According to the latest data of the World Health Organization, infectious diarrhea is still the third leading cause of death among all communicable diseases worldwide, especially affecting under-five children in developing countries (World Health Organization, 2020a). Moreover, common risk factors associated with these gastrointestinal infections, such as inappropriate changes in the host-gut microbiome (Sun et al., 2018), have been considered as a crucial precondition for several noncommunicable intestinal diseases, including colorectal cancer, which is the third leading cause of cancer death globally (Taddese et al., 2020). The developed countries are at the highest risk, but the incidence of gastrointestinal cancers in developing nations is steadily increasing (Rawla et al., 2019). Infectious and toxigenic strains of Bacillus cereus, Campylobacter jejuni, Clostridium difficile, Clostridium perfringens, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Salmonella spp., Shigella spp., Vibrio cholerae, Vibrio parahaemolyticus, and Yersinia enterocolitica are the major causes of bacterial diarrhea (Casburn-Jones, 2004). Among these, Enterococcus spp., Escherichia spp., and Shigella spp. were previously observed to be richer in the fecal microbiota of patients with colorectal cancer (Xu and Jiang, 2017).

Despite the advantages of treatment utilizing antibiotic agents, disruption of the gut microbiota is usually considered as one of the negative consequences of their use in infectious diarrhea (Francino, 2016). Moreover, the antimicrobial resistance rate among diarrheagenic bacteria recovered from human patients has significantly increased, especially in developing countries (Meng et al., 2011; World Health Organization, 2020b). Therefore, it is important to seek new sources of efficient antimicrobial agents against which the infectious bacteria are less prone to develop resistance (Sibanda and Okoh, 2007). Additionally, agents that are highly selective, thus less disruptive for human microbial ecology, should be preferred (Garrett, 2019). Therefore, an investigation on the in vitro antimicrobial effects of promising candidates is recommended to involve both representatives of diarrheagenic and probiotic bacteria. The most common bacteria recognized to date as probiotics are Lactobacillus spp. and Bifidobacterium spp., belonging to the dominant bacterial phyla that can be found in human intestines (Behnsen et al., 2013). Although it is a simplified representation of beneficial gut microbiota, the counterscreen of in vitro inhibitory activities on gut commensals appears to be an effective way to avoid unnecessarily promiscuous agents (Gavrish et al., 2014).

Additionally, assessment of interactions with intestinal epithelial cells and toxicity profiles is another important factor for the evaluation of antibacterial agents, which target the intestinal site (Maher and McClean, 2006). To lower the potential toxic responses of intestinal epithelial cells, the use of antibacterial agents with mechanisms enabling toxicity to be prokaryotic but not eukaryotic cells, such as in the case of the antibiotic ceftriaxone, should be prioritized (Neftel and Hübscher, 1987). However, the antiproliferative activity can be acknowledged in cases where the immune responses caused by ongoing intestinal infection and dysbiosis have already promoted the carcinogenesis of epithelial cells. Initially, colorectal cancer usually has an oligosymptomatic characteristic; thus, many cases are diagnosed only at advanced stages, at which stage the therapeutic outcomes are poor (Rogowski and Sulzyc-Bielicka, 2016). Antibacterial agents able to eliminate diarrheagenic pathogens and having the additional selective antiproliferative properties could potentially help to prevent progression of yet not diagnosed intestinal cancers. The use of antibiotics—such as quinolones and tetracyclines, which are both utilized in diarrheal infections—as anticancer drugs has been previously suggested (Onoda et al., 2005; Batalha et al., 2016).

