ORIGINAL RESEARCH

published: 26 November 2021

doi: 10.3389/fphar.2021.746808

In Vitro Selective Antibacterial and

Antiproliferative Effects of Ethanolic
Extracts from Cambodian and
Philippine Plants Used in Folk
Medicine for Diarrhea Treatment
Tomas Kudera 1, Barbora Fiserova 1, Marie Korytakova 1, Ivo Doskocil 2, Hana Salmonova 2,
Edgardo E. Tulin 3, Samnang Nguon 4, Marlito M. Bande 5 and Ladislav Kokoska 1*
1
Laboratory of Ethnobotany and Ethnopharmacology, Department of Crop Sciences and Agroforestry, Faculty of Tropical
AgriSciences, Czech University of Life Sciences Prague, Prague, Czechia, 2Faculty of Agrobiology, Food and Natural Resources,
Department of Microbiology, Nutrition and Dietetics, Czech University of Life Sciences Prague, Prague, Czechia, 3PhilRootcrops,
Edited by:
Visayas State University, Baybay, Philippines, 4Graduate School, Royal University of Agriculture, Phnom Penh, Cambodia,
Alessandra Durazzo, 5
Institute of Tropical Ecology and Environmental Management, Visayas State University, Baybay, Philippines
Council for Agricultural Research and
Economics, Italy
Reviewed by: Bacterial diarrhea remains a global health problem, especially in developing tropical
Abdul Majeed,
countries. Moreover, dysbiosis caused by diarrheagenic bacteria and inappropriate
Bahauddin Zakariya University,
Pakistan antimicrobial treatment has been associated with intestinal carcinogenesis. Despite the
Jamuna Aswathanarayan, rich tradition of the use of herbs for the treatment of gastrointestinal disorders in
JSS Academy of Higher Education
and Research, India
Cambodian and Philippine folk medicine, many of them have not yet been
Folorunso Oludayo Fasina, systematically studied for their in vitro selective inhibitory effects on intestinal bacteria
University of Pretoria, South Africa
and cells. In the present study, in vitro inhibitory activities of 35 ethanolic extracts derived
Hasanga Rathnayake,
University of Ruhuna, Sri Lanka from 32 Cambodian and Philippine medicinal plants were determined by broth
microdilution method against 12 pathogenic bacteria. Furthermore, cytotoxicity against
*Correspondence:
Ladislav Kokoska intestinal cancer cells (Caco-2 and HT-29) using thiazolyl blue tetrazolium bromide
[email protected] cytotoxicity assay and safety to six beneficial intestinal bacteria (bifidobacteria and
lactobacilli) and intestinal normal cells (FHs 74 Int) were determined for the
Specialty section:
This article was submitted to
antimicrobially active extracts. Selectivity indices (SIs) were calculated among the
Ethnopharmacology, averages of minimum inhibitory concentrations (MICs), half-maximal inhibitory
a section of the journal
concentrations (IC50), and 80% inhibitory concentrations of proliferation (IC80) for each
Frontiers in Pharmacology
type of the tested agents. The extracts of Artocarpus blancoi (Elmer) Merr. (Moraceae),
Received: 24 July 2021
Accepted: 08 October 2021 Ancistrocladus tectorius (Lour.) Merr. (Ancistrocladaceae), and Pentacme siamensis (Miq.)
Published: 26 November 2021 Kurz (Dipterocarpaceae) produced significant growth-inhibitory effects (MICs

Citation: 32–512 μg/ml) against intestinal pathogenic bacteria at the concentrations nontoxic to
Kudera T, Fiserova B, Korytakova M,
Doskocil I, Salmonova H, Tulin EE,
normal intestinal cells (IC80 values >512 μg/ml; SIs
0.11–0.2). Moreover, the extract of P.
Nguon S, Bande MM and Kokoska L siamensis (Miq.) Kurz was relatively safe to beneficial bacteria (MICs ≥512 μg/ml; SI
0.1),
(2021) In Vitro Selective Antibacterial
and together with A. blancoi (Elmer) Merr., they selectively inhibited intestinal cancer cells
and Antiproliferative Effects of
Ethanolic Extracts from Cambodian (IC50 values ≥51.98 ± 19.79 μg/ml; SIs
0.3 and 0.6). Finally, a strong selective
and Philippine Plants Used in Folk antiproliferative effect on cancer cells (IC50 values 37.89 ± 2.68 to 130.89 ± 13.99 μg/
Medicine for Diarrhea Treatment.
Front. Pharmacol. 12:746808.
ml; SIs
0.5) was exerted by Ehretia microphylla Lam. (Boraginaceae), Lagerstroemia
doi: 10.3389/fphar.2021.746808 cochinchinensis Pierre ex Gagnep. (Lythraceae), and Melastoma saigonense (Kuntze)

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Kudera et al. Antibacterial and Anticancer Plant Extracts

Merr. (Melastomataceae) (leaves with flower buds). The results suggest that the above-
mentioned species are promising materials for the development of new selective
antibacterial and antiproliferative agents for the treatment of infectious diarrhea and
associated intestinal cancer diseases. However, further research is needed regarding
the isolation and identification of their active constituents.
Keywords: diarrhea, Cambodia, Philippines, medicinal plant, antibacterial, anticancer

INTRODUCTION Additionally, assessment of interactions with intestinal

epithelial cells and toxicity profiles is another important factor
According to the latest data of the World Health Organization, for the evaluation of antibacterial agents, which target the
infectious diarrhea is still the third leading cause of death among intestinal site (Maher and McClean, 2006). To lower the
all communicable diseases worldwide, especially affecting under- potential toxic responses of intestinal epithelial cells, the use of
five children in developing countries (World Health antibacterial agents with mechanisms enabling toxicity to be
Organization, 2020a). Moreover, common risk factors prokaryotic but not eukaryotic cells, such as in the case of the
associated with these gastrointestinal infections, such as antibiotic ceftriaxone, should be prioritized (Neftel and
inappropriate changes in the host-gut microbiome (Sun et al., Hübscher, 1987). However, the antiproliferative activity can be
2018), have been considered as a crucial precondition for several acknowledged in cases where the immune responses caused by
noncommunicable intestinal diseases, including colorectal ongoing intestinal infection and dysbiosis have already promoted
cancer, which is the third leading cause of cancer death the carcinogenesis of epithelial cells. Initially, colorectal cancer
globally (Taddese et al., 2020). The developed countries are at usually has an oligosymptomatic characteristic; thus, many cases
the highest risk, but the incidence of gastrointestinal cancers in are diagnosed only at advanced stages, at which stage the
developing nations is steadily increasing (Rawla et al., 2019). therapeutic outcomes are poor (Rogowski and Sulzyc-Bielicka,
Infectious and toxigenic strains of Bacillus cereus, Campylobacter 2016). Antibacterial agents able to eliminate diarrheagenic
jejuni, Clostridium difficile, Clostridium perfringens, Enterococcus pathogens and having the additional selective antiproliferative
faecalis, Escherichia coli, Listeria monocytogenes, Salmonella spp., properties could potentially help to prevent progression of yet not
Shigella spp., Vibrio cholerae, Vibrio parahaemolyticus, and diagnosed intestinal cancers. The use of antibiotics—such as
Yersinia enterocolitica are the major causes of bacterial quinolones and tetracyclines, which are both utilized in
diarrhea (Casburn-Jones, 2004). Among these, Enterococcus diarrheal infections—as anticancer drugs has been previously
spp., Escherichia spp., and Shigella spp. were previously suggested (Onoda et al., 2005; Batalha et al., 2016).
observed to be richer in the fecal microbiota of patients with Plant-derived products provide novel chemical scaffolds for
colorectal cancer (Xu and Jiang, 2017). anti-infective drugs and leads that have chemically been modified
Despite the advantages of treatment utilizing antibiotic agents, and developed as antimicrobial agents. Several over-the-counter
disruption of the gut microbiota is usually considered as one of pharmaceuticals, dietary supplements, and herbal medicines
the negative consequences of their use in infectious diarrhea recommended for the support and maintenance of
(Francino, 2016). Moreover, the antimicrobial resistance rate gastrointestinal health, containing antibacterially active plant
among diarrheagenic bacteria recovered from human patients extracts and derivatives of their constituents, are already
has significantly increased, especially in developing countries available at the international market. The benzylisoquinoline
(Meng et al., 2011; World Health Organization, 2020b). alkaloid berberine (e.g., Hydrastis canadensis L.
Therefore, it is important to seek new sources of efficient [Ranunculaceae]); simple phenol bismuth subsalicylate, the
antimicrobial agents against which the infectious bacteria are analog of salicylic acid derived from salicin (Salix alba L.
less prone to develop resistance (Sibanda and Okoh, 2007). [Salicaceae]) (Kokoska et al., 2019); and picrosides, an iridoid
Additionally, agents that are highly selective, thus less glycoside of Picrorhiza kurroa Royle ex Benth. (Plantaginaceae),
disruptive for human microbial ecology, should be preferred are some examples (Rathee et al., 2016). The in vitro selective
(Garrett, 2019). Therefore, an investigation on the in vitro antibacterial effects of plant-derived products have also been
antimicrobial effects of promising candidates is recommended reported. For example, Chan et al. (2018) reported that the
to involve both representatives of diarrheagenic and probiotic phenolic-rich extracts from various dietary spices and
bacteria. The most common bacteria recognized to date as medicinal herbs (Cinnamomum burmannii Nees and T.Nees]
probiotics are Lactobacillus spp. and Bifidobacterium spp., Blume [Lauraceae], Cinnamomum cassia [L.] J.Presl [Lauraceae],
belonging to the dominant bacterial phyla that can be found Origanum vulgare L. [Lamiaceae], Punica granatum L.
in human intestines (Behnsen et al., 2013). Although it is a [Lythraceae], Reynoutria japonica Houtt. [Polygonaceae], and
simplified representation of beneficial gut microbiota, the Syzygium aromaticum [L.]. Merr. and L.M.Perry [Myrtaceae])
counterscreen of in vitro inhibitory activities on gut exerted in vitro growth-inhibitory effects against selected
commensals appears to be an effective way to avoid foodborne pathogenic bacteria but not against lactic-acid
unnecessarily promiscuous agents (Gavrish et al., 2014). bacteria. Selective in vitro antibacterial activity was also

