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Cistus Incanus from Strandja Mountain As a Source of Bioactive Antioxidants

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Cistus Incanus from Strandja Mountain As a Source of Bioactive Antioxidants Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 January 2018 doi:10.20944/preprints201711.0184.v2 Peer-reviewed version available at Plants 2018, 7, 8; doi:10.3390/plants7010008 1 Article 2 Cistus incanus from Strandja Mountain as a Source of 3 Bioactive Antioxidants 4 Vanya Dimcheva *, Maria Karsheva * 5 6 Department of Chemical Engineering 7 University of Chemical Technology and Metallurgy, 8 Kl. Ohridski bul., 1756 Sofia, Bulgaria 8 *Correspondence: [email protected]; [email protected] 9 10 11 Abstract: The purpose of the present study is survey of extraction conditions and exploring 12 antioxidant potential of the non-traditional for the Bulgarian ethno-medicine wild herb Cistus incanus 13 widespread in Strandja Mountain. The influence of the extraction time (0–500 min) and solvent 14 composition (0–50% ethanol in water) on the polyphenols, flavonoids yields and on antioxidant 15 capacity of the extracts of leaves, stalks (wood parts) and buds mixture were studied. The antioxidant 16 capacity (AOC) was evaluated by use of scavenging assays of 2,2-diphenyl-1-picrylhydrazyl (DPPH) 17 radicals. Total polyphenol and flavonoid contents were quantified using UV–vis spectrometry. 18 Optimal yield of desired components has been obtained with 30% ethanol in water solvent at 390th 19 min extraction time. In addition, the influence of the seasonality (winter and summer Cistus incanus), 20 and of the different areal parts - hard-coated seeds; buds, and mixture of leaves and stalks of the wild 21 plant on the presence of polyphenols, flavonoids and AOC were investigated. Present work revealed 22 the high values of the polyphenols, flavonoids, the high AOC not only in the summer leaves, but also 23 found in the winter leaves, hard-coated seeds, buds and stalks. Based on the obtained results the 24 Cistus incanus from Strandja mountain could be a new excellent source of natural antioxidants in food 25 and pharmaceutical industries. 26 27 Keywords: Cistus incanus; Strandja; antioxidants; polyphenols; flavonoids; seasonality; buds; hard- 28 coated seeds 29 30 31 1. Introduction 32 Medicinal plants, especially with antioxidant activity, are the main source of drugs for the treatment 33 of complications induced by oxidative stress. Today, about half of the available drugs are estimated to 34 come from plants [1]. The synthesized drugs may appear different adverse effects [2]. So, it is important 35 to look for new sources of phytomedicines in nature. 36 Cistus incanus L. within the habitat of temperate ericoid communities or European dry heaths and 37 in Bulgaria covers almost the whole most intriguing region – Strandja mountain [3]. The plant is not 38 included in the “Law of medicinal plants” and protected and declared as medicinal plant. 39 In the Quaternary, among unaffected by glaciations parts of Europe, only Strandja seaside rests 40 almost untouched and keeps climatic conditions similar to tertiary "eternal spring” [4]. Many plants 41 grown there with therapeutic action are not commonly used and popular in Bulgarian folk medicine, © 2017 by the author(s). Distributed under a Creative Commons CC BY license. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 January 2018 doi:10.20944/preprints201711.0184.v2 Peer-reviewed version available at Plants 2018, 7, 8; doi:10.3390/plants7010008 2 of 15 42 such as sub endemic Cistus incanus L. or “Pamukliyka” (local name). It is most known only as food for 43 goats and sheep in our lands, while the history of the “Holly rose” and its ethno-medicinal usage in 44 Mediterranean began in ancient times [5]. The wild herb has provided antibacterial, antimicrobial, 45 anti-inflammatory and strong gastroprotective beneficial effects [6]. Many research studies have 46 demonstrated that the main components of the leaves of the different Cistus species are polyphenolic 47 compounds from flavanols, flavan-3-ols family such as (+) - catechins, gallic acid, rutin, flavonoid 48 aglycones based on quercetin, kaempferol, and myricetin [7, 8]. It is well established that phenolics 49 content in plants is mainly responsible to their antioxidant activities and scavenging power. 50 This work contributes to establish the Cistus incanus beneficial properties of our geographical 51 longitude, due to its tendency to polymorphism or alteration of phytochemical composition under 52 different environmental factors, conditions and seasonality. An appropriate extraction of phenolic 53 compounds depends on multiple factors, such as their chemical nature, row material, storage time and 54 conditions. Not at least it depends on the extraction and quantification methods, choice of standards, 55 and presence of interferences [9, 10]. Thus, it is necessary to adjust sample preparation procedures to 56 achieve the optimal possible estimation of the phenolic compounds. Results on evaluation of 57 operational extraction conditions of Cistus incanus will provide a better understanding of the 58 antioxidant potential of the wild herb and will allow its use as high added value dietary antioxidant 59 additive. 