Chemical characterisation and the anti- 1 inflammatory, anti-angiogenic and antibacterial 2 properties of date fruit (Phoenix dactylifera L.) 3 4 Hajer Taleb1, Sarah E. Maddocks2*, R. Keith Morris2, and Ara D. Kanekanian1 5 1Department of Healthcare and Food. 2Department of Biomedical Sciences. 6 Cardiff School of Health Sciences, Cardiff Metropolitan University, Llandaff 7 Campus, Western Avenue, CF5 2YB, Wales, United Kingdom. * Sarah E. Maddocks. Department of Biomedical Sciences. Cardiff Metropolitan University, Llandaff Campus, Western Avenue, CF5 2YB, Wales, United Kingdom. Email: [email protected], telephone number: +442920415607 8 Abstract 9 Ethnopharmacological relevance: Date fruit, Phoenix dactylifera L. has 10 traditionally been used as a medicine in many cultures for the treatment of a 11 range of ailments such as stomach and intestinal disorders, fever, oedema, 12 bronchitis and wound healing. 13 Aim of the review: The present review aims to summarise the traditional use 14 and application of Phoenix dactylifera date fruit in different ethnomedical 15 systems, additionally the botany and phytochemistry are identified. Critical 16 evaluation of in vitro and in vitro studies examining date fruit in relation to anti- 17 inflammatory, anti-angiogenic and antimicrobial activities are outlined. 18 Key Findings: The ethnomedical use of Phoenix dactylifera in the treatment of 19 inflammatory disease has been previously identified and reported. 20 Furthermore, date fruit and date fruit co-products such as date syrup are rich 21 sources of polyphenols, anthocyanins, sterols and carotenoids. In vitro studies 22 have demonstrated that date fruit exhibits antibacterial, anti-inflammatory and 23 anti-angiogenic activity. The recent interest in the identification of the 24 numerous health benefits of dates using in vitro and in vivo studies have 25 confirmed that date fruit and date syrup have beneficial health effects that can 26 be attributed to the presence of natural bioactive compounds. 27 Conclusions: Date fruit and date syrup have therapeutic properties, which 28 have the potential to be beneficial to health. However, more investigations are 29 needed to quantify and validate these effects. 30 Keywords: Phoenix dactylifera, date fruit, polyphenols, antioxidant, anti- 31 inflammatory 32 2 33 Abbreviations 34 BCCAO; Bilateral common carotid artery occlusion 35 CD31; Cluster of differentiation 31 36 COX-2; Cycloxygenase-2 37 HBA; Hydroxybenzoic acid 38 HCA; Hydroxycinnamic acids 39 IL -1; Interlukin -1 40 IL -1β; Interlukin -1 beta 41 IL -6; Interlukin -6 42 LPS; Lipopolysaccharide 43 MIC; Minimum inhibitory concentration 44 ROS; Reactive oxygen species 45 TNF-α; Tumor necrosis factor alpha 46 VEGF; Vascular endothelial growth factor 3 47 Table of Contents 48 Abstract .......................................................................................................... 2 49 1. Introduction ................................................................................................ 5 50 2. Botanical nomenclature and classification ............................................. 6 51 3. Traditional relevance ................................................................................. 7 52 4. Phytochemical composition ..................................................................... 8 53 4.1 Polyphenols ............................................................................................. 9 54 4.1.1 Hydroxy benzoic acids (HBA) ............................................................ 10 55 4.1.2. Hydroxy cinnamic acids (HCA) ......................................................... 10 56 4.2 Carotenoids ............................................................................................ 12 57 4.3 Tannins ................................................................................................... 12 58 5. Medicinal Properties ................................................................................ 14 59 5.1 Anti-inflammatory activity ..................................................................... 14 60 5.2 Anti-angiogenic activity ........................................................................ 17 61 5.3 Antimicrobial activity ............................................................................ 19 62 6. Conclusions and future directions ......................................................... 21 63 References ................................................................................................... 23 64 List of Captions ................................................................................................. 