Introduction
Schistosomiasis or bilharzia is one of the most common parasitic neglected tropical diseases. It is caused by the flatworms in the genus Schistosoma. The World Health Organization (WHO) estimates that more than 230 million people from 78 countries including the Middle East, South America, East Asia and sub-Saharan Africa are at risk with about 200,000 deaths each year (World Health Organization, 2022). The main burden of the disease is in sub-Saharan Africa, which accounts for over 90% of all incident cases (Colley et al., Reference Colley, Bustinduy, Secor and King2014). The primary schistosome species infecting humans are Schistosoma mansoni, Schistosoma haematobium and Schistosoma japonicum and transmission is mainly by contact with contaminated freshwater, whereby infective larvae released from freshwater snails, for instance, Biomphalaria glabrata, penetrate the host skin, develop within circulation and mature into Schistosoma adult worms in the mesenteric or bladder veins of human host (Colley et al., Reference Colley, Bustinduy, Secor and King2014). The egg deposition within the liver and or bladder and in some cases the central nervous system and pulmonary systems of the human host evokes immunological responses with associated disease pathophysiology (Colley et al., Reference Colley, Bustinduy, Secor and King2014).
There is no vaccine for schistosomiasis treatment and the only existing drug recommended by the WHO for chemotherapy praziquantel (PZQ) has been utilized for the past four decades. The widespread use of PZQ monotherapy in African endemic communities has raised concerns about the selection of drug resistant schistosomes. Even though evidence of clinically relevant emergence of PZQ resistance is lacking, low cure rates in response to PZQ in the field after extensive mass drug administration in Egypt and Senegal have been reported (Simoben et al., Reference Simoben, Ntie-Kang, Akone and Sippl2018). Furthermore, S. mansoni isolates from Kenyan patients showed lower susceptibility to PZQ, including one isolate from a patient who was never fully cured after repeated PZQ treatment over several years and PZQ-resistance can be generated in the laboratory (Fallon & Doenhoff, Reference Fallon and Doenhoff1994; Bergquist et al., Reference Bergquist, Utzinger and Keiser2017). The continual administration of PZQ most likely will select for schistosomes with reduced susceptibility, which could accelerate the emergence of resistance as large reservoirs of untreated schistosomes become exposed to PZQ. There is, therefore, the need for alternative therapeutic options that could be used in place of or to complement PZQ.
Plants, bacteria and fungi are good sources of pharmacologically active natural products used in the treatment of various diseases (Simoben et al., Reference Simoben, Ntie-Kang, Akone and Sippl2018) and traditional, plant-based medicines are a potential source for new drugs. Research efforts have intensified in search of new, potent, affordable and effective drugs to treat parasitic diseases due to the limited treatment options and the lack of vaccines targeting the parasitic diseases. Plant parts have been used as traditional medicine for schistosomiasis for centuries in many African countries. Phytolacca dodecandra berries are used as a molluscicide to control schistosomiasis in Ethiopia (Esser et al., Reference Esser, Semagn and Wolde-Yohannes2003). Breonia decaryana, Citrus reticulata, Dalbergia monticola, Senna alata and Zingiber zerumbet are used for schistosomiasis treatment in Madagascar (Rakotoarivelo et al., Reference Rakotoarivelo, Rakotoarivony, Ramarosandratana, Jeannoda, Kuhlman, Randrianasolo and Bussmann2015). In Mali, Cissus quadrangularis and Stylosanthes erecta (Bah et al., Reference Bah, Diallo, Dembele and Paulsen2006), Rauvolfia vomitoria (Tekwu et al., Reference Tekwu, Bosompem and Anyan2017), Elephantorrhiza goetzei and Pilistigma thonningii are used as remedies for schistosomiasis (Maroyi, Reference Maroyi2013). Due to the enormous therapeutic properties shown by many plants, in this study we explored the potential of seven plants used in Kenya for treating malaria, pneumonia, and diarrhoea for their schistosomicidal activity: Artemisia annua (Anibogwu et al., Reference Anibogwu, Jesus, Pradhan, Pashikanti, Mateen and Sharma2021); Ajuga remota (Cocquyt et al., Reference Cocquyt, Cos, Herdewijn, Maes, Van den Steen and Laekeman2011; Yikna & Yehualashet, Reference Yikna and Yehualashet2021); Bredilia micranta (Maroyi, Reference Maroyi2017); Cordia africana (Lelamo, Reference Lelamo2021); Physalis peruviana (Kasali et al., Reference Kasali, Tusiimire, Kadima, Tolo, Weisheit and Agaba2021); Prunus africana (Kathambi et al., Reference Kathambi, Mutie, Rono, Wei, Munyao, Kamau, Gituru, Hu and Wang2020); and Senna didymobotrya (Schmelzer et al., Reference Schmelzer and Gurib-Fakim2008).