60 In this investigation we selected to follow the steps of extraction optimization of Cistus incanus by 61 total polyphenols, flavonoids and antioxidant capacity. Initially, the effect of the solvent (ethanol in 62 water mixtures) concentration was evaluated for a previously chosen extraction time. Once it has been 63 found it was evaluated the extraction time at constant chosen previously extractive parameters - 64 temperature, particle size, solid-to-solvent ratio. Also, it was followed the kinetic by total dry residue 65 of the extracts, kinetic by total dry mass, and kinetic by the final volume of the extracts received after 66 hand pressing the exhausted row material. The kinetics were done to establish equilibrium of the 67 extraction process likewise for better understanding the extraction process of the herb studied. In 68 addition, the influence of the seasonality and evaluation of the different areal parts of Cistus incanus on 69 the presence of polyphenols and flavonoids also was investigated. 70 The total polyphenol content (TPC) was determined through the method of Folin-Chiocalteau at 71 the wave length of 765 nm. The total flavanoid content (TFC) was measured by the aluminium 72 chloride colorimetric assay at wave length 510 nm. The AOC was studied by DPPH assay at 73 wavelength 517 nm. The total dry residue (TDR) was found gravimetrically after evaporation of 10 mL 74 of the extract and through drying of exhausted drug to constant weight in the oven at 105° C. 75 All extractions were made duplicate. In the proposed kinetics, the averaging values of the 76 analyses were used (<5% RSD). 77 78 2. Materials and methods 79 80 2.1. Chemicals 81 82 Ethanol 96% was supplied by „Valerus“, Bulgaria, methanol, HPLC grade; sodium carbonate (> 83 99%); gallic acid anhydride (> 99%), sodium nitrite, aluminium chloride hexahydrate - by „Merck“, 84 Germany, Folin-Ciocalteau reagent – 2M solution, 2,2-diphenyl-1-picrylhydrazyl (DPPH), rutin 85 hydrate, quercetin hydrate (≥ 95%), tannic acid (≥ 91%), pyrogallic acid (≥ 98%), (+) - catechin hydrate 86 (≥ 96%), sodium hydroxide, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (97%) 87 were supplied by „Sigma Aldrich“, Germany. Ammonia-iron alum - by „Sharlau“, Germany. 88 Deionized water from water deionizer – „Elix70C Gulfstream-Merck“. 89 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 2 January 2018 doi:10.20944/preprints201711.0184.v2 Peer-reviewed version available at Plants 2018, 7, 8; doi:10.3390/plants7010008 3 of 15 90 2.2. Plant material 91 92 For this study were used wild Cistus incanus L. leaves, stalks (wood parts) and buds collected 93 in the end of the May (2015) in the beginning of the flowering and hard-coated seeds of the plant 94 collected of September (2015); leaves and stalks collected in the end of the March (2016). The drugs 95 were gathered from the area „Parnara“ around the village „Varvara“ („Tsarevo“ municipality) 96 according to the rules of conservation of biodiversity of the „National Park Strandja“, Bulgaria. The 97 temperatures measured in the days of collecting of Cistus incanus were 25 ° C in May, 26° C in 98 September, and 7° C in March, respectively. For guaranty a representative sampling was collected 2 kg 99 from the wild plant. The Cistus incanus L. was identified by the experienced biologists from the 100 „National Park Strandja“. 101 102 2.3. Extraction procedure 103 104 For the experiments were used the following mixtures of Cistus incanus drugs - leaves, stalks, buds 105 (80:10:10, w:w); Cistus incanus stalks and leaves (50:50, w:w); hard-coated seeds; leaves and stalks 106 (90:10, w:w) gathered in summer and respectively in the winter harvest seasons. All samples were 107 dried at room temperature and kept at dry place for a year, before to be ground into grinder and 108 sieved. All samples were used with LOD (loss on drying) not more than 10%. For the experiments a 109 fraction of 0,5 - 2,0 mm particle size was used. The initial solid to solvent ratio was fixed to 1:20 (2 g 110 Cistus incanus in 40 mL solvent). The temperature used for the extraction was the room temperature 111 and was kept constant as far as possible. Extractions were done through magnetic stirring at 1411 RCF 112 (Relative Centrifugal Force) with a Magnetic stirrer (MS-H-Pro+, Dragon Lab). The influence of the 113 solvent composition water or water-ethanolic solution (10, 20, 30, 40, 50, v:v) were studied for the 80th 114 min extraction time. The extraction kinetics of Cistus incanus samples were followed during 8,3 h (5, 115 10, 30, 50, 80, 120, 180, 390, 500 min) with the chosen constant extraction condition. Each exhausted 116 raw material was carefully pressed, and the extract was filtered through cotton and filter paper, 117 measured and analyzed immediately after appropriate dilutions.
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