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Pentacyclic triterpenes in antimalarial plants, a new paradigm

May 8, 2016 - 07:48 -- Pierre Lutgen

Many plants have antiplasmodial properties but nobody really knows why some do and others do not. We have paid most of our attention to artemisinin and derivatives, to flavonoids and antioxidants, swamped and blinded by thousands of papers on these molecules, billions invested and earned in ACTs, the prohibition of clinical trials with Artemisia annua by WHO-Geneva, the Vatican of malaria, and the colonial ITG-Antwerp. And we have forgotten that there are other molecules in Artemisia which may play an equivalent or stronger role.

THE BREAKING NEWS PAPER OF MAVONDO

A recent paper opens a door (G Mavondo et al., Malaria Journal. May 2016, 15:226). Asiatic acid, a pentacyclic triterpene from Centella asiatica is strongly prophylactic, suppressive and curative against malaria. The main points of this study: Pre-infection administration of asiatic acid retards parasitaemia induction, averts the development of severe malaria, reduces inflammation, avoids anemia and weight loss and generates a persistent immunoprotection. The suppressive effect, even at a 3x lower dose, is stronger than for chloroquine,

Due to their low hydrophilicity, pentacyclic triterpenes were considered to be inactive; however, evidence regarding their wide range of pharmacological activities is emerging, and several studies have highlighted these activities. These compounds have incontestable biological activity, such as antibacterial, antiviral, and antiprotozoal effects. They display appreciable antiparasitic effects against Plasmodium falciparum, Toxoplasma gondii, Trypanosoma cruzi, and Leishmania sp. A paper from Brazil (L Duarte et al., Rev Instit Med Trop de SaoPaolo, 2002, ISSN 1678-9946) for example shows that some pentacyclic triterpenes from Austroplenckia populnea are as strong as primaquine or ketoconazole against Trypanonsoma cruzi , the Chagas disease.

Many triperpenes are found as glycosides (saponins, phytosterols) which refers to the attachment of various sugar molecules. These sugars can be cleaved off in the gut by bacteria, allowing the aglycone tripterpene to be absorbed into the bloodstream or to insert into cell membranes. A well known saponin with therapeutic properties is ginsenoside. The α-amyrin pentacyclic triterperpene saponin from Ilex asprella is widely used in South China as anti-influenza herbal drug (M Zheng et al., Int J Mol Sci 2014, 15, 5970-87). See also the documents “Saponin lowers iron, glucose, uric acid, cholesterol: key factors in malaria” and “Cholesterol, phytosterols and malaria” on www.malariaworld.org.

In Centella asiatica asiatic acid is mainly present in the form of the saponin asiaticoside, up to 2% DW (Viola Müller, Thesis 2013, Universität Bonn). Mavondo (op.cit) finds for asiatic acid a surprising elongation of the bioavailability in plasma, after oral administration, based on its amphiphilic nature. Asiatic acid human intestinal absorption was found to be as high 91.23% as well as a strong binding to plasma proteins which could account for its accumulation in plasma. This may explain how the parasitemia was suppressed even when high parasite inoculum was used, several times higher than human infection dose. A Chinese bioavailability study showed that all pentacyclic triterpenes are absorbed and adsorbed in their intact forms in plasma, brain, liver, kidney (Mei-Chin Yin et al., J Agric Food Chem 2012 60 7697-7701).

In their introduction Mavondo et al. (op.cit.) refer to the work of Patrick Ogwang from Uganda (Ogwang PE, et al. Trop J Pharm Res. 2012;11:445–53) who showed that an infusion of Artemisia annua consumed once weekly reduced the risk of Plasmodium falciparum episodes due to a yet unidentified constituent. IFBV-BELHERB has numerous anecdotic reports on this same prophylactic effects from our partners. Astonishing is the long lasting effect of asiatic acid which is similar to the effect noticed by Ogwang for one weekly cup of Artemisia infusion.