Plant-derived products provide novel chemical scaffolds for anti-infective drugs and leads that have chemically been modified and developed as antimicrobial agents. Several over-the-counter pharmaceuticals, dietary supplements, and herbal medicines recommended for the support and maintenance of gastrointestinal health, containing antibacterially active plant extracts and derivatives of their constituents, are already available at the international market. The benzylisoquinoline alkaloid berberine (e.g., Hydrastis canadensis L. [Ranunculaceae]); simple phenol bismuth subsalicylate, the analog of salicylic acid derived from salicin (Salix alba L. [Salicaceae]) (Kokoska et al., 2019); and picrosides, an iridoid glycoside of Picrorhiza kurroa Royle ex Benth. (Plantaginaceae), are some examples (Rathee et al., 2016). The in vitro selective antibacterial effects of plant-derived products have also been reported. For example, Chan et al. (2018) reported that the phenolic-rich extracts from various dietary spices and medicinal herbs (Cinnamomum burmannii Nees and T.Nees] Blume [Lauraceae], Cinnamomum cassia [L.] J.Presl [Lauraceae], Origanum vulgare L. [Lamiaceae], Punica granatum L. [Lythraceae], Reynoutria japonica Houtt. [Polygonaceae], and Syzygium aromaticum [L.]. Merr. and L.M.Perry [Myrtaceae]) exerted in vitro growth-inhibitory effects against selected foodborne pathogenic bacteria but not against lactic-acid bacteria. Selective in vitro antibacterial activity was also described in the study by Novakova et al. (2013), where the anticlostridial effect of 8-hydroxyquinoline (Microstachys corniculata [Vahl] Griseb. [Euphorbiaceae]) was higher than the activities revealed against different strains of bifidobacteria. In our previous study, we also reported that 8-hydroxyquinoline exerts selective in vitro antiproliferative activity against some intestinal cancer cell lines with a comparably lower effect on normal cells (Kudera et al., 2020). Quinoline alkaloids, such as camptothecin extracted from the bark of Camptotheca acuminata Decne. (Cornaceae), are already being used in chemotherapy for the treatment of colon cancer (Zeng et al., 2013). Anticancer activities of other antibacterially active phytochemicals (e.g., berberine) utilized against infectious diarrhea are currently studied (Lin et al., 2008). Based on these studies, it is evident that plant-derived products have great potential for the development of antibacterial and anticancer preparations for the treatment of infectious diarrhea and associated intestinal cancer diseases. Although many of such products work based on antidiarrheal activity (e.g., antisecretory and astringent effects) (Palombo, 2006), their antimicrobial effect is not an uncommon 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 of plant biodiversity (Duriyaprapan et al., 2005). Situated in the humid tropics with areas of high rainfall, Southeast Asia has one of the largest numbers of vascular plants species globally. For centuries, people living in this region have relied on traditional medicine using available plants for daily healthcare. Cambodia and the Philippines are two geographically distinct Southeast Asian countries, each having numerous plant biodiversity hotspots and a long tradition of herbalism (de Padua et al., 1999). While the former is situated in the mainland, having rich ecosystems, especially around the Mekong River (Chassagne et al., 2016), the latter is a huge archipelago consisting of approximately 7,107 islands, many of which are the center of endemicity and biodiversity (Guzman et al., 2016).

Diarrhea has been a significant issue in both Cambodia and the Philippines (Our World in Data, 2011). Therefore, plant resources in these countries have extensively been utilized medicinally to treat this ailment. The Philippines also has the highest estimated number of cases of colorectal cancer in Southeast Asia and the tenth highest number of deaths in the world (Rawla et al., 2019). In certain provinces of Cambodia, treatment of digestive disorders, such as abdominal pain (chhu poh), diarrhea (reak ach), and dysentery (reak muol), has particularly been based on herbal medicine. Alcohol maceration is a common method of preparation of antidiarrheal medicines, whereas a majority of the preparations are administered orally: drunk, eaten, or chewed (Kham, 2004). Grilling the plant part over a fire and then boiling it into a form of decoction is also common. As an example, Bunong people in Mondulkiri province treat diarrhea using a “step-by-step” process using a sole ingredient from one plant that is substituted by a different species if the condition becomes persistent. In the Philippines, conditions such as diarrhea (pagtatae) and dysentery (pagdidisenyo) have similarly been treated by orally administered herbal preparations that are processed by alcohol maceration, decoction, or infusion or eaten and chewed raw. According to the Philippine traditional medicinal system, the disease is usually conceptualized as a disruption (dys-krasia) of the balance of forces (whether germs or evil spirits), both external and internal to humans. Therefore, it can be assumed that the use of herbal preparations is intended to also defend the immunological mechanisms, helping the body to overcome the disease itself (Tan, 1980). Despite the existence of several reports on the antibacterial and antiproliferative effects of Cambodian and Philippine medicinal plants used for the treatment of diarrhea (Beloy et al., 1976; Chea et al., 2007), there are several species in both regions that have not yet been appropriately studied using modern scientific techniques. In this study, we, therefore, examine the in vitro selective antibacterial and antiproliferative effects of ethanolic extracts from various parts of plant species that have been used in Cambodian and Philippine traditional herbal systems the treatment of gastrointestinal disorders and determine which bioactive properties have not been properly tested in such form and degree before.

