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Anthocyanins and sickle cells

September 5, 2017 - 18:42 -- Pierre Lutgen

Anthocyanins are generally accepted as the largest and most important group of water-soluble pigments in nature. Research articles on anthocyanins have escalated dramatically in the last 20 years : 230 in the decade from 1982 through 1991, 753 from 1992 through 2001 and 3043 from 2002 through 2012 Anthocyanin pigments and associated flavonoids have demonstrated ability to protect against a myriad of human diseases.

        Mary Ann Lila, Anthocyanins and Human Health : an in vitro investigative Approach. J Biomed Biotechnol 2004, 5. 306-313

Anthocyanins had already fascinated botanists 200 years ago. Their findinds are summarized in a book of 340 pages published in 1916. Muriel Wheldale, The anthocyanin pigments of plants. Cambridge at the University Press, 1916.

A few importants findings or facts

- They are water soluble flavone derivatives, generally in the form of glucosides

- In acid solutions they are red and in alkaline solutions blue and green.

- Sugar enhances their production in the plants - The greatest production takes part in the illuminated parts, preferably in dry and sunny places

- The three major anthocyanidins are pelargonidin (orange/red), cyanidin (pink) and delphinidin (violet/blue)

Several people noticed that stems in the exterior part of an Artemisia annua plantations often have red stems, the plants in the interior not.. It is a way for the plant to protect itself from photoinhibitory stress. The anthocynidins are limited to the stem’s outer layer Anthocyanins can be detected and quantified by fluorescence.

         Giovanni Agati, Paolo Matteini, Joana Oliveira, Victor de Freitas, and Nuno Mateus. Fluorescence Approach for Measuring Anthocyanins and Derived Pigments in Red Wine. J. Agric. Food Chem., 2013, 61 (42), pp 10156–10162

         B. Pliszka, E Mieleszko. Application of fluorescence analysis in examination of anthocyanins in red cabbage. XVIIth Ars Separatoria- Borowno. Poland 202

In humans and animals, many of anthocyanins ingested are absorbed intact and circulate in the plasma. Animal model studies indicate that anthocyanins are rapidly absorbed, appearing in the bloodstream a few minutes after consumption.

         Matsumoto H, Takenami E, Iwasaki-Kurashige K, Osada T, Katsumura T, Hamaoka T. Effects of blackcurrant anthocyanin intake on peripheral muscle circulation during typing work in humans. Eur J Appl Physiol. 2005 May;94(1-2):36-45.

The bioavailability of the constituents of grape seed extract have been studied in the US. Gallic acid, epicatechin and proanthocyanidins were identified after gavage with these extracts. The absorption of small molecular weight contituents was enhanced by repeated dosing. Dimers and trimers of proanthocyanidins are also absorbed but not in their intact form. They are known to alter the microbial ecology in the lower intestine through antibiotic and prebiotic activities

         MG Ferruzzi, J Lobo, G Pasineti. Bioavailability of gallic acid and catechins from grape seed extract. J Alzheimers disease 2009, 18, 113-124

The detection of pure anthocyanins is often difficult because of strong molecular interactions with flavonoids. And the question remains open whether the health benefits stem from anthocynins alone, or from their synergistic interactions with other phenolic compounds. Anthocyanins frequently interact with other phytochemicals to potentiate biological effects, thus contributions from individual components are difficult to decipher. A study investigated the distribution of anthocyanins to various organs in rats fed with a blackberry anthocyanin-enriched diet for 12 days. The urinary excretion of total anthocyanins was negligable. The bladder contained the highest levels of anthocyanins followed by the prostate, testes, heart, kidneys, liver and adipose tissue. They are also taken up into human vascular endothelial cells. Anthocyanins have the ability to cross the blood-brain barrier. They have positive cardiovascular properties. Thus, anthocyanin feeding in rats resulted in a wide distribution of anthocyanin derivatives to several organs. Identification of target tissues of anthocyanins may then help to understand the mechanisms of action of anthocyanins in vivo.

