Bioassay-guided isolation of antibacterial compounds from the leaves of Tetradenia riparia with potential bactericidal effects on food-borne pathogens

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Abstract

Ethnopharmacological relevance

Tetradenia riparia (commonly known as ginger bush) is frequently used in traditional African medicine to treat foodborne infections including diarrhoea, gastroenteritis, and stomach ache.

Aim of the study

The present study aims to identify in Tetradenia riparia the compounds active against foodborne pathogens.

Materials and methods

Dried Tetradenia riparia leaf powder was consecutively extracted with hexane, ethyl acetate, methanol and water. The hexane extract was counter-extracted with methanol:water (9:1), and after evaporation of the methanol, this phase was extracted with dichloromethane. The water extract was counter-extracted with butanol. All these fractions were tested against a panel of foodborne bacterial pathogens. A bioassay-guided purification was performed to isolate antimicrobial compounds using Staphylococcus aureus as a target organism. Further, antibiofilm activity was evaluated on S. aureus USA 300.

Results

The dichloromethane fraction and ethyl acetate extract were the most potent, and therefore subjected to silica gel chromatography. From the dichloromethane fraction, one active compound was crystalized and identified using NMR as 8(14),15-sandaracopimaradiene-7alpha, 18-diol (compound 1). Two active compounds were isolated from the ethyl acetate extract: deacetylumuravumbolide (compound 2) and umuravumbolide (compound 3). Using a microdilution method, their antimicrobial activity was tested against eight foodborne bacterial pathogens: Shigella sonnei, S. flexneri, Salmonella enterica subsp. enterica, Escherichia coli, Micrococcus luteus, S. aureus, Enterococcus faecalis, and Listeria innocua. Compound 1 had the strongest activity (IC50 ranging from 11.2 to 212.5 μg/mL), and compounds 2 and 3 showed moderate activity (IC50 from 212.9 to 637.7 μg/mL and from 176.1 to 521.4 μg/mL, respectively). Interestingly, 8(14),15-sandaracopimaradiene-7alpha, 18-diol is bactericidal, and also showed good antibiofilm activity with BIC50 (8.8 ± 1.5 μg/mL) slightly lower than for planktonic cells (11.4 ± 2.8 μg/mL).

Conclusions

These results support the traditional use of this plant to conserve foodstuffs and to treat gastrointestinal ailments, and open perspectives for its use in the prevention and treatment of foodborne diseases.

Introduction

Foodborne pathogens are mainly bacteria, but also viruses or even parasites, that can be present in food, causing a range of diseases with major effects on human health and the economy (Green-Johnson 2006; Bintsis 2017). According to the U.S. Food and Drug Administration, foodborne illness is often caused by consuming food contaminated by bacteria and/or their toxins, parasites, viruses, chemicals, or other agents (FDA, 2020). Over 200 diseases are caused by foodborne pathogens. Each year worldwide, unsafe food causes 600 million cases of foodborne disease, and 420,000 deaths. Over 30% of foodborne deaths occur in children under 5 years of age, Foodborne pathogens can cause severe diarrhoea or debilitating infections, including meningitis (WHO, 2020). In the European Union (EU) for the year 2018, 26 member states reported 5146 foodborne and waterborne outbreaks, 48,365 cases of illness, 4588 outbreak-related hospitalizations and 40 deaths, Salmonella was the most commonly detected agent, with S. enterica causing one in five outbreaks. In the United States, foodborne infections trigger an estimated 76 million illnesses, with 5000 deaths each year (Mead et al., 1999). The top five foodborne pathogens are Norovirus, Salmonella, Clostridium perfringens, Campylobacter and Staphylococcus aureus. Moreover, some other foodborne germs do not cause illness frequently, but more likely lead to hospitalization, including Clostridium botulinum, Listeria, E. coli, and Vibrio (CDC 2020).