Material and methods
Collection and preparation of plant material
The botanical information on the traditional medicinal plants of Kenya used in this study are presented in table 1. Dry powdered material of seven plants (A. remota, B. micrantha, S. didymobotrya, C. africana, P. peruviana, P. africana, and A. annua) were obtained from the Department of Pharmacognosy, Pharmaceutical Chemistry and Pharmaceutical & Industrial Pharmacy, Kenyatta University, Nairobi, Kenya. The plant material was first extracted with boiling water by decoction and then sequentially extracted with methanol and hexane in a Soxhlet apparatus. Plant material was also extracted non-sequentially using acetone with Soxhlet extraction. The extraction solvents (methanol, acetone and hexane) were evaporated under nitrogen gas, and water extracts were dried by lyophilization. Depending on sample availability and extraction yield, the samples were tested for anti-schistosomal activity. The yields of crude extracts are shown in table 2.
Table 1. Botanical information of medicinal plants used for anti-schistosomal study.
Table 2. Crude extraction yield from Kenyan medicinal plants.
Evaluation of schistosomicidal activity
Preparation of schistosomula
Biomphalaria glabrata (strain NMRI) infected with S. mansoni (strain NMRI) were obtained from the National Institute of Allergy and Infectious Diseases Schistosomiasis Resource Center of the Biomedical Research Institute. After infections were patent, snails were exposed to bright light for 1 h to obtain cercariae. Cercariae were mechanically transformed to schistosomula as described (Lombardo et al., Reference Lombardo, Pasche, Panic, Endriss and Keiser2019). Briefly, cercariae were placed on ice for 30 min and then centrifuged at 350 × g for 10 min. The supernatant was decanted and 2 ml of serum-free M199 medium was added to cercarial pellets and vortexed for 1 min until cercarial tails were detached. Newly transformed schistosomula (NTS) were purified by layering on 4°C Percoll gradient suspension containing Eagle's minimum essential medium, penicillin–streptomycin (10,000 U per ml penicillin/10,000 U per ml streptomycin), and 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid in 0.85% sodium chloride with cercariae suspension and centrifuged at 500 × g for 15 min. Cercarial pellets were resuspended and washed thrice in serum-free M199 medium and collected at 100 × g for 5 min. NTS (240) were transferred to U-bottom 96 well assay plates containing 200 μl of M199 medium supplemented with 5.5 mm D-glucose, penicillin–streptomycin and 5% heat inactivated fetal bovine serum and incubated at 37°C in a 5% carbon dioxide (CO2) incubator overnight.
Preparation of adult worms
Swiss Webster mice (Charles River) housed in the Comparative Research Center of Rush University Medical Center were infected by percutaneous tail exposure to about 200 S. mansoni cercariae through natural transdermal penetration of the cercariae for one hour (Tucker et al., Reference Tucker, Karunaratne, Lewis, Freitas and Liang2013). Mice were euthanized 7-weeks post infection using a lethal dose of 0.018 ml of Euthasol and 5.85 mg/ml heparin to prevent blood coagulation (injection volume of 400 μl). Perfusion was performed by flushing pre-warmed Roswell Park Memorial Institute (RPMI) containing phenol red and L-glutamine through a 25- and 3/8-gauge needle placed into the aorta attached to Tygon® tubing aided by the Masterflex™ L/S perfusion pump as described (Tucker et al., Reference Tucker, Karunaratne, Lewis, Freitas and Liang2013). Adult worms were carefully washed in phenol red free RPMI medium and subsequently incubated in phenol red free RPMI medium supplemented with 5.5 mm D-glucose, penicillin–streptomycin and 5% heat inactivated fetal bovine serum and at 37°C in a 5% CO2 incubator overnight.
