*4.4. Rotavirus*

Rotavirus infections represent the leading cause of dehydrating gastroenteritis among children less than 5 years of age. Despite the worldwide vaccinations against rotavirus, it is still a major cause of fatality (>200,000 deaths annually) especially in low-income countries [198]. These viruses usually infect enterocytes, thus induce diarrhea via the decline in the absorptive capacity of enterocytes, increase intestinal secretion stimulated by viral non-structural protein 4 and activation of the enteric nervous system [199]. It has been observed that supplementation *Nelumbo nucifera* Gaertn., *Aspalathus linearis* (Burm.f.) R.Dahlgren, *Urtica dioica* L., *Glycyrrhiza glabra* L. and *Olea europaea* L. extracts are effective in the managmen<sup>t</sup> of rotavirus induced diarrhea [200,201]. Moreover, aqueous extracts from *Nelumbo nucifera*, *Urtica dioica*, *Aspalathus linearis*, *Glycyrrhiza glabra* and *Olea europaea* leaves were reported to exhibit substantial antiviral potentials with IC50 of <300 μg/mL. Likewise, 18β-glycyrrhetinic acid and luteolin isolated from *G. glabra* and *A. linearis* exhibited IC50 of 46 μm and 116 μm, respectively [201]. *Rubia cordifolia* L. extracts, isolated compounds including xanthopurpurin and vanillic acid were quite effective against rotavirus and inhibited its multiplication by augmenting virus-mediated apoptosis in MA-104 cells [202]. The antirotavirus activity of *Bauhinia variegata* L. was examined, which showed antiviral activity against rotavirus in vitro with therapeutic index ranged from 0.2 to 23 and a reduction in virus titers ranged from 0.25 log10 to 4.75 log10. These results demonstrated that *B. variegata* has a potential for the pharmacotherapeutic managemen<sup>t</sup> of rotavirus induced gastroenteritis [203]. The antiviral activity of different extracts from *Calliandra haematocephala* Hassk. leaves against rotavirus (RV) infection was evaluated both in vitro and in vivo. *C. hematocephala* at non-cytotoxic concentrations exhibited antirotavirus activities at a different magnitude of potency with therapeutic index ranging from 1.3 to 32 and a reduction in virus titer ranging from 0.25 log10 to 5.75 log10. In the in vivo study, oral administration of the methanol extract at 50 mg and 100 mg/kg/day significantly reduced mortality, virus titers, duration and severity of diarrhea, as well as alleviation of lesion in the small intestine in rotavirus infected mice [204]. The leaf of Japanese big-leaf magnolia (*Magnolia obovate* Thunb.) has long been used as a natural packaging material for traditional foods in Japan. The extract significantly inhibited cytopathic effects and mRNA expression of rotaviral proteins in SA11-infected MA104 cells

and thus can be used as a medicine or food additive to prevent and ameliorate rotavirus-induced diarrhea in individuals that may have difficulty in benefitting from the rotavirus vaccines [205].

### **5. Plant Extracts and Phytochemicals against Parasites Causing Foodborne Diseases**

### *5.1. Giardia lambia*

Giardiasis is a human parasitic infection most commonly transmitted through the ingestion of infected food and is associated with significant morbidity [206]. The most common cause of giardiasis is the protozoan, *Giardia lamblia* commonly called *G. intestinalis* and *G. duodenalis*. Several treatment strategies are devised for its eradication which includes nutritional intervention, ingestion of probiotics and phytotherapeutic agents [207]. Several natural products including *Allium sativum*, berberine and flavonoid-rich herbs, *Piper longum* L., *Butea monosperma* (Lam.) Taub. and various isolated compounds have been found effective in the eradication of giardiasis [208,209].

Natural products based therapeutics play a significant role in the treatment and managemen<sup>t</sup> of giardiasis, mediated through eradication of parasite and thus relieving the unwanted symptoms of infection. Several herbs are extensively reported for their effectiveness in *Giardia* infections; *Allium sativum* is a well-known household remedy against numerous human diseases including microbial and parasitic infections [210]. In a study, Harris et al. reported the anti-giardial potentials of crude *A. sativum* and its isolated compounds; A. *sativum* crude extract exhibited IC50 of 0.3-mg/mL against *Giardia intestinalis*, whereas, the isolated compounds allyl mercaptan, diallyl disulfide, diallyl sulfide, allyl alcohol and dimethyl disulfide showed IC50 values of 0.037, 0.1, 1.3, 0.007 and 0.2 mg/mL, respectively [33].

