A review of efficacy and safety of Ugandan anti-malarial plants with application of RITAM score

Artemisia annua (Fig. 1) is known as sweet wormwood, sweet annie or annual wormwood in English. Botanically, it has been classified as Family—Asteraceae, Genus—Artemisia, Species: annua [30, 31].

As shown in Fig. 1, the plant is a large vigorous weedy annual shrub often reaching more than 2 m tall, usually ribbed single-stemmed with alternate branches and stem covered with fine, silky grey-green hairs [46]. It naturally grows to 30–100 cm high but cultivated plants may reach 200 cm high, and is widely distributed in the temperate, cool temperate and subtropical zones (mainly in Asia) of the world [47]. It originated from China and grows mainly in the middle, eastern and southern parts of Europe and in the northern, middle and eastern parts of Asia. However, a few countries are currently cultivating A. annua on both large and small scale, such as China, Kenya, the United Republic of Tanzania, and other countries in Africa (including Uganda), and altitudes ranging 1000–1500 m are favourable for its growth [48].

Artemisia annua (called Qinghao—Chinese) has long been used in China as a herbal remedy with its first documentation dating 168 BC [48]. Its first record for malaria was made by Ge Hong in 341 AD. Li Shizen also wrote in his Pharmacopoeia in 1596 that qinghao cures cold and hot fevers. Based on this, the Chinese scientists led by Zhenxing Wei and You-Tou Li developed extraction methods to finally isolate its active compound, artemisinin (Fig. 2) in 1972 [45].

Since the isolation of the active ingredient, artemisinin has influenced the current treatment of malaria. In most African countries, ACT was introduced right on time when the parasite had already developed resistance against the previous drugs, including chloroquine, quinine and others. The trends over the years on this novel plant is fascinatingly filled with disagreements among various researchers on some issues and agreements on other issues, especially between researchers from herbal regimen and those from synthetic regimen. The anti-malarial literature on the plant ranges from in vitro, in vivo in animals to several human clinical trials.

The earliest clinical study conducted on A. annua leaf infusion was performed 20 years ago as a pilot trial in a rural primary health care scheme, involving a district hospital and three health centres, in the eastern Democratic Republic of the Congo (DRC) from February to December 2001 [49]. In the study, extract groups (2) received 5 g (A5) and 9 g (A9) of the leaf powder prepared in 1 L per day and the control group were on quinine and later changed to chloroquine. A 77% cure rate was reported in A5 group and 70% in A9 while the positive control led to 91% by day 7. Parasite recrudescence was observed in the infusion groups after day 7 though there was no clear method used to distinguish recrudescence from new infections. However, there was a rapid improvement of malaria symptoms in artemisia groups, leading to a conclusion that, “malaria monotherapy with tea preparation cannot be recommended for treatment because of recrudescence as well as concerns of possible artemisinin resistance. A similar study was done in Tanzania in 2002—2003 with same doses of A. annua infusion but sulfadoxine/pyrimethamine used as control [50]. Compared to the previous trial [49], the number of participants available by day 7 was lower (4–8, compared to 39–43). However, the cure rate reported in this work (70% & 77.7% for 5 g and 9 g doses, respectively) was similar to the study from DRC. Another similarity with both studies is that the plant materials were all collected from Germany and a high rate of recrudescence.

Contrary to the findings in the two clinical studies above, a cure rate of 91.8% was reported in an Ethiopian survey that assessed the use of the plant for malaria treatment among locals with no major adverse effects [51]. The researchers recommended that a policy and regulatory mechanisms to integrate herbal medicines to modern health care system should be established and adopted. But these findings were only based on experiences of people, with no scientific methods to prove the cure and may not be applicable for informing policy. Interestingly, these findings were corroborated by a pharmacovigilance study in Kenya and Uganda which revealed that, over 3000 cases of presumed malaria (250 children and 54 pregnant women in 1st trimester) were treated using A. annua [52]. However, the latter study reported poor compliance by children to the infusion because of bitterness and vomiting. Two miscarriages were reported among the pregnant patients. In a randomized Ugandan clinical trial [53], consumption of the A. annua tea infusion (5 g) once a week significantly reduced the risk of suffering more than one episode of malaria in 9 months by 55%. No serious side effects were reported except the bitter taste associated with the plant. Another study [54] supported the efficacy reported of A. annua tea infusion for malaria treatment. The researchers reported a potent activity demonstrated by the infusion in vitro against both chloroquine resistant (W₂) and sensitive (D10) strains (IC50 (ug/ml): 1.11 ± 0.21 for D10 and 0.88 ± 0.35 for W₂). With the minimal concentration of artemisinin in the infusion (0.18 ± 0.02% of the leaf powder), which was too low to be responsible for the observed activity, they postulated the possibility of artemisinin acting synergistically with other ingredients in the extract to give such an impressive effect.