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Kudera et al. Antibacterial and Anticancer Plant Extracts

described in the study by Novakova et al. (2013), where the processed by alcohol maceration, decoction, or infusion or
anticlostridial effect of 8-hydroxyquinoline (Microstachys eaten and chewed raw. According to the Philippine traditional
corniculata [Vahl] Griseb. [Euphorbiaceae]) was higher than medicinal system, the disease is usually conceptualized as a
the activities revealed against different strains of bifidobacteria. disruption (dys-krasia) of the balance of forces (whether germs
In our previous study, we also reported that 8-hydroxyquinoline or evil spirits), both external and internal to humans. Therefore, it
exerts selective in vitro antiproliferative activity against some can be assumed that the use of herbal preparations is intended to
intestinal cancer cell lines with a comparably lower effect on also defend the immunological mechanisms, helping the body to
normal cells (Kudera et al., 2020). Quinoline alkaloids, such as overcome the disease itself (Tan, 1980). Despite the existence of
camptothecin extracted from the bark of Camptotheca acuminata several reports on the antibacterial and antiproliferative effects of
Decne. (Cornaceae), are already being used in chemotherapy for Cambodian and Philippine medicinal plants used for the
the treatment of colon cancer (Zeng et al., 2013). Anticancer treatment of diarrhea (Beloy et al., 1976; Chea et al., 2007),
activities of other antibacterially active phytochemicals (e.g., there are several species in both regions that have not yet been
berberine) utilized against infectious diarrhea are currently appropriately studied using modern scientific techniques. In this
studied (Lin et al., 2008). Based on these studies, it is evident study, we, therefore, examine the in vitro selective antibacterial
that plant-derived products have great potential for the and antiproliferative effects of ethanolic extracts from various
development of antibacterial and anticancer preparations for parts of plant species that have been used in Cambodian and
the treatment of infectious diarrhea and associated intestinal Philippine traditional herbal systems the treatment of
cancer diseases. Although many of such products work based gastrointestinal disorders and determine which bioactive
on antidiarrheal activity (e.g., antisecretory and astringent effects) properties have not been properly tested in such form and
(Palombo, 2006), their antimicrobial effect is not an uncommon degree before.
feature.
The Southeast Asian region is one of the world’s major sources
of useful plant resources and has long been recognized as a center MATERIALS AND METHODS
of plant biodiversity (Duriyaprapan et al., 2005). Situated in the
humid tropics with areas of high rainfall, Southeast Asia has one Plant Materials
of the largest numbers of vascular plants species globally. For The criteria for selection of promising plant species included their
centuries, people living in this region have relied on traditional uses for the treatment of diarrhea, dysentery, abdominal pain, and
medicine using available plants for daily healthcare. Cambodia other gastrointestinal complaints in traditional herbal systems of
and the Philippines are two geographically distinct Southeast Southeast Asia, particularly Cambodia and the Philippines.
Asian countries, each having numerous plant biodiversity Therefore, the appropriate literature on ethnobotany and
hotspots and a long tradition of herbalism (de Padua et al., ethnomedicine of this region was primarily used (Chassagne
1999). While the former is situated in the mainland, having et al., 2016; de Padua et al., 1999; van Duong, 1993; Kham,
rich ecosystems, especially around the Mekong River 2004; Langenberger et al., 2008; Lemmens and Bunyapraphatsara,
(Chassagne et al., 2016), the latter is a huge archipelago 2003; Lim, 2012; Stuart, 2017; Tan, 1980; van Valkenburg and
consisting of approximately 7,107 islands, many of which are Bunyapraphatsara, 2001). Additionally, several species were
the center of endemicity and biodiversity (Guzman et al., 2016). identified through meetings with local herbalists in Cambodia
Diarrhea has been a significant issue in both Cambodia and (2) and the Philippines (1), assembled by local experts Dr. Nguon
the Philippines (Our World in Data, 2011). Therefore, plant and Dr. Bande, respectively. Overall, more than 100 plant species
resources in these countries have extensively been utilized were selected, referring to a limited number of previous studies
medicinally to treat this ailment. The Philippines also has the testing their bioactivity in vitro. A total of 35 samples from
highest estimated number of cases of colorectal cancer in different parts (bark, fruit, leaves, or roots, one per plant
Southeast Asia and the tenth highest number of deaths in the except three of the species) of 13 Cambodian and 19
world (Rawla et al., 2019). In certain provinces of Cambodia, Philippine medicinal plant species were collected from various
treatment of digestive disorders, such as abdominal pain (chhu locations in the Republic of the Philippines in April–May 2017
poh), diarrhea (reak ach), and dysentery (reak muol), has and 2018 and in the Kingdom of Cambodia in March–April 2019
particularly been based on herbal medicine. Alcohol (Table 1). The collected fresh samples were subsequently air-
maceration is a common method of preparation of dried for several days and sent to the Czechia for further
antidiarrheal medicines, whereas a majority of the processing and bioactivity testing. Ethnobotany expert Prof.
preparations are administered orally: drunk, eaten, or chewed Kokoska and local experts Dr. Bande and Dr. Nguon
(Kham, 2004). Grilling the plant part over a fire and then boiling authenticated the species. Their voucher specimens have been
it into a form of decoction is also common. As an example, deposited in the herbarium of the Department of Botany and
Bunong people in Mondulkiri province treat diarrhea using a Plant Physiology of the Faculty of Agrobiology, Food and Natural
“step-by-step” process using a sole ingredient from one plant that Resources of the Czech University of Life Sciences Prague
is substituted by a different species if the condition becomes (Prague, Czechia). The scientific names of the collected species
persistent. In the Philippines, conditions such as diarrhea were reviewed using (The Plant List, 2013), and their local names
(pagtatae) and dysentery (pagdidisenyo) have similarly been were verified with data from literature and local herbalists (Tan,
treated by orally administered herbal preparations that are 1980; van Duong, 1993; de Padua et al., 1999; van Valkenburg and

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Kudera et al. Antibacterial and Anticancer Plant Extracts

TABLE 1 | Ethnobotanical data on Cambodian and Philippine medicinal plants.

Latin name GPS coordinates Local name Voucher Tested part(s) Extract yield Ethnomedicinal use
(family) (country) specimen (%)