Furthermore, the incubation of *Giardia* trophozoites in the presence of whole *A. sativa* exhibited considerable decline in the parasite motility, flagellar movement and ultimate swelling of the trophozoite. The mechanisms underlying these events involve the loss of osmoregulation of parasite and loss of transmembrane electrochemical gradient. Microscopic studies revealed morphologic changes in the ventral disc of the parasite which can be responsible for its inability to attached to its host cell [33]. In another clinical study, Soffar and Mokhtar administered fresh *A. sativum* distilled water extract (5 mL) or 0.6 mg commercially available capsules to 26 children infected with *G. lamblia* for three days and observed its beneficial effects against giardiasis [211]. The symptoms of giardiasis were effectively subsided in all the groups of children with thirty-six hours of therapy and the stool examination revealed complete eradication of parasite [211]. Allicin isolated from *A. sativum* is reported to mediate its action against the parasite via inhibition of giardia's cysteine proteases and inhibition of products responsible for unwanted symptoms of giardiasis [212,213]. Moreover, *A. sativa* also increases the mucosal nitric oxide (NO) production via stimulation of nitric oxide synthase, which subsequently increases the liberation of NO from enterocytes and exhibits direct giardicidal effects [33].

*Piper longum* L. is a well-known folk medicine for the treatment of gastrointestinal disorders and helminthesis [214]. In a study, Tripathi et al. investigated the giardicidal actions of crude extracts of *P. longum*. Treatment with crude ethanolic extract (125 μg/mL) and aqueous extracts (250 μg/mL) showed 100% lethality against *Giardia lamblia*. In an animal model of giardiasis, *P. longum* crude ethanolic extract, aqueous extract and fruit powder at doses of 250, 450 and 900 mg/kg, respectively significantly (75%) diminished the live trophozoites count in the intestinal mouse aspirates after five days of therapy [215]. The two important gastroprotective herbs including *Piper longum* and *Butea monosperma* have been combined in a traditional herbal formulation called Pippali rasayana (PR). This formulation is very famous in folk medicine for the treatment of helminthesis and chronic dysentery. In a study, Agarwal et al. reported the anti-giardial effects of PR formulation in an animal model. The in vivo study revealed that PR administration in doses of 225, 450 and 900 mg/kg exhibited 62%, 79% and 98% parasitic clearance, respectively. Moreover, PR significantly augmented the macrophage phagocytic activity and macrophage migration index at all the tested doses. As the PR formulation is inactive against the parasite in vitro, so it was proposed that the formulation may mediate its anti-giardial activity through boosting of host immune system and thereby increasing clearance mechanisms [216]. To further explore the clinical significance of PR formulation, Agarwal et al. extended the study and included fifty human subjects with all signs and symptoms of giardiasis including cysts and Giardia trophozoites in the stool samples. The subjects were divided into two groups; the treated group was maintained on PR (1 g T.D.S) for fifteen days, whereas the second group served as a placebo control. After the completion of therapy, stool samples were analyzed for *G. lamblia*, frequency of diarrhea and mucous content of the stool. In the treated group, 92% inhibition of *Giardia* and a significant decline in diarrhea and mucous were observed. A boost in the cell-mediated immune system indicated by a decline in leukocyte migration was also observed [217].

