AÃ§ai (Euterpe oleracea) - ACS Publications - American

Chapter 13

Downloaded by STANFORD UNIV GREEN LIBR on May 13, 2013 | http://pubs.acs.org Publication Date (Web): March 18, 2010 | doi: 10.1021/bk-2010-1035.ch013

Açai (Euterpe oleracea) An Amazonian Palm Fruit with Broad Antioxidant and Anti-Inflammatory Activities Alexander G. Schauss,*,1 Gitte S. Jensen,2 and Xianli Wu3 1Natural

and Medicinal Products Research, AIBMR Life Sciences, Puyallup, Washington 98373 2NIS Labs, Klamath Falls, Oregon 97601 3USDA Arkansas Children’s Nutrition Center, Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72202 *[email protected]

The pulp of the Amazonian palm fruit, açai (Euterpe oleracea Mart.), has been found to have exceptional antioxidant capacity in vitro, particularly against the superoxide, peroxyl, and hydroxyl radicals. Studying foods for their potential health benefits to humans can benefit from in vitro assays that can correlate with in vivo outcomes. Using the cell-based protection of erthrocytes (CAP-e) assay can determine if red blood cells exposed to foods allow antioxidants to enter into cells and protect cells from oxidative damage. Using this assay, the reduction in fluoresecence has been shown to be proportional to the level of intracellular antioxidant protection. In comparing results from blood samples taken during a randomized, double-blind, placebo-controlled, cross over study involving an açai-based fruit juice taken by subjects in a state of oxidative stress, the CAP-e assay correlated well with levels of antioxidant compounds in cells and reduction in lipid peroxidation. A dose dependent reduction in formation of reactive oxygen species in polymorphonuclear (PMN) cells has shown açai pulp to be active even when diluted down to 0.1 picogram/mL, suggestive of cellular signaling, and indicative that the pulp may contain compounds with the potential to be a © 2010 American Chemical Society In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

food able to exhibit potent anti-inflammatory activity in vivo. A pilot study on range of motion and joint pain in older adults supports this hypothesis, when studied in in older subjects with mild to moderate inflammation of the knees and lumbar spine.


Introduction The palm fruit, Euterpe olearace Martius (E. oleracea), is one of three species of edible palm fruit that are members of the genus Euterpe. These three species, E. olearacea, E. edulis, and E. precatoria, are only found growing within the boundaries of the Amazon region, particularly Brazil. Natives living in Brazil refer to these three fruit species by the same name, açai (pronounced ah sigh ee), although the fruit of E. edulis is also referred to as jussara or jucara. The most dominant fruit gathered commercially as a food or nutraceutical ingredient is that of E. oleracea. The pulp of this fruit is primarily processed for use as a juice for domestic consumption and export. The fruit is harvested in the Brazilian states of Para and Amapa, where it is found growing in abundance among the hundreds of islands within the complex floodplain ecosytem of the Amazon River and its tributaries. Due to its perishability, it is rapidly shipped after harvesting to regional processing facilities to be made into frozen pulp, or further processed into freeze-dried or spray-dried powders. These powders can be added to water or fruit/berry concentrates to produce açai-based juices. Generally, açai pulp is mixed with more flavorful fruits and/or berries to improve taste, as the pulp tastes slightly chalky. Fresh fruit is not available outside of the harvest season domestically, nor found sold in its whole fruit form outside of Brazil, due to its perishable nature. In Belem, the largest city within the Amazon basis, located in Para state, with a population exceeding one million residents, daily per capita consumption of açai pulp juice is estimated to be nearly two liters a day from July to December. Açai fruit stands that expel the seed and provide açai juice are found all over the city, sometimes with several retail açai juice stands no more than a few blocks apart. A characteristic of the species is its growth in clumps, a result of basal suckering. The number of these clumps varies and can reach 25, including suckers, depending on environmental conditions (1). The palm has a thin trunk and averages 15 to 20 meters in height. The number of fruit clusters varies, with three to five common. Each cluster of infloresence-producing fruit is at a different stage of development, allowing for several harvests of fruit from the same palm tree within a six month period. The fruit is round and between 1.5 to 2.5 cm in diameter; the edible mesocarp of the fruit is dark purple. Like most fruits found in the Amazon, it is highly perishable. Açai palm trees are found abundantly growing over a range of approximately 7 million square kilometers within the eastern portion of the Amazon, with the highest density of palms, up to 7,000 trees per hectare, located in the Brazilian states of Para and Amapa. Locals living in this region harvest the fruit from June through December. 214 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Harvesting the fruit requires climbing to within a few meters of the top of the palm to remove the mature fruit clusters, which are attached to the infloresence. Each cluster can weigh between one to two kilos. Accomplished harvesters can swing from one palm to another and remove several clusters before returning to the ground, where the fruit is stripped and placed in woven baskets for transport. Transport almost always is done by canoe or boat where baskets are aggregated into the hulls and then transferred into larger vessels that can take up to 2,000 fourteen kilo baskets to collection points where they are transferred to processing facilities, a cycle that goes on seven days a week throughout the dry season. Processing of the fruit to preserve it nutritional density, organoleptic qualities, and phytochemical composition, must be performed within 18-20 hours of harvesting. Thereafter the fruit pulp will diminish in quality and desirability. When processed, the skin is soaked for removal, leaving behind the pulp and seed. The seed constitutes between 82% to 89% of the fruit, and is expelled, leaving only the pulp to be further processed for various food uses, the most common of which is as a juice. The percentage of solids in the pulp following processing varies between 14% to 6%, with the higher percentage considered the highest grade of pulp. The nutrient composition of de-seeded açai fruit was first reported in 1945 (2). Açai and farinha (Manihot esculenta) (derived from cassava – an edible starchy tuberous root), can constitute up to two-thirds of the calories found in the diet of natives that live in thousands of rural villages and communities within the flood plains (varzea) where the fruit grows abundantly. In 2006, the nutritional composition of açai pulp for its vitamins, minerals, amino acids, and fatty acids, and phytochemical composition, particularly of polyphenols, was reported by chemists working in several labs in the United States (3). The pulp was found to have a surprisingly high concentration of monoand poly-unsaturated fatty acids; 82% of its unsaturated fatty acid composition in freeze-dried samples of açai pulp due to the presence of oleic acid and linoleic acid, higher in unsaturated fatty acids than found in olive oil or avocado oil, but likely comparable in concentration given variability of these oils and açai pulp (3).

