Brief chemistry of PTs

As shown in Fig. 1A–E, the PTs of the quinone methides, oleanane and ursane groups generally have five fused six-membered rings (designated a–e), while the lupane and hopane types have four six-membered and one five-membered rings. The distinguishing feature between the oleanane and ursane types is the localization of the methyl group on the ‘e’ ring, whereas the taraxasteranes differ in the orientation of substituents and the positions of double bonds in the backbone. Also, the lupane and the hopane skeletons differ on the localization of the isopropenyl group on the ‘e’ ring. In plants, all these groups of PT (except the nortriterpenoids quinone methides) commonly originate from cyclization of squalene and oxidosqualene via multiple enzymatic and redox stages involving formation of carbocations (Xu et al. Reference Xu, Fazio and Matsuda2004; Vincken et al. Reference Vincken, Heng, de Groot and Gruppen2007). Moreover, in the PT possessing the oleanane and ursane backbone, the C4, C17 and C20 appear to show the highest diversity and unsaturation as well as formation of epoxides, whilst oxygen bridges are formed between the various carbon atoms (Vincken et al. Reference Vincken, Heng, de Groot and Gruppen2007). On the other hand, the C3 and the substituent at C17 have been the primary target for synthetic modification. Finally, the saponins of the various PT are formed via attachment of diverse sugar subunits (ranging from 1 to 8 subunits) to the parent skeleton especially at C3 and C17 and rarely on C4, C16, C20, C21 and C22 (Vincken et al. Reference Vincken, Heng, de Groot and Gruppen2007). Although the physico-chemical properties of saponins as well as the non-glycosylated PT are as diverse as the compounds themselves, the sugar moiety on saponins tend to make them more soluble than the corresponding aglycone (Güçlü-Üstündağ and Mazza, Reference Güçlü-Üstündağ and Mazza2007).

Fig. 1. Representative skeletons of the different classes of pentacyclic triterpenes showing the carbon numbers and ring annotations. (A) Quinone methides, (B) oleananes, (C) ursanes, (D) lupanes, and (E) taraxastanes.

Anti-plasmodial activities of PTs

Perhaps the most in vitro active anti-plasmodial plant derived PT belong to the small group of quinone methides. Almost all the compounds belonging to the group isolated from different sources were highly active against both chloroquine sensitive and chloroquine resistant strains of Plasmodium falciparum. The compounds are pristimerin ( 1), isoiguesterol ( 2 ), celastrol ( 3 ), 28-hydroxyisoiguesterin ( 4 ), 17-(methoxycarbonyl)-28-nor-isoiguesterin ( 5 ), 28-nor-isoiguesterin-17-carbaldehyde ( 6 ) and Tingenin B ( 7 ) which all possessed very low IC₅₀ values (<0·5 µg mL⁻¹) against P. falciparum (Supplementary Table S1, available from http://journals.cambridge.org/PAR) (Figueiredo et al. Reference Figueiredo, Räz and Séquin1998; Maregesi et al. Reference Maregesi, Hermans, Dhooghe, Cimanga, Ferreira, Pannecouque, Vanden Berghee, Cose, Maese, Vlietincka, Apersa and Pieters2010; Ruphin et al. Reference Ruphin, Baholy, Emmanue, Amelie, Martin and Koto-te-Nyiwa2013).

The oleanane PT also showed high to low activity against Plasmodia. Epi-Oleanolic acid (OA) ( 11) from Viola verecunda inhibited the growth of the chloroquine sensitive D10 strain of P. falciparum with a very low IC₅₀ of 0·018 µg mL⁻¹ which was close to that of artemisinin (0·015 µg mL⁻¹) (Moon et al. Reference Moon, Jung and Lee2007). However, the same compound isolated from Celaenodendron mexicanum had moderate activity against multidrug resistant K1 strain of the parasite (IC₅₀ 12·92 µg mL⁻¹) (Camacho et al. Reference Camacho, Mata, Castaneda, Kirby, Warhurst, Croft and Phillipson2000). Another oleanane PT with potent anti-P. falciparum activities is 1-O-[α-L-(rhamnopyranosyl)]-23-acetoimberbic acid 29-methyl ester ( 12 ) from Pittosporum mannii (IC₅₀ 1·2 µg mL⁻¹) (Nyongbela et al. Reference Nyongbela, Lannang, Ayimele, Ngemenya, Bickle and Efange2013). Furthermore, OA ( 13 ) isolated from different plant species has been shown to possess anti-plasmodial activities with IC₅₀ ranging from 2·1 µg mL⁻¹ against chloroquine sensitive clone D6 (He et al. Reference He, Ma, Zhang, Tan, Tamez, Sydara, Bouamanivong, Southavong, Soejarto, Pezzuto and Fong2005) to 88 µg mL⁻¹ against multidrug resistant K1 strain of P. falciparum (Steele et al. Reference Steele, Warhurst, Kirby and Simmonds1999). Large variation in IC₅₀ values for the same compound often reflects the differences in the parasite strain or sometimes different experimental procedures. Moreover, the variations in the documented anti-plasmodial activities of OA might suggest that strain differences are critical for the anti-plasmodial effects of the oleananes.