Despite the efficacy of the crude plant extracts reported above, the WHO in 2012 ruled out the use of A. annua plant material in any form including capsules and tea for malaria treatment or prevention [55]. The international regulatory body decision was based exclusively on two trials described previously [35, 36], stating that the reported clinical outcomes were unsatisfactory with malaria recrudescence, low dose of the infusions compared to the recommended ACT dose may promote anti-malarial resistance, and reported interactions between artemisinin and other compounds in the infusions are unsatisfactory. The WHO recommended that an extensive fundamental and clinical research be done to demonstrate that the non-pharmaceutical dosage forms of the plant are safe and effective for malaria, and that their use would not lead to development of artemisinin-resistant parasite. A follow-up study on the possibility of synergism among various compounds in the plant was conducted [56], different varieties of the plant were tested in comparison with pure artemisinin. It was observed that the IC₅₀ of all the A. annua tea infusions were not significantly different from that of pure artemisinin, and thus in agreement with the WHO analysis. The researchers, therefore, concluded that, “artemisinin seems to be the only active anti-malarial agent in A. annua, but they did not comprehensively explain the similarity in the IC₅₀’s between the extract and the pure drug since the extract obviously has lower quantity of the active and would be expected to show lower activity if their conclusion is to be justified.

In a comprehensive literature review on the use of A. annua for malaria management [37], reviewers highlighted discrepancies in the studies used for informing the above WHO decision. They also presented several studies that provided evidence of efficacy of the plant infusion for malaria especially those that proved biopharmaceutical interaction viz., a number of compounds from flavonoids, terpenes polysaccharides and others that improved absorption of artemisinin and some exhibiting anti-plasmodial activities. They suggested that the tea could play a critical role in malaria prophylaxis to reduce incidence of malaria in different communities or in temporary relief of malaria as the patient buys time to access a hospital or a clinic stocked with ACT. More scientific evidences have also emerged recently all supporting the synergism between artemisinin and other compounds. For example, in a combination of artemisinin with three other components in high contents in the A. annua (arteannuin B, arteannuic acid, and scopoletin), a sharper reduction in parasitaemia (93%) compared to the pure artemisinin (30%) in animal model was reported [17]. This indicated clear synergism among the different compounds. The pharmacokinetic studies showed increased absorption in the combination groups. The findings imply that specific components in the plant might offer a possibility to develop new artemisinin-based natural combination therapy for malaria treatment. In a similar study, arteannuin B was reported to inhibit biotransformation of artemisinin through inhibition of CYP3A [57]. This observed effect of arteannuin B explains the enhanced anti-plasmodial potency of the plant extract, but the synergism did not reduce the rate of recrudescence. Therefore, there is still the need to combine the extract with other anti-malarial agents to prolong activity and alleviate recrudescence.

The limitation of A. annua tea infusion in malaria treatment by parasite recrudescence was further evidenced in another randomized controlled trial, which used artemether-lumefantrine as control. The study reported negative parasitaemia in the tea infusion within few days but recrudescence surfaced on day 14 and 28. The researchers also attributed the observed activity to interaction between artemisinin and other components (possibly flavonoids), and recommended combination of the extract with other agents to extend therapeutic action [31]. In contrast to a large-scale randomized, double blind and controlled study showing 91% and 100% cure rates in children and adults, respectively, compared to 50 and 30% (children and adults respectively) for the standard control (artesunate-amodiaquine – ASAQ) [58], no recrudescence was reported after 28 days in the extract group. Due to strong criticism of the study findings [35], the publication was retracted. Despite continued challenges in acceptability of A. annua extract for malaria prophylaxis or treatment, more evidence on its effectiveness continues to surface. Another study in 2020 [59] reported that, the malaria prophylaxis provided by the plant infusion that was thought to last for few weeks actually extends for months and years. This was explained by an observation that the IgE induced Artemisia consumption remained for months on the skin. Another researcher described therapeutics based on isolated molecules such as quinine or its derivative chloroquine, artemisinin or its derivatives (artesunate), as having higher tendencies to cause drug resistance than crude plant extracts having complex chemical composition [60]. Therefore, standardization of crude extract formulations will not only lead to introduction of phytopharmaceuticals into the conventional health care mainstream, but also prevent any possible drug resistance which is common with synthetic drugs.