In the present study, 16 of 35 tested extracts revealed in vitro growth-inhibitory effect on the diarrheagenic bacterial pathogens, especially A. blancoi (Elmer) Merr., A. camansi Blanco, A. tectorius (Lour.) Merr., and P. siamensis (Miq.). Except A. camansi Blanco, the antibacterially active concentrations of the three were nontoxic to normal intestinal cells. Among the 16 extracts, A. blancoi (Elmer) Merr., E. microphylla Lam., L. cochinchinensis Pierre ex Gagnep., M. saigonense (Kuntze) Merr., and P. siamensis (Miq.) Kurz also revealed a strong selective antiproliferative effect against intestinal cancer lines. The extract of P. siamensis (Miq.) Kurz exhibited activities combining selective inhibition of pathogenic bacteria and intestinal cancer cells without affecting beneficial bacteria and normal intestinal cells. To the best of our knowledge, this is the first study on antibacterial and antiproliferative activities of A. polymorphum Spire, B. vitis-idaea (Burm.f.) C.E.C.Fisch., I. nigricans R.Br. ex Wight and Arn., L. cochinchinensis Pierre ex Gagnep., P. siamensis (Miq.) Kurz, and M. saigonense (Kuntze) Merr. Moreover, there are no previous studies on the cytotoxic effects of A. blancoi (Elmer) Merr. Although the cytotoxic effect of products isolated from B. malabarica Roxb. was described previously (Kittakoop et al., 2000), its antibacterial activity is herein reported for the first time. Our results correspond with those of previous studies on antibacterial and antiproliferative activities of A. grandis Benth. (Bradacs et al., 2009), D. esculentum (Retz.) Sw. (Mackeen et al., 1997; Rahmat et al., 2003), and P. javanica Blume (Khan et al., 2001; Win et al., 2015). The above highlighted seven plant extracts with promising activities have mainly been discussed.

Two of the four tested species of the genus Artocarpus (Moraceae), namely, A. blancoi (Elmer) Merr. and A. camansi Blanco, exhibited strong antibacterial and antiproliferative activities in our study. Artocarpus spp. are rich in phenolic compounds, such as flavonoids, stilbenoids, and arylbenzofurans, which are known to possess a wide range of biological activities, including antibacterial and anticancer effects (Hafid et al., 2017). Our study is the first to report on anticlostridial activities of Artocarpus spp. As flavonoids have been reported to have potent in vitro inhibitory effect on some clostridia (Wu et al., 2013), these compounds might be responsible for significant antibacterial activities revealed by A. blancoi (Elmer) Merr. and A. camansi Blanco against C. difficile and C. perfringens. Beloy et al. (1976) isolated the flavonoid 5,7,4′-trihydroxyflavanone-3-O-α-L-rhamnopyranoside from the bark extract of A. blancoi (Elmer) Merr., showing antibacterial activity against Mycobacterium tuberculosis. Ante et al. (2016) showed that bark essential oil of A. camansi Blanco produced antibacterial activity against some diarrheagenic bacteria. Our results show that both of these plants inhibited gram-positive bacteria only. Beside their anticlostridial effect, this selectivity probably also contributed to their relative toxicity to beneficial bacteria. In vitro inhibitory effect against lactobacilli was previously reported for Artocarpus lacucha Buch.-Ham. (Teanpaisan et al., 2014). An example of a compound isolated from the plant of this genus and showing similar activities is artocarpin. In the study by Sato et al. (1996), this flavonoid exhibited strong inhibition of all gram-positive bacteria, including L. casei, whereas in another study, it produced higher MICs against E. coli and Pseudomonas aeruginosa (Septama and Panichayupakaranant, 2015). The absence of antibacterial action of A. odoratissimus Blanco found herein will correlate with rather low levels of phenolic content detected in its fruit methanolic extract (Abu Bakar et al., 2015), compared to antibacterially active species (Jalal et al., 2015). Although hexane bark extract of A. elasticus Reinw. ex Blume exhibited activity against B. cereus and E. coli in the study by Ramli et al. (2016), its lack of activity in the present study could be influenced by the use of different extraction procedures. According to our results, A. blancoi (Elmer) Merr. and A. camansi Blanco had a selective cytotoxic effect on intestinal cancer cells, whereas the former did not show cytotoxicity to normal cells at the inhibitory concentrations against several pathogens. Various terpenoids and phytosterols were previously isolated from methanolic and dichloromethane extract of stem and leaves of A. camansi Blanco, respectively. Among them, friedelinol, cycloartenol, and cycloartenol acetate inhibited the growth of HT-29 cells; squalene has profound chemopreventive activity against colon carcinogenesis; and ß-sitosterol has been shown to induce apoptosis in human colon tumors (Tsai et al., 2013). Regarding cytotoxic compounds isolated from other Artocarpus spp., the prenylated flavone artelastin revealed strong in vitro activity against five colon cancer cell lines (COLO 205, HCT 116, HCT 15, HT-29, and SW 620) in the study by Pedro et al. (2005).