Another moderately active anti-plasmodial oleanane PT and the most extensively investigated is maslinic acid (MA) ( 61 ). Incubation of different concentrations of the compound obtained from the fruits of Olea europaea with P. falciparum (at different growth stages) showed that the compound arrests the maturation of the intraerythrocytic parasites from early-ring to schizont stages. The IC₅₀ for the chloroquine sensitive and chloroquine resistant strains of the parasite were 15·13 and 12·29 µg mL⁻¹, respectively (Moneriz et al. Reference Moneriz, Marín-García, Bautista, Diez and Puyet2011a ). The proposed mechanism of the anti-plasmodial activity of oleanane-type PT is via incorporation into the erythrocytes membrane thereby modifying accessibility of the parasites into the cells (Sairafianpour et al. Reference Sairafianpour, Bahreininejad, Witt, Ziegler, Jaroszewski and Stærk2003). Indeed, other studies have demonstrated that PT exert their anti-parasitic activities via an interaction with the host cell membranes (Ziegler et al. Reference Ziegler, Staalsø and Jaroszewski2006; Broniatowski et al. Reference Broniatowski, Flasiński and Wydro2012).

On the other hand, some oleanane PT such as β-amyrin ( 19 ), arjun glucoside ( 73 ), sericoside ( 74 ), and maytensifolin B ( 22 ) were shown to possess very low or no anti-plasmodial activity (Supplementary Table S3, available from http://journals.cambridge.org/PAR). However, it is also noteworthy that these low active anti-plasmodial oleanane PT totally lack an acid group and/or the C3 hydroxyl or these groups are derivatized/sterically hindered (Cunha et al. Reference Cunha, Martins, Ferreira, Crotti, Lopes and Albuquerque2003). This signifies the role of the polar groups at C27 and C3 in the anti-plasmodial activity of this class of PT.

In the ursane group, ursolic acid (UA) ( 88 ) isolated from Baccharis dracunculifolia had the highest reported in vitro activity against chloroquine sensitive P. falciparum with IC₅₀ of 1 µg mL⁻¹ (da Silva Filho et al. Reference da Silva Filho, Resende, Fukui, Santos, Pauletti, Cunha, Silva, Gregório, Bastos and Nanayakkara2009). Additionally, UA from the leaves of Mimusops caffra showed an IC₅₀ of 6·8 µg mL⁻¹ against chloroquine sensitive D10 strain of P. falciparum (Simelane et al. Reference Simelane, Shonhai, Shode, Smith, Singh and Opoku2013). The activity was boosted by derivatization of the compound to 3β-O-acetylursolic acid ( 89 ) and 3-oxo-ursolic acid ( 99 ) with IC₅₀ of 1·9 and 7·3 µg mL⁻¹ respectively, using the same organism. However, other reports on the anti-plasmodial activity of UA contradict the above findings. For instance, Suksamrarn et al. (Reference Suksamrarn, Tanachatchairatana and Kanokmedhakul2003) and Graziose et al. (Reference Graziose, Rojas-Silva, Rathinasabapathy, Dekock, Grace, Poulev, Lila, Smith and Raskin2012) reported UA to be inactive against multidrug resistant and chloroquine sensitive strains of P. falciparum respectively. It is pertinent to note that the authors used either different methods or compound dilutions in the anti-plasmodial assay protocol which highlights the need for harmonization of protocols from different laboratories for easier comparison. Other ursanes with potent anti-plasmodial activity are uvaol ( 92 ) and 2α-hydroxy-ursolic acid ( 90 ) isolated from Baccharis dracunculifolia with IC₅₀ of 1·9 and 3 µg mL⁻¹ respectively against a chloroquine resistant K1 strain as well as 3-acetylpomolic acid ( 101 ) (IC₅₀ 2·1 µg mL⁻¹) and pomolic acid (100) (IC₅₀ 3·47 µg mL⁻¹) both isolated from Markhamia tomentosa (da Silva Filho et al. Reference da Silva Filho, Resende, Fukui, Santos, Pauletti, Cunha, Silva, Gregório, Bastos and Nanayakkara2009; Tantangmo et al. Reference Tantangmo, Lenta, Boyom, Ngouela, Kaiser, Tsamo, Weniger, Rosenthal and Vonthron-Senecheau2010). Hence, ursane-type PT also provide a promising class of anti-plasmodials for future research.