38 65 4 66 1. Introduction 67 Fruits have always been a major constituent of the human diet. Recently, 68 human food selections, dietary lifestyles and patterns have become 69 increasingly governed by economic necessity, availability and promotion by 70 industry and governments (Heber and Bowerman, 2001). These factors are 71 having a significant impact on diet selection and food intake rather than 72 nutritional significance or health benefits. This has led in some cases to 73 increase in morbidity and mortality associated with food related diseases such 74 as obesity and diabetes (Kris-Etherton et al. 2002). 75 There is growing epidemiological evidence coupled with clinical and scientific 76 studies strongly supporting the assertion that diets rich in fruits, vegetables, 77 whole grains and fish have a protective role in preventing a wide-range of 78 diseases including type 2 diabetes, cancers, atherosclerosis and 79 cardiovascular diseases. As a result there has been a growing interest in 80 assessing the role of food-based bioactive compounds in preventing the 81 development and the incidence of these diseases. 82 The health benefits of medicinal foods, plants and herbs are subject to 83 immense interest amongst the public, pharmaceutical companies and health 84 professionals. This interest has resulted in the global health market becoming 85 flooded with products claiming to prevent, reduce symptoms and cure diverse 86 ailments or improve health and prevent chronic diseases (Raskin et al. 2002). 87 Due to this increased commercial exploitation of medicinal foods, almost all 88 varieties of fruit and vegetables are being re-evaluated for their health 89 benefits and phytochemical composition in both clinical settings and under 90 laboratory conditions. 91 Where access to modern medicines is limited, plants have become 92 increasingly important as a source of alternative medicinal compounds 93 (Raskin et al. 2002). Many plant-based medicines are extracted from diverse 94 sources (Evans, 2009). Primary and secondary metabolites in fruits are 95 numerous with primary metabolites including amino acids, sugars, and 96 chlorophylls whilst secondary metabolites include carotenoids, tannins, 5 97 flavonols, phenols, alkaloids and saponins (Evans, 2009). The metabolites in 98 fruits conferring specific appearance, colour, taste, aroma and astringency. 99 Secondary metabolites have been associated with a wide-range of bioactive 100 behaviour, believed to have significant beneficial effects for human health 101 (Balasundram et al. 2006) such as antimicrobial (Taleb et al. 2016a), anti- 102 inflammatory and anti-angiogenic activities (Taleb et al. 2016b). The present 103 review aims to assess the traditional use and application of Phoenix 104 dactylifera L. date fruit in different ethnomedical systems, additionally the 105 botany and phytochemistry are identified. Critical evaluation of in vitro and in 106 vitro uses of date fruit in relation to anti-inflammatory, anti-angiogenic and 107 antimicrobial activities are outlined. 108 2. Botanical nomenclature and classification 109 According to Tropicos (Tropicos, 2016), date palm belongs to the Kingdom 110 Plantae, the class Equisetopsida C. Agardh, the subclass Magnoliidae Novák 111 ex Takht, the superorder Lilianae Takht, the order Arecales Bromhead, the 112 family Arecaceae Bercht. & J. Presl and the genus Phoenix L. Furthermore, 113 The Plant List identifies that Phoenix dactylifera L. is the only accepted name 114 for the date palm tree, with available synonyms such as Phoenix dactylifera L. 115 var. costata Becc., P. dactylifera var. cylindrocarpa Mart., P. dactylifera var. 116 gonocarpa Mart., P. dactylifera var. oocarpa Mart., P. dactylifera var. 117 oxysperma Mart., P. dactylifera var. sphaerocarpa Mart., P. dactylifera var. 118 sphaerosperma Mart., and P. dactylifera var. sylvestris Mart. (The Plant List, 119 2013). 120 The date palm (Phoenix dactylifera) and its fruits are cultivated in dry and 121 semi-arid regions of the world and is the dominant constituent upon which the 122 sustainable biophysical and socio-economic structures of the oasis 123 ecosystem are based (Barreveld, 1993). Furthermore, date palm is the only 124 indigenous wild desert plant definitively domesticated in its native harsh 125 environment (Jaradat and Zaid, 2004). Phoenix dactylifera is composed of 126 genetically discrete clones representing thousands of cultivars without the 127 benefits of a dynamic mutation-recombinant system (Chao and Krueger, 6 128 2007). It thrives alongside numerous wild palms distributed across the desert 129 belt in the Middle East and North Africa (Zaid and Arias-Jimenez, 1999). 