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The strong prophylactic and antimalarial properties of polyunsaturated fatty acids.

February 3, 2016 - 13:08 -- Pierre Lutgen

ABSTRACT

Artemisia plants are rich in polyunsaturated fatty acids (PUFA) which generate prostaglandins and stimulate monocytes. PUFAs possess well documented antimalarial and prophylactic properties. Their half-life in plasma is several days and in adipose tissue several weeks. This may explain the prophylactic effect of regular consumption of Artemisia infusion or powder.

INTRODUCTION

In a previous document on www.malariaworld.org it was shown that fish oils rich in polyunsaturated fatty acids (PUFAs) cause a marked in vitro growth inhibition of Plasmodia. Similar effects were seen in vivo on mice infected with Plasmodium berghei and treated during 4 days with these acids. The effect is not only suppressive but also prophylactic (L Kumaratilake et al., J Clin Invest 1992, 89 , 961-967). A very recent thesis from the French CNRS confirms that PUFAs derived from human phospholipids exert a potent in vitro anti-plasmodium activity primarily by hydrolyzing lipoproteins from plasma, thereby releasing PUFAs that are toxic to the parasite (C Guillaume et al., Infect Immun March 2015 accepted manuscript).

An infant is usually born with a deficient immune system, and the long chain polyunsaturated fatty acids (LC-PUFA) in breast milk play an important role in the development and maturation of infant’s immune system. Foetal cord blood contains higher portions of n-3 and n-6 long-chain PUFA than maternal blood. (W Schlörman et al., Nutr Res. 2015. doi: 10.3402).

It is assumed that during the blood stage of malarial infection the Plasmodium falciparum reproduces in a safe haven, the food vacuole, totally isolated from the hostile outside world of host defenses. Nevertheless the parasite must import a multitude of substances and export others. To this effect he grossly alters the structure and composition of the host erythrocyte. The phospholipid and fatty acid compositions change. The unsaturation index is considerably lower than in uninfected erythrocytes (L Hsiao et al., Biochem J 1991, 274, 121-132). Large increases in palmitic acid from 21.9 to 31.2, in oleic acid from 14.6 to 24.6 and major decreases in arachidonic acid from 17.4 to 7.8, in docosahexaenoic (DHA) from 4.9 to 1.8 %. The parasite, during intra-erythrocyte maturation, markedly refashions the composition of the phospholipids.

The analysis of residual erythrocyte plasmas, food vacuoles and hemozoin identifies predominantly saturated palmitic and stearic acids, and smaller amounts of oleic and linoleic unsaturated fatty acids. Arachidonic acid is absent (JM Pisciotta et al.,Biochemical Journal 2007, 402, 197-204). This confirms the results of another paper (C Fitch et al ., Biochimica and Biophysica Acta 2000, 1535, 45-49). In Plasmodium infected mice erythrocytes palmitic acid increased from 161 to 830 nmol/ml, stearic acid from 35 to 256, oleic acid from 73 to 260 and linoleic acid from 6 to 211 nmol/ml. When radioactive carbon labeled palmitic, stearic and oleic acids were incubated with infected rhesus monkey Plasmodium knowlesi these were incorporated into lipids of the erythrocytes, the intracellular parasites. Another author found similar increases in saturated fatty acids and decreases in PUFAs for Plasmodium iopurae in ducks, Plasmodium knowlesi in monkeys and Plasmodium berghei in rats (GG Holz , Bulletin of the WHO, 1977, 55, 237-248). Since malaria parasites lack the capacity to synthesize fatty acids an expanded pool of short chain acids probably supplies the building blocks for those classes of lipids needed for parasite survival, and more particularly for hemozoin crystallization.