The lupane-type PT investigated for anti-plasmodial activity include betulinic acid (BA) ( 129 ) isolated from Harungana madagascariensis and Zizyphus vulgaris with IC₅₀ values of 2·33 and 6·3 µg mL⁻¹ against W2 and 3D7 strains of P. falciparum respectively (Lenta et al. Reference Lenta, Ngouela, Boyom, Tantangmo, Tchouya, Tsamo, Gut, Rosenthal and Connolly2007; de Sá et al. Reference de Sá, Costa, Krettli, Zalis, de Azevedo Maia, Sette, Câmara, da Silva Filho, Giulietti-Harley, dos Santos and Soares2009). A structural analogue of BA isolated from Diospyros quaesita highlights the importance of derivatization of the compound at C3 (true also for OA and UA) for potentiation of anti-plasmodial activity. The 3-caffeate derivative of BA ( 122 ) isolated from the plant was active against both chloroquine sensitive D6 and chloroquine resistant W2 strains of P. falciparum with IC₅₀ of 0·86 and 0·61 µg mL⁻¹ respectively. This activity was enhanced with double acetylation of the caffeate ( 121 ) (IC₅₀ 0·45 and 0·42 µg mL⁻¹, respectively) (Ma et al. Reference Ma, Musoke, Tan, Sydara, Bouamanivong, Southavong, Soejarto, Fong and Zhang2008). Similar results were obtained with other derivatives such as messagenic acid A ( 123 ) and messagenic acid B ( 124 ) (trans and cis C27 coumaroyl derivatives of BA, respectively) isolated from Gardenia saxatalis which possessed IC₅₀ of 1·5 and 3·8 µg mL⁻¹ respectively against a multidrug resistant strain of P. falciparum while the non-derivatized BA (also OA and UA) was inactive (Suksamrarn et al. Reference Suksamrarn, Tanachatchairatana and Kanokmedhakul2003). Moreover, cis and trans C3 coumaroyl derivatives of BA ( 138 and 139 ) isolated from Cornus florida were also highly active derivatives against P. falciparum D10 with IC₅₀ of 6·03 and 9·22 µg mL⁻¹ respectively (Graziose et al. Reference Graziose, Rojas-Silva, Rathinasabapathy, Dekock, Grace, Poulev, Lila, Smith and Raskin2012). Other naturally occurring lupane-type PT active against the K1 strain of P. falciparum are betulone ( 126 ) and lupenone ( 127 ) with IC₅₀ of 1·32 and 2 µg mL⁻¹, respectively (Gachet et al. Reference Gachet, Kunert, Kaiser, Brun, Zehl, Keller, Munoz, Bauer and Schuehly2011). However, synthetic modifications of BA did not lead to profound increase in activity. Ziegler et al. (Reference Ziegler, Franzyk, Sairafianpour, Tabatabai, Tehrani, Bagherzadeh, Hägerstrand, Stærk and Jaroszewski2004) reported the activities of methyl betulinate ( 125 ), betulinic aldehyde ( 131 ), betulinic acid amide ( 132 ), lupeol ( 128 ) and betulin ( 134 ) which were IC₅₀ of 3·3, 6·2, 6·4, 11·8, and <12 µg mL⁻¹ respectively against P. falciparum. The more interesting finding of the study, however, was that BA, 131 and 134 resulted in a dose-dependent structural change in the membrane of non-parasitized erythrocytes. The compounds consequently prevented entry of P. falciparum merozoites into non-parasitized erythrocytes. These findings demonstrated that lupane-type PT may also restrict parasites’ erythrocyte invasion in vitro via a mechanism that involves modulation of the erythrocytic membrane.

Few PT were investigated for possible in vivo anti-plasmodial activity based on their promising in vitro activities. The only oleanane-type PT that was investigated for in vivo anti-plasmodial activity was MA ( 61 ). Mice were infected with the lethal strain of Plasmodium yoelii and treated with a daily single intraperitoneal dose of 40 mg kg⁻¹ body weight (bw) MA. As found in the in vitro studies, MA demonstrated a static effect on the parasite with accumulated schizonts in the erythrocytes of the infected mice. However, the treated mice consistently maintained lower levels of parasitaemia and remained immunoprotected against further infection with the parasite after 40 days (Moneriz et al. Reference Moneriz, Marín-García, García-Granados, Bautista, Diez and Puyet2011b ). Further analysis of the possible mechanism of action of MA suggested a multi routed mechanism involving the inhibition of a number of proteases necessary for the growth of the parasite. Other binding sites for MA, which include the Plasmodium phospholipase, were putatively proposed in an in silico analysis (Moneriz et al. Reference Moneriz, Mestres, Bautista, Diez and Puyet2011c ). This remarkable in vivo activity demonstrated by MA calls for similar investigation on other oleanane-type PT especially those with even lower in vitro IC₅₀ than MA such as epi-OA ( 11 ).

On the other hand, 3β-O-acetylursolic acid ( 89 ) was shown to suppress 94·01% of circulating Plasmodium berghei in mice (effective concentration not clear in the report). The compound was also less cytotoxic against HEK293 and HepG2 cell lines (Simelane et al. Reference Simelane, Shonhai, Shode, Smith, Singh and Opoku2013). Furthermore, taraxasterol acetate ( 115 ) isolated from Pluchea lanceolata at 10 mg kg⁻¹ bw suppressed 52·20% of circulating P. berghei in mice and showed 7 days extension of mean survival time (Mohanty et al. Reference Mohanty, Srivastava, Maurya, Cheema, Shanker, Dhawan, Darokar and Bawankule2013). In another study, in vivo evaluation of betulinic acid revealed that the compound was ineffective in reducing P. berghei in mice even at 250 mg kg⁻¹ bw day⁻¹ (Steele et al. Reference Steele, Warhurst, Kirby and Simmonds1999).

One of the greatest limitation on the in vivo activity of PT, especially the less polar among them, is the hydrophobicity. Moreover, another limitation is the high cytotoxicity of some classes. For instance, Pristimerin ( 1 ) isolated from Salacia leptoclada was shown to have a selective index of <1 for P338 leukaemia cell lines (Ruphin et al. Reference Ruphin, Baholy, Emmanue, Amelie, Martin and Koto-te-Nyiwa2013), whereas 17-(methoxycarbonyl)-28-nor-isoiguesterin ( 5 ) at 10 mg kg⁻¹ bw was toxic to mice after just one day of administration (Figueiredo et al. Reference Figueiredo, Räz and Séquin1998). The latter compound which was isolated from S. kraussii although being the most active of all PT against chloroquine-resistant P. falciparum in vitro (IC₅₀ 0·037 µg mL⁻¹), was unable to clear P. berghei in mice treated with 1 and 5 mg kg⁻¹ bw (Figueiredo et al. Reference Figueiredo, Räz and Séquin1998). Therefore, bioavailability and cytotoxicity should be taken into account when further developing PT as possible anti-plasmodial agents is considered especially if the oral route is to be used. Moreover, these compounds could at least serve as structural backbones for synthesis of less toxic and more efficient compounds.