130 The fruit of the date palm is processed and utilised in various ways but the 131 purported medicinal properties remain largely unknown in the Far East and 132 the West, essentially due to its lack of growth potential and use in these 133 regions but importantly, due to insufficient scientific and clinical data (Vayalil, 134 2012). 135 3. Traditional relevance 136 The historical and religious significance of Phoenix dactylifera and date fruit 137 are well documented, they were utilised as anthropomorphic symbols in early 138 as Mesopotamian civilisations, including Sumer and Babylonia, and by the 139 ancient Egyptians in the Nile valley, in the pre-Dynastic era and the Greco- 140 Roman Period (350 AD) (Manickavasagan et al. 2012). 141 The health and medicinal use of date fruit expanded originally from Middle 142 Eastern folklore to Indian traditional medicine. Phoenix dactylifera and date 143 fruit are used as alternative medicine in countries such as Algeria, Egypt, 144 India, Iran and Iraq (Table 1). Ayurveda medicine, a medicinal system with 145 historical roots in the Indian subcontinent uses date fruit as a medicinal 146 application for the treatment of lower respiratory tract infections, sciatica, 147 oedema, microbial infections and alcohol intoxication (Kunte and Navre, 1939; 148 The Wealth of India, 1952; Nadkarni, 1976). In the Middle East and across 149 Arabia a decoction of dates with salt is used as a remedy for dehydration 150 associated with diarrhoea (Al-Qarawi et al. 2005). Additionally date products 151 such as date syrup and date paste are administered for treating sore throat 152 and inflammation of the mucus membranes and intestinal disturbances (Souli 153 et al. 2014). Alternative and various uses of Phoenix dactylifera in different 154 ethnomedical systems are outlined in Table 2. Despite widespread use, there 155 is limited scientific and clinical evidence to support the aforementioned claims. 156 However the increased understanding of functional composition and 157 phytochemistry of date fruit has begun to provide scientific rationale for date 158 fruit’s medicinal ability, which are outlined below. 7 159 4. Phytochemical composition 160 As previously mentioned, secondary metabolites are known to mediate some 161 of the health benefits associated with date fruit. Secondary metabolites form 162 an integral component of a fruit’s structural and cellular integrity (Macheix and 163 Fleuriet, 1990) and have gained importance for their potential cancer 164 prevention, diet related disease prevention and cardiovascular associated risk 165 minimisation. Date fruit at the Tamr stage consist of a very thin pericarp 166 containing pigments, a colourless thick mesocarp, and a thin endocarp 167 surrounding a single seed. Date fruit, are also sugar rich (Al-Shahib and 168 Marshal, 2002) and the amount of sugar is dependent upon type of cultivar 169 and degree of maturation, with some varieties attaining reducing sugar 170 concentrations of up to 78% (Al-Farsi et al. 2007). Dates are a good source of 171 fibre in particular insoluble fibre approximating 11.5 g /100 g at complete 172 maturation (Al-Shahib and Marshall, 2002). The protein content in date fruit is 173 relatively low 2.5 – 6.5 g /100 g (Chaira et al. 2009), despite this date fruit 174 contain proportionally high levels of essential amino acids including arginine 175 and histadine which are vital to human health (Al-Aswad 1971; Auda et al. 176 1976; Auda and Al-Wandawi, 1980). Furthermore date fruit are a source of 177 minerals, in particular potassium (864 mg /100 g), calcium (70.7 mg /100 g), 178 sodium (32.9 mg /100 g), iron (0.3 – 6.03 mg /100 g), zinc (0.5 mg /100 g) and 179 magnesium (64.2 mg /100 g) (Al-Farsi et al. 2005; Al-Farsi and Lee, 2008). 180 These micronutrients are essential for physiological functions such as 181 respiration (Na+), functioning of the immune system (Zn) and physical fatigue 182 (Fe) (Vayalil, 2012). Phytochemical analyses on Phoenix dactylifera have 183 revealed the presence of various phytochemicals including phenolic acids, 184 flavonoids, tannins, anthocyanins and carotenoids (Oni et al. 2015). The 185 active constituents of Phoenix dactylifera date fruit are volatile compounds 186 (alcohols, esters, aldehydes, lactones, ketones and terpenoids) (Guido et al. 187 2011; El Arem et al. 2012), phenolic acids (cinnamic acid derivatives, caffeic 188 acid, vanillic acid and protocatechuic acid) and flavonoids 189 (proanthocyanidines, flavonoid glycosides and anthocyanins) (Al-Farsi et al. 190 2005; Mansouri et al. 2005; Hong et al. 2006). The following chapter 191 summarises the three major phytochemicals characterized for date fruit: 192 polyphenols, carotenoids and tannins. 