Aganonerion polymorphum 12.3966000N, Vor Thneung 02559KBFRC Whole plant 19.7 Diarrhea (Chassagne et al. (2016))
Spire (Apocynaceae) 107.1934975E (C)
Acalypha grandis Benth. 10.7623297N, Unknown 02537KBFR8 Leaves 23.6 Diarrhea and dysentery; sapped/crushed
(Euphorbiaceae) 124.8062889E (P) into water/food (van Valkenburg and
Bunyapraphatsara (2001))
Acanthus ebracteatus Vahl 10.6888289N, Diluario 02505KBFR3 Whole plant 17.9 Abdominal pain; decoction of 30–60
(Acanthaceae) 124.7956483E (P) gDW (Stuart. (2017))
Ancistrocladus tectorius 13.7333775N, Khan Maa 02560KBFR4 Leaves 16 Dysentery; decoction (Lemmens and
(Lour.) Merr. 107.0151108E (C) Bunyapraphatsara (2003); interviewed
(Ancistrocladaceae) herbalist)
Aporosa villosa (Lindl.) Baill. 12.3965292N, Krong 02561KBFR5 Leaves 6.1 Diarrhea and abdominal pain; decoction
(Phyllanthaceae) 107.1938597E (C) (Chassagne et al. (2016))
Artocarpus blancoi (Elmer) 10.7435833N, Antipolo 02538KBFR9 Fruit 25.7 Diarrhea; cooked (Tan. (1980); Stuart.
Merr. (Moraceae) 124.8020564E (P) (2017))
Artocarpus camansi Blanco 10.6819242N, Kamansi 02512KBFR1 Bark 13.3 Diarrhea; cooked (Tan. (1980); Stuart.
(Moraceae) 124.8001064E (P) (2017))
Artocarpus elasticus Reinw. ex 10.7435939N, Terap 02539KBFRA Bark 11.8 Dysentery (Lim, 2012; interviewed
Blume (Moraceae) 124.8019275E (P) herbalist)
Artocarpus odoratissimus 10.7436072N, Marang 02540KBFR2 Fruit 22.9 Diarrhea (Lim. (2012); interviewed
Blanco (Moraceae) 124.8017989E (P) herbalist)
Bauhinia malabarica Roxb. 12.4428908N, Choeung Koo 02562KBFR6 Bark and leaves 14.7 and 11.2 Diarrhea and abdominal pain; alcohol
(Leguminosae) 107.1592217E (C) maceration or decoction (Chassagne
et al. (2016))
Breynia cernua (Poir.) Müll.Arg. 9.8153556N, Mutang-Ulang 02541KBFR3 Bark 10.6 Dysentery; infusion (van Valkenburg and
(Phyllantaceae) 124.3597258E (P) Bunyapraphatsara (2001))
Breynia vitis-idaea (Burm.f.) 11.5627122N, Phnek Preab 02563KBFR7 Wood with bark 9.9 Dysentery; infusion (Kham, 2004)
C.E.C.Fisch. (Phyllanthaceae) 104.9167906E (C)
Commelina communis L. 10.6159294N, Alibangon 02542KBFR4 Whole plant 13.3 Diarrhea (van Valkenburg and
(Commelinaceae) 124.9272431E (P) Bunyapraphatsara (2001))
Cyathula prostrata (L.) Blume 10.7433806N, Dayang 02543KBFR5 Whole plant 12.8 Dysentery and cholera; decoction or
(Amaranthaceae) 124.8001225E (P) infusion (van Valkenburg and
Bunyapraphatsara (2001); Stuart. (2017))
Diplazium esculentum (Retz.) 10.7577433N, Paco 02545KBFR7 Rhizome 5.4 Diarrhea and dysentery; pulverization and
Sw. (Athyriaceae) 124.7975153E (P) cold water maceration (Stuart. (2017);
interviewed herbalist)
Ehretia microphylla Lam. 10.7442369N, Tsaang-Gubat 02489KBFRE Leaves 15.3 Diarrhea, dysentery, and abdominal pain;
(Boraginaceae) 124.7897825E (P) decoction or infusion (8 tbsp of chopped
leaves in 2 glasses) (de Padua et al.
(1999); Stuart. (2017))
Emilia sonchifolia (L.) DC. ex 10.7407072N, Tagulinaw 02520KBFR0 Whole plant 20.9 Diarrhea, dysentery, and enteritis;
DC. (Compositae) 124.8002914E (P) decoction (6–15 gDW) (Tan. (1980);
Stuart. (2017))
Helicteres angustifolia L. 12.3963028N, Sambok 02564KBFR8 Root 9.2 Diarrhea, dysentery, and abdominal pain;
(Malvaceae) 107.1938622E (C) Cheas decoction (Chassagne et al. (2016))
Hyptis capitata Jacq. 10.7590292N, Botonesan 02546KBFR8 Whole plant 10.1 Gastrointestinal problems; decoction
(Lamiaceae) 124.8020589E (P) (Lemmens and Bunyapraphatsara
(2003))
Ixora nigricans R.Br. ex Wight 13.7291931N, Phka Mochul 02565KBFR9 Leaves 10.8 Dysentery and abdominal pain (Kham.
and Arn. (Rubiaceae) 107.0113667E (C) Pich (2004))
Kyllinga brevifolia Rottb. 11.0610592N, Pugo-Pugo 02544KBFR6 Whole plant 11.4 Diarrhea (de Padua et al. (1999); Stuart.
(Cyperaceae) 124.7009597E (P) (2017))
Lagerstroemia 13.4692872N, Sralao 02566KBFRA Bark 2.8 Diarrhea; decoction (Chassagne et al.
cochinchinensis Pierre ex 105.8909203E (C) (2016))
Gagnep. (Lythraceae)
Leea indica (Burm. f.) Merr. 11.5627122N, Kdaing Baay 02567KBFRB Root 8.3 Diarrhea, dysentery, digestive and
(Vitaceae) 104.9167906E (C) intestinal complaints; decoction or
infusion (Kham. (2004))
Melastoma dodecandrum 12.4089644N, Unknown 02568KBFRC Bark and leaves 12.7 and 9.9 Diarrhea (van Duong. (1993))
Lour. (Melastomataceae) 107.3133011E (C) with flower buds
Melastoma saigonense 11.5627122N, Baay Nhenh 02569KBFRD Wooden stem 7.3 and 17.3 Diarrhea (Chassagne et al. (2016))
(Kuntze) Merr. 104.9167906E (C) and leaves with
(Melastomataceae) flower buds
(Continued on following page)

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Kudera et al. Antibacterial and Anticancer Plant Extracts

TABLE 1 | (Continued) Ethnobotanical data on Cambodian and Philippine medicinal plants.

Latin name GPS coordinates Local name Voucher Tested part(s) Extract yield Ethnomedicinal use
(family) (country) specimen (%)

Parkia javanica (Lam.) Merr. 10.7448892N, Kupang 02547KBFR9 Bark 25.7 Diarrhea and dysentery; decoction (Tan.
(Leguminosae) 124.8059375E (P) (1980); Stuart. (2017))
Pentacme siamensis (Miq.) 13.4474300N, Raing Phnom 02571KBFR6 Bark 5.8 Diarrhea (Chassagne et al. (2016)
Kurz (Dipterocarpaceae) 105.8756317E (C)
Picrasma javanica Blume 10.7438825N, Manunggal 02548KBFRA Bark 6.3 Digestive and abdominal pain; decoction
(Simaroubaceae) 124.8039956E (P) (Langenberger et al. (2008))
Pseudelephantopus spicatus 9.8110686N, Kokunbanog 02553KBFR6 Whole plant 12.5 Diarrhea; decoction (Langenberger et al.
(Juss. ex Aubl.) Rohr 124.3551231E (P) (2008))
(Compositae)
Rourea minor (Gaertn.) Alston 12.3965372N, Unknown 02570KBFR5 Leaves 11.4 Diarrhea (Chassagne et al. (2016))
(Connaraceae) 107.1933392E (C)
Tabernaemontana 14.1667808N, Pandakaking- 02503KBFR1 Bark 10.1 Gastroenteritis (Tan. (1980))
pandacaqui Lam. 121.2143336E (P) Puti
(Apocynaceae)
Triumfetta bartramia L. 10.7467864N, Kulutkulutan 02554KBFR7 Root 14.9 Diarrhea and intestinal ulcers (van
(Malvaceae) 124.8152500E (P) Valkenburg and Bunyapraphatsara
(2001); Stuart. (2017))

C, Cambodia; P, Philippines.

