Food and Nutrition Sciences, 2011, 2, 35-46 35 doi:10.4236/fns.2011.21005 Published Online january 2011 (http://www.SciRP.org/journal/fns) Antioxidant and Antimicrobial Activities of Larrea Divaricata Cav. Aqueous Extract on Vitamin C from Natural Orange Juice Patricia Micucci1, Maria Rosario Alonso1, Sebastian Turner1, Roberto Davicino1, Claudia Anesini1,2 1IQUIMEFA-CONICET-UBA, Buenos Aires, Argentina; 2Pharmacognosy Unit, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina Email: [email protected] Received August 10th, 2010; revised October 9th, 2010; accepted October 17th, 2010 ABSTRACT Larrea divaricata Cav. is a South American plant widely distributed in Argentina with reported antioxidant and antim- icrobial activities. Vitamin C (Vit C) the most important water-soluble antioxidant, is degraded upon factors such as oxygen, heat, light, storage time and temperature, during cooking and in presence of microorganisms. The aim of this work was: to study the antioxidant and antimicrobial activity of an aqueous extract of L. divaricata (E) on Vit C pre- pared in water and on Vit C from natural orange juice, submitted to different store conditions such as temperatures and oxygen. The antioxidant activity was determined by DPPH method and used to obtained the relative Vit C concentra- tion; Vit C decreased its activity and concentration under temperature and air oxygen, but E allowed Vit C to recover its antioxidant activity and prevented Vit C microorganism contamination. These studies were performed in view to es- tablish whether L. divaricata can be used as a supplement on orange juices capable to prevent Vit C decay and micro- bial growth. Keywords: Vitamin C, Larrea Divaricata Cav., Antioxidant Activity, Antimicrobial Activity 1. Introduction food additive [2]. The North American Dietary Reference Intake recom- Vitamin C (Vit C) is considered as a most important wa- mends 90 milligrams per day and no more than 2 grams ter-soluble antioxidant. It is an electron donor and there- per day (2000 milligrams per day) [3]. It is generally fore a reducing agent, therefore, it has antioxidant prop- agreed that a balanced diet without supplementation co- erties preventing other compounds from being oxidized. ntains enough Vit C to prevent scurvy in an average It is known that the physiological and biochemical ac- healthy adult, while those who are pregnant, smoke toba- tions of vitamin C are due to its antioxidant capacity. cco, or are under stress require slightly more. However, Vit C by itself is oxidized during these proc- esses. Beside, Vit C is an essential nutrient for humans. The richest natural sources of Vit C are fruits and As it can directly scavenge superoxide radical, singlet vegetables, several fruit juices are fortified with Vit C, oxygen, hydrogen peroxide and hydroxyl radical and including apple, cranberry and grape juices; it is also protects compounds in extracellular and intracellular present in some cuts of meat, especially liver. Vit C in- spaces in most biological systems and participates in creases quality and technological properties of food as numerous metabolic reactions in all animals and plants, well as nutritional value [4]. therefore, is required for bone formation to scar tissue Orange juices are a rich source of Vit C [5]. Concen- repair and also for the synthesis of collagen in humans. tration of Vit C is a significant indicator of orange juice The major role of Vit C is the prevention of scurvy, this quality, and it may serve as an indicator that all processes, disease leads to the formation of spots on the skin, which ensure a high quality of the product, have been spongy gums, and bleeding from the mucous membranes applied in the production processes. [1]. Because of its antioxidant activity, Vit C is widely However, Vit C is an unstable compound and under used as supplement in different form such as tablets, less desirable conditions it decomposes easily [6]. Deg- drink mixes, supplemented orange juices and also, as a radation of Vit C proceeds by both aerobic and anaerobic Copyright © 2011 SciRes. FNS 36 Antioxidant and Antimicrobial Activities of Larrea Divaricata Cav. Aqueous Extract on Vitamin C from Natural Orange Juice pathways [7] and depends upon many factors such as tected from light and freshly prepared. Buffer TRIS. HCl oxygen, heat, light, storage temperature and storage time containing 1.2 g % P/V of TRISMA (SIGMA) and HCl [8] and also during cooking. Factors that affect the Vit C 0.1 M to reach pH 7.4. Agar Müeller Hinton (34 g/l) was contents of citrus fruits include, production factors, cli- prepared in distilled water. mate conditions, maturity state and position on the tree, 2.2. Natural Orange Juice type of fruits (species and variety), type of container, handling and storage [9,10]. Other causes include chemical Fresh fruits of Citrus sinensis (orange) were purchased changes from ripening and senescence (aging) processes from vegetable stores in Buenos Aires city. These fruits occurring in the fruit. Both low and high temperatures were washed thoroughly with water and the juice was can also cause decay on Vit C content. In addition, oxy- extracted by mechanical pressure. Then filtered to re- gen has shown to be the most destructive ingredient in move pulp and seeds and used immediately for the study. juice causing Vit C degradation. [11]. Physical store 2.3. Plant Material conditions do not only alter Vit C but also biological conditions, such as the presence of microorganisms. It is Leaves of Larrea divaricata Cav. (Zygophyllaceae), ide- known that, the growth of microorganisms, such as bac- ntified by employing morphological, anatomical and hist- teria and fungi, conduce to an important spoilage and ochemical techniques, where used during this study. One decay of Vit C. In turn, microorganisms generate free voucher specimen was deposited at CEFYBO-CONICET radicals species which are involved in the rapidly oxida- and another at the Museum of Pharmacobotany, School tive destroy of the vitamin. of Pharmacy and Biochemistry, University of Buenos So, the loss of Vit C might be a critical factor for the Aires. Aqueous extract was prepared from air-dried leaves shelf life of some products as citrus juice concentrates as follows: 750 mg were infused with 10 ml of sterilized [12] since Vit C content of citrus juices undergoes de- boiling distilled water and heated with stirring for 45 mm struction during storage. at 56˚C. After maceration at 5˚C for 72 h and centrifuga- By other way, Larrea divaricata Cav. is a South tion (20 mm at 1500 rpm), the supernatant was lyophi- American plant widely distributed in Argentina. In pop- lized [15]. ular medicine it is used for treatment of tumors, inflame- 2.4. Incubation Conditions matory disease, rheuma and fever. It has been demon- strated that a metanolic extract presented antioxidant The samples were prepared as follows: L. divaricata ex- activity [13] and that an aqueous extract exerts antim- tract (E) and vitamin C in water alone or associated; or- icrobial activity [14] but there are not studies about the ange juice; orange juice with L. divaricata extract; or- antioxidant activity of the aqueous extract and about the ange juice with vitamin C (for antimicrobial experiments) antimicrobial and antioxidant activities of the aqueous and orange juice with both L. divaricata extract and vi- extract applied to food conservation. tamin C (for antimicrobial experiments). Both vitamin C Based on this information, the aim of this work was: 1) and E were added to sample solutions on concentrations to study the antioxidant activity of an aqueous extract of of 1 mg/ml. The incubations were performed under dif- ferent conditions such as temperatures (5, 45 and 100˚C) L. divaricata, 2) to determine the capacity of the extract and air oxygen to mimic stores conditions. For 5˚C the to protect the oxidation of Vit C (from a water solution or samples were conserved in glass containers with her- from a natural orange juice) by the exposition at different metic close in refrigerator. For 45˚C or 100˚C samples temperatures and air oxygen, studying the variation of were maintained in glass containers with hermetic close the antioxidant activity and 3) to study the antimicrobial in a stove maintained under the selected temperature. activity of the extract upon the natural orange juice. When air oxygen was studied, samples were placed in no These studies were performed in view to establish wheth- hermetic close glass containers at room temperature. All er L. divaricata can be used as a supplement on orange the samples were protected from light. Also, the activity juices capable to prevent Vit C decay and microbial was determined under different incubation times 2, 7 and growth under stores conditions. 10 days for 5˚C and air oxygen and 1, 10 and 20 h for 45˚C in the case of Vit C in water or 1, 2, 3 and 4 days of 2. Materials and Methods incubation in the case of Juice. 2.1. Reagents 2.5. Measurement of DPPH Free Vitamin C L(+)—ascorbic acid (Anedra) was dissolved Radical-scavenging Activity in water and orange juice. 2, 2-diphenyl-1-picrylhydrazyl (6 mg) were diluted on 30ml of absolute ethanol, pro- The antioxidant activity of the samples was determined Copyright © 2011 SciRes. FNS Antioxidant and Antimicrobial Activities of Larrea Divaricata Cav. Aqueous Extract 37 on Vitamin C from Natural Orange Juice analyzing the scavenger activity of the free radical di- prepared. Then the samples submitted to different store phenylpicrylhydrazyl (DPPH) (Sigma, St Louis, Mo, conditions as temperature 45˚C and 5˚C and air oxygen USA) in accordance with the method described by Wang during 24 h, were inoculated onto the dish under sterile [16].