For the culture of Plasmodia plasma can be completely replaced by the saturated stearic acid (WA Siddiqui et al., Science, 1967, 156, 1623). This has been confirmed by PI Trigg (Parasitology, 1969, 59, 915 and 925). A fatty acid mixture, rich in stearic acid was also found to be an ideal growth medium for eggs for Schistosoma mansoni eggs in vitro (GR Newport et al., Am J Trop Med Hyg 1982, 31, 349-57). In 1960 already it had been found that only stearic acid and oleic acid were satisfying the growth needs for Paramecia. Unsaturated fatty acid are inactive (CA Miller et al., J Protozoology 1960 7, 297-301). Similar results were found in 1962 at Elizabethville for Trypanosoma cruzi. Stearic acid is the essential growth factor. None of the longer chain fatty acids, saturated or unsaturated, had a growth promoting effect ((GJ Boné et al., J Gen Microbiol 1963, 31, 261-266). These are important facts in light of the absence of a stearic acid in Artemisia annua (WE Nibret et al., Phytomedicine, 2009, PHYMED 50735).

PUFAs IN PLANTS

Not only PUFAs from fish oil but also from plants have an effect. When comparing palm oil and corn oil with fish oil in malaria induced by Plasmodium berghei in Swiss mice (Blok WL et al., J Infect Dis. 1992;165(5):898-903) it was found that cerebral malaria occurred in only 23% of fish oil-fed mice, in 61%, in the corn oil group and 81% in the palm oil group. Survival in the fish oil group was 78%, 38% in the corn oil group and 20% in the palm oil group.

According to Brisibe et al., (Food Chemisitry, 2009, 115, 1240-1246) Artemisia annua leaves contain up to 10% of fatty acids in dry mater. Another author finds 6% (S Iqbal et al., Molecules 2012, 17, 6020-32).This is extremely high, at the same level as for plants which are used for oil production, as for example flax i.e. Linum usitatissimum (A de Aguiar et al., Ciencias agrotec Lavras 2010, 34, 1500.1506). Another study on 39 aromatic plants finds values ranging from 0.7 to 4.5% (C Pereira et al., Thesis CIMO, Bragança). In forage leaves the content is 2.3% dw (DL Palmquist et al., J Anim Sci 2003,81, 3250-3254). In the medicinal plant Panax ginseng the total concentration of fatty acids is 5.1 % dw and 62% of these are PUFAs (X-J Zhang et al., Chemistry Central Journal, 2013, 7:12). The concentration in periwinkle (Cantharantus roseus) is 2,3 % (S Mishra et al., Braz J Plant Physiol 2006, 18, 447-454), in switchgrass (Panicum virgatum) 5.1 %, in the herb Brachypodium distachyon 3.9%, in the cress Arabidopsis thaliana 5.3 % (Zhenle Yang et al., Plant physiology, 2009, 150, 1981-89), in Neem flowers 3.06%. A study on edible plants finds 1.70 for spinach, 0.60 in lettuce, 1.10 in mustard mg/g wet weight which would correspond to 1% fatty acids in dry material (AP Simopoulos, Biological Res., 2004, 37,263-277).

Care must be taken because leaves of medicinal plants lose most of their fatty acids during senescence (Zhenle Yang, op cit).

Nuts and seeds are particularly rich in PUFAs: pumpkin seeds 20.9%, sunflower seeds 23.1%, almonds, 12.1%, pistachios 13.2%, chestnuts 0.9%, hazelnuts 7.9% dw. In breast milk 90% of the fatty acids are PUFAs.

Unfortunately there aren’t many documents in the scientific literature which analyzed Artemisia annua for the content of saturated and unsaturated fats. The analysis is not very easy (DL Palmquist, op cit) The high content of polyunsaturated fatty acids (PUFA) in Artemisia herba alba is confirmed by feeding studies. Dietary supplementation with essential oil from A herba alba increases the content of these fats in the muscles of lambs much more than essential oil from Rosemary (V Vasta et al., Meat Sci2013 95, 235-41).