Bunyapraphatsara, 2001; Lemmens and Bunyapraphatsara, 2003; Brno, Czechia), German Collection of Microorganisms and
Kham, 2004; Langenberger et al., 2008; Lim, 2012; Chassagne Cell Cultures (DSMZ, Braunschweig, Germany), and National
et al., 2016; Stuart, 2017). For all assayed species, the scientific Collection of Type Cultures (NCTC, London, United Kingdom).
names, families, local names, voucher specimen codes, GPS In accordance with the diversity of diarrheagenic gram-
coordinates, collected parts (plant samples), and their uses in positive and gram-negative bacteria responsible for globally
folk medicine are presented in Table 1. distributed foodborne, waterborne, and nosocomial infections
(Diniz-Santos et al., 2006; Rajkovic et al., 2020), the following 12
Preparation of Plant Extracts strains were used in this study: B. cereus (ATCC 14579), C.
Although the most common procedures of processing difficile (DSMZ 12056), C. perfringens (DSMZ 11778), E. faecalis
antidiarrheal plants in Cambodia and the Philippines are (ATCC 29212), E. coli (ATCC 25922), E. coli 0175:H7 (NCTC
decoction and infusion (Table 1), ethanol was selected for the 12900), L. monocytogenes (ATCC 7644), Shigella flexneri (ATCC
extraction of plant samples since it is an efficient solvent for 12022), Salmonella enterica ssp. enterica serovar Enteritidis
herbal drugs with a well-established tradition in herbal medicine (ATCC 13076), S. enterica ssp. enterica serovar Typhimurium
(Kelber et al., 2016). With the aim of preventing possible loss or (ATCC 14028), V. parahaemolyticus (ATCC 17802), and Y.
changes of active constituents due to storage of plant samples, the enterocolitica (ATCC 9610). The above-mentioned strains were
extraction was performed immediately after their arrival in the considered as obligate or facultative pathogens. The following six
Czechia. Each dried sample was homogenized into powder using bacterial strains, which belong to the dominant bacterial phyla in
Grindomix mill (Retsch, Haan, Germany), and 15 g of dry matter the human gut and exhibit probiotic functions (Behnsen et al.,
was extracted in 450 ml 80% ethanol (Penta, Prague, Czechia) for 2013), were used in this study: Bifidobacterium adolescentis
24 h at room temperature using a laboratory shaker (GFL3005, (DSMZ 20087), Bifidobacterium animalis spp. lactis (DSMZ
GFL, Burgwedel, Germany). Therefore, the drug extract ratio was 10140), Bifidobacterium breve (ATCC 15700), Lactobacillus
1:30. Extracts were subsequently filtered and concentrated using a casei (DSMZ 20011), Lactobacillus reuteri (CCM 3625), and
rotary vacuum evaporator (R-200, Buchi Labortechnik, Flawil, Lactobacillus rhamnosus (CCM 7091). All these strains were
Switzerland) in vacuo at 40°C. According to the considered beneficial gut bacteria.
recommendations of Cos et al. (2006), the dried residue was As the maintenance and growth medium, Mueller-Hinton
finally diluted in 100% dimethylsulfoxide (DMSO) (Penta, Broth (Oxoid, Basingstoke, United Kingdom) was used for the
Prague, Czechia) to obtain stock solutions with a final majority of bacteria that grow aerobically (E. faecalis supp. 1%
concentration of 51.2 mg/ml and stored at −20°C until their glucose, V. parahaemolyticus supp. 3% NaCl). Y. enterocolitica
use. Some of the extracts were not completely soluble in other was stored and cultured in Brain Heart Infusion Broth (Oxoid,
solvents, such as distilled water. Yields (%) of the dried residues Basingstoke, United Kingdom). Bifidobacteria and lactobacilli
are shown in Table 1. were maintained and cultured in Wilkins-Chalgren Broth
(Oxoid, Basingstoke, United Kingdom) supplemented with 5 g/
Bacterial Strains and Media L soya peptone and 0.5 g/L cysteine. Although the same growth
The intestinal bacterial type strains were obtained from the medium was used for clostridia, they were stored in cooked meat
American Type Culture Collection (ATCC, Rockville, MD, medium (both from Oxoid, Basingstoke, United Kingdom) at
United States), Czech Collection of Microorganisms (CCM, room temperature. The standard safety guidelines for handling

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Kudera et al. Antibacterial and Anticancer Plant Extracts

microorganisms were followed. Therefore, all items, such as The lowest concentration (μg/ml) of the extracts that inhibited
culture tubes, syringes, and gloves, were discarded in the the bacterial growth by ≥80% was defined as the minimum
biohazard autoclave bag after every use. inhibitory concentration (MIC). Ciprofloxacin (Sigma-Aldrich,
Prague, Czechia), an antibiotic commonly recommended for the
Cell Cultures treatment of infectious diarrhea (Casburn-Jones, 2004), was
One representative of normal intestinal cell line (FHs 74 Int dissolved in distilled water and used as a positive control drug.
[ATCC CCL 241]) and two of cancer intestinal cell lines (Caco-2 All tests were performed as three independent experiments, each
[ATCC HTB 37]) and HT-29 [ATCC HTB 38]) were purchased conducted in triplicate. The mode and median were used for the
from ATCC (Rockville, MD, United States). Normal cells were final MIC value calculation when the triplicate endpoints were
cultured in Hybri-Care medium supplemented with 10% fetal within the two- and three-dilution ranges, respectively. The
bovine serum, 1% sodium bicarbonate, 1% nonessential amino antibacterial activities were classified as strong (MICs ≤64 μg/
acids, 30 ng/ml of epidermal growth factor, and 1% penicillin- ml), moderate (MICs
128–256 μg/ml), and weak (MIC

streptomycin solution (10,000 units/ml and 100 mg/ml, 512 μg/ml) (Kokoska et al., 2019). As a result of experiments
respectively). The cancer cells were cultured in Eagle’s performed without dissolved extracts and ciprofloxacin (Sigma-
Minimum Essential Medium (EMEM) supplemented with 1% Aldrich, Prague, Czechia), their respective solvents, namely,
sodium pyruvate, 10% fetal bovine serum, 1% sodium DMSO (Sigma-Aldrich, Prague, Czechia) and distilled water,
bicarbonate, 1% nonessential amino acids, and 1% penicillin- did not inhibit bacterial growth of any strain at the tested
streptomycin solution (10,000 units/ml and 100 mg/ml, concentrations (≤1%).
respectively) (all purchased from Biowest, Nuaille, France).
The cultures were incubated at 37°C and 5% CO2. The culture Cytotoxicity Assay
medium was replaced every 2–3 days, and cells were passaged The antiproliferative activities of the extracts that showed some
every 7 days. inhibitory action against the tested bacteria were further assessed
using the modified thiazolyl blue tetrazolium bromide (MTT)
Antibacterial Assay cytotoxicity assay developed by Mosmann (1983). Cancer (2.5 ×
Initially, all 35 extracts (Table 1) were evaluated for their 103) and normal intestinal (2.5 × 105) cells were seeded in a 96-
antibacterial activities against the pathogenic strains. Those well microtiter plate for 24 h. Cells were incubated with twofold
showing any inhibitory action were subsequently tested against serially diluted plant extracts (0.25–512 μg/ml) for 72 h. Next, the
the probiotic strains. The growth-inhibitory activities against cells were incubated with MTT reagent (1 mg/ml) (Sigma-
aerobic and anaerobic bacteria were evaluated using the broth Aldrich, Prague, Czechia) in EMEM or Hybri-Care medium
microdilution method using 96-well microtiter plates, following for an additional 2 h at 37°C and 5% CO2. The medium with
the protocols of (Clinical and Laboratory Standards Institute, MTT was removed, and the intracellular formazan product was
2021) and Hecht (1999), respectively. For the effective assessment dissolved in 100 μl DMSO. The absorbance was measured at
of the anti-infective potential of natural products, slight 555 nm using a Tecan Infinite M200 spectrometer (Tecan,
modifications were implemented as described by Cos et al. (2006). Männedorf, Switzerland), and the percentage of viability was
Prior to testing, the strains that grow aerobically were calculated when compared to an untreated control.
subcultured in the appropriate media at 37°C (Y. enterocolitica The antiproliferative activity of the tested plant extracts was
at 30°C) for 24 h. Bifidobacteria, clostridia, and lactobacilli represented as half-maximal inhibitory concentration (IC50; μg/
were cultured at 37°C for 48 h using Whitley A35 Anaerobic ml). The colon cancer chemotherapeutic drug 5-fluorouracil
Workstation (Don Whitley Scientific, Bingley, United Kingdom). (Sigma-Aldrich, Prague, Czechia) was used as a positive
The anaerobic conditions were created by supplying a standard control (Fuente et al., 2020). Three independent experiments
anaerobic gas mixture of 10% H2, 10% CO2, and 80% N2 (Linde (two replicates each) were performed for every test. Data are
Gas, Prague, Czechia). presented as mean ± standard deviation. The antiproliferative
The extracts were diluted twofold in appropriate growth media activity was evaluated as follows: cytotoxic (IC50 values ≤100 μg/
(initial concentration of 512 μg/ml) using the Freedom EVO 100 ml), moderately cytotoxic (IC50 values
100–400 μg/ml), and
automated pipetting platform (Tecan, Männedorf, Switzerland) weakly cytotoxic (IC50 values
401–512 μg/ml) (Srisawat et al.,
and multichannel pipette (Eppendorf, Hamburg, Germany) in 2013). The solvents did not affect the viability of normal and
case of aerobic and anaerobic bacteria, respectively. After the cancer intestinal cell lines at the tested concentration (≤1%).
optimalization of bacterial cultures to inoculum density of 1.5
× 10 8 CFU/ml by 0.5 McFarland standard using Densi-La- Calculations
Meter II (Lachema, Brno, Czechia), the cultures were For comparison of microbiological and toxicological data, 80%
inoculated in 96-well plates (5 μl/well). Bacterial cultures bacterial growth inhibition (IC80) was calculated as equivalent to
in microplates were incubated by employing the same the MIC endpoint (Houdkova et al., 2018). Subsequently, x-MIC,
protocols as used for their cultivation prior to the test. x-IC50, and x-IC80 values (±standard deviations) were calculated
The optical density of the cultures was measured at to quantify the inhibitory activity of the tested plant extracts
405 nm (OD 450 nm ) using a Cytation 3 Imaging Reader against pathogenic/beneficial bacteria and intestinal
(BioTek, Winooski, VT, United States) before and after the cancer/normal cells. Subsequently, the selectivity index (SI)
growth period. was calculated between normal intestinal cells and pathogenic

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Frontiers in Pharmacology | www.frontiersin.org

Kudera et al.
TABLE 2 | In vitro selective inhibitory activities of ethanolic extracts of Cambodian and Philippine plants against intestinal bacteria and cells.