Artemisia plants contain a lot of acids and in high concentrations: for example, rosmarinic acid as we had found for Artemisia annua from Luxembourg, Brazil and Cameroon in 2010 at Celabor-Herve. In her thesis B Ivanescu in Romania found a high content of ferulic acid in Artemisia annua (Chemistry of Natural Compounds, Vol. 46, No. 3, 2010), Artemisia afra contains betulinic acid, α-amaryn (G More et al., Evid Based Complement Alternat Med. 2012: 252758) and β-amyrin (Yusra Kriel, Thesis, Western Cape University 2010). Elizabeth Hsu in her « Reflections on the discovery of the antimalarial qinghao” (Br J Clin Pharmacol 61(6), June 2006) writes:”Shen gua, and after him Shizhen valued Artemisia apiacea more than Artemisia annua”. This plant apparently doesn’t contain any artemisinin. But Artemisia apiacea is rich in α-amyrin and β-amyrin (S Lee Arch Pharm Res, 2003, 26, 902-5). In a study in Brazil (Gabriela Hrckova et al., Book. Springer 2012) on plant extracts containing several pentacyclic triterpenes it was found that α-amyrin had the greatest antiplasmodial activity (IC50:0.96 µg/ml).

SURPRISING SYNERGIES : OLIVES, MILLET, PEANUTS

But other plants also contain pentacyclic triterpenes.

The University of Firenze has studied the antimalarial properties of olive leaf extract (Olea europea) and found that 200 µg/ml gave a 73% reduction of parasitemia. They also found that this extract is strongly synergistic with artemisinin (B. Isacchi , Thesis 17 oct. 2013 -" Studio sul sinergismo dell'artemisina e dei suoi derivati con sostanze naturali di origine vegetale per una chemioterapia antimalarica). Teas from olive leaves are known and used against malaria since centuries. In 1820 doctors developed a blend of strong olive leaf tea and wine known as Tinctura Olea Foliorum. The pentacyclic triterpene maslinic acid and oleanolic acid are major constituents of olive leaves and oil (G Lozano-Mena et al., Molecules, 2014, 19, 11538-59). The Egyptions even found four pentacyclic triterpenes in their olives: maslinic, betulic, oleanolic and ursolic acid (S Fiad et al, Am J Food Technol, 2015, 10, 1-13). A surprising paper comes from Nigeria. Using the ethanolic extract of local Olea europea subs. africana on Plasmodium falciparum infected mice they find that the dose of 120 mg/kg bwt extract was more potent than the antimalarial drug artesunate at 50 mg/kg) (O Okere et al., Int J Biomed Res 2014, 05 doi 10.7439).

We have many anecdotic reports on the antimalarial properties of peanut butter and oil, especially as enhancers of antimalarial properties of artemisinin (Congrès Maladies Tropicales, Luxembourg, 6-7 avril 2009). They could be related to the pentacyclic sophoradiol present in peanuts.

In some African countries Artemisia leaves are mixed with millet (Panicum miliaceum) porridge as first-aid for children with malaria. One of the main reasons is to hide the bitter taste. An Italian team has studied this mixture, used with success in Kenya and is called Uji. They found that the artemisinin content is stable over 14 days (M Bonati et al., J Alternat Complement Medicine, 2011, 4, 371-73). But it is possible that the efficacy is related to a large extent to the pentacyclic tripernoid miliacin present in millet. One of the features of this plant ; rice doesn’t contain any (N Bossard et al., Organic Geochemistry, 2013).

One of the items where I disagree with Mavondo (op.cit.) is the claim that because it is tasteless asiatic acid does not have the effect of bitter antimalarials which as they say induce satiety, reduce food and water intake. To the contrary, as somebody wrote “Tous les aperitifs qu’on nous vend avant les repas sont amers: Campari, Cynar, Martini, Absinthe, Fernet Branca, Quinquina et si les restaurateurs les proposent systématiquement au client qui débarque, ce n’est pas pour qu’il bouffe et pote moins.”

Nevertheless the fact that asiatic acid stabilizes food intake and bodyweight, reduces inflammation, maintains hemoglobin at a high level while all other antimalarials and commercial drugs have a negative effect on these parameters, as well as frequent side effects noticed for these commercial drugs. It is possible that asiatic acid leads also to some minor side effects but they are hidden by the above mentioned positive effects. Eventually this applies also to Artemisia herbal infusions where in all the small and large clinical trials we run over the years, the patients noticed no side effects. Surprising indeed but eventually the small negative effects are overshadowed by the remarkable positive effects of the treatment.

PENTACYCLIC TRITERPENES: MANY OTHER HEALTH BENEFITS

The anti-inflammatory properties of asiatic acid are related to inhibition of IL-6 and NFκB. In our work with the University of Louvain (Pedro Melillo de Magalhaes et al., Food Chemistry, 2012) it was found that Artemisia annua with low artemisinin content had similar strong anti-inflammatory properties. The methanolic extract of Costus igneus showed anti-inflammatory effects stronger than standard drug Diclofenac (K Krishnan et al., Inflammopharmacology, 2014, 22, 373-85). α, β-amyrine have anti-inflammatory properties of the same level as Indomethacin (P de Almeida et al., Evid Based Complement and Alternat Med., 2015, ID 293768).

