Antimicrobial Agents

Identifying and characterizing new active ingredients in African medicinal plants to combat phytopathogenic fungi in agricultural crops

Wheat germ infection assay
© Fraunhofer IZI
Wheat germ infection assay.

Phytopathogenic fungi and their resistance to conventional fungicides are increasingly becoming a problem and compromising the global supply of agricultural products. This is why researchers around the world are searching for effective alternatives. In Africa, all kinds of plant extracts have long since been used here, especially to combat phytopathogenic fungi and to modulate the rhizosphere.

The objective of this project was to extract fungicidal agents from African medicinal plants and test them in vitro and in planta using a suitable technology platform in order to develop products based on these extracts that could then be marketed in the future. The substantive focus here was placed in particular on plants that could be administered orally in human and veterinary medicine. This already implies low off-target toxicity in humans and animals, which is a key prerequisite if the approval procedure is to be a success.

Extracts were first gained from the barks, leaves and roots of various eastern African plants with the aid of different solvents from the eluotropic series before their toxicity was tested against the conidia and mycelia of relevant phytopathogenic fungi (e.g. Botrytis cinerea, Fusarium graminearum, Colletotrichum graminicola). Toxicity testing was conducted both in vitro (microdilution assay) and in planta (leaf infection assay, stem infection assay, fruit infection assay). The toxicity of the extracts was compared to that of conventional fungicides (e.g. tebuconazole). The results showed that plant extracts have an entirely comparable fungicidal effect. The plant extracts actually demonstrated a much better fungicidal effect in the case of fungicide-resistant, harmful fungi species (e.g. Fusarium graminearum, PH-1 strain). This is due to the fact that, over the course of evolution, several different fungicidal ingredients acting independently of each another were generally formed in plants as part of a co-evolutionary adaptation process to changing environmental conditions.

The plant extracts studied could be used, among other things, in seed dressing, as a fungicide in conventional crop protection and to treat the surface of citrus fruits. In addition to having a fungicidal effect, several plant extracts also demonstrated root growth induction in the case of wheat germ. Several plant extracts could therefore also be used as plant strengtheners.

Evaluation of effective antimicrobial compounds taken from plant tissues for the treatment of bacterial infections in preclinical immunotoxicological models

Nowadays, the treatment of nosocomial infections, especially those comprising multidrug-resistant bacteria and fungi, presents one of the greatest challenges to modern medicine. Hygiene measures that are in place yet not consistently observed in hospitals, the overuse of antibiotics and lack of strict regime on the part of patients taking them as well as the exorbitant use of antibiotics in large-scale, industrial livestock farming have all led to a situation that can barely be kept under control. The problem of multidrug resistance will only get worse in the long term due to increasing average life expectancy and the associated decrease in the strength of the immune system in older people. This project was initiated to test the effect of antimicrobial peptides and active substances isolated from plants on typical human pathogenic germs. These include Methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), bacteria that could produce extended-spectrum beta-lactamases (ESBL) and several human pathogenic fungi from the candida and aspergillus genera. The focus here was placed in particular on plants from tropical parts of Africa, as plants growing under these climatic conditions often form highly efficient, antimicrobial active substances during the course of their co-evolutionary adaptation. The antibiotic effect of plant-based secondary substances (AMPS) was first examined on attenuated laboratory strains of the respective human pathogenic bacteria and/or fungi species. Moreover, toxicity tests were conducted on the plantbased active substances with regard to different cells from the immune system. This is necessary in order to detect immunomodulatory effects at an early stage. A mouse model was set up to evaluate the effect of the AMPS in vivo. The efficacy of the AMPS in vivo was determined using several clinical parameters (e.g. survival rates, release of specific immune and activation markers, improvement in clinical score, etc.). An extensive histological organ analysis was carried out alongside these investigations. In all, active substances were isolated from several plants which could, in future, be utilized for a broad range of applications such as antibiotics, wound dressings and nutraceuticals.

Application of antimicrobial peptides for increasing the shelf life of food products

The effect of antimicro­bial peptides on the growth of Candida albicans
© Fraunhofer IZI
The effect of antimicro­bial peptides on the growth of Candida albicans.