Cultures tested Plant species with their parts and positive antibiotic and anticancer control

AP(w) AG(l) AT(l) AB (f) AC(b) BM(b) BV(wb) DE(r) EM(l) IN(l) LC(b) MD(b) MD(lf) MS(lf) PJ(b) PS(b) CIP 5-FU

Bacterial strain/ BC -a 512 64 64 256 512 - - 512 - - - - 512 - 256 1 nd

MIC CD 512 512 512 32 128 - - - 512 512 - - 512 512 - - 16 nd
(µg/ml) CP - 512 512 32 256 - - - - - - - - - - - 1 nd
EF - - - 128 - - - - - - - - - - - - 2 nd
EC 512 - - - - 256 256 512 - - - 256 - - 256 256 0.062 nd
ECS - - - - - - - - - - - - - - - - 0.016 nd
LM - - 128 256 - - - - - - - - - 512 - 512 4 nd
SF - - - - - - - - - - - - - - - - 0.016 nd
SE - - - - - - - - - - - - - - - 256 0.031 nd
ST - - - - - - 256 - - - - - 512 - - - 0.031 nd
VP - 256 - - - 512 - - 512 - 512 512 512 512 - - 0.062 nd
YE - - - - - - - - - - - - - - - - 0.125 nd
x-PB ± 938.7 ± 832 ± 784 ± 640 ± 821.3 ± 874.7 ± 896 ± 981.3 ± 896 ± 981.3 ± 981.3 142± 917.3 ± 896 ± 853.3 960 ± 789.3 ± 2±6 nd
SD 191 279 390 458 352 266 286 142 222 142 244 222 ± 241 212 338
BA 64 512 256 16 - - - - 512 512 - - 256 512 - - 8 nd
BB - 64 64 16 512 - - - 128 128 - - - 256 128 - 64 nd
BLC - - 256 16 - - - - 256 512 - - - 512 - - 32 nd
LC 512 256 128 16 256 - - - 128 128 - - - 512 512 512 32 nd
LR - - 256 16 - - - - - - - - - - - - 32 nd
LRM - - 128 16 512 - - - 512 - - - - - - - 4 nd
x-BB ± 778.7 ± 650.7 ± 181.3 ± 16 ± 0 725.3 ± 1,024 ± 0 1,024 ± 0 1,024 ± 0 426.7 ± 554.7 ± 1,024 ± 0 1,024 ± 0 896 ± 640 ± 789.3 ± 938.7 ± 29 ± 20 nd
SD 370 395 78 311 311 367 286 286 350 191

Cell line IC50 ± HT-29 130.52 ± 96.53 ± 82.19 ± 53.70 ± 84.77 ± 35.195 ± 81.79 ± - 130.89 ± 125.55 ± 37.89 ± 248.56 ± 210.85 ± 49.75 ± 155.87 ± 51.98 ± 19.79 88.81 ± 6.35
7

(µg/ml) SD 2.57 12.41 17.22 16.02 4.20 5.32 20.79 13.99 13.92 2.68 23.13 16.83 3.53 41.79 13.44 ± 2.07
Caco- 148.96 ± 78.42 ± 33.82 ± 79.41 ± 48.40 ± - - - 52.49 ± 135.60 ± 122.86 ± 193.73 ± 77.57 ± 87.40 ± 121.26 ± - 90.87 ± 181.79
2 17.14 23.18 10.57 6.90 0.93 8.81 3.11 13.16 1.61 10.22 19.18 15.34 17.98 ± 151.51
x-CC ± 139.7 ± 87.5 ± 9 58 ± 66.5 ± 66.5 ± 529.6 ± 552.9 ± - 91.7 ± 130.6 ± 5 80.4 ± 221.1 ± 144.2 ± 68.575 ± 138.6 ± 538 ± 486 89.84 ± 94
SD 9.2 24.2 12.8 18 494 471 39.2 42.5 27.4 66.6 18.8 17.3 1 ± 88
FHs 297.39 ± 118.76 ± 45.50 ± 273.32 ± 68.23 ± 158.36 ± 68.75 ± - 303.41 ± 243.50 ± 282.05 ± - 368.07 ± 195.19 ± 342.62 ± - 58.90 ± 492.43
74 Int 22.54 36.04 7.29 7.50 12.39 23.75 8.89 18.00 21.92 0.57 30.00 8.94 3.54 4.27 ± 22.92
IC80 ± HT-29 130.50 ± - 498.17 ± - 356.20 ± - - - - - - 351.26 ± 378.50 ± 143.70 ± - - 181.53 ± 367.26
SD 12.06 16.74 19.04 43.24 34.93 16.74 15.05 ± 0.57
Caco- - - - - - - - - - - - - - - - - - -
2
x-CC ± 577.25 ± - 761.1 ± - 690.1 ± - - - - - - 687.63 ± 701.25 ± 583.85 ± - - 602.77 ± 695.63 ±
SD 446.75 263 333.9 336.37 322.75 440.15 421.23 328.37
FHs - - - - 264.64 ± - - - - - - - - - - - 83.37 ± -
74 Int 6.82 8.60
November 2021 | Volume 12 | Article 746808

SI (a) 0.04 0.09 0.12 0.2 −0.49 0.07 0.06 0.02 0.06 0.02 0.02 0.05 0.06 0.08 0.03 0.11 1.62 nd

Antibacterial and Anticancer Plant Extracts

(b) −0.1 −0.1 −0.6 −1.6 −0.1 0.1 0.1 0.02 −0.3 −0.2 0.02 0.05 0 −0.1 −0.04 0.1 1.2 nd
(c) 0.3 0.1 −0.1 0.6 0.01 −0.5 −0.9 0 0.5 0.3 0.5 0.7 0.4 0.5 0.4 0.3 −0.2 0.4
(d) 0.13 −0.2 −0.62 −1.81 0.02 0 0 0 −0.38 −0.27 0 0.17 0.11 0.04 −0.11 −0.04 −1.32 nd

MIC, minimum inhibitory concentration; IC50, half-maximal inhibitory concentration; IC80, 80% inhibitory concentration of proliferation; SD, standard deviation; aNot active (MIC/IC50/80 >512 μg/ml, the value 1,024 μg/ml was used for average calculation); nd:
no data. AP(w), Aganonerion polymorphum Spire (whole plant); AG(l), Acalypha grandis Benth. (leaves); AT(l), Ancistrocladus tectorius (Lour.) Merr. (leaves); AB (f), Artocarpus blancoi (Elmer) Merr. (fruit); AC(b), Artocarpus camansi Blanco (bark); BM(b),
Bauhinia malabarica Roxb. (bark); BV(wb), Breynia vitis-idaea (Burm.f.) C.E.C.Fisch. (wood with bark); DE(r), Diplazium esculentum (Retz.) S roots); EM(l), Ehretia microphylla Lam. (leaves); IN(l), Ixora nigricans R.Br. ex Wight and Arn. (leaves); LC(b),
Lagerstroemia cochinchinensis Pierre ex Gagnep. (bark); MD(b), Melastoma dodecandrum Lour. (bark); MD(lf), Melastoma dodecandrum Lour. (leaves with flower buds); MS(lf), Melastoma saigonense (Kuntze) Merr. (leaves with flower buds); PJ(b), Picrasma
javanica Blume (bark); PS(b), Pentacme siamensis (Miq.) Kurz; CIP, ciprofloxacin; 5-FU, 5-fluorouracil. BC, Bacillus cereus; CD, Clostridium difficile; CP, Clostridium perfringens; EF, Enterococcus faecalis; EC, Escherichia coli; ECS, E. coli 0,175:H7; LM,
Listeria monocytogenes; SF, Shigella flexneri; SE, Salmonella Enteritidis; ST, Salmonella Typhimurium; VP, Vibrio parahaemolyticus; YE, Yersinia enterocolitica; BA, Bifidobacterium adolescentis; BB, Bifidobacterium breve; BLC, Bifidobacterium animalis spp.
lactis, LC, Lactobacillus casei; LR, Lactobacillus reuteri; LRM, Lactobacillus rhamnosus; x-PB, mean MIC for pathogenic bacteria; x-BB, mean MIC for beneficial bacteria; x-CC, mean IC50/80 for intestinal cancer cells; FHs 74 Int (intestinal normal cells); SI
(selectivity index): (a) normal cells/diarrheagenic bacteria, (b) beneficial bacteria/diarrheagenic bacteria, (c) normal cells/cancer cells, and (d) beneficial bacteria/cancer cells.
Kudera et al. Antibacterial and Anticancer Plant Extracts