Mavondo et al., also notice an inhibition of gluconeogenesis. Important fact because Plasmodium feeds on glucose. This antidiabetic effect for pentacyclic triterpenes was confirmed in China (Mei-Chin Yin, Biomedicine, 2015, 5-3, 1-8)

But the most important effect noticed is the total disappearance of parasites on day 21 and the absence of recurrence. The same effect was noticed in the large scale, randomized, double blind clinical trials run by our partners with Artemisia annua and afra in Maniema-RDC in 2015. A true hope for malaria eradication.

The paper of Mavondo et al., is in line with earlier results obtained with maslinic acid (C Moneriz et al., Malaria Journal2011, 10.103). In vivo treatment of Plasmodium yoelii-infected mice with maslinic acid increased the survival rate from 20 to 80%. The authors relate it to the parasitostatic effect of the acid. Complete clearance was evident on day 20 and the mice remained immunoprotected. No prophylactic effect was evidenced in this trial because the mice were treated only a few hours after infection. The results of Moneriz (op.cit) were confirmed in a thesis of the University of Kwazulu-Natal in 2014 by Tshidi Thaane. Treatment with maslinic acid (40 mg/kg per os) significantly reduced parasitemia in Plasmodium berghei infected mice, however this dose did not eliminate the malaria parasites until the end of the 21 days experimental period. The higher doses (80 and 160 mg/kg) cleared the malaria parasites from systemic circulation by 9 days following treatment, like chloroquine (30 mg/kg) did. Compared to the results of Mavondo (op.cit) the efficacy of maslinic acid, although excellent, is inferior to asiatic acid. But the author highlights that no secondary effects are noticed for the maslinic acid treatment. Chloroquine however, in the same trial, had many: hypoglycaemia, deposition of chloroquine in tissues, intravascular clumps…

Some other properties of these pentacyclic triterpenes have been studied. Asiatic acid is a strong antibacterial agent against E coli, S typhi, Pseudomonas, Enterococcus faecalis etc (WH Liu et al., Biomedicine, 2015 Aug, 16). Rosmarinic acid has an IC50 against Plasmodium strains of the same level as artemisinic acid (J Suberu et al., PLOS ONE 2013, 8-11, e80790). The University of Louvain and Liege (J Bero et al., J Ethnopharmacol. 2013, 149,176-183) have found for ursolic acid and oleanic acid good antiplasmodial activities. Betulinic acid from Artemisia afra was demonstrated to have antiplasmodial properties in vivo (MS Sa et al., Parasitol Res 2009, 105, 275-9). Olea europea is also rich in the pentacyclic oleanolic acid which has known antimalarial properties (JD Habile et al., Afric J Pharmacy and Pharmacology 2011, 5, 2667-75).

The antiplasmodial activity of oleanolic acid is apparently due to its incorporation into the erythrocyte membrane (M Sairafianpour et al., Planta Med 2003 69, 846-50). Loading the erythrocyte membrane with pentacyclic triterpenes inhibits Plasmodium falciparum invasion (HL Ziegler et al., Planta Med 2006, 72, 640-2). Oleanolic acid also has demonstrated antileishmanial activities (TS Melo et al., Parasitol int 201665 227-237) and its numerous positive health effects have been patented for several pharmaceutical drugs in China (C Lin et al., Expert Opin Ther Pat 2016 Apr 2016).

A paper from Panama found that the triperpenes α-amyrine, β-amyrine, ursolic acid, oleanolic acid, betulinic acid from Pelliciera rhizophorane inhibit α-glucosidase and are 100 times more potent than the drug acarbose commonly used against diabetes. In these trials kaempferol, quercetin, quercetrin showed no effect (D Lopez et al., Chemistry Central Journal, 2015, 9:53). The same paper studied the effect of these triterpenes and found that oleanic acid and betulinic acid are strongest against Leishmania donovani, oleanolic acid strongest against Plasmodium falciparum and α-amyrin strongest against Trypanosoma cruzi. Often the crude extract has better properties than isolated molecules. Ursolic acid from Minuscops caffra inhibits hemozoin and has an in vitro IC50 of 6.8 µg/ml; the crude extract 2.14 µg/ml (Mthokozisi S, University of Zululand, 2014). Miliacin is present in Azadirachta indica (Neem) leaves and seeds, alongside many other triterpenoids, which could explain the antimalarial properties of this plant.

