Total phenolics extracted from the skin of fuji apple and incorporated ...

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Romanian Biotechnological Letters Copyright © 2011 University of Bucharest

Vol. 16, No.6, 2011, Supplement Printed in Romania. All rights reserved ORIGINAL PAPER

Total phenolics extracted from the skin of fuji apple and incorporated by liposome in galenic bases: an alternative to use by-products of food industry Received for publication, September 25, 2011 Accepted, November 27, 2011 ALINE CARDOSO1, LIDIANE LEMOS2, MONICA FRIGHETTO3, ESTELA NUNES4* 1 Pharmacy academic, Biological and Health Sciences Center, Santa Catarina West University - UNOESC-Videira, Santa Catarina, Brazil 2,3 Biological and Health Sciences Center, Santa Catarina West University - UNOESCVideira, Santa Catarina, Brazil 4 Biotechnological Nucleus, Santa Catarina West University - UNOESC-Videira, Santa Catarina, Brazil. CEP:89.560-000. *Corresponding author: phone: +55(49)3533-4477 fax:+55(49)3533-4445; E-mail: [email protected]

Abstract The apple, Malus domestica Borkhausen has therapeutic potential by its action as free radical scavengers preventing oxidation of chemicals and their harmful effects on cells. Liposomes use as controlled releasing system of active ingredients is a new possibility. Santa Catarina produces 59% of Brazilian apples,therefore a study for an alternative use of the skin as a by-product from apple processing has been carried out. The work involved the extraction and quantification (Folin-Ciocalteau method) of total phenolics from Fuji apple skin with or without preservative addition; preparation of liposome with apple skin extract. Galenic bases were tested (Polavax®, Lanete and Gel cream) with the incorporation of the liposome with extract without preservative (A) and with preservative (B), in subsequent application primary stability test. Extraction performed with preservative had higher concentration of phenolics, suggesting the delay of oxidation process. However, should rule out the possibility of any interference of the preservatives in the quantification method used. It was well succeeded in the preparation and incorporation of liposome with phenolics from apple skin on the galenic bases, Polavax® showed the best performance. By-product “apple skin” proved to be a good source of phenolics, tests of chemical contaminants must be conducted in the future studies.

Keywords: apple skin, total phenolics, food industry, by-product.

1 Introduction The apple, Malus domestica Borkhausen (ROSS & al. [18]), has low caloric value and a considerable antioxidant activity. Phenolic compounds play an important role in the biotransformation processes of apple, fermentation processes, as well as contributing to the flavour of products and control the activity of microorganisms (Cowan [8]). Phenolic compounds are unevenly distributed during fruit development, being higher in skin than in the edible portions, due to the availability of metabolites and minerals, and exposure to solar radiation, since many enzymes involved in synthesis of such compounds are induced by light. Apples with high amount of phenolic compounds tend to have higher antioxidant activity; can act as scavengers of free radicals. In this way they provide anti-aging properties and confer some degree of photoprotection. (ZARDO & al. [20], OLIVEIRA & al. [11], PESCHEL & al. [12], AWAD & al. [2], LANCASTER & al. [9]). Phenolic compounds are classified as phenolic acids and flavonoids (AWAD & al. [2]). The last, synthesized from the phenylpropanoid pathway, are found in different structural forms, mostly formed by 15 carbon atoms in its fundamental nucleus. They may be in a free form (aglycone or genin) or as conjugated sugars (heterosides), with sugar component bound to an hydroxyl (Oheterosides) or a carbon atom (C-heterosides) (SIMÕES [17]). The basic structure (Figure 1) consists of diphenylpropane (C6C3C6) with two benzene rings (A and B) connected to a pyran ring (C) (BEHLING & al. [3]). In apple, the flavonoids include the following compounds: 40

ALINE CARDOSO, LIDIANE LEMOS, MONICA FRIGHETTO, ESTELA NUNES

flavonols, quercetin; dihydrochalcons (Phloretin and floredzina), anthocyanins and flavan-3ols, which include catechins, epicatechin and procyanidins (LANCASTER & al. [9]).

Font: Behling et al., 2004.

