diverse set of primary and secondary structures described for host defense peptides. Peptides with antifungal and antiparasitic activities are discussed in less detail, although the broad-spectrum activities of such peptides indicate that they are important host defense molecules. Knowledge regarding the relationship between peptide structure and function as well as their mechanism of action is being applied in the design of antimicrobial peptide variants as potential novel therapeutic agents.

Peptide Antimicrobial Agents

https://dspace.library.uu.nl/bitstream/handle/1874/1590/PMPP-van+Loon-1999.pdf?sequence=1

The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins

Various mechanisms to fend off microbial invaders have been devised by all living organisms, including microorganisms themselves. The most sophisticated of these mechanisms relies on the synthesis of immunoglobulins directed against specific microbial targets. However, immunoglobulin-based immunity operates only in a relatively minor subset of living species, namely the higher vertebrates. A much more ancient and widespread defense strategy involves the production of small peptides that exert antimicrobial properties. As products of single genes, antimicrobial peptides can be synthesized in a swift and flexible way, and because of their small size they can be produced by the host with a minimal input of energy and biomass. Wellknown examples of antimicrobial peptides are the cecropins that accumulate in the hemolymph of many invertebrates in response to injury or infection (reviewed by Boman and Hultmark, 1987) and the magainins that are secreted by glands in the skin of amphibians (reviewed by Bevins and Zasloff, 1990). Cecropins and magainins are small (20-40 residues) basic peptides displaying an amphipathic a-helical structure that can integrate in microbial membranes to form ion channels (Duclohier, 1994). Another class of antimicrobial peptides is formed by the Cys-rich peptides, which in contrast to cecropins and magainins, have a complex cystine-stabilized three-dimensional folding pattern often involving antiparallel ,3-sheets. Defensins are one class among the numerous types of Cys-rich antimicrobial peptides, which differ in length, number of cystine, bonds, or folding pattern (reviewed by Boman, 1995). Insect defensins (34-43 residues, three disulfide bridges) are, like cecropins, produced in a pathogeninducible manner by the insect fat body and secreted in the hemolymph (reviewed by Hoffmann and Hetru, 1992). Mammalian defensins (29-34 amino acids, three disulfide bridges) are produced by various specialized cells in the mammalian body (reviewed by Lehrer et al., 1993; Ganz and Lehrer, 1994). For example, they are very abundant in granules of phagocytic blood cells. These granules fuse with phagocytosis vesicles containing microorganisms, where the defensins are thought to contribute, together with other antimicrobial proteins and active oxygen species, to killing of the engulfed microorganisms. Defensins are also secreted by epithelial cells of the intestines and airways, where they may help maintain the normal microbial flora in a steady state. In addition, the expression of defensins in the airway epithelium has been shown to be up-regulated after exposure to bacterial lipopolysaccharides (Diamond et al., 1993). The importance of defensins in innate immunity of humans is underscored by the observation that certain disorders characterized by recurrent infections are associated with a lack of defensins in blood phagocytes (Ganz et al., 1988). Moreover, transposon mutants of a pathogenic Salmonella strain known to infect and grow inside phagocytes simultaneously lost their resistance to defensins (and other antimicrobial peptides) and their virulence (Groisman et al., 1992). Recently, we characterized a novel class of plant peptides whose structural and functional properties resemble those of insect and mammalian defensins. Hence, we termed this family of peptides "plant defensins" (Terras et al., 1995).

Plant defensins: novel antimicrobial peptides as components of the host defense system.

In the S locus-controlled self-incompatibility system of Brassica, recognition of self-related pollen at the surface of stigma epidermal cells leads to inhibition of pollen tube development. The female (stigmatic) determinant of this recognition reaction is a polymorphic transmembrane receptor protein kinase encoded at the S locus. Another highly polymorphic, anther-expressed gene, SCR, also encoded at the S locus, fulfills the requirements for the hypothesized pollen determinant. Loss-of-function and gain-of-function studies prove that the SCR gene product is necessary and sufficient for determining pollen self-incompatibility specificity, possibly by acting as a ligand for the stigmatic receptor.

https://science.sciencemag.org/content/sci/286/5445/1697.full.pdf

The male determinant of self-incompatibility in Brassica.

Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds.

We have developed a simple protocol to allow the production of transgenic banana plants. Foreign genes were delivered into embryogenic suspension cells using accelerated particles coated with DNA. Bombardment parameters were optimized for a modified particle gun resulting in high levels of transient expression of the beta-glucuronidase gene in both banana and plantain cells. Bombarded banana cells were selected with hygromycin and regenerated into plants. Molecular and histochemical characterization of transformants revealed the stable integration of the transferred genes into the banana genome.

Genetic Transformation of Banana and Plantain (Musa spp.) via Particle Bombardment

Although thionins and 2S albumins are generally considered as storage proteins, both classes of seed proteins are known to inhibit the growth of pathogenic fungi. We have now found that the wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) thionin concentration required for 50% inhibition of fungal growth is lowered 2- to 73-fold when combined with 2S albumins (at sub- or noninhibitory concentrations) from radish (Raphanus sativus L.) or oilseed rape (Brassica napus L.). Furthermore, the thionin antifungal activity is synergistically enhanced (2- to 33-fold) by either the small subunit or the large subunit of the radish 2S albumins. Three other 2S albumin-like proteins, the barley trypsin inhibitor and two barley Bowman-Birk-type trypsin inhibitor isoforms, also act synergistically with the thionins (2- to 55-fold). The synergistic activity of thionins combined with 2S albumins is restricted to filamentous fungi and to some Gram-positive bacteria, whereas Gram-negative bacteria, yeast, cultured human cells, and erythrocytes do not show an increased sensitivity to thionin/albumin combinations (relative to the sensitivity to the thionins alone). Scanning electron microscopy and measurement of K+ leakage from fungal hyphae revealed that 2S albumins have the same mode of action as thionins, namely the permeabilization of the hyphal plasmalemma. Moreover, 2S albumins and thionins act synergistically in their ability to permeabilize fungal membranes.

Synergistic Enhancement of the Antifungal Activity of Wheat and Barley Thionins by Radish and Oilseed Rape 2S Albumins and by Barley Trypsin Inhibitors

On the basis of an extensive screening of seeds from various plant species, we have isolated and characterized several different antimicrobial peptides They were all typified by having a broad antifungal activity spectrum, a relatively low molecular weight (3-14 kDa), a high cysteine content and a high isoelectric point (pI > 10) With respect to their amino acid sequence, these peptides can be classified into six structural classes Synergistic enhancement (up to 73-fold) of antimicrobial activity was demonstrated in some combinations of peptides belonging to different classes cDNA clones corresponding to different antifungal peptides were isolated and used to transform tobacco plants Extracts of these transgenic plants showed higher (up to 16-fold) antifungal activity than untransformed control plants Such antimicrobial peptides may find applications in molecular breeding of plants with increased disease resistance

Hilde M. E. Schoofs

Papers

Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds.

Genetic Transformation of Banana and Plantain (Musa spp.) via Particle Bombardment

Synergistic Enhancement of the Antifungal Activity of Wheat and Barley Thionins by Radish and Oilseed Rape 2S Albumins and by Barley Trypsin Inhibitors

Gene-encoded antimicrobial peptides from plants.