A phytochemical analysis of five Artemisia species in Turkey shows that saturated fatty acids in these plants represent on the average 40 % of the total and the unsaturated fatty acids 60 %, including those with antimalarial activities like linoleic acid, arachidonic acid and linolenic acid (M Kursat et al., Notulae Scientia Biologicae, 2015, 7, 495-499). These three PUFAs act as precursor for the synthesis of longer chain PUFAs like EPA and DHA in liver and brain (W Smink et al., Animal 2012, 6, 262-70)

Fatty acids are known as self-defensive agents in organism and possess various biological activities including anti-inflammation. It is known that NF-kB activation involved in the inflammation development and the inhibition of this transcriptional factor is considered as a therapeutic target for inflammation treatment (B Xue et al., PloS One, 2012. 7.e45990). An extensive review (J Schumann et al., PLoS One, 2011 1;6 e24066) describes how PUFAs modulate the macrophage membrane domains, promote the phagocytosis rate as well as bactericidal capacity of macrophages by increasing the concentration of ROS and NO inside of the macrophage, impact their respiratory burst and down-regulate the synthesis of the pro-inflammatory cytokine IL-1β, IL-6 and TNF-α.

The invasion of blood by Plasmodium triggers inflammation and increases in IL-1β and TNF-α, leading to a systemic inflammatory cascade, renal failure, hypoglycemia, lactic acidosis and death. During the development of the parasites large increases of palmitic, myristic, stearic and oleic saturated acids and major decreases in polyunsaturated acids like DHA and EPA occur. These modifications must be a result of parasite metabolism. Plasmodium tries to obtain in the infected erythrocyte and in the food vacuole a fatty acid composition required to obtain maximal down-regulation of TNF-α output and protection against damaging effects. These favorable products are released into the medium during schizont rupture and merozoite release (F Debierre et al., Infection and Immunity, 2006, 74, 5487-96).

All this is described in detail in an obsolete patent filed in 1992 (US005604258A). Docosahexaenoic (DHA) acid, eicosapentaenoic (EPA), acid arachidonic acid (AA) and linoleic acid (LA) had a strong inhibitory effect on various Plasmodium falciparum isolates and in vivo on Plasmodium berghei in mice, at daily intraperitoneal doses of 10 µg. The antimalarial activity was concentration dependent. The monounsaturated oleic acid had a week effect, but none of the saturated fatty acids had any effect, even long chain ones. After oxidation PUFAs had a stronger effect. Antioxidants like Vit E, BHT, SOD, CAT reduced the antimalarial effect of PUFAs up to 70%. Importantly, the PUFAs showed no toxic effects in mice but, in fact, prevented malaria-induced anemia.

Polyunsaturated fatty acids (PUFAs) lead to a 40% lower risk of type 2 diabetes (J Salmeron et al., Am J Clin Nutr, 2001, 73, 1019-26). In male Wistar rats intake of fish oil containing DHA and EPA acids leads to increase in glucose utilization and insulin sensitivity. Breast-fed infants have a significantly higher percentage of DHA and lower plasma glucose concentrations than do formula fed-infants (LA Baur et al., Metabolism, 1998, 47. 106-12). And as a lower glucose content in blood leads to less malaria infections. Plasmodia also need cholesterol as food. After a malaria infection the plasma concentration of cholesterol is lower. It is well known that PUFAs lower plasma cholesterol (DF Horrobin et al., Lipids. 1983, 18, 558-62).

In summary, PUFAs have a strong starving action on the malaria-causing pathogen, Plasmodium spp. PUFAs,

PROSTAGLANDINS AND PROPHYLAXIS

There are many anecdotal reports on the prophylactic effect of Artemisia annua and this has been documented in several peer reviewed papers PE Ogwang et al., Brit J Pharm Res 2011, 1, 124-131 and Trop J Pharm Res. 2012, 13, 445-453). But the mechanism of this action has not been elucidated. The prophylactic effect is not due to artemisinin and derivatives. If this was the case, it would have been broadcasted in all the media since years. Artemisinin is even immunodepressive. The advantage of Artemisia herbal polytherapy over artemisinin monotherapy in ACTs is significant.