Antibiotics used for human treatment are increasingly prohibited for other applications such as food, agriculture or veterinary use, in part to decrease the development of resistance. Therefore, research on natural products could yield sustainable alternatives for chemically synthesized antimicrobials (Panda et al., 2019). Resistant foodborne pathogens represent one of the most important public health problems related to the emergence of antibacterial resistance in the food supply chain. Indeed, several foodborne pathogens developed a tolerance or resistance to different antibiotics (Olsen et al., 2004; Hummel et al., 2007; Alfredson and Korolik, 2007; Werner et al., 2013). This can result in treatment failure, increased mortality as well as treatment costs, reduced infection control efficiency, and spread of resistant pathogens from hospitals to the community (Hashempour-Baltork et al., 2019). Therefore, many research projects try to find new alternative approaches to control and prevent this problem. Plant extracts have long been considered as a natural source of antimicrobial agents, that may be nutritionally safe and easily degradable. Many potential antibacterial agents against foodborne pathogens have been purified from plants (Ma et al., 2018; Pereira et al., 2008; Bajpai et al., 2017a; Bajpai et al., 2017b). Natural preservatives such as herbal extracts and essential oils, as well as their components, are used increasingly as alternatives for inhibiting pathogenic and spoilage microorganisms (Schirone et al., 2019).

Tetradenia (T.) riparia (Hochst.) Codd (Lamiaceae), is an African medicinal herb, widely distributed throughout Eastern and tropical Africa (Gairola et al., 2009). This plant is well known for its medicinal properties against a number of infectious diseases (malaria, yaws, gastroenteritis, gonorrhoea, dental abscesses), chest pain (angina), several kinds of fever and aches, and for treating stomach-related ailments (Van Puyvelde et al., 1987, 2018; Van Wyk and Wink, 2004). Interestingly, the leaves are used as a spice in foods, for the conservation of food products in traditional silos, as well as for dry storage of crops, mostly to repel insects (Van Puyvelde et al., 1975; Xaba 2009).

Therefore, we used bioassay-guided purification to identify compounds from this plant which could be used against foodborne pathogens and help to reduce the emergence of drug resistance.

Section snippets

Plant material

“T. riparia plant was harvested in Mukoni, Huye, Rwanda and identified by an expert botanist (Vedaste Minani). A voucher specimen (No. 86) was deposited in the National Herbarium at the National Industrial Research and Development Agency (NIRDA), Huye, Rwanda” (Van Puyvelde et al., 2018). The leaves were air-dried, ground to a powder using a mechanical grinder, and stored in a cold room (4 °C) till use.

Extraction of plant and isolation of active compounds

Briefly, powdered air-dried leaves (10 g) were successively extracted (for 5 × 30 min each)

Antimicrobial activity against foodborne bacteria

Six different dried residues were tested against bacterial foodborne pathogens (Table 1). The dichloromethane fraction and ethyl acetate extract had the broadest activity, inhibiting both Gram-positive and Gram-negative bacteria when tested at a final concentration of 250 μg/mL (stock 5 mg/mL in DMSO).

Consequently, these were further fractionated on silica gel. Those fractions were analysed by TLC, similar fractions were pooled, and tested for antibacterial activity. From each of three active

Discussion

T. riparia is well known for its medicinal properties (Demarchi et al., 2015; Cardoso et al., 2015; Campbell et al., 1997; de Melo et al., 2015a,b). It has good anti-parasitic as well as antispasmodic and antimicrobial activity (Van Puyvelde et al., 1987), strong acaricidal activity against Rhipicephalus (Boophilus) microplus (Gazim et al., 2011), anthelmintic activity (Van Puyvelde et al., 2018), antidermatophytic activity (of leaf extract; Endo et al., 2015), anti-mycobacterium activity (

Conclusion

In conclusion, crude extracts of T. riparia leaves from Rwanda showed antimicrobial activity against a wide range of foodborne pathogens, due essentially to the presence of 8(14),15-sandaracopimaradiene-7e,18-diol. This supports the traditional use of this plant to conserve foodstuffs and to treat stomach-related ailments and opens perspectives for its use in combating foodborne illnesses.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author contribution

Study conception and design: LVP, SKP, WL.

Acquisition of data: LVP, AA, WDB.

Analysis and interpretation of data: LVP, AA, MJM, WDB, WL.

Drafting of manuscript: LVP, AA, SKP.

Critical revision: SKP, WL.

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgements

Luc Van Puyvelde, Sujogya Kumar Panda and Walter Luyten largely supported themselves. We are thankful to Dr. Purity Kipanga for her technical assistance in performing antimicrobial assay.

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