Schistosomicidal activity of extracts against S. mansoni schistosomula and adult worms
The dimethyl sulphoxide (DMSO) formulated plant extracts were diluted with phenol red free M199 medium or RPMI medium for NTS and adult worms, respectively, at <1% DMSO final concentrations. NTS and adult worms from overnight cultures were tested against extracts in triplicate at 100 μg/ml. Controls were treated with DMSO alone or 5 μM auranofin as a positive control in appropriate medium (Kuntz et al., Reference Kuntz, Davioud-Charvet, Sayed, Califf, Dessolin, Arnér and Williams2007). NTS and adult worm viability was assessed at 24 h by measuring adenosine triphosphate (ATP) content of worms using the CellTiter-Glo® Assay (Promega) following manufacturer's instructions as described (Lalli et al., Reference Lalli, Guidi, Gennari, Altamura, Bresciani and Ruberti2015).
Statistical analysis for schistosomicidal activity
Schistosome viability in the presence of the crude extracts were assessed using this formula:
Phytochemical analysis
Qualitative phytochemical characterization was performed by placing 5 μl of solution onto a silica plate and then subjecting to standard reagents used for phytochemical analysis. The following reagents were used for the phytochemical analysis: anisaldehyde/sulphuric acid reagent for steroids (Xu & Liu, Reference Xu and Liu2021); Dragendorff reagent for alkaloids (Dube et al., Reference Dube, van Heerden, Zachariades, Uyi and Munyai2021); potassium hydroxide (Bornträger reaction) (Henzelyová et al., Reference Henzelyová, Antalová, Nigutová, Logoida, Schreiberová, Kusari and Čellárová2020) for coumarins (365 nm) and anthraquinones (vis. and 365 nm); 5% ethanolic solution of sulphuric acid (H2SO4) for cardiac glycosides (Onyema, Reference Onyema2019); aluminium chloride (AlCl3) solution (1% ethanolic AlCl3) for flavonoids (Gwatidzo et al., Reference Gwatidzo, Dzomba and Mangena2018); iron (III) chloride (FeCl3) reagent (3% FeCl3) for tannins and phenolic compounds (Fu & Chen, Reference Fu and Chen2019); ninhydrin for amino acids, amines and amino sugars (0.2% ethanolic ninhydrin solution) (Tyagi et al., Reference Tyagi, Dwivedi and Bagchi2019); phenol/ H2SO4 solution for carbohydrates (Su et al., Reference Su, Lu, Lu, Lai and Ng2020; Oh et al., Reference Oh, Lee and Kim2021); and vanillin/H2SO4 solution for terpenes/terpenoids (Jiang et al., Reference Jiang, Kempinski and Chappell2016).
Results
Schistosomicidal activity of crude extracts of Kenyan medicinal plants
Seven Kenyan medicinal plants were extracted with various solvents resulting in 22 different crude extracts (table 2). These were evaluated for schistosomicidal activity against S. mansoni NTS. Following 24-hour exposure of NTS to 100 μg/ml of each extract worm viability was determined by ATP quantitation. We observed variable viabilities among the crude extracts analysed. Greater than 80% reduction in NTS viability was detected after treatment with A. annua (acetone) – 97.9%, P. africana (acetone) – 96.4% B. micrantha (water) – 89.9%, P. peruviana (acetone) – 89.5%, B. micrantha (methanol) – 88.6%, A. remota (hexane) – 82.5%, and S. didymobotrya (acetone) – 82.4% compared to the DMSO treated NTS negative control as shown in fig. 1. While treatment with most of the extracts resulted in moderate viability reductions, treatment with A. remota (water) (4.2% reduction) and B. micrantha (hexane) (9.2% reduction) had minimal activity against NTS.
Fig. 1. Schistosoma mansoni newly transformed schistosomula viability against crude extracts from Kenyan medicinal plants. The error bars represent the standard deviation of three independent experiments.