Anti-trypanosomal activities of PTs

Tingenin B ( 7 ), a quinone methide, is the most active reported PT against Trypanosoma brucei brucei and Trypanosoma cruzi with IC₅₀ < 0·25 µg mL⁻¹ against each of the species. However, as observed with other compounds belonging to the same class, the compound was highly cytotoxic on MCR-5 cells (IC₅₀ 0·45 µg mL⁻¹) (Maregesi et al. Reference Maregesi, Hermans, Dhooghe, Cimanga, Ferreira, Pannecouque, Vanden Berghee, Cose, Maese, Vlietincka, Apersa and Pieters2010). On the other hand, UA ( 88 ) has been reported in many studies to possess anti-trypanosomal activity with low IC₅₀. The compound isolated from Strachynos spynosa possessed an IC₅₀ of 1 µg mL⁻¹ against T. brucei brucei (Hoet et al. Reference Hoet, Pieters, Muccioli, Habib-Jiwan, Opperdoes and Quetin-Leclercq2007). Furthermore, in a study by Abe et al. (Reference Abe, Yamauchi, Nagao, Kinjo, Okabe, Higo and Akahane2002), UA from Rosmanirus officinalis with an MC₁₀₀ of 40 µg mL⁻¹ was shown to be 86% more effective than the natural trypanocidal compound gossypol (Abe et al. Reference Abe, Yamauchi, Nagao, Kinjo, Okabe, Higo and Akahane2002). Other structural analogues of UA were less effective or inactive against trypanosomes. The carboxylic group at C17 appears to be important for the anti-trypanosomal action of UA as evident in lower activities of uvaol ( 92 ) (aldehyde group replacing carboxyl) with an IC₅₀ of 12·3 µg mL⁻¹ and α-amyrin ( 95 ) (methyl group replacing carboxyl) with IC₅₀ of 48 µg mL⁻¹. Likewise, β-amyrin ( 19 ) (IC₅₀ 54·2 µg mL) with CH₃ in place of COOH at C17 of OA (IC₅₀ 2·9 µg mL⁻¹) lost anti-trypanosomal activity against T. brucei brucei (Hoet et al. Reference Hoet, Pieters, Muccioli, Habib-Jiwan, Opperdoes and Quetin-Leclercq2007). The OH at C3 also appears to be equally important in the trypanosomal action of both UA and OA (Cunha et al. Reference Cunha, Martins, Ferreira, Crotti, Lopes and Albuquerque2003; Taketa et al. Reference Taketa, Gnoatto, Gosmann, Pires, Schenkel and Guillaume2004). This is confirmed by the loss in the activity against T. cruzi of UA in a mixture with OA with addition of acetyl group at C3 ( 14 and 89 , respectively) of both compounds (Cunha et al. Reference Cunha, Martins, Ferreira, Crotti, Lopes and Albuquerque2003). Moreover, oleanonic acid ( 66 ) and 3,11-dioxoolean-12-en-28-onic acid ( 67 ) (IC₅₀ 113·62 and 173·9 µg mL⁻¹ against T. cruzi, respectively) which are similar in structure with OA but with adulterated C3 possessed no activity against the parasite (Cunha et al. Reference Cunha, Martins, Ferreira, Crotti, Lopes and Albuquerque2003; Hoet et al. Reference Hoet, Pieters, Muccioli, Habib-Jiwan, Opperdoes and Quetin-Leclercq2007; Leite et al. Reference Leite, Ambrozin, Fernandes, Vieira, Silva and Albuquerque2008). However, replacement of the C3 OH of OA with a polar group in saponin ( 18 ) did not lead to a loss in activity (Taketa et al. Reference Taketa, Gnoatto, Gosmann, Pires, Schenkel and Guillaume2004). From these findings, it is evident that the presence (and/or property) of the C3 hydroxyl group and C17 COOH group are significant for the trypanocidal activity of the ursane and oleanane-type PT. The role of C3 OH may be, in part, to increase the polarity of the compound because glycosylation of the group in OA with a disaccharide (3-O-[β-D-glucopyranosyl-(1–2)-β-D-galactopyranosyl]) ( 18 ) or addition of potassium in UA ( 93 ) were found to maintain the activity against T. brucei brucei (IC₅₀ 3·05 µg mL⁻¹) and T. cruzi (IC₅₀ 4·26 µg mL⁻¹), respectively (Taketa et al. Reference Taketa, Gnoatto, Gosmann, Pires, Schenkel and Guillaume2004; Cunha et al. Reference Cunha, Crevelin, Arantes, Crotti, Silva, Furtado, Albuquerque and Ferreira2006). However, substitution of the neighbouring carbon (C4) to C3 appears to counteract the effect despite the presence of additional polar groups as seen in the loss of activity of both brevicuspisaponin 1 and 2 ( 102 and 103 ) against T. brucei brucei where UA was most potent (Taketa et al. Reference Taketa, Gnoatto, Gosmann, Pires, Schenkel and Guillaume2004). This suggests that in addition to increasing the polarity of the compounds, the nature and orientation of substituents at positions C3 and C17 may be involved directly in the activities of PT. Furthermore, the double bond between C12 and C13 may also play a role in the activity because friedelanol ( 85 ) lacking any double bond was inactive despite the presence of a C3 OH (da Silva Filho et al. Reference da Silva Filho, Bueno, Gregório, Silva, Albuquerque and Bastos2004).