8 193 4.1 Polyphenols 194 Polyphenols are divided into flavonoids and non-flavonoids. Flavonoids share 195 a common carbon skeleton of diphenyl propanes or two benzene rings joined 196 by a linear 3-carbon chain (Fang et al. 2002). Flavonoids are further 197 subdivided on the basis of their chemical structure, including benzene and 198 pyran rings, examples include flavonols, flavones, anthocyanidins and 199 isoflavones. Non-flavonoids include phenolic acids, which are divided into 200 derivatives of benzoic acids and derivatives of cinnamic acid (Harborne and 201 Baxter, 1993). 202 The phenolic content and subsequent polyphenol content in date fruit is 203 correlated with cultivar, growth and development stages, health and exposure 204 of date fruit to environment and pests (El-Hadrami et al. 2011). The phenolic 205 accumulation is a result of tissue browning involved in the maturation process 206 of date fruit and is biosynthesised by the shikimate pathway. 207 Phenylalanine is the most common precursor in the biosynthesis of 208 polyphenols, and itself is an intermediate in the shikimate pathway (Tsao, 209 2010). The hydroxycinnamic acids, in particular have an important role due to 210 their abundance and diversity as the common structural elements of other 211 phenolic compounds such as flavonoids, condensed tannins, lignin and 212 hydroxycinnamic derivatives (Macheix and Fleuriet, 1990; Rice-Evans et al. 213 1996). Date fruits are rich source of phenolics that vary among different 214 varieties. Phenylalanine concentrations vary significantly during fruit 215 maturation, and increased during dried date cultivars, however, the amounts 216 of protein in date fruit are too low to be considered a vital nutritional source, 217 date fruit contains essential amino acids such as phenylalanine, leucine and 218 threonine (Al-Farsi and Lee, 2008). 219 Date fruits typically show a decline in phenolic compounds with ripening, but 220 an increase in response to stress such as bruising and fungal infection (El- 221 Hadrami et al. 2011). Date palm cultivars exhibit distinct levels and profiles of 222 polyphenol compounds such as gallic, protocatechuic, p-hydroxybenzoic, 223 vanillic, caffeic, syringic, p-coumaric, ferulic, o-coumaric acid, 3-caffeoylquinic 224 acid and 3-O-caffeoylshikimic acid (dactylifiric acid) (Harborne and Baxter, 9 225 1993; Duke, 2001; Duke and Beckstrom-Sternberg, 2015). The characteristic 226 polyphenols in date fruit are further subdivided into two primary classes 227 hydroxyl benzoic acids and hydroxyl cinnamic acids, which are represented 228 below. 229 4.1.1 Hydroxy benzoic acids (HBA) 230 Hydroxy benzoic acids are derived directly from benzoic acid and structural 231 variations are a result of hydroxylations and methoxylations of the aromatic 232 ring. The most common HBAs identified in date fruit include p- 233 hydroxybenzoic, vanillic, syringic, protocatechuic and gallic acid (Fig. 1a). The 234 first three acids are constituents of lignin and it is generally assumed that 235 plants lacking lignin lack these acids (Macheix and Fleuriet, 1990). The 236 benzoic acids are often present in bound form, thus making them insoluble as 237 they are often covalently bound to cell wall structural components such as 238 lignin and cellulose (Acosta-Estrada et al. 2014). Furthermore, more than 239 often HBAs constitute hydrolysable tannins or simple molecules by combining 240 with sugars and organic acids (Harborne and Baxter, 1993). p- 241 Hydroxybenzoic and vanillic acids are present in numerous fruits and are 242 found as simple combinations with glucose in soft fruits (Robards et al. 1999). 243 Protocatechuic acid has also been detected in date fruit and a number of soft 244 fruits in the form of glucosides (Waterhouse et al. 2000; Hong et al. 2006). 245 Quantitatively, HBA content is generally low in date fruit and other fruits, 246 constituting approximately 24% of the total phenolics, with the exception of 247 blackberry and the Rosaceae family (apples, pears, quinces, apricots) 248 (Haslam, 1989; Acosta-Estrada et al. 2014). However, they should not be 249 overlooked since HBAs have a role in the organoleptic qualities of fruits by 250 interaction to form hydrolysable tannins (condensed), which are later 251 discussed in section 4.3. 252 4.1.2. Hydroxy cinnamic acids (HCA) 253 Hydroxy cinnamic acids are derived from cinnamic acid and are present as 254 combined forms of four basic molecules: p-Coumaric, caffeic, ferulic and 255 sinapic acids (Fig. 1b). Coumarins are also derived from HCAs. There are 10