We further tested all nine extracts that showed >80% decrease in viability against NTS for activity against S. mansoni adult worms. Viability was determined for each extract after 24-hour and 48-hour exposure to adult worms. After 24-hour exposure, all extracts showed ≤36% viability against adult worms except A. annua (acetone) (48%) and B. micrantha (methanol) (51.5%) (fig. 2). Upon 48-hour exposure, about 10% increased reduction in viability was observed among the seven tested extracts with all compounds resulting in >55% mortality. The most active extract against S. mansoni adult worms was B. micrantha (water) with 81.6% reduction in viability (fig. 2).
Fig. 2. Schistosoma mansoni adult worm viability after treatment with crude plant extracts with potent newly transformed schistosomula-killing activity. Viability (%) after 24 h (
) or 48 h (
) treatment with extract. The error bars represent the standard deviation of three independent experiments.
Qualitative phytochemical screening results
Qualitative analysis of crude plant extracts showed the presence of various phytochemicals (table 3). The level of phytochemicals was categorized as high, moderate, low, or absent. Artemisia annua plant extracts showed high levels of steroids, tannins, anthraquinones and terpenes/terpenoids. Moderate levels of glycosides and flavonoids were present in A. annua extracts. Steroids and tannins were present at high levels in B. micrantha extracts. Alkaloids, anthraquinones and glycosides were moderately present in B. micrantha plant extracts. Cordia africana plant extracts showed steroids, tannins and proteins at high levels whereas glycosides and anthraquinones were scarcely present. Ajuga remota plant extracts showed steroids, flavonoids, terpenes/terpenoids, glycosides, proteins and anthraquinones eminently whereas tannins were scarcely present. Prunus africana plant extracts showed steroids, glycosides and anthraquinones at high levels whereas tannins, terpenes/terpenoids and flavonoids were present at low levels. Senna didymobotrya plant extracts showed steroids, glycosides and anthraquinones at high levels whereas tannins, alkaloids and flavonoids were present at low levels or absent. Physalis peruviana plant extracts showed high levels of steroids and terpenes/terpenoids whereas tannins, flavonoids, and anthraquinones were present at low levels or absent.
Table 3. Phytochemical analysis from seven Kenyan plant extracts.
+++ = high, ++ = moderate, + = low, − = absent.
Discussion
The lack of a vaccine or alternative drugs to PZQ for the management of schistosomiasis has necessitated the need to search for novel anti-infective agents. Even though the efficacy of PZQ for the past four decades has not been in doubt, concerns about the possible emergence of resistance due to selective drug pressure are inevitable, reinforcing the urgency towards the discovery of new schistosomicidal agents. Furthermore, PZQ has poor activity against migrating juvenile worms and treatment often results in incomplete cures (Pica-Mattoccia & Cioli, Reference Pica-Mattoccia and Cioli2004). Plants have served as active components of many pharmacological products due to their broad spectrum of biological activity including cytotoxic, antiparasitic and antimicrobial properties (Heinrich & Lee Teoh, Reference Heinrich and Lee Teoh2004; Wright, Reference Wright2010). In most healthcare resource-limited settings, indigenes rely extensively on plant medicines for primary healthcare services. We, therefore, explored the therapeutic potential of seven Kenyan herbal plants commonly used in the treatment of malaria, pneumonia and chest pains. We report herein the schistosomicidal properties of A. annua, B. micrantha, P. peruviana, A. remota, S. didymobotrya and P. africana, with a demonstrated potential to be repurposed as treatment for schistosomiasis.