Similar to Artocarpus spp., the leaf extract of A. tectorius (Lour.) Merr. exhibited growth-inhibitory effects only against gram-positive bacteria. This corresponds with the study by Wiart et al. (2004), where its methanolic leaf extract produced antibacterial activity against B. cereus but not E. coli. We also found that the overall cytotoxic effect of this plant was strong. Although the antiproliferative effect on cancer cells was not selective, the extract concentrations inhibiting the pathogens were generally nontoxic to normal intestinal cells. Previous phytochemical analysis of leaf ethanolic/methanolic extracts of this plant showed the presence of various naphthylisoquinoline alkaloids, such as 7-epiancistrobrevine D, ancistrocladinine, ancistrotectoquinone A-B, ancistrotectoriline A–C, and hamatinine (Anh et al., 1997; Tang et al., 2000; Tang et al., 2010; Bringmann et al., 2016). Since these isoquinoline alkaloids are known to possess various biological activities, including antimicrobial and cytotoxic effects, we suspect them to be responsible for the growth-inhibitory effects revealed by A. tectorius (Lour.) Merr. in the present study. For example, in the study by Mihalyi et al. (2014), michellamine B isolated from Ancistrocladus korupensis D.W.Thomas and Gereau inhibited B. subtilis. Jiang et al. (2013) showed that naphthylisoquinolines isolated from A. tectorius (Lour.) Merr. exhibited cytotoxic effect against three leukemia cells in vitro. In another study, 7-epiancistrobrevine and ancistrotectoriline exhibited activity against pancreatic cancer cells (Shang et al., 2020). The present study is the first to report on in vitro selective antiproliferative activity of A. tectorius (Lour.) Merr. against intestinal cells.

Regarding P. siamensis (Miq.) Kurz, there are no comparable studies dealing with species of the same genus. However, our results showing a noticeable combination of selective antibacterial and cytotoxic effects of its bark extract can be compared to the data available for closely related genus Shorea (Dipterocarpaceae). For example, Marandi et al. (2016) showed that bark ethanolic extract from Indian antidiarrheal and antidysenteric medicinal plant Shorea robusta Gaertn. exhibited inhibitory action against B. cereus, B. subtilis, E. faecalis, E. coli, S. Typhi, and V. cholerae. Stilbene derivatives isolated from barks of Shorea spp. previously showed strong antibacterial effects against some of these strains (Nitta et al., 2002; Sudto et al., 2019). Some polyphenols, such as stilbenes, can inhibit several nonbeneficial bacteria from the human microbiota, with no noticeable effects on the growth of probiotic bacteria (Requena et al., 2010). Therefore, we suggest that some of these agents could also contribute to the selective antibacterial activities of P. siamensis (Miq.) Kurz shown in the present study. Regarding cytotoxic effect, oligostilbenoids were usually the constituents derived from Shorea spp., with reported antiproliferative action against various cancer cell lines (Rohaiza, 2011; Zawawi et al., 2012; Moriyama et al., 2016). Among them, ampelopsin E exhibited obvious antiproliferative properties on COLO205 and HT-29 cells (Tian et al., 2019), whereas α-viniferin showed selective inhibition of colon cancer cells (HCT-116, HT-29, and Caco-2) with twofold lower IC₅₀ compared to normal colon cells (CCD-18Co) (Gonzalez-Sarrias et al., 2011). To identify phytochemicals responsible for the in vitro selective inhibitory actions shown by P. siamensis (Miq.) Kurz in the present study, an accurate chemical analysis of this plant is needed.