In a study, Miyares et al. evaluated the giardicidal potential of propolis in 136 subjects with well-known symptoms of giardiasis. In the five days' trials, propolis was administered in 20, 30% solutions (adults) and 10% solution (children) and duodenal aspirates were evaluated for giardicidal activity. Results of the study revealed 60% giardicidal activity for 30% solution and 40% giardicidal activity for 20% solution in adults whereas, 52% giardicidal activity was observed in children after administration of 10% solution. The standard drug, tinidazole exhibited a 40% e fficacy [218]. Several studies have confirmed the e fficacy of berberine and berberine rich plants including *Coptis chinensis* Franch., *Berberis vulgaris* L., *Berberis aquifolium* Pursh, *Berberis aristata* DC. and *Hydrastis canadensis* L. in the managemen<sup>t</sup> of gastrointestinal disorders like parasitic infestation and diarrhea [219,220]. Berberine is highly e ffective against Giardia trophozoites and mediates its beneficial e ffects via morphologic changes in the trophozoites including changes in the shape of vacuoles, trophozoites swelling and deposition of glycogen deposits [221]. Berberine hydrochloride at a dose of 5 mg/kg showed a 68% decline in the stool–*Giardia* content and a significant decline in giardiasis symptoms in a clinical study [222]. In a clinical trial, berberine administered in doses of 5, 10 mg/kg for 5–10 days showed a 47%, 55%, 68% and 90% decline in the Giardia content in all the treated groups of children. The standard drugs, furazolidone and metronidazole exhibited a 92% and 95% e fficiency against *Giardia* [223]. In vitro studies have revealed high e fficacy of crude extracts from these plants in comparison to pure berberine, which can be attributed to the synergistic interactions of berberine with other isoquinoline alkaloids present in these plants [224]. Natural flavonoids including epicatechin, quercetin, epigallocatechin, apigenin and kempferol, which are abundant in several natural products like *Quercus robur* L., *Hamamelis virginiana* L. and *Croton lechleri* Müll.Arg. are extensively reported for giardicidal potentials [225–227]. In a study, flavonoids and tannins rich plants including *Origanum vulgare* L., *Psidium guajava* L., *Mangifera indica* L. and *Plantago major* L. showed high anti-giardial activity in comparison to the standard drug, tinidazole [228]. Barbosa E et al. reported the antigiardial activity of flavonoids isolated from *Geranium mexicanum* Kunth, *Helianthemum glomeratum* (Lag.) Lag. ex Dunal, *Cuphea pinetorum* Benth. and *Rubus coriifolius* Liebm. Among the isolated compounds, kempferol, tiliroside and epicatechin exhibited IC50 values of 2.057, 1.429 and 0.072 μmol/kg, respectively against *G. lambia* [229].

### *5.2. Entamoeba histolytica*

Amoebiasis is a ubiquitous gastrointestinal disorder most prevalent in less developed countries with poor sanitation and socioeconomic status, a ffecting about 12% of global population [230]. It is known to be the third leading cause of mortality [231–233]. The causative agen<sup>t</sup> of amoebiasis is *Entamoeba histolytica,* which is associated with typical symptoms of amoebic dysentery, abdominal cramps, bloating or tenderness and stomachache [234,235]. Among the currently available chemotherapeutics, metronidazole is an e ffective amoebicide, but is associated with unwanted side-e ffects like carcinogenesis, mutagenesis, nausea and vomiting [236,237]. The stratagem of utilizing natural drugs in antiamoebic therapy traces back to the pre-historic era. For instance, emetine isolated from *Cephaelis ipecacuanha* (Brot.) A.Rich. is used as a front line anti-amoebic drug [238]. Currently, a large population is dependent on traditional therapeutics for the managemen<sup>t</sup> of various diseases [18,25,239]. Medicinal plant and herbal drugs represent an indispensable part of the traditional

medicine practiced in many countries owing to low costs, frequent availability, biosafety and ancestral knowledge [240–242]. Hence, in search of more effective drugs, traditionally used natural products are of grea<sup>t</sup> importance. Several natural products and isolated compounds have been scientifically validated for effective eradication of *Entamoeba histolytica*, for potential drug development. For instance, bruceantin, a potent amoebicide from *Brucea antidysenterica* J.F.Mill. exhibited IC50 of 0.018 μg/mL [243], parthenin from *Parthenium hysterophorus* L. [244], extracts and isolated compounds from *Brucea javanica* (L.) Merr. fruits and *Simarouba amara* Aubl. [245] and alkaloids from *Alstonia angustifolia* Wall. ex A.DC. roots [246] were extensively studied against *E. histolytica*. Cimanga et al. investigated the effect of *Morinda morindoides* (Baker) Milne-Redh. leaves extracts and isolated compounds against *E. histolytica* [247]. The crude methanolic extract and aqueous decoction showed significant anti-amoebic action with IC50 values of 1.7 and 3.1 μg/mL, respectively. Among the isolated compounds, kempferol, apigenin, luteolin, apigenin-7-*O*-glucoside and luteolin-7-*O*-glucoside exhibited IC50 values of 10.3, 12.7, 17.8, 22.3 and 37.4 μg/mL, respectively against *E. histolytica*.