The antimalarial, anti-inflammatory, antibacterial and analgesic properties of these extracts are known; however, their schistosomicidal properties are unclear. Our findings show that 100 μg/ml of acetone extracts of A. annua, P. africana, P. peruviana and S. didymobotrya, 100 μg/ml of water and methanol extracts of B. micrantha and 100 μg/ml of hexane extracts of A. remota had significant worm killing activity (>90%) against S. mansoni NTS after 24-hour exposure. These extracts further showed ≥69.7% and ≥72.5% killing activity in S. mansoni adult worms after 24 and 48-hours of exposure, respectively. The extracts show promise as possible leads by their killing potential on both the larval stage and adult worms of S. mansoni. Since PZQ is primarily active against only adult worms, combination therapy with the extracts may inhibit all the developmental stages of the parasite and may overcome the problem of drug resistance. Ethanolic extracts of A. annua has been shown to have schistosomicidal activity at 2 mg/ml (Ferreira et al., Reference Ferreira, Peaden and Keiser2011). In China, artemisinin extracted from A. annua and its derivatives artemether, artesunate and dihydroartemisinin, have efficacy against S. japonicum. Multiple doses of artemisinin at 6 mg/kg body weight showed preventive efficacies as high as 65–97% (Liu et al., Reference Liu, Wu, Liang, Jie, Wang, Wang and Huang2014). The extract dose of 100 μg/ml used in this study was comparatively lower in relation to similar studies where doses within the ranges of 1.25–2.5 mg/mL of different plant extracts resulted in 90% of the killing of Paramphistomum cervi, the causative agent of enteritis and anaemia in livestock mammals (Elango & Rahuman, Reference Elango and Rahuman2011).
Phytochemical analyses of the extracts detected different classes of compounds comprising steroids, tannins, anthraquinones, glycosides and terpenoids mediating the observed S. mansoni worm killing. Bioactive components of plants display variable activities against different stages of pathogens (Ferreira et al., Reference Ferreira, Peaden and Keiser2011). The acetone extracts of A. annua which contained steroids, anthraquinones and terpenoids and P. africana with steroids, anthraquinones and glycosides as major compounds showed complete killing of NTS. However, worm killing activity was reduced after 48-hours of exposure against S. mansoni adult worms in some of the extracts. The reduced activity influenced by variable susceptibilities of the bioactive components could be as a result of poor uptake or efflux of the compounds by the adult worm. We observed a strong correlation in the worm killing activity of a water decoction extract of B. micrantha. After 24-hour exposure to extracts, 90.1% of NTS were killed while 72.2% and 81.6% adult worms were killed after 24 and 48-hour exposure, respectively. This suggests that tannins, the major phytochemical component in B. micrantha, possess schistosomicidal properties. Tannins obtained from Lotus corniculatus, Hedysarum coronarium and Onobrychis viciifolia, caused a remarkable reduction in Trichostrongylus colubriformis hatched eggs and inhibited the development of eggs in gastrointestinal nematodes (Molan et al., Reference Molan, Hoskin, Barry and McNabb2000, Reference Molan, Waghorn and McNabb2002). Tannins have been shown to inhibit parasites by minimizing the formation of infective stage larvae, reduction of eggs excretion by the adult worms and reduction of eggs’ development (Athanasiadou et al., Reference Athanasiadou, Kyriazakis, Jackson and Coop2001). Simple tannins such as (-)-epicatechin are orally bioavailable (Zhu et al., Reference Zhu, Chen and Li2000; Serrano et al., Reference Serrano, Puupponen-Pimia, Dauer, Aura and Saura-Calixto2009) suggesting that they may be active against blood dwelling schistosome worms; similar tannins were identified in this study.
In summary, this study has found that extracts of seven plants used for the treatment of a variety of conditions in Kenya have schistosomicidal activity against cultured S. mansoni worms. Further studies of these extracts to identify the active components will provide promising lead compounds that can be developed to meet the urgent need for new drugs for the treatment of schistosomiasis.
Acknowledgements
Biomphalaria glabrata snails infected with Schistosoma mansoni were provided by the National Institute of Allergy and Infectious Diseases (NIAID) Schistosomiasis Resource Center of the Biomedical Research Institute (Rockville, MD) through National Institutes of Health–NIAID Contract HHSN272201000005I for distribution through Biodefense and Emerging Infections Research Resources.
Financial support
The authors acknowledge Northern Illinois University for supporting and funding this work. The study was partially funded by National Institutes of Health (D.L.W. AI127635).
Conflicts of interest
No conflict of interest was reported by the authors.
Ethical standards
This study was approved by the Institutional Animal Care and Use Committee of Rush University Medical Center (20-069; Department of Health and Human Services animal welfare assurance number A-3120−01).