One hypothesis for this prophylactic effect is resting on the high concentration of fatty acids in medicinal plants. The prophylactic effect of PUFAs is well documented in the literature. Preliminary studies had indicated to a research team in Norway a suppressive influence of fish oils on rodent malaria (Fevang P et al., Lipids. 1995, 30(5):437-41). In a subsequent work, for two or four week groups of weaning male mice were fed a standard laboratory diet or one of eight purified diets containing various amounts of fish oil (providing 6-21% of energy). The diets were prepared with and without vitamin E. After the two- or four-week feeding period, the mice were injected intraperitoneally with Plasmodium yoelii-infected erythrocytes. Six months after the primary infection (four months after discontinuing fish oil feeding), the surviving mice were again injected intraperitoneally with parasitized red blood cells (or even better--erythrocytes, erythrocytes are used elsewhere). Primary malaria infection was lethal in mice fed standard diet alone or with fish oil and vitamin E added. In contrast, feeding a fish oil-based diet without vitamin E improved survival to at least 70% if the mice had been fed these diets for four weeks. Protection against malaria did not seem to be related to the fish oil dose used. Regardless of the previous fish oil dose, all the mice surviving the primary infection survived the rechallenge infection with low parasitemia. A similar effect was found with menhaden-fish oil (OA Levander et al., J Parasitol. 1995 Feb;81(1):99-103). Feeding 20% menhaden-fish oil in a standard laboratory chow diet for 4 wk partially protected mice from the central nervous system consequences of infection with Plasmodium berghei.

The effect of one of the PUFAs, arachidonic acid, ARA, on bilharzia has been extensively studied at the University of Cairo (R El Ridi et al., Antimicrob Agents and Chemother. 2010, 54, 3383-3389). They have demonstrated that 5 nM ARA leads to irreversible killing of ex vivo 1-, 3-, 4-, 5- and 6- weeks old Schistosoma manzoni within 3 to 4 hours. This efficiency could be duplicated in vivo in mice indicating that using ARA in pure form or included in an infant formula consistently led to a 40 to 80% decrease in total worm burden.

A possible link with malaria is that the addition of ARA to several Plasmodium falciparum strains grown in vitro led to a significant increase in prostaglandins PGD, PGE and PGF, molecules which are detrimental for the parasite. In the absence of ARA the production of prostaglandins was insignificant (K Kubata et al., JEMM, 1998, 188, 1197-1202). It is well known that prostaglandins are lipids mainly derived from arachidonic acid which is one of the major constituents of peanut oil. But DHA end EPA play a role too (K Shane Broughton et al., Nutrition Research, 2009, 29, 510-18); DHA intake alone increases PGE 1.5 fold. Linoleic acid intake also increases PGE₂ (W Schepp et al., Gastroenterology, 1988 95 18-25). Prostaglandins both sustain homeostatic functions and mediate pathogenic mechanisms, including the inflammatory response. But that does not clearly explain why they play a role in malaria. In children with cerebral malaria their production is impaired and this often leads to adverse outcomes, whereas elevated levels of PGE₂ in asymptomatic parasitemia suggest a potential role for PGs in protective immunity (DJ Perkins et al., JID, 2005, 191, 1548-57).

Already in 2000 it had been demonstrated in a study on Gabonese children with and without malaria that prostaglandins are important pro-inflammatory mediators of the host-immune response to infection. (JB Weinberg et al., Blood. 2000, 96). The authors postulate that PGE₂ levels in healthy malaria-exposed children protects against malaria, while decreases in PGE₂ during acute malaria increase susceptibility to severe disease. This has been confirmed in Tanzanian children (GJ Perkins et al., op cit). The authors suggest that high levels of PGE₂ in children with asymptomatic parasitemia may contribute to the maintenance of malaria tolerance, which is the ability to tolerate circulating parasites without fever.

In 1983 already it was claimed that PGE₂ derived from arachidonic acid plays a clear role in the regulation of cellular and humoral responses (JS Godwin et al., J Clin Immunol. 1983 3, 295-315). PGE₂ regulates macrophage derived TNF-α. (S Kunkel et al., J Biol Chem. 1988, 11, 5380-84). These studies confirmed that this prostaglandin can regulate macrophage activity. PUFAs promote the phagocytosis of bacteria (S Adolph et al. Curr Microbiol 2012 65, 649-55).

