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Published in final edited form as: J Helminthol. 2022 Mar 3;96:e16. doi: 10.1017/S0022149X22000074

Traditional Kenyan Herbal Medicine: Exploring Natural Products Therapeutics against Schistosomiasis

Fidensio K Ndegwa 1,†, Chaitanya Kondam 2,†, Samuel Y Aboagye 3,†, Taiwo E Esan 2, Zohra Sattar Waxali 2, Margaret E Miller 2, Nicholas K Gikonyo 1, Paul K Mbugua 4, Paul O Okemo 5, David L Williams 3,*, Timothy J Hagen 2,*
PMCID: PMC10030042  NIHMSID: NIHMS1880855  PMID: 35238288

Abstract

Praziquantel (PZQ) remains the only drug of choice for the treatment of schistosomiasis, caused by parasitic flatworms. The widespread use of PZQ in schistosomiasis endemic areas for about four decades raises concerns about the emergence of resistance of Schistosoma spp. to PZQ under drug selection pressure. This reinforces the urgency in finding alternative therapeutic options that could replace or complement PZQ. We explored the potential of medicinal plants commonly used by indigenes in Kenya for the treatment of various ailments including malaria, pneumonia, and diarrhea for their antischistosomal properties. Employing Soxhlet extraction method with different solvents, seven medicinal plants Artemisia annua, Ajuga remota, Bredilia micranta, Cordia africana, Physalis peruviana, Prunus africana and Senna didymobotrya were extracted. Qualitative phytochemical screening was performed to determine the presence of various phytochemicals in the plant extracts. Extracts were tested against S. mansoni newly transformed schistosomula (NTS) and adult worms and the schistosomicidal activity was determined by using ATP quantitation assay. Phytochemical analysis of the extracts showed different classes of compounds such as alkaloids, tannins, terpenes, etc., in plant extracts active against S. mansoni worms. Seven extracts out of 22 resulted in < 20% viability against NTS in 24 hours at 100 μg/ml. Five of the extracts with inhibitory activity against NTS showed >69.7% and ≥72.4% reduction in viability against adult worms after exposure for 24 and 48 hours, respectively. This study provides encouraging preliminary evidence that extracts of Kenyan medicinal plants deserve further study as potential alternative therapeutics that may form the basis for the development of the new treatments for schistosomiasis.

Keywords: traditional medicine, Artemisia annua, Ajuga remota, Bredilia micranta, Cordia africana, Physalis peruviana, Prunus africana, Senna didymobotrya, schistosomiasis

Introduction

Schistosomiasis or bilharzia is one of the most common parasitic neglected tropical disease. It is caused by the flatworms in the genus Schistosoma. 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 (WHO, 2022). The main burden of the disease is in sub-Saharan Africa, which accounts for over 90% of all incident cases (Colley et al., 2014). The primary schistosome species infecting humans are S. mansoni, S. haematobium and S. 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 an adult worms in the mesenteric or bladder veins of human host (Colley et al., 2014). 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., 2014).

There is no vaccine for schistosomiasis treatment and the only existing drug recommended by 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., 2018). 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 lab (Bergquist et al., 2017; Fallon & Doenhoff, 1994). 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., 2018) 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 molluscicide to control schistosomiasis in Ethiopia (Esser et al., 2003). Breonia decaryana, Citrus reticulata, Dalbergia monticola, Senna alata, and Zingiber zerumbet are used for schistosomiasis treatment in Madagascar (Rakotoarivelo et al., 2015). In Mali, Cissus quadrangularis and Stylosanthes erecta (Bah et al., 2006), Rauvolfia vomitoria (Tekwu et al., 2017), Elephantorrhiza goetzei and Pilistigma thonningii are used as remedies for schistosomiasis (Maroyi, 2013). 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 diarrhea for their schistosomicidal activity: Artemisia annua (Anibogwu et al., 2021), Ajuga remota (Cocquyt et al., 2011; Yikna & Yehualashet, 2021), Bredilia micranta (Maroyi, 2017), Cordia africana (Lelamo, 2021), Physalis peruviana (Kasali et al., 2021), Prunus africana (Kathambi et al., 2020), and Senna didymobotrya (Schmelzer et al., 2008).

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, A. annua were obtained from 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.

Family Name Botanical Name Common Name Part Used Preparation Traditional use
Asteraceae Artemisia annua Sweet wormwood Leaf, flower Decoction Malaria, Psoriasis, Infections
Lamiaceae Ajuga remota Bugleweed Leaf Decoction Malaria, Chest pains
Solanaceae Physalis peruviana Cape gooseberry Leaf Decoction Typhoid, Pneumonia
Rosaceae Prunus africana Red stinkwood Stem bark Decoction Pneumonia / chest pain, loss of appetite
Boraginaceae Cordia africana Giku Bark, leaf Decoction fatigue, anti-inflammatory
Fabaceae Senna didymobotrya Candelabra tree Leaf Decoction, steam Pneumonia
Euphorbiacece Bridelia micrantha Mitzeeeri Stem bark Chew Chest pains
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Table 2.