         Felgines C, Texier O, Garcin P, Besson C, Lamaison JL, Scalbert A. Tissue distribution of anthocyanins in rats fed a blackberry anthocyanin-enriched diet Mol Nutr Food Res. 2009 Sep;53(9):1098-103. doi: 10.1002/mnfr.200800323.

The anthocyanidin metabolites also are bioactive. They alter the expression of inflammatory mediators like IL-6.

         HP Amin, C Czank, CD Kay, Anthocyanins and their physiologically relevant metabolites alter the expression of IL-6. Mol Nutr Food Res 2015, 59, 1095-1106

Some plants rich in anthocyanins and gallic acid have a pronounced antimalarial effect. This is the case for example for Phyllantus amarus used in traditional medicine.

        T Wulandari Kesetyangsih and Sri Sundari. Effect of bioactive Phyllantus niruri before and during infection with Plasmodium berghei in mice: Kne Life Sciences 2015, 2, 497-499

But gallic acid itself has no antimalarial effect. Recent assays of Mutaz Akkawi at Al Quds university fail to show any inhibition of beta-hematin even at high concentrations (personal communication).

Cocoa (Theobroma cocoa) contains phenolic acids, most notably anthocyanin. It is known for its antimalarial properties in many countries like Ghana or Mexico

        Andújar, M. C. Recio, R. M. Giner, and J. L. Ríos. Cocoa Polyphenols and Their Potential Benefits for Human Health Oxid Med Cell Longev. 2012; 2012: 906252.

It raises the CD4+ count as we found for Artemisia afra

        O Alademesemipe, B Solomon, O Afolabi. Antiplasmodial efficacy of crude cocoa powder extract on CD4+ count. Open J of Med Microbiology, 2013, 3,178-184

        Constant Kansango Tchandema, Pierre Lutgen In Vivo Trials on The Therapeutic Effects of Encapsulated Artemisia annua and Artemisia afra. GJRA Global Journal for Research analysis 2016 June, ISSN No2277-8160

Pomegranate rind has strong beta-hematin inhibitory properties and is rich in anthocyanins. It is sold as antimalarial drug in India under the tradename OMARIA.

        D. Bhattacharya ; American Journal of Tropical Medicine and Hygiene , Vol. 69 – No. 3 , Sep. 2003 (Philadelphia), pp 484. (Supplementary – Abstract Book ), Abstract No. 640.

The oral administration of Annona muricata aqueous leaf extract (100–1000 mg/kg) to mice for 4 days significantly reduced parasitemia in experimental mice, with nontoxicity. The plant is rich in anthocyanin and luteolin.

        Voravuth Somsak, Natsuda Polwiang, and Sukanya Chachiyo. In Vivo Antimalarial Activity of Annona muricata Leaf Extract in Mice Infected with Plasmodium berghei. Journal of PathogensVolume 2016 (2016), Article ID 3264070,

        José G. A. S. Júnior, Henrique D. M. Coutinho, Chemical Characterization and Cytoprotective Effect of the Hydroethanol Extract from Annona coriacea Mart. (Araticum). Pharmacognosy Res. 2016 Oct-Dec; 8(4): 253–257.

In the first weeks following harvest the anthocyanin content may increase. This was observed for red grapes or for pomegranate fruits.

        G Miguel, C Fontes, D Antunes, Anthocyanin concentration of Pomegranate fruits during different cold storage conditions. J Biomed. Biotech, 2004, 5, 338-342.

        A Marquez, M Serratosa, J Merida. Anthocyanin evolution and color changes in red grapes during their chamber drying. J Agric Food Chem. 2011, 61, 9908-9914.

The medicinal value of plants depends on phenolics and flavonoids. Exposure of plants to UV‐B causes flavonoid productIon. The higher temperatures in sun drying may inhibit the enzymatic degradadation of flavonoids and other constituents. The increase of flavonoids go in parallel and are not antagonistic. Both are five times higher in a plant grown under high light than in the shade.