We have several anecdotal reports indicating that prophylaxis with Artemisia annua is more reliable in people leaving in endemic areas. It is well documented that phagocytosis is present at higher levels in hyperimmune people than in people suffering their first attack (S Khusmith et al., Infection and Immunity, 1982, 874-879). PUFA’s may enhance this immune effect. This is in line with the fact that in endemic areas the concentration of IL-1β, which is released by macrophages, is much higher in the blood of residents than in non-endemic areas (D Prakash et al., JID 2006-194, 198-206).

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) reduce outward potassium currents in ventricular myocytes. The efflux of potassium from infected erythrocytes is a critical issue in malaria (Li HX1, Lipids. 2011 46:163-70). Arachidonic acid has an effect on ion channels, stronger than oleic acid and much stronger than stearic acid (H Meves, Brit J Pharmacol. 2008, 155, 4-16). PGE₁ activates ATP-sensitive potassium channels, which induces vasorelaxation ( S Eguchi et al., J Cardiovasc Pharmacol 2007, 50, 686-91).

Heme impairs PGE₂ formation. Patients with severe disease have lower concentrations than those with mild disease. Antimalarial treatments reverse the trend and PGE₂ see a 2 to 10 fold increase. This may be related to the crystallization of heme into hemozoin (B Andrade et al., J of Immunology , 2010, 185 1196-1204).

ARTAVOLᴿ FROM UGANDA

PGs are important soluble mediators involved in the immune response to invading pathogens and this may explain the strong prophylactic effect of ARTAVOL, a drug developed by Dr Patrick Ogwang from the University of Makerere and the Ministry of Health in Uganda. The powder contains lemongrass (Cymbopogon citratus), avocado (Persea americana) and Artemisia annua. Avocados contain 9% of fatty acids and 71 % of these are monounsaturated. The fatty acid of lemongrass is very rich in linoleic and oleic acid. Linoleic acid generates a strong PGE₂ production and oleic acid a weaker one (GM Denning et al., J Lipid Res. 1982, 23 584-90). The prophylactic effect of lemongrass and neem is known and well described (Mgebena IC et al., J Amer Sci. 2010 6). In ARTAVOLᴿ artemisinin has been removed from the Artemisia annua powder to avoid its immunodepressive effect. Artemisinin, dihydroartemisinin, arteanuin B, artemisinic acid strongly inhibit PG2 in vitro (XX Zhu et al., Pharmacol Reports 2013, 65, 410-420). The inhibitory effect of Artemisia annua extracts and of artemisinin has been confirmed in a recent paper from Korea (Wan-Su Kim et al., Korean J Physiol Pharmacol. 2015 19. 21-27). Artesunate also suppresses PGE₂ production in vitro (Uchechukwu P et al., Bioorganic & Medicinal Chemistry, 2014 17, 4726-34).

The effect may be less pronounced in vivo where the metabolism of these peroxides is very rapid. And the complexity of prostaglandin generation and inhibition is large. It is known that scopoletin and phytosterols also interfer (AB Awad et al., PLEFA, 2004, 70, 511-520). Acetaminophetamin (Paracetamol) inhibits the PGE₂ production. The hypocholesterolemic agent simvastatin activates the formation of AA from LA (Risé P et al., Prostaglandins Fatty Acids, 2002. 6, 85-9). Prostaglandins also alter the activity of the Na-K ATPase (Lockete et al., Circulation Research, 1980, 46, 718-720). Host genetic factors and blood characteristics (sickle cell, O blood group, thalassemia, G6DP, PKD) certainly play a role.

An effect Patrick Ogwang had noticed in his trials, was an increase in monocytes. This is in line with a thesis “The role of Omega-3 fatty acids in determining monocyte and macrophage phenotypes” (AL Brown, Wake Forest University, 2011). As fatty fish or fish oil is consumed in low quantities in the U.S. the author tried to determine whether the botanical oil from Echium plantagineum will alter macrophage phenotypes in a murine model. It had been noticed that echium oil reduced inflammation in a waysimilar to fish oil. Using flow cytometry it was found that PUFAs promote macrophage phenotype shifting from the more inflammatory M1 to a less inflammatory M2 phenotype.