strains (SIa), beneficial and pathogenic strains (SIb), normal and C.E.C.Fisch. inhibited S. Typhimurium; and Acalypha grandis
cancer intestinal cells (SIc), and beneficial strains and cancer Benth. (Euphorbiaceae) inhibited V. parahaemolyticus. Finally,
intestinal cells (SId) using the following formulas where X1
IC80 Aganonerion polymorphum Spire (Apocynaceae), Diplazium
against normal intestinal cells; X2
x-MIC against beneficial esculentum (Retz.) Sw. (Athyriaceae), Ehretia microphylla Lam.
strains; X3
IC50 against normal intestinal cells; Y1
x-MIC (Boraginaceae), Ixora nigricans R.Br. ex Wight and Arn.
against pathogenic strains; Y2
x-IC50 against cancer intestinal (Rubiaceae), Lagerstroemia cochinchinensis Pierre ex Gagnep.
cells; and Y3
IC80 against cancer intestinal cells: (Lythraceae), Melastoma dodecandrum Lour.
(Melastomataceae) (leaves with flower buds), and Melastoma
SIa
log (X1/Y1), (1)
saigonense (Kuntze) Merr. (Melastomataceae) (leaves with
SIb
log (X2/Y1), (2) flower buds) produced only weak inhibitory actions at MICs
SIc
log (X3/Y2), (3) of 512 μg/ml (Table 2).
SId
log (X2/Y3). (4) The remaining 19 extracts of Acanthus ebracteatus Vahl
(Acanthaceae), Aporosa villosa (Lindl.) Baill. (Phyllanthaceae),
The SI values >0 and <0 indicate selective toxicity against Artocarpus elasticus Reinw. ex Blume (Moraceae), Artocarpus
pathogenic strains/cancer cell lines and beneficial strains/normal odoratissimus Blanco (Moraceae), B. malabarica Roxb. (leaves),
cell lines, respectively. Breynia cernua (Poir.) Müll.Arg. (Phyllantaceae), Commelina
communis L. (Commelinaceae), Cyathula prostrata (L.) Blume
(Amaranthaceae), Emilia sonchifolia (L.) DC. ex DC.
RESULTS (Compositae), Helicteres angustifolia L. (Malvaceae), Hyptis
capitata Jacq. (Lamiaceae), Kyllinga brevifolia Rottb.
Antibacterial Activity (Cyperaceae), Leea indica (Burm. f.) Merr. (Vitaceae), M.
Diarrheagenic Bacterial Pathogens saigonense (Kuntze) Merr. (wooden stem and leaves), Parkia
Considering the antibacterial activity against the pathogens, 16 of javanica (Lam.) Merr. (Leguminosae), Pseudelephantopus
35 tested extracts revealed a growth-inhibitory effect on at least spicatus (Juss. ex Aubl.) Rohr (Compositae), Rourea minor
one of these bacterial strains. While B. cereus, C. difficile, E. coli, (Gaertn.) Alston (Connaraceae), Tabernaemontana pandacaqui
and V. parahaemolyticus were the most susceptible bacteria Lam. (Apocynaceae), and Triumfetta bartramia L. (Malvaceae)
inhibited by the highest number of extracts, none of the did not show any inhibitory action; thus, they have not been
extracts exerted activity against E. coli O157:H7 and S. further discussed.
flexneri. The MICs (32–512 μg/ml) of all 16 plant extracts for
the diarrheagenic bacterial pathogens are presented in Table 2. Beneficial Gut Bacteria
There were four extracts showing promising antibacterial Subsequently, 16 extracts that exerted growth-inhibitory effect
actions against multiple pathogenic bacteria, especially the against diarrheagenic pathogens were verified for their safety to
gram-positive strains. Namely, the fruit extract of Artocarpus beneficial bacteria. The final MICs are presented in Table 2. Five
blancoi (Elmer) Merr. (Moraceae) inhibited B. cereus and both extracts, namely, B. malabarica Roxb., B. vitis-idaea (Burm.f.)
clostridia at MICs 64 and 32 μg/ml, respectively. This plant was C.E.C.Fisch, D. esculentum (Retz.) Sw., L. cochinchinensis Pierre
also moderately active against E. faecalis (MIC
128 μg/ml) and ex Gagnep., and M. dodecandrum Lour. (bark), did not have any
L. monocytogenes (MIC
256 μg/ml). Similarly, the leaf extract of inhibition of these strains (MICs >512 μg/ml), suggesting their
Ancistrocladus tectorius (Lour.) Merr. (Ancistrocladaceae) harmless effect on gut commensals.
revealed a strong inhibitory effect on B. cereus (MIC
64 μg/ The remaining 11 extracts affected to some degree the growth
ml) and moderate activity against L. monocytogenes (MIC
of beneficial gut bacteria, particularly of bifidobacteria and L.
128 μg/ml). However, it produced only weak inhibitory action casei (Table 2). The single strain was inhibited by P. siamensis
against both clostridia (MICs
512 μg/ml). Next, bark extract of (Miq.) Kurz (L. casei) and leaf with flower bud of M.
Artocarpus camansi Blanco (Moraceae) inhibited B. cereus and dodecandrum Lour. (B. adolescentis) at MICs of 256 and
both clostridia at MICs ranging from 128 to 256 μg/ml. Although 512 μg/ml, respectively. Moreover, P. javanica Blume inhibited
the antibacterial activities of bark extract of Pentacme siamensis B. breve (MIC
128 μg/ml) and L. casei (MIC
512 μg/ml).
(Miq.) Kurz (Dipterocarpaceae) were rather moderate, it exerted Although A. polymorphum Spire significantly affected the growth
inhibitory action against several gram-positive as well as gram- of B. adolescentis (MIC
64 μg/ml), the remaining probiotic
negative pathogenic strains. Namely, it inhibited B. cereus, E. coli, strains were rather resistant toward this extract (MICs ≥512 μg/
and S. Enteritidis at MICs of 256 μg/ml and L. monocytogenes at ml). Three and four probiotic bacteria were inhibited (MICs

MIC of 512 μg/ml. 256–512 μg/ml) by A. camansi Blanco and M. saigonense
Additionally, there were five more plant extracts exerting (Kuntze) Merr., respectively. At MICs ranging from 128 to
moderate activity (MIC
256 μg/ml) against a single gram- 512 μg/ml (Table 2), E. microphylla Lam. and I. nigricans
negative strain (Table 2). Namely, Bauhinia malabarica R.Br. ex Wight and Arn. affected the growth of the majority
Roxb. (Leguminosae) (bark), Breynia vitis-idaea (Burm.f.) of beneficial strains. Although half of the bacteria were not
C.E.C.Fisch. (Phyllanthaceae), Melastoma dodecandrum Lour. inhibited by A. grandis Benth., this extract inhibited B. breve
(Melastomataceae) (bark), and Picrasma javanica Blume at low MIC (64 μg/ml). Finally, all six strains were inhibited by A.
(Simaroubaceae) inhibited E. coli; B. vitis-idaea (Burm.f.) blancoi (Elmer) Merr. and A. tectorius (Lour.) Merr. Whereas the

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Kudera et al. Antibacterial and Anticancer Plant Extracts

former uniformly affected the growth at very low MICs (16 μg/ beneficial strains was shown by B. malabarica Roxb., B. vitis-
ml), the latter inhibited B. breve (MIC
64 μg/ml) only. idaea (Burm.f.) C.E.C.Fisch., and P. siamensis (Miq.) Kurz
(Table 2). However, none of the selective effects were as
Cytotoxic Effect significant as in the case of ciprofloxacin (SIb
1.2). Other
The outcomes of the MTT assay for all 16 antibacterially active extracts did not show any noticeable selectivity or were
plant extracts against normal and cancer intestinal cells are comparably more harmful to beneficial bacteria, especially A.
presented in Table 2. With the exception of D. esculentum blancoi (Elmer) Merr., and A. tectorius (Lour.) Merr. (SIb values

(Retz.) Sw. (IC50 values > 512 μg/ml), all the 16 extracts −1.6 and −0.6, respectively). Regarding the selective
produced a certain antiproliferative effect on at least one of antiproliferative effects against cancer intestinal cells, A.
the tested cell lines (IC50 values
33.82 ± 10.57–368.07 ± blancoi (Elmer) Merr., E. microphylla Lam., L. cochinchinensis
30.00 μg/ml). Pierre ex Gagnep., M. dodecandrum Lour. (bark), and M.
saigonense (Kuntze) Merr. revealed higher selectivity (SIc
Normal Intestinal Cells values
0.5–0.7) than that of 5-fluorouracil (SIc
0.4)
Considering the toxicity to normal intestinal cells (FHs 74 Int), (Table 2). Other extracts produced either the same or lower
M. dodecandrum Lour. (bark) and P. siamensis (Miq.) Kurz did degree of selective effects than that of this cytotoxic drug, whereas
not show inhibitory action at the concentrations tested (IC50 > A. tectorius (Lour.) Merr., B. malabarica Roxb., and B. vitis-idaea
512 μg/ml) (Table 2). Moderate toxicity was shown by A. (Burm.f.) C.E.C.Fisch. were relatively more toxic to normal
polymorphum Spire, A. grandis Benth., A. blancoi (Elmer) intestinal cells (SIc values
−0.9 to −0.1). The probiotic
Merr., B. malabarica Roxb., E. microphylla Lam., I. nigricans strains were not affected by the antiproliferative
R.Br. ex Wight and Arn., L. cochinchinensis Pierre ex Gagnep., M. concentrations of A. polymorphum Spire (SId
0.13), mainly
dodecandrum Lour. (leaves with flower buds), M. saigonense because of moderate inhibition of HT-29 (IC80
130.50 ±
(Kuntze) Merr., and P. javanica Blume at IC50 values ranging 12.06 μg/ml) (Table 2). Interestingly, the extract of P.
from 118.76 ± 36.04 to 368.07 ± 30.00 μg/ml. Finally, the extracts siamensis (Miq.) Kurz produced noticeable selective actions
of A. tectorius (Lour.) Merr., A. camansi Blanco, and B. vitis-idaea combining antibacterial and antiproliferative effects on
(Burm.f.) C.E.C.Fisch. were shown to be cytotoxic (IC50 values
pathogenic bacteria and intestinal cancer cells without
45.50 ± 7.29, 68.23 ± 12.39 and 68.75 ± 8.89 μg/ml, respectively). affecting beneficial bacteria and normal intestinal cells.