        E Goto. K Hayashi, Effect of UV light on phytochemical accumulationof anthocyanin biosynthesis genes in red leaf lettuce. ISHS, doi 10.17660/Acta Hortic.2016.1134.24

        A Arakawa, Effect of UV light on anthocyanin synthesis in sweet cherry. J Japan Hort Sci, 1993, 62-3 543-546.

        NW Albert, DH Lewis, KM Davies. Light-induced vegetative anthocyanidin pigmentation in Petunia J Exp Botany. 2009, 60, 2191-2202

A large study on Mediterranean herbs shows that air dried herbs contain more flavonoids and polyphenols. This was for example noticed for Ginkgo biloba leaves.

        M Sun, X Gu J Tian, Change of secondary metabolites in leaves of Ginkgo biloba in response to UV-B irradiation. Innov Food Sci and Emerging Technologies 2010 11, 672-676

This increase was also noticed for phenolic compounds in blueberries

       I Eichholz, S Huyskens-Keil, A Keller. UV-B induced metabolites and phenolic compounds in blueberries (Vaccinium corymbosum). Food Chemistry 2011, 126 60-64.

and in cherries, apple, pomegranate UV light has more effect than white or red light

       AR Vicente, C Pineda, L Lemoine. UC-C treatments reduce decay, retain quality and alleviate chilling injury in pepper. Postharvest Biol and Technol 2005, 35, 69-78

       Photoregulation of anthocyanin synthesis in apple fruit under UV-B and red light. Thesis Leclercq, 1992, Wageningen

       H Borochov-Neori. S Judeinstein, D Holland.  J Agric Food Chem2011, 59, 5325-34

Climate effects on anthocyanin accumulation and composition in the pomegranate (Punica granatum) fruit arils..

In Artemisia annua blue light increases artemisinin and anthocyanin.

      Gao-Jie Hong, Wen Li Hu, Inreased accumulation of artemisinin and anthocyanins in Artemisia annua under blue light. Plant Molecular Biology reporter, 2009, 27,3, 334-341

The strong effect of light on anthocyanidin content may explain the differences seen for the same plant Artemisia in different papers. A study from 1985 claims that only stems contain anthocyaninins, leaves not. Another paper from Iran finds much higher concentrations in leaves than in stems.

       N P Todaria, Changes in Pigments and Total Phenolics in Artemisia species grown at different Altitudes. Biologia Plantarum 1985, 28, 307-309.

       M Mazandarani, Z Majidi, F Fathiazad, Essential oil Composition, Total Phenol, Flavonoid, Anthocyanin in different parts of Artemisia annua. J Medicinal Plants and By-Products 2012 1, 13-21.

A study in the US showed that the water extract of Cynodon dactylon rich in anthocyanins showed a good correlation between anthocyanin quantity and antimalarial properties.

       Khlifi D, Hayouni E, Valentin A, Cazaux S. LC-MS analysis, anticancer, antioxidant and antimalarial activities of Cynodon dactylon extracts. 2013, Industrial Crops and Products. Volume 45, February 2013, Pages 240-247

Pomegranate (Punica granatum) skin and leaves also are rich in anthocyanins and strongly inhibit beta hematin formation (Mutaz Akkawi, personaal communication)

                                                               Sickle cell disease and anthocyanins

Sickle cell disease is an inherited genetic disorder that affects the hemoglobin within the red blood cells. Drepanocytosis or sickling anemia is responsible for a great mortality in those affected by tropical diseases. The relationship between this blood disease and malaria is complex. On one hand, heterozygotes for the sickle gene are relatively protected against malaria. The plausible mechanism is that in heterozygotes Plasmodium falciparum infected red cells sickle preferentially and are then removed by macrophages. On the other hand, patients who are homozygous for the sickle gene and therefore suffer from sickle cell anemia, are highly susceptible to the lethal effects of malaria.

        Makani J, Cox SE, Soka D, Komba AN, Mortality in sickle cell anemia in Africa: a prospective cohort study in Tanzania. PLoS One. 2011 Feb 16;6(2):e14699. doi: 10.1371/journal.pone.0014699.