Finally, E. microphylla Lam., L. cochinchinensis Pierre ex Gagnep., and M. saigonense (Kuntze) Merr. revealed a strong selective antiproliferative effect against intestinal cancer lines. It has been reported that triterpenes urs-12-en-24-oic acid, 3-oxo-, methyl ester, and ß-amyrin are involved in anticancer activities of products derived from leaves of E. microphylla Lam. (Rajkumar et al., 2019). Our study corresponds with other studies dealing with these chemicals. For example, in the study by Kuete et al. (2018), ß-amyrin produced a selective cytotoxic effect against Caco-2 compared to that on normal cell line HEK293. In another study, extract of Alstonia macrophylla Wall. ex G.Don containing ß-amyrin produced a selective cytotoxic effect against HT-29 compared to that on normal cell line HDFn (Tan et al., 2019). The present study is the first to report the antiproliferative activities of E. microphylla Lam. against intestinal cell lines. Compounds such as triterpenes, tannins, ellagic acids, glycosides, and flavones were previously attributed to bioactive properties of Lagerstroemia spp. (Chan et al., 2014). In previous studies, triterpenes isolated from species of this genus produced significant in vitro activity against colon cancer cells, for instance, betulinic acid and 3β-acetoxyolean-12-en-28-acid against HCT15 (Woo et al., 2016) and corosolic acid against HCT116 (Sung et al., 2014). Regarding M. saigonense (Kuntze) Merr., there are various previously published studies on related species showing corresponding results. For example, the methanolic leaf extract of Melastoma malabathricum L. produced an antiproliferative effect on HT-29 in the study by Kamsani et al. (2019). Asiatic acid, caffeic acid, p-coumaric acid, kaempferol, quercetin, rutin, and ursolic acid were isolated compounds with previously profound antiproliferative action to this cell line. In the study by Karakurt et al. (2020), p-coumaric acid exhibited selective inhibition of Caco-2 and HT-29 cells compared to that of healthy colon epithelial cells (CCD-18Co). Since the decoction from the leaves of M. malabathricum L. is also traditionally consumed to treat diarrhea, we suggest a similar composition 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 bud extracts of M. dodecandrum Lour., three pentacyclic triterpenoids (ursolic acid, asiatic acid, and terminolic acid) and one tannin (casuarinin) were previously isolated from this plant and found to significantly decrease interleukin-8 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-inhibitory effects against diarrheagenic bacterial pathogens at concentrations nontoxic to normal intestinal cells. Except the strong anticlostridial actions of A. blancoi (Elmer) Merr., the MICs determined for these plant extracts in the present study reflect rather moderate antibacterial activities. However, the discrimination of specific cell toxicity indicates that higher amounts of these products necessary to acquire the appropriate efficiency may still be safe to use (Cos et al., 2006). A long tradition of their use in folk medicinal systems supports this assumption. Moreover, it has been reported that microorganisms are less likely to develop resistance to phytochemicals with anti-infective potential, mainly because of their high diversity in plants. Some were even considered as antibiotic resistance modifying compounds (Sibanda and Okoh, 2007). Additionally, our study showed that the extract of P. siamensis (Miq.) Kurz was relatively safe for probiotic bacteria, and together with A. blancoi (Elmer) Merr., they exerted selective anticancer activities in vitro. Similar to the cytotoxic activities revealed by E. microphylla Lam., L. cochinchinensis Pierre ex Gagnep., and M. saigonense (Kuntze) Merr., the inhibitory effect of A. blancoi (Elmer) Merr. on cancer cell line Caco-2 and the selectivity of its overall antiproliferative actions were generally higher than those of anticancer drug 5-fluorouracil.

These results suggest that extracts from the above-mentioned Cambodian and Philippine plant species are promising materials for further research focused on the development of new plant-derived selective antibacterial and antiproliferative agents used in the treatment of infectious diarrhea and associated intestinal cancer diseases. For instance, the combination of strong anticlostridial and anticancer actions of A. blancoi (Elmer) Merr. may in the future be utilized in the treatment of digestive cancers associated with C. difficile infections (Han et al., 2013). However, further phytochemical and pharmacological research is needed for the isolation and proper identification of their bioactive constituents. Referring to studies dealing with taxonomically related plants to estimate the presence of their bioactive principles is a very limited approach as their composition can vary greatly. On the other hand, our results could serve as an indicator of bioactive potentials of products derived from species of the same taxa. This is mainly the case of P. siamensis (Miq.) Kurz that exhibited selective inhibition of pathogenic bacteria and intestinal cancer cells without affecting beneficial bacteria and normal intestinal cells. Future research combining the ethnomedicinal and chemotaxonomic approaches might help to identify more plants with promising bioactivities (Hao and Xiao, 2020).