Cancer Intestinal Cells

Regarding the antiproliferative activities against cancer intestinal DISCUSSION
cells (Table 2), the plants producing strong effects on Caco-2
(IC50 values
33.82 ± 10.57–87.40 ± 19.18 μg/ml) have been In the present study, 16 of 35 tested extracts revealed in vitro
ordered as follows: A. tectorius (Lour.) Merr., A. camansi Blanco, growth-inhibitory effect on the diarrheagenic bacterial
E. microphylla Lam., M. dodecandrum Lour. (leaves with flower pathogens, especially A. blancoi (Elmer) Merr., A. camansi
buds), A. grandis Benth., A. blancoi (Elmer) Merr., and M. Blanco, A. tectorius (Lour.) Merr., and P. siamensis (Miq.).
saigonense (Kuntze) Merr. With the exception of moderately Except A. camansi Blanco, the antibacterially active
cytotoxic E. microphylla Lam. and M. dodecandrum Lour. (leaves concentrations of the three were nontoxic to normal intestinal
with flower buds), the same plant extracts with the addition of B. cells. Among the 16 extracts, A. blancoi (Elmer) Merr., E.
malabarica Roxb., B. vitis-idaea (Burm.f.) C.E.C.Fisch., L. microphylla Lam., L. cochinchinensis Pierre ex Gagnep., M.
cochinchinensis Pierre ex Gagnep., and P. siamensis (Miq.) saigonense (Kuntze) Merr., and P. siamensis (Miq.) Kurz also
Kurz also produced strong antiproliferative effect on HT-29 revealed a strong selective antiproliferative effect against
(IC50 values
35.195 ± 5.32–96.53 ± 12.41 μg/ml) (Table 2). intestinal cancer lines. The extract of P. siamensis (Miq.) Kurz
A moderate cytotoxic effect on both these cancer cell lines was exhibited activities combining selective inhibition of pathogenic
then shown by A. polymorphum Spire, I. nigricans R.Br. ex Wight bacteria and intestinal cancer cells without affecting beneficial
and Arn., M. dodecandrum Lour. (bark), and P. javanica Blume bacteria and normal intestinal cells. To the best of our knowledge,
(IC50 values
121.26 ± 15.34–248.56 ± 23.13 μg/ml). The this is the first study on antibacterial and antiproliferative
majority of extracts revealed higher activities against Caco-2 activities of A. polymorphum Spire, B. vitis-idaea (Burm.f.)
than that of 5-fluorouracil (IC50
181.79 ± 151.51 μg/ml). C.E.C.Fisch., I. nigricans R.Br. ex Wight and Arn., L.
cochinchinensis Pierre ex Gagnep., P. siamensis (Miq.) Kurz,
Selective Toxicity and M. saigonense (Kuntze) Merr. Moreover, there are no
The calculated mean values for pathogenic/beneficial bacteria, previous studies on the cytotoxic effects of A. blancoi (Elmer)
cancer cells (x-MIC, x-IC50, and x-IC80), and derived SIs are Merr. Although the cytotoxic effect of products isolated from B.
presented in Table 2. Comparing the concentrations inhibiting malabarica Roxb. was described previously (Kittakoop et al.,
80% of growth for pathogenic bacteria and normal intestinal cells, 2000), its antibacterial activity is herein reported for the first
the antibacterially active extracts were shown to be relatively safe time. Our results correspond with those of previous studies on
(SIa values
0.02–0.2; IC80 values >512 μg/ml) except A. camansi antibacterial and antiproliferative activities of A. grandis Benth.
Blanco (SIa
−0.49; IC80
264.64 ± 6.82 μg/ml). Selective (Bradacs et al., 2009), D. esculentum (Retz.) Sw. (Mackeen et al.,
antibacterial effect (SIb values
0.1) with relative safety for 1997; Rahmat et al., 2003), and P. javanica Blume (Khan et al.,

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Kudera et al. Antibacterial and Anticancer Plant Extracts

2001; Win et al., 2015). The above highlighted seven plant Similar to Artocarpus spp., the leaf extract of A. tectorius
extracts with promising activities have mainly been discussed. (Lour.) Merr. exhibited growth-inhibitory effects only against
Two of the four tested species of the genus Artocarpus gram-positive bacteria. This corresponds with the study by Wiart
(Moraceae), namely, A. blancoi (Elmer) Merr. and A. camansi et al. (2004), where its methanolic leaf extract produced
Blanco, exhibited strong antibacterial and antiproliferative antibacterial activity against B. cereus but not E. coli. We also
activities in our study. Artocarpus spp. are rich in phenolic found that the overall cytotoxic effect of this plant was strong.
compounds, such as flavonoids, stilbenoids, and Although the antiproliferative effect on cancer cells was not
arylbenzofurans, which are known to possess a wide range of selective, the extract concentrations inhibiting the pathogens
biological activities, including antibacterial and anticancer effects were generally nontoxic to normal intestinal cells. Previous
(Hafid et al., 2017). Our study is the first to report on phytochemical analysis of leaf ethanolic/methanolic extracts of
anticlostridial activities of Artocarpus spp. As flavonoids have this plant showed the presence of various naphthylisoquinoline
been reported to have potent in vitro inhibitory effect on some alkaloids, such as 7-epiancistrobrevine D, ancistrocladinine,
clostridia (Wu et al., 2013), these compounds might be ancistrotectoquinone A-B, ancistrotectoriline A–C, and
responsible for significant antibacterial activities revealed by A. hamatinine (Anh et al., 1997; Tang et al., 2000; Tang et al.,
blancoi (Elmer) Merr. and A. camansi Blanco against C. difficile 2010; Bringmann et al., 2016). Since these isoquinoline alkaloids
and C. perfringens. Beloy et al. (1976) isolated the flavonoid are known to possess various biological activities, including
5,7,4′-trihydroxyflavanone-3-O-α-L-rhamnopyranoside from antimicrobial and cytotoxic effects, we suspect them to be
the bark extract of A. blancoi (Elmer) Merr., showing responsible for the growth-inhibitory effects revealed by A.
antibacterial activity against Mycobacterium tuberculosis. Ante tectorius (Lour.) Merr. in the present study. For example, in
et al. (2016) showed that bark essential oil of A. camansi Blanco the study by Mihalyi et al. (2014), michellamine B isolated from
produced antibacterial activity against some diarrheagenic Ancistrocladus korupensis D.W.Thomas and Gereau inhibited B.
bacteria. Our results show that both of these plants inhibited subtilis. Jiang et al. (2013) showed that naphthylisoquinolines
gram-positive bacteria only. Beside their anticlostridial effect, this isolated from A. tectorius (Lour.) Merr. exhibited cytotoxic effect
selectivity probably also contributed to their relative toxicity to against three leukemia cells in vitro. In another study, 7-
beneficial bacteria. In vitro inhibitory effect against lactobacilli epiancistrobrevine and ancistrotectoriline exhibited activity
was previously reported for Artocarpus lacucha Buch.-Ham. against pancreatic cancer cells (Shang et al., 2020). The
(Teanpaisan et al., 2014). An example of a compound isolated present study is the first to report on in vitro selective
from the plant of this genus and showing similar activities is antiproliferative activity of A. tectorius (Lour.) Merr. against
artocarpin. In the study by Sato et al. (1996), this flavonoid intestinal cells.
exhibited strong inhibition of all gram-positive bacteria, Regarding P. siamensis (Miq.) Kurz, there are no comparable
including L. casei, whereas in another study, it produced studies dealing with species of the same genus. However, our
higher MICs against E. coli and Pseudomonas aeruginosa results showing a noticeable combination of selective
(Septama and Panichayupakaranant, 2015). The absence of antibacterial and cytotoxic effects of its bark extract can be
antibacterial action of A. odoratissimus Blanco found herein compared to the data available for closely related genus Shorea
will correlate with rather low levels of phenolic content (Dipterocarpaceae). For example, Marandi et al. (2016) showed
detected in its fruit methanolic extract (Abu Bakar et al., that bark ethanolic extract from Indian antidiarrheal and
2015), compared to antibacterially active species (Jalal et al., antidysenteric medicinal plant Shorea robusta Gaertn.
2015). Although hexane bark extract of A. elasticus Reinw. ex exhibited inhibitory action against B. cereus, B. subtilis, E.
Blume exhibited activity against B. cereus and E. coli in the study faecalis, E. coli, S. Typhi, and V. cholerae. Stilbene derivatives
by Ramli et al. (2016), its lack of activity in the present study could isolated from barks of Shorea spp. previously showed strong
be influenced by the use of different extraction procedures. antibacterial effects against some of these strains (Nitta et al.,
According to our results, A. blancoi (Elmer) Merr. and A. 2002; Sudto et al., 2019). Some polyphenols, such as stilbenes, can
camansi Blanco had a selective cytotoxic effect on intestinal inhibit several nonbeneficial bacteria from the human
cancer cells, whereas the former did not show cytotoxicity to microbiota, with no noticeable effects on the growth of
normal cells at the inhibitory concentrations against several probiotic bacteria (Requena et al., 2010). Therefore, we suggest
pathogens. Various terpenoids and phytosterols were that some of these agents could also contribute to the selective
previously isolated from methanolic and dichloromethane antibacterial activities of P. siamensis (Miq.) Kurz shown in the
extract of stem and leaves of A. camansi Blanco, respectively. present study. Regarding cytotoxic effect, oligostilbenoids were
Among them, friedelinol, cycloartenol, and cycloartenol acetate usually the constituents derived from Shorea spp., with reported
inhibited the growth of HT-29 cells; squalene has profound antiproliferative action against various cancer cell lines (Rohaiza,
chemopreventive activity against colon carcinogenesis; and ß- 2011; Zawawi et al., 2012; Moriyama et al., 2016). Among them,
sitosterol has been shown to induce apoptosis in human colon ampelopsin E exhibited obvious antiproliferative properties on
tumors (Tsai et al., 2013). Regarding cytotoxic compounds COLO205 and HT-29 cells (Tian et al., 2019), whereas α-viniferin
isolated from other Artocarpus spp., the prenylated flavone showed selective inhibition of colon cancer cells (HCT-116, HT-
artelastin revealed strong in vitro activity against five colon 29, and Caco-2) with twofold lower IC50 compared to normal
cancer cell lines (COLO 205, HCT 116, HCT 15, HT-29, and colon cells (CCD-18Co) (Gonzalez-Sarrias et al., 2011). To
SW 620) in the study by Pedro et al. (2005). identify phytochemicals responsible for the in vitro selective