Globally 20-50 million people are affected by the disease and more than 50% of children born with the disease die before the age of 5 years. In sub-Saharan Africa it is estimated that annually 240 000 children are born with the disease. Several studies have been published on research conducted in the Democratic Republic of Congo. The molecule which in all these studies appears to be the most efficient in the antisickling effect are anthocyanidins, even at concentrations of 10 mg/L

        P. T. Mpiana, V. Mudogo, Y. F. Kabangu, D.S. T. Tshibangu, K.N. Ngbolua, E. K. Atibu, K.P. Mangwala, M. B. Mbala, L. K. Makelele and M. T. Bokota, 2009. Antisickling Activity and Thermostability of Anthocyanins Extract from a Congolese Plant, Hymenocardia acida Tul. (Hymenocardiaceae). International Journal of Pharmacology, 5: 65-70.

        D.D. Tshilanda, D.V. Onyamboko, D. T. Mwanangombo1, P.V. Tsalu, N.K. Misengabu, D.S.T. Tshibangu, K.N. Ngbolua, P.T. Mpiana, In vitro Antisickling Activity of Anthocyanins from Ocimum canun (Lamiacea), J of Advancment in Medical and Life Sciences, 2015, 3.2, ISSN 2348-294X

Roselle (hibiscus) also is effective against drepanocytose FB Mojiminiyi.

        Effect of Hibiscus sabdrariffa on erythrocyte indices in sckle cell anemia. 2016.

The polar extracts of Artemisia plants show the same antisickling effect

        Nessrin Ghazi Alabdallat, In vitro anti-sickling activity of Artemisia herba-alba (chih) methanolic extract on sickle cell disease. Asian J Pharmaceutical and Clinical Res 2016. 9, Online 2455-3891

Among green medicinal plants those of the Artemisia family appear among the highest in anthocyanins. Artemisia herba alba compared with two other medicinal herbs, Ruta chalepensis and Peganum harmala, contains up to ten times more anthocyanins.

        D.Khlifi, J Bouajila, Composition and anti-oxidant, anti-cancer and anti-inflammatory activities of Artemisia herba-alba, Ruta chalpensis L. and Peganum harmala L. Food and Chemical toxicology, 2013, 55, 202-208.

        Mehdi Mojarab, Seyyed Ahmad Emami, Mohammad Hassanzadeh-Khayyat. Antioxidant activity of methanol extracts of different species of Artemisia from Iran. Pharmacologyonline 2: 797-807 (2009)

It was found that the percentage of infected mosquitoes by gametocyte carriers of sickle cell hemoglobin was almost double that for normal hemoglobin.

        Robert V, Tchuinkam T, Mulder B, Bodo JM, Verhave JP, Carnevale P, Nagel RL. Effect of the sickle cell trait status of gametocyte carriers of Plasmodium falciparum on infectivity to anophelines. Am J Trop Med Hyg. 1996 Feb;54(2):111-3.

It appears that it is easier for sickle cell hemoglobin than for normal hemoglobin to succeed in the competitive interactions between the two alleles, despite the former’s higher blood disorder cost.

        Bronner P. Gonçalves, Sunetra Gupta, and Bridget S. Sickle haemoglobin, haemoglobin C and malaria mortality feedbacks Malar J. 2016; 15: 26.

This higher transmission of parasites in sickle cell affected patients has dramatic consequences for Africa.

In Burkina Faso a drug was developed based on the plants Calotropis procera and Fagara xanthoxyloides. The development was financed by the local government and the product is approved by the Ministry of Health. It is manufactured under the trade name FACA and sold at the price of 7.5 euro which is much lower than the standard pharmaceutical drug at 39 euro.

The benefits of similar developments are dramatic for Africa. They are fully in line with the WHO traditional medicine strategy: 2014-2023. They are also in line with WHO/EDM/TRM/2000.1 Traditional use refers to documentary evidence that a substance has been used over three or more generations of recorded use for a specific health related or medicinal purpose. In this case WHO maintains its position that there is no requirement for pre-clinical toxicity testing. Pre-clinical toxicity testing is only required for new medicinal herbal products which contain herbs of no traditional history of use