Investigations on the multifunctional roles of quinones derived from natural sources

Abstract:

Prenylated and non-prenylated naphthoquinones were the main components in D. undulata while similar 1,4-naphthoquinones and 9,10-anthraquinones were found in P. lanceolata. Four of these natural products were described for the first time. Main stress factor for both species was found to be infestation by microorganisms, which was hence primarily studied. To combat these, both plant species release the above-mentioned natural products into their immediate environment in the soil in measurable quantities. For some selected compounds an inhibition of the growth of various microorganisms as well as a germination-inhibiting effect on seeds of other plant species was found. To better understand this allelopathic effect on molecular basis, the redox activities of several isolated 1,4-naphthoquinones and anthraquinones were investigated by cyclic voltammetry. This revealed numerous redox potentials, dependent on the molecular structure and environmental conditions, e.g. pH value. All investigated potentials could be traversed multiple times, either reversibly or quasi-reversibly.

Implementation Period:

01/2025 – 12/2025

Project:

1. Brief description of the original project and its objectives

In this project, the organ-specific accumulation of 1,4-naphthoquinones in the unrelated plants Diospyros undulata Wall. Ex G.Don (Ebenaceae) native to Thailand and Pentas lanceolata, native to Tanzania, was investigated and compared from various chemical and botanical perspectives. The primary focus planned was on the significance of the constituents for the plants, with particular emphasis on the influence of stress (e.g. drought, sunlight) on the plants. The second central focus was on the possible use of some of the natural products in pharmaceutical applications, with justified hope for anti-malaria activity.

2. Scientific results, key findings and conclusions

In the first part of the project, the molecular structures of all isolated natural products were examined and compared between the two plants. The results showed that prenylated and non-prenylated naphthoquinones were the main components in D. undulata. Comparable but not identical natural substances were found in P. lanceolata, which occur in association with a couple of anthraquinones. Four of the natural compounds were described for the first time.

An unexpected observation led to refocusing and expansion of stress factors investigated in the investigation. Both plant species, D. undulata and P. lanceolata release most likely small molecules containing quinone moieties into their immediate environment in the soil in measurable quantities. To investigate this allelopathic effect in further detail, plant cultures of both species were grown in agar and the distribution of 1,4-naphthoquinone in the agar was examined. This was particularly successful with the herbaceous growth of P. lanceolata within a few weeks. The woodier D. undulata, however, showed significantly slower growth.

This allelopathic effect suggests that positive effects of the 1,4-naphthoquinone and anthraquinone bearing molecules for the plant species are based on an impact on various organisms in the direct environment of these plants and not on regulatory properties, e.g. relating to water balance or sun exposure within the plant. Based on this, the effects of 1,4-naphthoquinones on different bacterial and fungal strains were determined and investigated, whereby an inhibition of the growth of various microorganisms was observed. Furthermore, a germination-inhibiting effect of the 1,4-naphthoquinones and anthraquinones investigated on the seeds of other plant species (salad, Lactuca sativa L.) was found.

In the second part of the project no spectroscopically and spectrometrically detectable non-covalent bond formation between the isolated 1,4-naphthoquinones and heme could be detected in preliminary experiments. Therefore, the investigations of anti-malaria activities planned for the second part of the project did not appear feasible within the planned project timeframe. Hence, the results from the allelopathic investigations had a direct impact on the design of further experiments in the second part of the project, with focus on broader use in pharmaceutical or agricultural applications as well as in animal breeding. Therefore, antifungal, antibacterial, and cytostatic effects of some selected 1,4-naphthoquinones and anthraquinones were investigated in more detail. In all these test series, biological activities were observed that not only make further research interesting but also promise possible applications in the pharmaceutical sector, in agriculture and in animal breeding.

To better understand the molecular basis of these biological activities, the redox activities of several isolated 1,4-naphthoquinones and anthraquinones were investigated in detail using cyclic voltammetry. This revealed a wide variety of redox potentials, dependent on the molecular structure and environmental conditions, particularly on the pH value. All investigated potentials could be traversed multiple times, either reversibly or quasi-reversibly. These results suggest that the natural products released into the environment by the investigated plant species P. lanceolata and D. undulata remain stable in the environment of the plant over extended periods and affect living organisms in their immediate vicinity through multiple redox cycles. These activities can also be applied to isolated natural products in other environments.

3. Scientific goals achieved, potential applications, and outlook

Allelopathic effects were detected in both investigated plant species. The 1,4-naphthoquinones and anthraquinones released are effective against a wide range of microorganisms and can therefore be regarded as a response to stress caused by threat of infestation by these organisms. However, the inhibition of germination of seed from other plant species caused by the released compounds also shows that phytochemical means are used to gain growth space from competing plants. Thus, the main accumulated constituents of both species serve more to inhibit growth of other (hostile) organisms in their immediate environment than to regulate their own metabolism in response to external influences.

As part of this project, a method was developed for investigating allelopathic properties in agar as a grows medium. This technique allows photochemical analysis of the substances released.

The redox-active effect and proven stability of the natural substances investigated allow further research into applications in the field of combating microorganisms in general, especially against various bacteria and fungi. Further research into their use against malaria also appears possible but is somewhat more complex to investigate. The understanding of the molecular basis that has been gained thus allows for better planning in researching possible areas of application.