Vernonia amygdalina (Fig. 3) is a popular African vegetable that grows as a shrub or small tree indigenous to Central and East African including Uganda [61]. Ecologically and botanically, the plant has been previously described as follows [51]. It grows up to 10 m tall along rivers and lakes, in forests margins, woodland and grassland up to 2800 m altitude, in regions where mean annual rainfall is 750- 2000 mm The bark is light grey or brown; fissured, brittle branches. Leaves lanceolate oblong; up to 28 × 0 cm, but usually 10–15 × 4–5 cm. Flower heads thistle like, small, creamy white, 10 mm long, grouped in dense heads, axillary and terminal, forming large flat clusters, 15 cm in diameter, sweetly scented.

The plant is commonly referred to as bitter leaf but in Uganda, it has various local names; Luganda—Mululuuza, Lunyankole—Mubirizi, Acholi—Labori, Lugishu—Mululisi, Madi—Okelo-okelo, Iteso—etutum, and Lugbara—Echero [63]. It is used for treatment of multiple diseases in Uganda including schistosomiasis, amoebic dysentery, and gastrointestinal problems. Masaba reported reported the chewing of the pith of the plant by Chimpanzees for treating parasitic infections and determined the in vitro anti-plasmodial activity of aqueous and acetone–water extract obtaining IC₅₀ of 76.7 µg/ml and 25.5 µg/ml respectively [64]. Another in vitro activity was tested in fresh isolated P. falciparum parasites from patients in Nigeria [65]. The researchers reported IC₅₀ of 11.2 µg/ml for ethanolic leaf extract and 13.6 µg/ml for aqueous extract. These anti-plasmodial activities were higher than the study by Masaba [64]. The difference could be attributed to geographical locations (Nigeria vs Uganda) and parasite strains (isolates from Nigeria vs school child chloroquine sensitive strain—Uganda).

Several in vivo animal anti-malarial studies have also been conducted to validate the above in vitro findings (Table 4). The variations in the activities from different studies may emanate from many factors including geographical locations, route of administration, different extraction methods, and antimalarial method (prophylactic, curative and suppressive). The highest activity reported so far with the standard Peter’s four-day suppressive test was from the V. amygdalina collected from Uganda, that is, 75.15% at 400 mg/kg [66]. A previous study in Uganda [61], gave a similar activity at an even lower dose but the extract was administered by IP (intraperitoneal) route.

In addition to the in vivo animal studies validating the anti-malarial efficacy of the plant, two clinical studies have also been conducted in Uganda [56, 57]. The first was done in Bushenyi at Rukararwe Partnership Workshop for Rural Development (RPWRD), and the product was coded AM—which refers to antimalaria V. amygdalina leaf powder [67]. Complete parasite clearance was achieved only in one case, but the geometric mean of parasite count declined significantly by day 7 (5540/µl day 0 to 511/ µl day 7). There was also marked symptomatic improvement in 17/19 patients. No severe side effects were observed, and the most common minor ones reported included vomiting, abdominal pain, nausea, and bitter taste. These side effects affected the compliance of patients leading to six (6) dropouts. However, the study recommended a larger randomized controlled trial to determine whether the symptomatic improvement was the result of AM treatment or of natural immunity to malaria. The second trial [32] conducted in Kasese district (Uganda) reported adequate clinical response (ACR) on day 14 in 67% of the cases. However, complete parasite clearance occurred in only 32% of those with ACR, and of these, parasite recrudescence occurred in 71%. Just like the previous study, no severe side effects were reported except nocturia, insomnia and cough. According to the researchers, V. amygdalina is moderately effective and further work is necessary to establish the optimum dosage regimen, possibly in combination with other anti-malarial agents. A follow up study was conducted to validate this claim [68], and it reported 80.71% chemosuppression from a combination of V. amygdalina (125 mg/kg) and chloroquine (5 mg/kg), in mice against chloroquine resistant clones of Plasmodium berghei strain ANKA. The study concluded that, V. amygdalina leaf extract dose – dependently restored the efficacy of CQ against CQ resistant P. berghei malaria in mice. In another study in Uganda, a combination of V. amygdalina with A. annua achieved 100% parasite clearance in mice model [39]. There was still a challenge of shorter survival time (10.67 ± 1.09 days) compared to more than 30.0 ± 0.0 days for the ACT, P = 0.000. The study concluded that, “the V. amygdalina – A. annua petroleum ether extract combination shows promise for use as an herbal artemisinin combination against malaria, however the survival times need improvement to match that of the ACT”.