The anti-trypanosomal activity of the lupane group appeared in very few reports. Hoet et al. (Reference Hoet, Pieters, Muccioli, Habib-Jiwan, Opperdoes and Quetin-Leclercq2007) reported anti-T. brucei brucei activity of betulin ( 134 ), betulinic acid ( 129 ) and lupeol ( 128 ) with IC₅₀ values of 4·0, 14·9 and 19·3 µg mL⁻¹, respectively.

In an in vivo setup, UA, OA and the potassium salt of UA ( 93 ) were potent against the lethal Y strain of T. cruzi in mice treated with daily intraperitoneal dose of 2 mg kg⁻¹ bw. The treatment led to a reduction of parasite load in the infected rats more markedly by UA and the salt (75·7 and 70·4%, respectively) (Supplementary Table S1, available from http://journals.cambridge.org/PAR) (Cunha et al. Reference Cunha, Crevelin, Arantes, Crotti, Silva, Furtado, Albuquerque and Ferreira2006). In another study, da Silva Ferreira et al. (Reference da Silva Ferreira, Esperandim, Toldo, Saraiva, Cunha and De Albuquerque2010) reported 60 and 40% reduction in Bolivia strain of T. cruzi after treatment of infected rats with UA and OA at doses of 20 mg kg⁻¹ bw day⁻¹ orally. Findings of a later study demonstrated that the effectiveness of UA and OA treatment in T. cruzi infected mice is dependent on the bioavailability of the compound. It was observed that oral administration of the compounds (50 mg kg⁻¹ bw day⁻¹) resulted in 79 and 76% decrease in parasitaemia respectively, while administration of the same concentration via the intraperitoneal route was not effective. Presumably, the intraperitoneal route achieved higher effective concentration of the compounds, which could have modulatory effects on pro and anti-inflammatory cytokines that resulted in an observed immunosuppression (da Silva Ferreira et al. Reference da Silva Ferreira, Esperandim, Toldo, Kuehn, do Prado Junior, Cunha and Albuquerque2013a ). These effects may hence essentially counter the destructive effects of the compounds on the parasites since the immune system at some point of T. cruzi infection participate in parasite clearance (Tarleton, Reference Tarleton2007). Hence, at low concentrations (which is achieved via the oral route due to low oral bioavailability of UA and OA or low intraperitoneal dose), the compounds are sufficient to destroy the parasites on their capacity or via other mechanisms.

On a final note, the anti-trypanosomal potential of PT is equally promising. Further research in this area should be directed towards screening more PT (especially the quinone methides) against various species of Trypanosoma. The need to investigate the compounds in animal models is also paramount because the compounds appear to facilitate parasite clearance via stimulation of host mechanisms which cannot be attained in vitro. In this regard, alternating the routes of administration is critical in order to provide a conclusive profile on the full potencies of PT as anti-trypanosomal agents.

Anti-leishmanial activities of PTs

A number of studies have been conducted on the activity of PT against promastigotes and amastigotes of various Leishmania species. A range of saponin glycosides belonging to the oleanane PT isolated from Maesa balansae were very active against Leishmania infantum amastigotes with very low IC₅₀. The most active among them designated maesabalide III ( 25 ) possessed an IC₅₀ of 0·007 µg mL⁻¹. Other maesabalides ( 23, 24, 26, 27 and 28 ) gave IC₅₀ values of 0·014–0·046 µg mL⁻¹ (Germonprez et al. Reference Germonprez, Maes, Van Puyvelde, Van Tri, Tuan and De Kimpe2005). Oleonolic acid ( 13 ) isolated from Salvia cilicica also possessed activity with IC₅₀ of 0·04 and 0·029 µg mL⁻¹ against promastigotes and amastigote of Leishmania donovani, respectively (Tan et al. Reference Tan, Kaloga, Radtke, Kiderlen, Öksüz, Ulubelen and Kolodziej2002). Here also, the C3 OH of OA appears to play a crucial role in the activity as a conformational change tends to decrease the anti-leishmanial activity of the compound. This is because the activity of epi-OA ( 11 ) isolated from Celaendendron maxicanum was hundred-fold lower against the same parasite (IC₅₀ 8·59 µg mL⁻¹) (Camacho et al. Reference Camacho, Mata, Castaneda, Kirby, Warhurst, Croft and Phillipson2000). However, acetylation of the C3 OH group of OA may not cause a greater loss in activity. This is evident with acetylation of OA to form 3-OA acetate ( 14 ) which possessed an IC₅₀ 2·49 µg mL⁻¹ against Leishmania amazonensis (Gnoatto et al. Reference Gnoatto, Vechia, Lencina, Dassonville-Klimpt, Da Nascimento, Mossalayi, Gullon, Gosmann and Sonnet2008). Other oleanane triterpenes with potent activities include hederacolchiside A₁ ( 32 ) (IC₅₀ 0·061 µg mL⁻¹), β-hederin ( 16 ) (IC₅₀ 0·26 µg mL⁻¹) and α-hederin ( 15 ) (IC₅₀ 0·3 µg mL) from two hedera species against amastigotes of Leishmania mexicana (Ridoux et al. Reference Ridoux, Di Giorgio, Delmas, Elias, Mshvildadze, Dekanosidze, Kemertelidze, Balansard and Timon-David2001; Tantangmo et al. Reference Tantangmo, Lenta, Boyom, Ngouela, Kaiser, Tsamo, Weniger, Rosenthal and Vonthron-Senecheau2010). On the other hand, glycyrrhitinic acid (GRA) ( 33 ), a derivative of β-amyrin ( 19 ) was also potent against L. donovani promastigotes in vitro with an IC₅₀ of 4·6 µg mL⁻¹ (Ukil et al. Reference Ukil, Biswas, Das and Das2005).