Antimicrobial peptides (AMP) are an integral component of the defense systems of animals and plants. Their range of activity comprises bacteria, fungi and viruses. The intended project aims at the development of suitable peptides that effectively kill germs, in particular those associated with putrefaction, during production processes in the food industry. Thereby, for instance, the shelf life of fresh salads is intended to be increased by at least two days. On the basis of preliminary studies, sequence motifs have been produced from AMP having a known antimycotic / antibacterial activity and their effectiveness against yeasts, mildew and enterococci has been tested in an in vitro assay. We plan to concentrate, in particular, on short-chain antimicrobial peptides (<20 amino acids) as there are no immunological complications to be expected in case of a later application in association with food products. Five AMP could be identified that have a potent inhibitory effect on the growth of fungi and bacteria. No measurable toxicity to eukaryotic cells could be observed.

Development of therapeutically effective peptides for the treatment of contagious and tumor diseases

The need for new, effective drugs based on bioactive substances has increased greatly in recent years. The predictions for the future also reveal strong growth in this segment. This trend was picked up on in the Vascular Biology Unit and a technology platform was created which is able to develop and evaluate peptides both against multi-resistant hospital germs and also against tumor cells. This DNA-based technology allows an appropriate, antibiotically effective peptide to be developed against every relevant hospital germ by means of a high-throughput technique. Some of these antimicrobial peptides have a broad-spectrum effect and could thus be applied against a number of different types of bacteria or also pathogenic fungi (e.g. candida albicans).

During the course of 2012, several sequence libraries were established with partly differing ranges of efficacy, e.g. against human-pathogenic oral germs (cariogenic germs such as streptococcus mutans, streptococcus sobrinus or pathogens associated with paradontitis such as actinobacillus actinomycetemcomitans, porphyromonas gingivalis), germs found in the gastrointestinal tract (heliobacter pylori) and also against germs found in the respiratory tract (haemophilus influenzae).

The use of bioactive substances from plants, insects and amphibians for the treatment of inflammations and also tumor diseases (e.g. the so-called ”frog vaccine”) has been common practice for a number of years, particularly among the indigenous peoples of Central and South America. This is why, together with the Immunotherapy – Oncology Unit, peptides from the skin secretion of tropical frog species (e.g. phyllomedusa bicolor) were cloned in an additional experimental approach and several amino acids from these peptides were mutated at defined positions. Compared with the original peptides, it could be demonstrated in vitro that the cytotoxicity of these peptides, in comparison with tumor cells, could be increased by modifying the amino acid sequence, while control cells showed a comparably high level of resistance. Although the mechanism of action of these peptides was not yet able to be clarified, the assumption suggests that the different composition and net charge of the cell membrane of tumor cells and non-tumor cells play a decisive role here. Moreover, some of these peptides may have an additional, immunomodulatory effect.

Development of tumor therapeutics and antibiotics from plant extracts

Bark of an African plant from which ingredients were isolated
© Fraunhofer IZI
Bark of an African plant from which ingredients were isolated.

About 80 percent of the tumor therapeutics currently in use originate from substances of the secondary plant metabolism. Many tumor diseases (e. g. brain tumors, pancreatic carcinoma) are still difficult or even impossible to cure despite the progress that has been made in developing new chemotherapeutic agents. In close cooperation with naturopathic scientists from Africa substances / extracts from about 20 plant species were prepared which, to some extent, have a potential as new anti-tumor drugs for a potential application in palliative care. First examinations on various cancer cell lines attest the selective effect of the plant ingredients on tumor cells. These data could be verified in experiments in a mouse model. This gives rise to new options for tumor therapy, in particular for brain tumors. Moreover, some plants hold highly effective antibiotic ingredients, in particular in bark and roots, which also have the potential for applications in human medicine.

Development of silicon surfaces for the directed differentiation of murine stem cells

Cultivation of murine stem cells on microstructured silicon surfaces with the aim of induc­ing directed differentiation
© Fraunhofer IZI
Cultivation of murine stem cells on microstructured silicon surfaces with the aim of induc­ing directed differentiation.

In most cases, stem cells are located in tissue niches and exhibit a low metabolic activity. Their differentiation is only initiated by altered conditions (stimuli) in the microenvironment. The exact sequence of processes, however, is mostly unknown. Stem cell differentiation is significantly determined by intrinsic cellular signaling and extrinsic stimulators (cell-to-cell contact, contact between cell and extracellular matrix). Extrinsic signaling can be of a chemical (e.g. growth factors and cytokines) and also of a mechanical (expansion forces acting on cells due to interactions with micro- or nanostructured surfaces) nature. In the present project, a cell culture matrix prototype with a nanostructured surface (e.g. covalent peptide binding on cell culture basis, mechanical structuring patterns) shall be developed which is capable of inducing the differentiation behavior of stem cells in a directed manner.