Assessment of Safety Based on Historical Consumption and Toxicology Studies A study of the diet of paleoindian cave dwellers in the Amazon has confirmed that a significant energy source for humans living during the Pleistocene and Holecene occupations included palm fruit (4–6). The use of açai as a food source in the Amazon was chronicled by British, Portuguese and American explorers beginning with the 18th century account of noted botanist, Joseph Banks, who visited Brazil in 1768. In an entry to his diary dated December 7, 1768, Banks described the Açai fruit as “palm berries that appear much like black grapes but for eating have scarcely any pulp covering a very large stone” (7). Renowned American naturalist, William H. Edwards, lived 215 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


in Para state in 1846. In describing the importance of the fruit in the local diet, he wrote: “From various palm fruits are prepared substances in great request among difference classes of people … The fruit is covered by a thick skin, beneath which, imbedded in a very slight pulp, is the stone. Warm water is poured on, to loosen the skin, and berries are briskly rolled together in a large vessel. The stones are thrown out, the liquid strained off the skins, and there is left a thick, cream-like substance, of a purple color…To a stranger, the taste is, usually, disagreeable, but soon, it becomes more prized than all fruits beside, and is as much a necessity as one’s dinner” (8). A more recent account of how little has changed since 1846 in terms of local preparation of the fruit as a juice is described by researchers affiliated with the Nature Conservancy who reported in 2004 that, “The rind (mesocarp) of the açai fruit is ground and mixed with water to form the thick, purple vinho, which forms a staple part of the ribeirinho diet. In towns and cities, açai is processed into vinho at small stands or shops and sold by the litre...”((9), p. 318) A series of unpublished toxicology studies have been performed of açai pulp either in the form of freeze-dried pulp powder or as found in a juice product whose ingredient is predominantly acai pulp (freeze-dried and frozen) (Monavie Active®, Monavie LLC, Salt Lake City, UT, USA). In an acute oral toxicology study by gavage, administration of single oral doses of 5,000 mg/kg body weight (bw) and 20,000 mgkg bw, observed for lethality and toxic symptoms for 14-days post treatment, resulted in no lethality, clinical symptoms, or gross pathology. Hence, the single oral LD50 is determinated to be higher than 20,000 mg/kg bw for the juice. An acute toxicology study in rats given 2,000 mg/kg bw of freeze-dried açai pulp (OptiAcai®, K2A, Springville, UT, USA) by intubation, with a 14-day post-treatment observation period, resulted in no lethality, adverse effects, or gross pathology. Toxicity studies were also performed to assess mitogenicity, mutagenicity and clastogenicity of the açai-based juice. Cell cytotoxicity was not observed following 48 hours of incubating mulitplates containing L929 or Balb/3T3 clone A31 cells using the cytotoxicity test model. Negative findings were reported for the following assays of açai pulp and/or the açai-based fruit juice: bacterial reverse mutagenicity assay (AMES test), mammalian chromosomal aberration test in Chinese hamster V79 cells, mouse peripheral blood micronucleus assay, in vivo genotoxicity test by bone marrow micronucleus assay in adult mice BALB/c strain, and the mammalian cell gene mutation (L5178Y/TK mouse lymphoma) assay (unpublished). In the latter, no mutagenic effect was observed with and without metabolic activation. In the mammalian chromosomal aberration assay, when tested up to cytotoxic concentrations, both with and without metabolic activation, it did not induce structural chromosome aberrations in Chinese hamster lung cells. Therefore, it was not found to be clastogenic in this sytem. In the in vivo bone marrow micronucleus assay neither oral nor intraperitoneal administration showed an ability to induce an increase of micronuclei frequency in polychromatic erthrocytes in the bone marrow of BALB/c mice. The Ames test showed that neither the juice was mutagenic in using either Salmonella (TA98, TA100, TA1535, and TA1537) or E. coli {WP2 (uvrA)] strains tested in triplicate. There were no revertants exceeding the background average either 216 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