Among the ursanes, UA ( 88 ) isolated from Salvia cilicica appears to be the most active against both promastigotes and amastigotes forms of L. donovani and Leishmania major with low IC₅₀ values of 0·0032–0·042 µg mL⁻¹ (Tan et al. Reference Tan, Kaloga, Radtke, Kiderlen, Öksüz, Ulubelen and Kolodziej2002). However, other studies with UA reported much higher IC₅₀ of 2·28 µg mL⁻¹ against L. amazonensis (Torres-Santos et al. Reference Torres-Santos, Lopes, Rodrigues Oliveira, Carauta, Bandeira Falcao, Kaplan and Rossi-Bergmann2004), 3·7 µg mL⁻¹ against L. donovani (da Silva Filho et al. Reference da Silva Filho, Resende, Fukui, Santos, Pauletti, Cunha, Silva, Gregório, Bastos and Nanayakkara2009) and 4·55 µg mL⁻¹ against Leishmania tarentolae (Graziose et al. Reference Graziose, Rojas-Silva, Rathinasabapathy, Dekock, Grace, Poulev, Lila, Smith and Raskin2012). Some structural modification of UA led to reduction in activity as reported for 2α-hydroxy-ursolic acid ( 90 ) and uvaol ( 92 ) (IC₅₀ 19 and 15 µg mL⁻¹ respectively against L. donovani) (da Silva Filho et al. Reference da Silva Filho, Resende, Fukui, Santos, Pauletti, Cunha, Silva, Gregório, Bastos and Nanayakkara2009). On the other hand, a bis-(3-aminopropyl) piperazine moiety added to the carboxylic acid of 3β-acetylursolic acid ( 89 ) in compounds 106–108 retained the activity of UA against promastigotes of Leishmania infantum and L. amazonensis (IC₅₀ 6–17 µg mL⁻¹) (Gnoatto et al. Reference Gnoatto, Vechia, Lencina, Dassonville-Klimpt, Da Nascimento, Mossalayi, Gullon, Gosmann and Sonnet2008). From the above findings, UA appears to be a potent anti-leishmanial agent against multiple species of the parasites. Because the investigated structural modification did not lead to an increase in activity, further modifications of the parent UA may be an experimental strategy for further development of ursane-type PT as anti-leishmanial agents. Other ursane-type PT with promising in vitro anti-leishmanial activity include pomolic acid ( 100 ) and 3-acetyl pomolic acid ( 101 ) from Markhamia tomentosa (IC₅₀ 0·31 µg mL⁻¹ and 3·4 µg mL⁻¹, respectively) against L. donovani and synthetic N-{3-[4-(3-Aminopropyl)piperazinyl]propyl}−3-O-acetylursolamide ( 105 ) (IC₅₀ 3·7 µg mL⁻¹ against L. infantum) (Gnoatto et al. Reference Gnoatto, Vechia, Lencina, Dassonville-Klimpt, Da Nascimento, Mossalayi, Gullon, Gosmann and Sonnet2008; Tantangmo et al. Reference Tantangmo, Lenta, Boyom, Ngouela, Kaiser, Tsamo, Weniger, Rosenthal and Vonthron-Senecheau2010).

In the lupane group, a few derivatives of BA were active against Leishmania although the parent compound was inactive in multiple studies. Betulinic acid acetate ( 130 ) and trans and cis 3-coumarol derivatives of BA ( 138 and 139 ) isolated from Cornus florida had IC₅₀ values of 0·45, 5·14 and 1·36 µg mL⁻¹ respectively against L. tarentolae (Graziose et al. Reference Graziose, Rojas-Silva, Rathinasabapathy, Dekock, Grace, Poulev, Lila, Smith and Raskin2012). Moreover, dihydrobetulinic acid (DHBA) ( 143 ) from Bacopa monniera possessed an IC₅₀ of 2·6 and 4·1 µg mL⁻¹ against L. amazonensis promastigotes and amastigotes, respectively (Chowdhury et al. Reference Chowdhury, Mandal, Goswami, Ghosh, Mandal, Chakraborty, Ganguly, Tripathi, Mukhopadhyay, Banyopadhyay and Majumder2003). Although a number of structural modification of the lupane-type PT led to loss in anti-leishmanial activity (Supplementary Table S3, available from http://journals.cambridge.org/PAR), future research on the group may be targeted towards different synthetic classes of the compounds and species of the parasite.