Three possible applications are currently being pursued in parallel or follow-up projects. An ingredient isolated from D. undulata is being investigated in fish farming to explore the possibility of combating a common and threatening bacterial infection. The results from P. lanceolata are being incorporated into an MS-based study of metabolome in several species from the genera Rhodopentas and Pentas, which, among other targets, provides a more detailed overview of the occurrence of 1,4-naphthoquinones and anthraquinones in these genera. The developed method for investigating allelopathic properties is currently being applied to another plant species in order to find completely different molecular lead structures that nature uses for this yet only barley studied purpose.

4. Development and expansion of cooperation

The funded project has been used intensively to involve additional colleagues at the University of Vienna, Kasetsart University, and another university in Thailand. The broad scientific and technical orientation of the methods used has thus laid the foundation for jointly developing further projects with more comprehensive technical and personnel framework. In addition to the interdisciplinary and international orientation of the research topics, this also includes the possibility of more effectively acquiring third-party funding for these research projects across disciplines and countries. This focus is not only on funding for mutual visits, but also on the further financing of joint research projects.

This is to be achieved, on the one hand, by raising funds from public sources (e.g. EU Horizon in 2028-2034). The potential applications emerging from the technical results also allow for the planning of commercialization of the innovations and the involvement of companies that can utilize them. This should also be extended to future joint application-oriented research in selected areas. Interesting opportunities are opening, particularly considering the direct utilization of research results by Thai universities.

For the two younger researchers (W. Songoen and E. Mahambo) involved in the funded project, the networks developed in the project also open up opportunities for them to pursue their own future careers in the respective partner countries. The cooperation that has been initiated is currently being intensified and the corresponding financing is being examined.

5. Lessons learned and final assessment of the project

The organization of the project makes it clear that precise planning and the involvement of additional local cooperation partners before the project begins are important for its success. Basing cooperation mainly on joint work during mutual visits of the researchers involved does not seem very promising. However, involving local colleagues with additional expertise at an early stage leads to better scientific success and outcome within the relatively short time frame of twelve months. Furthermore, this results in significantly better networking opportunities for all participants, and possible applications for project results and outcomes can be identified more effectively.

This was successfully implemented in this funded project. The scientific results achieved are being incorporated into three joint publications, with the younger project participants benefiting from this for the next stages of their careers. The establishment of a scientific network of all participants is leading to the involvement of further cooperation partners from other faculties at University of Vienna and Kasetsart University as well as other Universities in Thailand. This growing network provides a good basis for the future acquisition of third-party funding for joint projects. Initial trials are also being conducted within the framework of the funded project to test the application and possible future commercial use of the jointly achieved results.

6. Oral presentations and planned scientific publications

Oral presentations given during mutual visits as part of the project:

–   Weerasak Songoen. “Structurally and stereochemical identifications of rarely found flavan-benzofuranes from Arthocarpus lacuccha using NMR and molecular modeling” NMR Valtice – 38^th Central European NMR Meeting, Valtice, Czech, May 18^th-21^st, 2025.

–   Johann Schinnerl. “Plant-derived specialized (secondary) metabolites—Biosynthesis & Biological Activities (with focus on current research)”, Kasetsart University, Central Laboratory and Greenhouse Complex, Faculty of Agriculture at Kamphaeng Saen, Thailand. September 15^th, 2025.

–   Lothar Brecker. “The Functions of Natural Products: Looking at their Advantages for Plants and at Possible Utilizations”, Kasetsart University, Central Laboratory and Greenhouse Complex, Faculty of Agriculture at Kamphaeng Saen, Thailand. November 27^th, 2025.

Planned publications resulting from the project:

–   Weerasak Songoen, Morakot Thabpho, Dominik Wenisch, Witthawat Phanchai, Lothar Brecker, Wanchai Pluempanupat, Nutthawat Chuanopparat, Michael A. Jakupec, Manakorn Sukmak, Sumet Kongkiatpaiboon, Akkharadet Piyasaengthong, Siriphan Sukkhaeng, Johann Schinnerl. “Potent phytochemicals from roots of Diospyros undulata targeting agricultural pathogens: antibacterial, cytotoxic, antioxidant, and molecular docking insights” Manuscript in preparation for submission at Journal of Agricultural and Food Chemistry (Q1).

–   Emanuel Thomas Mahambo, Dejana Paponjak, Martin Zehl, Dominik Wenisch, Morakot Thabpho, Johann Schinnerl, Lothar Brecker. “Constituents and bioactivities of Rhodopentas bussei and Pentas lanceolata var. usambarica” Manuscript in preparation for submission at Phytochemistry (Q1).

–   Weerasak Songoen, Emanuel Thomas Mahambo, Dominik Wenisch, Morakot Thabpho, Siriphan Sukkhaeng, Porntip Sangsil, Sunipa Detpitthayanan, Johann Schinnerl, Johannes Theiner, Michael A. Jakupec, Manakorn Sukmak, Lothar Brecker. “Investigations on the multifunctional roles of plant-derived 1,4-naphthoquinones and 9,10-anthraquinones” Manuscript in preparation for submission at Metabolites (Q2).