The phytochemicals responsible for above-described activities of V. amygdalina have been identified, isolated and their anti-plasmodial effect determined. They include (with their IC₅₀) sesquiterpene lactones: vernodalin (4.0 µg/ml), vernolide (8.4 µg/ml), vernodalol (4.2 µg/ml), and hydroxyvernolide (11.4 µg/ml); and also steroid glycoside: vernonioside B1 (46.1 µg/ml) [38, 70]. The bitter taste of the plant leaf decoction is attributed to the steroid glycosides (vernoniosides A₁ – A₄ and B₁ – B₄). The LD₅₀ of the leaf decoction has been reported to be > 2000 mg/kg [61, 62].

Microglossa pyrifolia (Fig. 4) is an erect or scandent shrub that grows up to 5 m high, occurring throughout tropical Africa and Asia [71]. The plant is locally known as Kafugakande in Uganda – Luganda [72], Nyabungodide in Kenya [73]. Diola in Senegal and Gambia, and Bulom in Sierra Leon [71]. The leaves are simple alternate, carried by a short petiole (10–15 mm long). The leaf blade is oval, 5 to 10 cm long and 2.5 to 4 cm wide.

The plant leaf is used locally in Uganda for treatment of malaria and several in vitro anti-plasmodial studies have provided evidence supporting this use (Table 5). The plant leaves have demonstrated very potent (IC₅₀ < 2 µg/ml) anti-plasmodial activity according to the RITAM ranking of in vitro efficacy across different Plasmodium strains and countries. Interestingly in Uganda, the aqueous leaf extract also showed a very potent activity [72]. A similar finding was reported earlier in a Kenyan study, though the extract used in the latter was methanol [73]. The promising anti-plasmodial activity of this plant has been attributed to presence of several terpenoids, such as E-phytol, 1,3- hydroxyoctadeca-9Z, 11E, 15-trien-oic-acid and 6E-geranylgeraniol-19-oic-acid, which exhibited IC₅₀ values between 2.5 and 13.7 µg/ml [74]. The higher IC₅₀ values shown by these active compounds compared to the crude extracts is pointing to possible combined effects (synergism or additive) among the compounds in the extract against the malaria parasite. In a recent mini-review on the anti-plasmodial activities of various plants around the world, M. pyrifolia leaf extracts (ethyl acetate and aqueous solvents) ranked among the best three in terms of their lower IC₅₀ indicating higher potency [75]. The researchers attributed the potency of the plant to 6E-geranylgeraniol-19-oic-acid, tannins and other polar compounds. The 6E-geranylgeraniol-19-oic-acid is also known to be present in aqueous extracts despite its lipophilic character [74].

In terms of safety, the plant leaf methanolic extract showed a higher cytotoxicity antiplamodial ratio (CAR) of 1578.0 (chloroquine sensitive strain, D-6) and 946.8 (chloroquine resistant strain, W-2), which are indicative of weak toxicities [73]. In a similar study, 89.7% reduction of ATP levels have been reported with the methanolic extract of the plant indicating a hepatotoxic effect [76]. This finding also agreed with the study [43], that listed the plant among those associated with liver fibrosis in Rakai—Uganda. The study mentioned that plants of Microglossa family contain diterpenoids known to cause liver toxicity. In addition, herbs in Asteraceae family contain pyrrolizidine alkaloids which are associated with veno-occlusive liver disease [77]. Unfortunately, there are no in vivo toxicity studies on the plant to determine its LD₅₀ for guiding dose selection for efficacy studies.

Most of the anti-malarial studies on the plant stopped at in vitro despite its extremely potent anti-plasmodial activity. Therefore, there is a need to conduct in vivo anti-malarial activities for this plant since its anti-plasmodial potency has already been demonstrated in different countries and strains of the malaria parasite. It is also possible that the plant (in combination) may successfully improve anti-malarial efficacy of A. annua and/or V. amygdalina, especially the challenge of parasite recrudescence. Addition of the plant extract in small doses in such a combination may also control its potential liver toxicity.