In an in vivo study, the anti-leishmanial activity of GRA ( 33 ) was further assessed where rats were treated with 50 mg kg⁻¹ bw day⁻¹ (given three times, 5 days interval for 45 days) of the compound. The compound cleared the amastigotes form of the parasite from the liver and spleen of infected animals with a mechanism that involves decrease in the expression of mRNA for anti-inflammatory cytokines [interleukin (IL)-10 and IL-4] and an increase in the level of interferon-γ (IFN-γ) and tumor necrosis factors alpha (TNF-α) (Ukil et al. Reference Ukil, Biswas, Das and Das2005). This comprehensively resulted in an increased immune response to the infection and clearance of the parasite via an nuclear factor kappa-B (NF-κB)-mediated mechanism. The mechanism through which GRA upregulate NF-κB was further described to involve multiple kinases and phosphatases (Ukil et al. Reference Ukil, Kar, Srivastav, Ghosh and Das2011). Indeed, stimulation of the immune system has been deemed a rational strategy for the development of anti-leishmanial drugs (Santos et al. Reference Santos, Coutinho, Madeira, Bottino, Vieira, Nascimento, Bernardino, Bourguignon, Corte-Real, Pinho, Rodrigues and Castro2008). In a different study, oral and intraperitoneal administration of 10 mg kg⁻¹ bw DHBA to infected golden hamsters caused >90% reduction in parasite load in the spleen and liver of the infected animals. The compound was proposed to exert its effect via a mechanism that involves inhibition of DNA topoisomerases thereby essentially destroying the parasites (Chowdhury et al. Reference Chowdhury, Mandal, Goswami, Ghosh, Mandal, Chakraborty, Ganguly, Tripathi, Mukhopadhyay, Banyopadhyay and Majumder2003, Reference Chowdhury, Mukherjee, Sengupta, Chowdhury, Mukhopadhyay and Majumder2011). As observed with other PT, the chemical entities on the C3 and C28 position of the lupeol-type PT is critical for the anti-parasitic activity of the group. Further analysis of the existing members of the group and structural manipulations to enhance activity is recommended.

Anti-nematodal activities of PTs

PTs were also investigated in a number of studies as possible therapeutic agents against lymphatic filariasis, onchocerciasis as well as conditions caused by other parasitic nematodes. Antifilarial activities of oleanane PT were reported against both human lymphatic filaria Brugia malayi and the rodent infective species. Oleanonic acid ( 66 ) and OA ( 13 ) isolated from the stem of Lantana camara were active against B. malayi in vitro with an LC₁₀₀ of 31·25 and 62·50 µg mL⁻¹ respectively (Misra et al. Reference Misra, Sharma, Raj, Dangi, Srivastava and Misra-Bhattacharya2007). Glycyrrhetinic acid ( 33 ) and its analogs ( 34–37 ) were also shown to be effective against the adult and microfilarial forms of B. malayi. The acyl derivatives ( 38 and 39 ) were inactive against both growth stages of the parasite, while the others showed IC₅₀ values in the of 0·56–28·63 µg mL⁻¹ range against the microfilariae. However, against the adult worms, only the benzyl amide ( 34 ) and octyl amide ( 35 ) derivatives were active (IC₅₀ 5·95 and 12·04 µg mL⁻¹ respectively) (Kalani et al. Reference Kalani, Kushwaha, Verma, Murthy and Srivastava2013).

In animal studies, the OA and oleanonic acid each administered at oral and intraperitoneal doses of 200 and 100 mg kg⁻¹ bw respectively had no effect on the circulating microfilariae of B. malayi in infected mastomys. However, against the adult worms, both compounds showed approximately 56% female worm sterility, although only OA had a filaricidal activity of 18·18% (Misra et al. Reference Misra, Sharma, Raj, Dangi, Srivastava and Misra-Bhattacharya2007). Moreover, B. malayi infected jirds were treated with 100 mg kg⁻¹ bw doses of the in vitro active amide derivatives of GRA ( 34 and 35 ). The result showed that only the benzyl amide derivative possessed macrofilaricidal activity (54%) while the other was inactive (Kalani et al. Reference Kalani, Kushwaha, Verma, Murthy and Srivastava2013).

The oleanane-type PT, 3-O-acetyl aleuritolic acid ( 75 ) isolated from Discoglypremna caloneura was active against Onchorcerca gutturosa worms. The compound was found to reduce the motility and viability of the worms up to 57·1 and 64·8%, respectively. The reduction in viability was found to be 33·3% more than that of amocarzine and hence compound 75 was considered an interesting compound against filarial infections (Nyasse et al. Reference Nyasse, Ngantchou, Nono and Schneider2006).

Although the volume of research on the anti-filarial activities of PT is not large, available data suggest them to be potent against different filariid. Hence, future screening of other PT against filariasis may be worthwhile.

Betulin ( 134 ) from Schefflera vinosa as well as OA and UA from Miconia langsdorfii were tested for schistomicidal activities. Among the three compounds, only 134 led to the mortality of the adult worms of Schistosoma mansonii at concentrations of 100 µ m (25% mortality) and 200 µ m (50%) after 120 h of incubation (Cunha et al. Reference Cunha, Uchôa, Cintra, de Souza, Peixoto, Silva, Magalhaes, Gimenez, Groppo, Rodriguez, da Silva Filho, e Silva, Cunha, Pauletti and Januário2012). Further research on this subject area should focus on testing newly isolated and available PT on different species of Schistosoma to compliment the library of biological activities of the group as future anti-parasitic agents.

OA isolated from Calendula officinalis was investigated for possible nematocidal activity against the mice intestinal parasite, Heligmosomoides polygyrus. The compound alongside other derivatives exhibited >50% growth inhibition of the larvae incubated with 70 µg mL⁻¹ of the compounds in vitro. The mechanism through which OA and related PT reduces the viability of H. polygyrus was later shown to involve modulation of the pattern of larval antigen glycosylation which appears to lead to a robust increase in cytokine production in mice infected with larvae incubated with the compound (Doligalska et al. Reference Doligalska, Joźwicka, Laskowska, Donskow-Łysoniewska, Pączkowski and Janiszowska2013). Because anti-filarials act via an immune-mediated mechanisms (Hoerauf et al. Reference Hoerauf, Pfarr, Mand, Debrah and Specht2011), and PTs were shown to modulate the immune system, PTs are logical candidates for in vivo screening as anti-filarial drugs.