Crude extraction yield from Kenyan medicinal plants

Plant Species Percent mass extracted per plant
Water Methanol Hexane Acetone
A. remota 14.20 % 8.15 % 10.65 % 8.90 %
A. annua 15.28 % 2.91 % 10.08 % 11.85 %
B. micrantha 23.28 % 3.77 % 0.63 % 21.15 %
C. africana 8.56 % 8.02 % 2.36 % 21.55 %
P. africana 17.16 % 2.57 % 9.79 % 6.24 %
P. peruviana 8.85 % 4.48 % 4.23 % 7.69 %
S. didymobotrya 10.29 % 10.56 % 8.03 % 10.49 %
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Evaluation of schistosomicidal activity

Preparation of schistosomula

Biomphalaria glabrata (strain NMRI) infected with S. mansoni (strain NMRI) were obtained from the NIAID Schistosomiasis Resource Center of the Biomedical Research Institute. After infections were patent, snails were exposed to bright light for 1 hour to obtain cercariae. Cercariae were mechanically transformed to schistosomula as described (Lombardo et al., 2019). Briefly, cercariae were placed on ice for 30 minutes and then centrifuged at 350 × g for 10 minutes. The supernatant was decanted and 2 ml of serum-free M199 medium was added to cercarial pellets and vortexed for 1 minute 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 HEPES in 0.85% NaCl with cercariae suspension and centrifuged at 500 × g for 15 minutes. Cercarial pellets were resuspended and washed thrice in serum-free M199 medium and collected at 100 × g for 5 minutes. 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% 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., 2013). 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 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., 2013). 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

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., 2007). NTS and adult worm viability was assessed at 24 hours by measuring ATP content of worms using Cell Titer Glo Assay (Promega) following manufacturer’s instructions as described (Lalli et al., 2015).

Statistical analysis for schistosomicidal activity

Schistosome viability in the presence of the crude extracts were assessed using this formula:

%viability=AveragesofTest/AveragesofDMSOControl×100

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/ sulfuric acid reagent for steroids (Xu & Liu, 2021); Dragendorff reagent for alkaloids (Dube et al., 2021); potassium hydroxide (Bornträger reaction) (Henzelyová et al., 2020) for coumarins (365 nm) and anthraquinones (vis. and 365nm); 5% ethanolic solution of H2SO4 for cardiac glycosides (Onyema, 2019); aluminum chloride solution (1% ethanolic AlCl3) for flavonoids (Gwatidzo et al., 2018); iron (III) chloride reagent (3% FeCl3) for tannins and phenolic compounds (Fu & Chen, 2019); ninhydrin for amino acids, amines, and amino sugars (0.2% ethanolic ninhydrin solution) (Tyagi et al., 2019); phenol / sulfuric acid solution for carbohydrates (Oh et al., 2021; Su et al., 2020); and vanillin / H2SO4 solution for terpenes/terpenoids (Jiang et al., 2016).

Results

Schistosomicidal activity of crude extracts of Kenyan medicinal plants

Seven Kenyan medicinal plants were extracted with various solvents resulting in twenty-two 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 analyzed. 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.

Fig. 1.

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S. mansoni NTS 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.

Fig. 2.

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S. mansoni adult worm viability after treatment with crude plant extracts with potent NTS-killing activity. Viability (%) after 24 hr (█) or 48 hr (█) 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. A. 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. C. africana plant extracts showed steroids, tannins, and proteins at high levels whereas glycosides and anthraquinones were scarcely present. A. remota plant extracts showed steroids, flavonoids, terpenes/terpenoids, glycosides, proteins, and anthraquinones eminently whereas tannins were scarcely present. P. africana plant extracts showed steroids, glycosides, and anthraquinones at high levels whereas tannins, terpenes/terpenoids, and flavonoids were present at low levels. S. didymobotrya plant extracts showed steroids, glycosides, and anthraquinones at high levels whereas tannins, alkaloids, and flavonoids were present at low levels or absent. P. 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

Plant Extract Steroids Alkaloids Tannins/Phenolic compounds Carbohydrates Proteins/Amino acids Glycosides Flavonoids Anthraquinones Terpenes/Terpenoid
Artemisia annua
Acetone + + + − + + + − + + + + + + +
Hexane + − − − − − − − + +
Methanol + + − + + + + + + + + + + + +
Bridelia micrantha
Hexane − − − − − − − − +
Acetone + + + − + + − − − − + −
Methanol + + + + + + + + + + + + + − + + + −
Water + + + + + + + + − − + −
Senna didymobotrya
Acetone + + + − − − − + + + −
Water + + + − + + + + + + + + + + −
Hexane − − − − − − − − +
Methanol + + + + + + + − + + + + + + + −
Physalis peruviana
Acetone + + + − + + + + + + + + + + +
Hexane + − − − − − − − + + +
Prunus africana
Methanol + − + + − + + + +
Hexane − − − − − − − − +
Acetone + + + − + + + − + + + + + + + +
Ajuga remota
Methanol + + − + + + + + + + + + + + +
Acetone + + − − − − + − + + +
Hexane + − − − − − − − + +
Water + + + − + + + + + + + + + + + + + + + + + + +
Cordia africana
Methanol + + + − − + − + − − −
Water + + − + + + + + + + − + −
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+++ = 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 of 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, 2004). 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, 2004; Wright, 2010). 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., 2011). 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., 2014). 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% the killing of Paramphistomum cervi, the causative agent of enteritis and anemia in livestock mammals (Elango & Rahuman, 2011).

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., 2011). 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 whilst 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., 2000; Molan et al., 2002). 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., 2001). Simple tannins such as (−)-epicatechin are orally bioavailable (Serrano et al., 2009; Zhu et al., 2000) suggesting 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 S. mansoni were provided by the NIAID Schistosomiasis Resource Center of the Biomedical Research Institute (Rockville, MD) through NIH-NIAID Contract HHSN272201000005I for distribution through BEI 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).

Footnotes

Disclosure statement

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).

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