Tona et al. evaluated forty-five Congolese plant extracts used in traditional medicine against *E. histolytica*. Among the tested samples, *Mangifera indica* L., *Rauwolfia obscura* K.Schm., *Carica papaya* L., *Euphorbia hirta* L., *Hymenocardia acida* Tull., *Jatropha curcas* L., *Maprounea africana* Mull. Arg., *Paropsia brazzeana* Baill., *Psidium guajava* L., *Cryptolepis sanguinolenta* (Lindl.) Schltr. and *Quassia africana* (Baill.) Baill. were highly active with IC50 values of 7.81, 31.5, 7.81, 31.25, 31.25, 31.25, 31.25, 7.81, 7.81, 7.81 and 31.5 μg/mL, respectively [248]. In another study, Sohni et al. investigated the antiamoebic potentials of an herbal formulation containing *Zingiber <sup>o</sup>*ffi*cinale*, *Berberis aristata*, *Boerhavia di*ff*usa* L., *Tinospora cordifolia* (Willd.) Miers and *Terminalia chebula* extracts. Among these plant extracts, *Berberis aristata* and *Tinospora cordifolia* showed IC50 values of 100 and 1000 μg/mL, respectively. All these plants in a combined formulation exhibited an IC50 of 1000 μg/mL. The herbal formulation was also tested against some enzymes of *E. histolytica* including DNase, RNase, aldolase, alkaline, acid phosphatases and α-amylase that are known to play a significant role in the virulence and invasiveness of the parasite. Results confirmed various degrees of inhibition of these enzymes [249]. Owing to the traditional use of *Salvia polystachya* Cav. for the treatment of dysentery in the Mexican traditional medicine, Calzada et al. investigated various isolated compounds from *Salvia polystachya* against *E. histolytica* and *G. lamblia* trophozoites. Among the tested compounds, Linearolactone was most active showing IC50 value of 22.9 and 28.2 mM against *E. histolytica* and *G. lamblia,* respectively. Whereas, polystachynes A, B and D, exhibited modest antiprotozoal potentials with IC50 values ranging from 117.0–160.6 mM against *E. histolytica* and 107.5–134.7 mM against *G. lamblia* [250].

In the search for new antiprotozoal drugs from natural products, Calzada et al. studied the inhibitory effects of twenty-six traditional Mexican drugs against *E. histolytica* and *G. lamblia*. Among the tested samples, *Chiranthodendron pentadactylon* Larreat., *Annona cherimola* Mill., *Punica granatum, Dichondra argentea* Humb. & Bonpl. ex Willd., *Chenopodium ambrosioides* L. and *Chrysactinia mexicana* A.Gray were most active showing IC50 values of 2.5, 14.8, 29.5, 38.3, 45.2, 45.3 μg/mL, respectively against *E. histolytica*. Whereas, *Dorstenia contrajerva* L., *Senna villosa* (Mill.) H.S. Irwin & Barneby, *Ruta chalepensis* L., *Cocos nucifera* L. and *Chiranthodendron pentadactylon* Larreat. exhibited IC50 values of 23.3, 32.1, 37.8, 44.1, 44.2 μg/mL, respectively against *G. lamblia* [251]. In another study, the same group investigated the antiprotozoal efficiency of crude extracts and isolated compounds from the roots of *Geranium mexicanum* against *E. histolytica* and *G. lamblia*. Among the crude samples, dichloromethane–methanol, ethyl acetate, fraction 13 (from ethyl acetate) and aqueous fractions showed IC50 values of 79.2, 66.7, 51.5 and 221.6 μg/mL, respectively against *E. histolytica* and 100.4, 63.7, 59.7 and 215.9 μg/mL, respectively against *G. lamblia*. Among the isolated compounds, epicatechin, catechin, β-sitosterol 3-O-β-D-glucopyranoside and tyramine revealed IC50 values of 1.9, 65.6, 82.2 and 54.2 μg/mL, respectively against *E. histolytica* and 1.6, 33.9, 61.5 and 68.9 μg/mL, respectively against *G. lamblia* [252]. The antiprotozoal potential of another traditional plant, *Rubus coriifolius* and its isolated compounds were investigated by Alanís et al. against *E. histolytica* and *G. lambia*. In the crude fractions, dichloromethane–methanol was most active with IC50 values of 11.6 and 55.6 μg/mL, respectively against *E. histolytica* and *G. lamblia*. Whereas, the isolated compounds including epicatechin, catechin, nigaichigoside, β-sitosterol-3- *O*-β-D-glucopyranoside, hyperin, gallic acid and ellagic acid exhibited IC50 value of 1.9, 65.5, 111.9, 82.16, 143.6, 220 and 56.5 μg/mL, respectively against *E. histolytica* and 1.6, 34.0, 123.6, 61.5, 49.2, 70.3 and 24.9 μg/mL, respectively against *G. lamblia* [253].