Figure 1: Flavonoids fundamental nucleus

Worldwide fruits production increased 26% in the last decade, according to the data from the United Nations Food and Agriculture Organization (FAO/UN), and a performance factor was the increasing demand for healthy foods (VITTI [19]). Brazil despite having less than 40 years of tradition occupies the 13th position among the largest apple producers of the world, the main varieties cultivated are Fuji, Gala and its clones. The Brazilian apple production is concentrated on Southern Region and Santa Catarina State is responsible for 59% of the national production (BITTENCOURT & MATTEI [4]). The fruit processing generates significant quantities of peel, seed, among others, all subject to waste recovery and reuse for its nutritional and functional properties. Bioactive compounds derived from food extracts have greater acceptance by the consumer when compared to synthetic substances are awakening scientific and economic interest in developing new technologies (COELHO & al. [7]). In accordance with PESCHEL & al. [12] the residues of certain fruits can serve as raw materials and additives for the cosmetic and pharmaceutical industry, especially apple, and suggesting its use in galenic formulations as an alternative to promote sustainability. Even with consumer acceptance, the incorporation of food extracts derived from plants in topical formulations may be restricted by physical and chemical properties, which can cause instability of the product or skin irritation. During the development of new formulations stability studies are necessary because they help to monitor and verify in advance the performance of the product against various conditions of shelf-life, a time interval, providing data on the maintenance of the characteristics from manufacturing to the expiration date. The preliminary stability study in galenic formulations is also known as screening and consists of testing in the initial phase of the product development (BRAZIL [6], SILVA [16]). This work aimed to develop liposomes with total phenolics, extracted from the skin of Fuji apples (as an alternative of to add value to a byproduct of food industry) and to determine the primary stability when incorporated into galenic bases.

2 Materials and methods 2.1 Total phenolic extraction The variety of apple chosen for this study was the Fuji, being one of the most consumed in Brazil and according to studies by BOYER & LIU [5] it has the highest content of total phenolics and flavonoids, when compared, for example, with the cultivars Gala and Golden delicious. Peels of ripe apples were used, selected according to the appearance of the fruit on colour uniformity and damages free. The extracting solvent was defined in accordance with PESCHEL & al. [12], which demonstrated the advantage of ethanol in the extraction of total phenolic compounds when compared to other organic solvents; in addition it offers less risk to the environment. The extraction was performed (25oC, 10 days, without mixing) with and without addition of synthetic preservatives: Sample A, with Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011)

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Total phenolics extracted from the skin of fuji apple and incorporated by liposome in galenic bases: an alternative to use by-products of food industry

butylhidroxitoluene (BHT) 0.02% and sodium bisulfite 0.1%, and Sample B, without the addition of such preservatives. The extracts were concentrated in Fisatom ® - 802D Rotaevaporator, between 92 to 94 rpm at 55 ° C, until complete solvent evaporation. The choice of preservative was based on TAGLIARI & al. [18] in antioxidants for aqueous systems, such as NaHSO3 in combination with antioxidants for oily systems, such as BHT, typically are used in preparations containing hydroquinone, in order to stabilize the substance which is easily oxidized. All tests were done in triplicate and the experiment performed at the Biotechnology Nucleus, Santa Catarina West University, UNOESC-Videira. 2.2 Total phenolics Total phenolics were determined by Folin-Ciocalteau method, adapted by AMERINE & OUGHT [1]. The standard used for the construction of the calibration curve was gallic acid. The extracts were diluted in the proportions of 1:5 (those which were not concentrated) and 1:50 (concentrated samples). The quantification was performed in Spectrophotometer Pro-Analise® UV-3000, the reading at λ= 765 nm and total phenolics concentration expressed in mg.L-1 of gallic acid. 2.3 Galenic bases formulation The choice criteria was the use of self-emulsifying bases, which are among the most commonly used and are described below: • Polavax®: Base nonionic (12%), Cetiol V (2.5%), Nipagim (0.15%), Nipazol (0.15%), BHT (0.1%), Natrosol (0.5 %), propylene glycol (5%), EDTA (0.005%), distilled water (30 g qsp). • Lanette Cream: Base anionic Lanette N (12%), Nipagin (0.15%), Nipazol (0.15%), BHT (0.1%), Natrosol (0.5%), propylene glycol (5%) , EDTA (0.05%), distilled water (qsp 30 g). • Gel-Cream Facial Serum Gel: Sepigel 2% (9g), Polavax ® Cream (30 g qsp), and Sepigel formulation: Sepigel (2%), Nipagin (0.02%), Imidazonidil urea (0.3 %), Nipazol (0.1%), BHT (0.1%), distilled water (qsp 90ml). 2.4 Liposomes formulation Prepared as described by MERTINS [10], with the following modification: phosphatidylcholine 75% (Gerbras®) and 2.5 mL of phenolics from apple skin for each sample. Both were dissolved in ethyl acetate, followed by the addition of 2mL of chitosan solution (chitosan 1% solubilised in acetic acid at 2%) and submitted to sonication in a ultrasonic mixer Unique® for 5 min. The organic solvent was evaporated in a rotaevaporator at 25 rpm and 40°C, until organogel formation. Distilled water was added with intense hand stirring until the organogel detachment, followed by homogenization in Disperser Extratur Quimis® at 8000 rpm for 10 min. The samples A and B of phenolic liposomes were added at a concentration of 10% to the galenic bases used for testing. 2.5 Stability preliminary study The samples were packed in a vessel of glass neutral, transparent, well sealed (screw cap) and 50 mL of capacity. The study was adapted from the methodology proposed in the Guide to Stability of the National Agency of Sanitary Surveillance - ANVISA (BRAZIL [6]): Centrifuge Test: All samples were subjected to centrifugation at 3,000 rpm for 30 min in order to observe instability or evidence, such as coalescence, flocculation, etc. (PRESTES & al. [13]). It is a pre-test where the product must remain stable and without any sign of instability so that sample can be submitted to Preliminary stability test Storage Conditions: The Test-samples and Reference-sample of each of the bases (without liposome incorporation) were submitted to complete cycles (24 hours in the oven at a temperature of 45 ° C ± 2 ° C, and 24 hours in refrigerator to 2 ° C ± 2° C) for a period of 12 days. At the same time every standard galenic base (without liposome incorporation) was maintained at room temperature in an environment protected from light and humidity (normal storage conditions). 42