with or without metabolic activation, nor any dose-related increase over the range tested. The freeze-dried acai pulp similarly failed to show the test item was mutagenic in either of two experiments in the presence or absence of metabolic activation, tested in triplicate, at concentrations up to 5000 micrograms per plate under the conditions employed.


Comparative Antioxidant Activity Considerable scientific interest in this fruit arose following the 2006 publication by multiple investigators and laboratories that açai had stronger antioxidant capacity compared to other antioxidant-rich fruits, berries and vegetables tested by the U.S. Department of Agriculture’s Agricultural Resesarch Services (USDA-ARS), based on the oxygen radical absorbance capacity (ORAC) assay. The ORAC assay had been used extensively by USDA-ARS, to compare various freeze-dried fruits, vegetables and nuts, for their scavenging capacity against the peroxyl free radical in vitro (10–12). Initial evidence reported by Pozo-Insfran and colleagues in 2006, found that açai polyphenolics in their glycoside and aglycone forms induced apoptosis of (HL-60) leukemia cells due to caspase-3 activation. This paper stimulated further investigation of the fruit’s bioactivity in vitro and in vivo (13). The author’s present findings report on the use of a novel assay to document the bioavailability of antioxidants in açai pulp, both at the cellular level, and in human subjects, after acute or chronic daily consumption.

Antioxidant and Anti-inflammatory Activity of Açai Pulp Reactive oxygen species (ROS) are produced in aerobic organisms and are widely believed to play a pivotal role in aging and a number of degenerative diseases. Freeze-dried de-seeded açai pulp powder has been shown to have exceptional antioxidant properties against superoxide (O2•−) by the SOD assay, as well as against the peroxyl radical (RO2•) by the ORACFL assay. Freeze-dried açai pulp had the highest reported superoxide scavenging capacity in vitro of any food tested at the time of publication, at 1,614 SOD units/g (14). The ORAC-hydrophilic/lipophilic score of 1026.9 µmole Trolox equivalent (TE)/g represented a scavenging capacity that exceeded any other freeze-dried fruit or vegetable tested by USDA-ARS, with the exception of certain spices, while also showing moderate squenching capacities against both the peroxynitrite radical (ONOO–) and hydroxyl radical (OH•) (14). Based on the content of potent antioxidant compounds found in the fruit reported in 2006 (14), açai pulp was tested in biological assays, to evaluate whether the antioxidants played a specific role in biological systems in vitro.

217 In Flavor and Health Benefits of Small Fruits; Qian, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


Testing of Pulp in Fresh Human Cells As a direct extension of the tests of açai pulp for antioxidant capacity, the pulp was tested in the CAP-e bioassay, which specifically measures to what extent antioxidants can enter into and protect living cells (15). It was found that antioxidants in açai pulp were able to cross the lipid bilayer cell membrane of fresh human red cells and provide intracellular protection from oxidative damage (15) (Figure 1). An open trial was conducted, followed by a randomized, double-blind, placebo-controlled cross-over study. The primary antioxidants in the açai-based juice tested (Monavie Active®, Monavie LLC, Salt Lake City, UT) were in the form of polyphenols, predominantly flavonoids such as anthocyanins. In the first open pilot study, consumption of a single acute dose (120 mL) of the açai-rich juice, following an overnight fast to induce oxidative stress, resulted in a significant increase in antioxidant protection in vivo, confirmed by testing blood samples before drinking the juice and at 1 and 2 hours following ingestion. The cell-based antioxidant protection in erythcrocyte (CAP-e) assay was performed of the serum, according to the method of Honzel and colleagues (15), to see if the antioxidant compounds in açai were capable of providing cellular protection. The results reflect the antioxidants in serum that are actually able to enter into living cells and thereby potentially contribute to a reduction of oxidative damage within the cell. The CAP-e assay were compared to the thiobarbituric acid reactive substances (TBARS) assay which measures the rate of lipid peroxidation (15). In the randomized clinical trial, blood samples at baseline, 1-hour, and 2-hours following consumption of the juice or placebo were tested for antioxidant capacity using several antioxidant assays as well as the TBARS assay. A within subject post consumption comparison showed an increase in serum antioxidants at 1-hour (p

AÃ§ai (Euterpe oleracea) - ACS Publications - American

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