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Kudera et al. Antibacterial and Anticancer Plant Extracts

inhibitory actions shown by P. siamensis (Miq.) Kurz in the supports this assumption. Moreover, it has been reported that
present study, an accurate chemical analysis of this plant is microorganisms are less likely to develop resistance to
needed. phytochemicals with anti-infective potential, mainly because of
Finally, E. microphylla Lam., L. cochinchinensis Pierre ex their high diversity in plants. Some were even considered as
Gagnep., and M. saigonense (Kuntze) Merr. revealed a strong antibiotic resistance modifying compounds (Sibanda and Okoh,
selective antiproliferative effect against intestinal cancer lines. It 2007). Additionally, our study showed that the extract of P.
has been reported that triterpenes urs-12-en-24-oic acid, 3-oxo-, siamensis (Miq.) Kurz was relatively safe for probiotic bacteria,
methyl ester, and ß-amyrin are involved in anticancer activities of and together with A. blancoi (Elmer) Merr., they exerted selective
products derived from leaves of E. microphylla Lam. (Rajkumar anticancer activities in vitro. Similar to the cytotoxic activities
et al., 2019). Our study corresponds with other studies dealing revealed by E. microphylla Lam., L. cochinchinensis Pierre ex
with these chemicals. For example, in the study by Kuete et al. Gagnep., and M. saigonense (Kuntze) Merr., the inhibitory effect
(2018), ß-amyrin produced a selective cytotoxic effect against of A. blancoi (Elmer) Merr. on cancer cell line Caco-2 and the
Caco-2 compared to that on normal cell line HEK293. In another selectivity of its overall antiproliferative actions were generally
study, extract of Alstonia macrophylla Wall. ex G.Don containing higher than those of anticancer drug 5-fluorouracil.
ß-amyrin produced a selective cytotoxic effect against HT-29 These results suggest that extracts from the above-
compared to that on normal cell line HDFn (Tan et al., 2019). The mentioned Cambodian and Philippine plant species are
present study is the first to report the antiproliferative activities of promising materials for further research focused on the
E. microphylla Lam. against intestinal cell lines. Compounds such development of new plant-derived selective antibacterial and
as triterpenes, tannins, ellagic acids, glycosides, and flavones were antiproliferative agents used in the treatment of infectious
previously attributed to bioactive properties of Lagerstroemia spp. diarrhea and associated intestinal cancer diseases. For
(Chan et al., 2014). In previous studies, triterpenes isolated from instance, the combination of strong anticlostridial and
species of this genus produced significant in vitro activity against anticancer actions of A. blancoi (Elmer) Merr. may in the
colon cancer cells, for instance, betulinic acid and 3β- future be utilized in the treatment of digestive cancers
acetoxyolean-12-en-28-acid against HCT15 (Woo et al., 2016) associated with C. difficile infections (Han et al., 2013).
and corosolic acid against HCT116 (Sung et al., 2014). Regarding However, further phytochemical and pharmacological
M. saigonense (Kuntze) Merr., there are various previously research is needed for the isolation and proper identification
published studies on related species showing corresponding of their bioactive constituents. Referring to studies dealing with
results. For example, the methanolic leaf extract of Melastoma taxonomically related plants to estimate the presence of their
malabathricum L. produced an antiproliferative effect on HT-29 bioactive principles is a very limited approach as their
in the study by Kamsani et al. (2019). Asiatic acid, caffeic acid, composition can vary greatly. On the other hand, our results
p-coumaric acid, kaempferol, quercetin, rutin, and ursolic acid could serve as an indicator of bioactive potentials of products
were isolated compounds with previously profound derived from species of the same taxa. This is mainly the case of
antiproliferative action to this cell line. In the study by P. siamensis (Miq.) Kurz that exhibited selective inhibition of
Karakurt et al. (2020), p-coumaric acid exhibited selective pathogenic bacteria and intestinal cancer cells without affecting
inhibition of Caco-2 and HT-29 cells compared to that of beneficial bacteria and normal intestinal cells. Future research
healthy colon epithelial cells (CCD-18Co). Since the decoction combining the ethnomedicinal and chemotaxonomic
from the leaves of M. malabathricum L. is also traditionally approaches might help to identify more plants with
consumed to treat diarrhea, we suggest a similar composition promising bioactivities (Hao and Xiao, 2020).
of bioactive compounds to be present in M. saigonense (Kuntze)
Merr. (Ong and Nordiana, 1999). Regarding the moderate
selective antiproliferative activities of bark and leaf with flower DATA AVAILABILITY STATEMENT
bud extracts of M. dodecandrum Lour., three pentacyclic
triterpenoids (ursolic acid, asiatic acid, and terminolic acid) The original contributions presented in the study are included in
and one tannin (casuarinin) were previously isolated from this the article/Supplementary Material; further inquiries can be
plant and found to significantly decrease interleukin-8 directed to the corresponding author.
production in HT-29 (Yang et al., 2014).
In summary, A. blancoi (Elmer) Merr., A. tectorius (Lour.)
Merr., and P. siamensis (Miq.) Kurz produced significant growth- AUTHOR CONTRIBUTIONS
inhibitory effects against diarrheagenic bacterial pathogens at
concentrations nontoxic to normal intestinal cells. Except the TK collected the plant materials, coordinated antibacterial
strong anticlostridial actions of A. blancoi (Elmer) Merr., the activity testing and its related data analysis, and prepared the
MICs determined for these plant extracts in the present study article. BF and MK processed the voucher specimens and
reflect rather moderate antibacterial activities. However, the participated in the collection of plant materials and testing of
discrimination of specific cell toxicity indicates that higher antibacterial activity. ID conducted the cytotoxicity assays and
amounts of these products necessary to acquire the their related data analysis. HS was responsible for maintenance
appropriate efficiency may still be safe to use (Cos et al., and culturing of anaerobic bacteria tested. ET and MB
2006). A long tradition of their use in folk medicinal systems participated in the collection of plant materials and

Frontiers in Pharmacology | www.frontiersin.org 11 November 2021 | Volume 12 | Article 746808

Kudera et al. Antibacterial and Anticancer Plant Extracts

verification of the data on the ethnobotanical use of plants in the FUNDING

Philippines. SN participated in the collection of plant materials
and verification of the data on the ethnobotanical use of plants in This work was supported by the Internal Grant Agency of the
Cambodia. LK conceptualized and coordinated the whole study Faculty of Tropical AgriSciences (IGA 20213109), the
and provided the botanical identification of plant samples. All METROFOOD-CZ Research Infrastructure Project (MEYS
authors have read and agreed to the published version of the LM2018100), and the Strategic Setting of Human Resources
article. Development at CZU (CZ.02.2.69/0.0/0.0/18_054/0014642).

de Padua, L. S., Bunyapraphatsara, N., and Lemmens, R. H. M. J. (1999). Plant

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