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Control Parameters: Regarding the organoleptic characteristics the following parameters were observed: appearance, colour, odour, and the pH value as a physico-chemical parameter. The measurement of pH was performed with a pH meter coupled to the spectrophotometer Hach® DR/2500. The pH was measured at time zero, each change of the cycle of 24 hours and at the end of the stability tests. The Test-samples were analyzed in comparison to the Standardsample of each galenic base and are shown in Table 1. Table 1 – Organole ptic pa rameters and pH va lue of the Sta ndard sam ple. O rgan oleptic parameters

Gale nic b ase

pH

Aspe ct

Color

Odor

Homoge neous

White

Odorless

4,5

Lanette cr ea m

Homoge neous

White

Odorless

4,5

Gel-c ream

Homoge neous

White

Odorless

4,5

Polavax

®

3 Results and Discussions The amount of total phenolics was determined in the skin apple extracts: (A) with the BHT + NaHSO3 addition and (B) with no added preservatives. The obtained results are presented in Table 2. The total phenolics were 16.8% higher in the sample (A) as compared to the values obtained in the sample (B), indicating probable preservation of phenolics during the extraction. However, this does not mean that any interference in the quantification of total phenolics in the presence of synthetic antioxidants can be excluded. Table 2 – Total phenolics amounts determined in the extracts of Fuji apple skin. Gallic acid Concentrated samples (Extracts) -1 (mg.g ) Sample A* Sample B** 76,42 61,39 Total phenolics 77,48 62,45 72,45 64,05 Mean ± SD 75,45 ± 2,65 62,63 ± 1,34 *With added synthetic antioxidants; **Without added synthetic antioxidants.

The physico-chemical tests were done in duplicate and showed variations in pH in all Test samples, with Lanette Reference sample having the greatest variation, justifying the instability of the test samples A and B. Test samples of Lanette and Cream-gel (A and B) and Polavax ® (A and B) had no marked variations in their pH values (Table 3): Table 3 – pH values of standards and samples at: time zero and 12nd day after storage at the described conditions. pH Samples description Time zero 12nd Standard (1) 4,5 4,5 Standard (2) 4,5 4,5 Standard Samples Standard (3) 4,5 4,5 Reference Samples

Reference (1) Reference (2) Reference (3)

4,5 4,5 4,5

4,5 5,5 4,5

Sample A (Phenolics lipossome and BHT+NaHSO3)

Gel-cream Lanette Polavax®

4,5 5,0 4,5

4,9 5,4 4,8

Sample B (only Phenolics lipossome)

Gel-cream Lanette Polavax®

4,5 5,0 4,5

4,8 5,5 4,6

(1) Gel-cream, (2) Lanette, (3) Polavax®.