MA was also investigated for possible action against the Trichinella, the causative agent of trichinellosis in humans. Against the mammalian infective Trichinella zimbabwensis, the compound orally administered once on 25 dpi or twice on 25 and 32 dpi cleared >90% of the parasite's larvae. This was achieved at a lower dose (2·5 mg kg⁻¹ bw) compared with the anthelmic drug fenbendazole (7·5 mg kg⁻¹ bw) which gave similar efficacy (Mukaratirwa et al. Reference Mukaratirwa, Gcanga and Kamau2016). Hence, MA has shown promising activity against Trichinella and therefore screening of other PT against this parasite will be worthwhile.

Against the plant nematode Meloidogyne incognita, camarinic acid ( 110 ) activity was similar to that of a standard nematicidal drug, furadan, at the same concentration of 1 mg mL⁻¹. The compound which was isolated from Lantana camara led to 100% larval mortality after 24 h exposure (Supplementary Table S1, available from http://journals.cambridge.org/PAR) (Begum et al. Reference Begum, Wahab, Siddiqui and Qamar2000). Later studies on this plant showed it to be a repository of PT with varying degrees of nematicidal activities. Lantanilic acid ( 46 ), camaric acid ( 45 ) and OA ( 13 ) from the plant caused 98, 95 and 70% M. incognita larval mortality respectively at 5 mg mL⁻¹ concentration (Qamar et al. Reference Qamar, Begum, Raza, Wahab and Siddiqui2005). Furthermore, camarinin ( 43 ), lantanolic acid ( 44 ), UA, pomolic acid ( 100 ), lantacin ( 114 ), camarin ( 77 ) and lantoic acid ( 111 ) from the same plant all caused 100% larval mortality at 1 mg mL⁻¹ concentration after 48 h of exposure. Compounds 43, 44 and UA ( 88 ) proved to be comparatively more potent with 90, 10 and 10% larval mortality at 2 µg mL⁻¹ after 72 h exposure (Begum et al. Reference Begum, Zehra, Siddiqui, Fayyaz and Ramzan2008). In a different study with Cordia latifolia, cordinoic acid ( 112 ) isolated from the plant at 5 mg mL⁻¹ concentration led to 100% M. incognita larval mortality after 24 h exposure (Begum et al. Reference Begum, Perwaiz, Siddiqui, Khan, Fayyaz and Ramzan2011). On the other hand, polygalacic acid ( 48 ) and bayogenin ( 49 ) and their saponins 50–60 isolated from Microsechium helleri and Sicyos bulbosus were active against Meloidogyne javanica that also affects plants. Among the compounds, those with a xylose residue attached to the second rhamnose residue at the substituent on C28 ( 50–53 ) were found to be inactive while the others inhibited >74% of the parasite's larvae growth at various concentrations. Moreover, bayogenin which differs from polygalacic acid only in the absence of an OH group at C16 of the latter molecule was the most active together with saponin 58 . Both compounds immobilized 100% of the parasite's larvae at 0·5 µg mL⁻¹ concentration (Hernández-Carlos et al. Reference Hernández-Carlos, González-Coloma, Orozco-Valencia, Ramírez-Mares, Andrés-Yeves and Joseph-Nathan2011). From the above findings, it is clear that the activities of PT and their saponin against plant nematodes are promising and warrant further investigation.

Activities of PTs against other parasites

Toxoplasma: Maslinic acid ( 61 ) isolated from Olea europaea inhibited the infectivity of Vero cells by T. gondii tachyzoites with an ID₅₀ of 3·78 µg mL⁻¹ after incubation for 48 h. Moreover, the compound at a concentration of 50 µ m inhibited the motility of 100% of the parasites. The compound was also shown to inhibit key parasite proteases thereby effectively blocking parasite entry into the cells (De Pablos et al. Reference De Pablos, González, Rodrigues, García Granados, Parra and Osuna2010). This dual effect (inhibition of motility and entrance into cells) of MA on T. gondii is interesting as therapeutic approach and hence calls for further screening alongside other PT.

Trichomonas: Only one PT, hederagenin ( 47 ), isolated from Cussonia holstii was investigated for activity against Trichomonas viginalis. The result indicated high in vitro activity with an IC₅₀ of 1·32 µg mL⁻¹ (He et al. Reference He, Van Puyvelde, Maes, Bosselaers and De Kimpe2003). Hence, PT could be suitable candidates for future screening as anti-trichomonas agents.

Toxicity aspects

One of the disadvantages of using PT as therapeutic agents has been known to be associated with high cytotoxicity (Dzubak et al. Reference Dzubak, Hajduch, Vydra, Hustova, Kvasnica, Biedermann, Markoba, Urban and Sarek2006). However, at low concentrations, some of these PT have proved to be therapeutic (Liu, Reference Liu2005). Moreover, cytotoxicity studies of some PT, for example MA, reported in vivo safety both in acute and chronic treatments (Sánchez-González et al. Reference Sánchez-González, Lozano-Mena, Juan, García-Granados and Planas2013). In another study, BA was found to have selective toxicity against cancerous cells but not normal cells (Zuco et al. Reference Zuco, Supino, Righetti, Cleris, Marchesi, Gambacorti-Passerini and Formelli2002). Hence, since PT are selective to different cells lines, further toxicity assessments and in vivo safety studies of the most active compounds is warranted.