### *5.3. Toxoplasma gondii*

Toxoplasmosis is a parasitic infection caused by *Toxoplasma gondii* and transmitted from infected pregnan<sup>t</sup> women and contaminated food and is a major cause of foodborne hospitalization and death [254]. Meat from various sources including pork, cattle, wild game meat, poultry meat, lamb if not properly cooked and vegetables contaminated with oocysts, infected water and feces can cause transmission of toxoplama infection [255]. Proper cleansing, cooking of meat and washing of fruits and vegetables can significantly reduce the risk of toxoplasmosis. Immunocompromised and organ transplant individuals are at more risk to develop this infection. Several toxoplasmosis outbreaks in Korea are reported to be linked with the use of uncooked pork [256]. Toxoplasmosis is reported as a major cause of neurologic infections in HIV infected individuals [257]. Toxoplasmosis is reported as the second major cause of foodborne infections and the fourth major cause of hospitalization and deaths in the United States [4]. In Greece, toxoplasmosis has been reported among the top five contributors of foodborne infections, leading to major disabilities and deaths [258].

Several studies regarding the e fficacy of natural products against toxoplasmosis have been reported. In a study, Youn et al. reported the antiparasitic potentials of traditionally used plant extracts against *Toxoplasma gondii* and *Neospora caninum* [259]. Solvent extracts of *Sophora flavescens* Aiton, *Torilis japonica* (Houtt.) DC., *Ulmus macrocarpa* Hance, *Sinomenium acutum* (Thunb.) Rehder & E.H.Wilson and *Pulsatilla koreana* (Yabe ex Nakai) Nakai ex T. Mori collected from South Korea were tested. Among the tested herbs, *T. japonica* exhibited significant inhibitory potential against *T. gondii* by inhibiting parasite proliferation from 54% to 99% at 19.5–156 ng/mL; S. *flavescens* extracts restrain *T. gondii* growth by 27.2–98.7% at a concentration ranging from 39-ng/mL to 156 ng/mL. Extracts from other plants including *Pulsatilla koreana*, *U. macrocarpa* and *S. acutum* showed moderate antiparasitic activity against *T. gondii*. Wright et al. tested the e ffectiveness of isolated compounds from *Simarouba amara* and *Brucea javanica* fruits against *Toxoplasma gondii*, *E. histolytica* and *Giardia intestinalis*. Among the tested compounds, ailanthinone, bruceantin, bruceine B, bruceine C, bruceine D, brusatol, glaucarubinone and quassin exhibited IC50 values of 0.0251, 0.0115, 0.75, 0.842, 7.56, 0.179, 0.374 and 111 μM, respectively [260].

In a study, Choi et al. tested methanolic extracts from fifteen traditional herbs against *T. gondii*. Among the tested samples, *Sophora flavescens, Zingiber <sup>o</sup>*ffi*cinale*, Meliae Cortex, *Acorus gramineus* Aiton, *Dryopteris crassirhizoma* Nakai and *Glycyrrhiza glabra* were potent and exhibited EC50 values of 0.20, 0.18, 0.77, 0.11, 0.15 and 0.13 mg/mL, respectively against *Toxoplasma gondii* [261].