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Total phenolics extracted from the skin of fuji apple and incorporated by liposome in galenic bases: an alternative to use by-products of food industry

The centrifuge tests showed no phase separation, no coalescence or precipitates formation. Thus, all emulsions, with or without phenolics liposomes were stable. Organoleptic properties of the Test samples (A and B) are presented in Table 4. By comparison with the Standard samples, the homogeneity and the appearance of all samples were observed except for the Sample B – (cream-gel), where dark spots were detected. In relation to the colour in all galenic bases alterations were observed, from slightly modified to modified, as well as changes of the odour, where variation from slightly to intensely modified was noticed. Table 4 – Organoleptic parameters (appearance, color and odor) from analyzed samples at time zero and 12nd day after storage at the described conditions. Time zero 12nd day Standard/Reference samples

Standard1,2,3 Reference1,2,3

NA, NC, NO NA, NC, NO

NA, NC, NO NA, NC, NO

Sample A (Phenolics lipossome and BHT+NaHSO3

Gel-cream Lanette Polavax®

NA, SMC, SMO NA, SMC, SMO NA, SMC, SMO

NA, MC, SMO NA, MC, MO NA, SMC, SMO

Sample B (only phenolics liposome)

Gel-cream Lanette Polavax®

NA, SMC, SMO NA, SMC, SMO NA, SMC, SMO

MA*, SMC, MO NA, SMC, IMO NA, SMC, SMO

NA (normal aspect), SMA (slightly modified aspect), MA (modified aspect), NC (normal color), SMC (slightly modified color), MC (modified color), IMC (intensely modified color), NO (normal odor), SMO (slightly modified odor), MO (modified odor), IMO (intensely modified odor). *Dark spots presence. 1,2,3 Gel-cream, Lanette and Polavax® bases.

Polavax ® samples A and B showed better stability when compared to other test samples, since there were only few changes, including pH values. However, by comparing them with the samples and Standard Reference, there were slight changes in parameters related to the colour and the odour from zero time but keeping such characteristics until the end of the study. This was observed specially in the Samples A (with synthetic antioxidants added), suggesting that the combination of antioxidants derived from plants and synthetic antioxidants in low concentration is a good strategy to preserve the organoleptic quality of products. As PESCHEL & al. [12] describe in their study the concentrations of 0.1% and 0.5% of apple phenolic compounds are acceptable from cosmetic point of view, with regard to the characteristics of colour and odour. However, they show that concentrations higher than 0.1% are not recommended for general use in cosmetic formulations, which suggest that small changes in the colour and the odour, observed in the present study are acceptable, considering that the concentration here used was of 0.25%. To minimize the most probable adverse effects of the polyphenolic extract on galenic formulations, microencapsulation of phenolics technique (with and without combination of synthetic antioxidants) in liposome chitosan-based was tested here. Liposome chitosanassociated provide high encapsulation efficiency, bio-adhesion capacity and protective effect of the active principle (RIDOLFI [14]), while phenolics increases the liposome stability.

4. Conclusions The extraction of phenolics from Fuji apple skin with ethyl alcohol as an extraction solvent proved feasible, in function of the obtained concentrations, as well, as much less damaging to the environment. 44

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Through the preliminary stability study it was possible to show that among the used Galenic bases, Polavax® A and B presented better performance in comparison to the other Test samples. The samples with the addition of BHT + NaHSO3 showed higher concentrations of total phenolics, indicating probably phenolics preservation during the extraction. No study to evaluate any interference of additives in the quantification was done, so it is suggested to do HPLC analysis to rule out the possibility of interference of the preservatives in the quantification method used in this experiment. With the aim of minimize adverse effects of the action of natural phenolics in galenic formulations, maximizing the bioavailability and reducing the skin irritating effect, natural phenolics microencapsulation by liposome chitosan-based appeared as a promising alternative. The apple skin considered as a by-product of food industry, can become raw material for pharmaceutical and cosmetic industries, providing their beneficial effect to human and animal health.

5. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

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