Lupane type triterpenes from the bark of Zisiphus cambodiana have strong antiplasmodial and antimycobacterial properties (S Suksamrarn et al., Chem Pharm Bull 2006, 54, 535-537). Already in 1999 it was found that pentacyclic triterpenes from the Tanzanian tree Uapaca nitida had strong antimalarial properties (J Steele et al., Phytotherapy Res. 1999, 13, 115-119). Another stem bark from Spathodea campanulatu showed strong antiplasmodial properties related to ursolic acid (O Oluwole et al., Phytotherapy Res. 1996, 10, 692-693). At the University of Antioquia in Colombia it a was found that ursolic acid from Calophyllum inophyllum had strong antimalarial properties in vitro (A Mesa-Vanegas et al., Revista QuimicaViva, 2011 2, 118-126). The root of Glycyrrhiza glabra in India contain glycyrrhetinic acid with dose dependent antiplasmodial properties (K Kalani et al., PLOS ONE 2013, 8-9, e74761). Ampeloziziphus amazonicus is a plant widely used by the population of the Amazonian region to prevent and treat malaria. It contains several pentacyclic tripterpenes, the strongest among them against Plasmodium is betulinic acid (FM Dominique et al., Pharmacognosy Magazine, 2015 11, 244-250). The most interesting feature in this research is that the extract of the root bark has good hepatic antiparasitic properties. This deserves more attention because if the extract is sporozoitocidal this could explain the prophylactic properties described in the paper of Mavondo (op.cit.). As noted previously betulinic acid causes modifications in the cholesterol-rich erythrocyte membrane preventing Plasmodium falciparum from entering the erythrocyte which is preloaded with this triterpene.

Asiatic acid is mainly present in Centella asiatica, often consumed as vegetable. A recent in vivo trial with Plasmodium berghei-infected mice was run with alcoholic extracts. Compared to control parasitemia was reduced up to 75% (HS Banyal et al., Asian J Biolog Sc, 2014 ISSN 1996-3351).

But if further trials with asiatic acid, oleanolic acid or maslinic acid should be run to confirm the potential as prophylactic and therapeutic drug against malaria, infusions from olive leaves are probably of easier access.

THE MEDITERRANEAN DIET

Consumption of fruit and vegetables has been associated with a lower incidence of diseases. Diets, especially along the Mediterranean coast, are correlated with healthiness. Mediterranean spices and fruits contain pentacyclic triterpenes from the lupane, oleanane and ursane groups,. For example, they can be found in rosemary, basil, origanum, salvia and other spices of the Lamiaceae family as well as within olive leaves and fruit. Virgin olive oil contains up to 197 mg/kg triterpenes, The fractionation of several hundred extracts led to the isolation of betulinic acid (BA), oleanolic acid and ursolic acid as the active principles. Known sources for triterpenes are mainly plant surfaces such as stem bark, or leaf and fruit waxes. These waxy coatings protect leaves and fruits against predators, insects and molds. Plant extracts rich in pentacyclic triterpenes have been patented US6433010B2 to fight pathogens. Apples are among the fruit most consumed worldwide and anti-tumoral effects from apples are correlated. Fruit peel of apples contains oleanic acid, ursolic acid and maslinic acid (« An apple a day, keeps the doctor away »). The average concentration of these triterpes is 0,2% (A Szakiel et al., Phytochem Rev 2012 , 11, 263-284). The grape berry cuticle is also characterized by a high content of waxes, and 60% thereof are oleanolic acid. Red wine has the reputation to have more health benefits than white wine. The major difference between making red wine and white wine is that the juice is fermented without the grape’s skins when making white wine. Propolis bee wax is very rich in pentacyclic triterpenes, ursolic and oleanic acid. It is thus not surprising that it has antimalarial properties (J Hutagalung Airlangga University, Indonesia).

The pharmacological relevance of these molecules has increased during the last two decades demonstrating multi-target properties such as wound healing, anti-inflammatory, anti-bacterial, anti-viral, antimalarial, hepatoprotective and anti-tumoral effects, combined with low or no toxicity.

Pierre Lutgen 8 May 2016