PUFAs might also affect the invasion by sporozoites. A team from the University of Illinois (Project ILLU-888-918) identified a lipid fraction that inhibits Cryptosporidum parvum sporozoite invasion. Characterization of this lipid revealed it is a long-chain polyunsaturated fatty acid. Only PUFAs which are between 18-20 carbons long, have at least one double bond which is in the cis configuration, and have an unsubstituted carboxyl group are able to block sporozoite invasion. Preliminary data suggest these L-PUFAs inhibit invasion by blocking both sporozoite microneme secretion and gliding motility. Similar results were obtained for Eimeria tenella coccidiosis sporozoite invasion in vitro and in vivo (MS Crane et al., Parasitology 1991, 72, 219-222). The alteration affects the sporozoites and not the host cells. More recently it was confirmed (Jennifer Hanbyul Lee et al., FASEB J, 2009 23 Meeting abstract) that this inhibition of sporozoites by long chain fatty acids applies to many parasitic pathogens of the phylum Apicomplexa like Cryptosporidium parvum, Toxoplasma gondii, and Plasmodium gallinaceum. These parasites commonly infect a variety of vertebrate species and can produce life-threatening diseases, especially in the immunocompromised.

This inhibition of sporozoite invasion could be related to the production of the potent bactericidal protein BPI by EPA and DHA. In US patent 20010029245 it is claimed that BPI is also active against Leishmania, Trypanosoma, Plasmodium and Toxoplasma. Through its binding ability BPI derived from DHA and EPA may interfere with the binding of infectuous parasite forms to host cells. An in vivo study with mice, similar to the work of Patrick Ogwang has been made in Nigeria. They studied combinations of the plants Nauclea latifolia, Artocarpus altilis, Murraya koniigii, Enantia chlorantha. (AC Adebajo et al., Molecules, 2014, 19 doi :10.3390). In a prophylactic model the ethanol-water extracts were administred using oral cannula, daily 3 days before infection with Plasmodium berghei , in the chemosuppressive model during 3 days immediately after infection and in the curative model after 3 days of infection during 5 days. For all the plants and/or their mixtures a significant effect on parasitemia was noticed , although not as strong as for the control drug chloroquine. The most striking result is obtained for survival time in the prophylactic approach : on the average a doubling of the survival time compared to much lower effects in the chemosuppressive and curative approaches. This indicates that the herbal treatment has a strong effect on the first steps of the plaasmodial invasion and might be less effective on trophozoites ; it highlights the prophylactic and survival effect regular tea consumption.

Another trial combining several medicinal plants under the tradename SCAT is described in a thesis by Afzal Ahmad from Hamdard University Karachi in 2004 : Artemisia vulgaris, Sisymbrium irio, Tinsporia cordifolia, Caesalpina bonducela. The conclusion of a randomized clinical trial was that this herbal medicine is equivalent or even better than amodiaquine in reduction of parasitemia, but superior in avoiding side effects and relapses. Of considerable importance were the observations on gametocyte clearance with SCAT which showed a more rapid reduction in gametocyte numbers.

CONCLUSION

Artemisia plants are rich in linolenic, linoleic acid and arachidonic acid but lack EPA and DHA present in fish oil. However alpha linolenic acid is easily converted in a healthy human into EPA and later into DHA. Linoleic acid is converted into arachidonic acid. This metabolism is slow and half-life of AA, DHA and EPA in human infants is longer than 4 days (N Salem et al., Proc Natl Acad Sci). A more elaborate study on DHA and EPA supplementation shows that these fatty acids are rapidly incorporated into plasma lipids. The DHA concentration in plasma plateaus at 350 mmol/ml. The half-life in plasma is 4 days and in whole body 100 days (M Plourde et al., AJCN et al., 2014, doi 10.074708). In adipose tissue it even has a half-time of 680 as demonstrated by the use of radiactive PUFAs (A Baylin Am J Epidemiol., 2004,162, 373-381) This explains why the consumption of a few cups of Artemisia infusion or capsules per week might generate a prophylactic effect. As during malaria infection the concentration of unsaturated fatty acids rapidly declines, their presence and replenishment by the consumption of medicinal herbs may play an important role in prophylaxis and therapy.

Pierre Lutgen

2 February 2016.