Guanidine-modified cyclometalated iridium(III) complexes for mitochondria-targeted imaging and photodynamic therapy.

Accepted Manuscript Phytochemical and pharmacological attributes of piperine: A bioactive ingredient of black pepper Sergey Shityakov, Ehsan Bigdelian, Aqeel A. Hussein, Muhammad Bilal Hussain, Yogesh Chndra Tripathi, Muhammad Usman Khan, Mohammad Ali Shariati PII: S0223-5234(19)30301-0 DOI: https://doi.org/10.1016/j.ejmech.2019.04.002 Reference: EJMECH 11237 To appear in: European Journal of Medicinal Chemistry Received Date: 6 January 2019 Revised Date: 16 March 2019 Accepted Date: 1 April 2019 Please cite this article as: S. Shityakov, E. Bigdelian, A.A. Hussein, M.B. Hussain, Y.C. Tripathi, M.U. Khan, M.A. Shariati, Phytochemical and pharmacological attributes of piperine: A bioactive ingredient of black pepper, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/ j.ejmech.2019.04.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 1 Phytochemical and pharmacological attributes of piperine: A bioactive ingredient of black 2 pepper 3 Sergey Shityakova*, Ehsan Bigdelianb, Aqeel A. Husseinc,d, Muhammad Bilal Hussaine, Yogesh 5 Chndra Tripathif, Muhammad Usman Khang,h, Mohammad Ali Shariatii* RI PT 4 6 a 7 Department of Anesthesia and Critical Care, University of Würzburg, 97080 Würzburg, Germany 8 b SC Department of Food Science and technology, Faculty of Agricultural Engineering and 9 Technology, University of Tehran 10 School of Chemistry, University of Southampton, Highfield, Southampton, SO171BJ, UK d 12 e 13 M AN U c 11 School of Medicine, University of Al-Ameed, Karbala P.O No: 198, Iraq Institute of Home and Food Sciences, Government College University, Faisalabad, Pakistan 14 f 15 Chemistry Division, Forest Research Institute, P. O. New Forest, Dehradun - 248 006, Uttarakhand, India 16 g Richland, WA 99354, USA 18 h 19 24 25 26 27 Laboratory of Biocontrol and Antimicrobial Resistance, Orel State University Named After I.S. EP 23 i Turgenev, 302026 Orel, Russia Corresponding authors email: Correspondence: Shityakov_S@ukw.de (S.S.); AC C 22 Department of Energy Systems Engineering, Faculty of Agricultural Engineering and Technology, University of Agriculture, Faisalabad 38000, Pakistan 20 21 TE D Bioproducts Sciences and Engineering Laboratory (BSEL), Washington State University, 17 shariatymohammadali@gmail.com (M.A.S.); Tel.: +49-931-201-30016 28 29 30 31 1 ACCEPTED MANUSCRIPT Graphical abstract RI PT 32 SC 33 34 Abstract 36 Plants are vital for the wellbeing of humankind in a variety of ways. Some plant extracts contain 37 antimicrobial properties that can treat different pathogens. Most of the world’s population relies 38 on medicinal plants and natural products for their primary health care needs. Therefore, there is a 39 growing interest in natural products, medicinal plants, and traditional medicine along with a 40 desire to design and develop novel plant-based pharmaceuticals. These plant-based 41 pharmaceuticals may address the concerns of reduced efficacy of synthetic antibiotics due to the 42 emergence of drug-resistant pathogens. In this regard, some plant extracts from black pepper 43 (Piper nigrum) with antimicrobial properties, including piperine, have the potential to be used as 44 natural dietary supplements together with modern therapeutic approaches. This review highlights 45 possible applications of piperine as the active compound in the fields of rational drug design and 46 discovery, pharmaceutical chemistry, and biomedicine. We discuss different extraction methods 47 and pharmacological effects of the analyzed substance to pave the way for further research 48 strategies and perspectives towards the development of novel herbal products for better 49 healthcare solutions. TE D EP AC C 50 M AN U 35 51 Keywords: black pepper; piperine; bio-active compounds; chemical synthesis; extraction; 52 medicinal chemistry; rational drug design; pharmacology 53 54 55 56 2 ACCEPTED MANUSCRIPT 1. Introduction 58 For as long as humans and animals have existed, they have depended on plants for nourishment 59 and other health benefits. Therefore, there has always been growing attention from the scientific 60 community to plants and their products as additional supplements to synthetic antimicrobials to 61 treat various multidrug-resistant pathogens [1]. Different plant species have been widely used as 62 food flavoring agents, colorant and preservative substances for many centuries across the globe 63 [2]. These plant components were applied in industry and research to extend food shelf-life or to 64 prevent it from spoilage and food-borne diseases [3]. As bioactive plant compounds, they have 65 strong antimicrobial and insecticidal properties widely used in traditional medicine to inhibit or 66 eradicate some infectious pathogens [4]. The well-known antibacterial efficacy of some species, 67 such as black seed (Nigella sativa), garlic bulb (Allium sativum), thyme (Thymus vulgaris), onion 68 (Allium cepa), clove (Syzygium aromaticum), oregano (Origanum vulgare), cinnamon bark 69 (Cinnamomum verum), cumin (Cuminum cyminum) and many more have been extensively tested 70 and verified [5]. 71 Recent scientific findings, concerning the medicinal applications of bioactive substances from 72 plant extracts, have sparked more interest for further development of novel plant-based 73 pharmaceuticals [6]. This initiative might be very important for more than 80% of the world’s 74 population, who are still largely reliant on plant-based medicines and natural products as a 75 primary source of treatment [7]. Additionally, it has been shown by the previous publications 76 that approximately 25% of all medications are derived from plants [8-10]. 77 About 500 various herbal species have been used in modern medicine to treat various illnesses 78 [11] based on anti-inflammatory [12], antioxidant [13] and spasmolytic [14] properties of plant- 79 derived drug-like substances [14]. 80 The last decade has witnessed an unprecedented growth of herbal medicine all over the world 81 [12]. Black pepper, which is widely used in the seasoning, contains bioactive ingredients in its 82 oleoresin fraction, such as essential oils and alkaloid piperine [14]. 83 substance can be considered as the main ingredient of black pepper, possessing diuretic and anti- 84 asthmatic effects [14]. As a GIT (Gastrointestinal Tract)-active agent, piperine can facilitate the 85 activation of pancreatic enzymes in the gut [15]. 86 However, piperine has been proven to be only slightly soluble in water [16], restricting its 87 therapeutic effects and biomedical applications. Therefore, this chemical substance should be AC C EP TE D M AN U SC RI PT 57 3 Moreover, the latter ACCEPTED MANUSCRIPT administered in high therapeutic doses due to its poor dissolution and gut absorption rates, which 89 might be toxic for the reproductive and central nervous systems [17, 18]. Some attempts have 90 been made to develop novel piperine formulations to enhance its bioavailability, using piperine- 91 encapsulated nanosize liposomes [19], which might be inefficient due to their hydrophobic 92 nature. Therefore, the aim of this review is to give a comprehensive outlook on the 93 phytochemical and phytopharmacological aspects of piperine as an active ingredient, and to 94 discuss future perspectives, considering all the aforementioned effects of piperine important for 95 modern herbal medicine. 96 SC RI PT 88 2. Piperine applications in traditional medicine 98 Black paper or Piper nigrum is associated with black peppercorns and berries used for seasoning 99 of different dishes. In general, black pepper mainly contains various alkaloids, volatile oils, 100 carbohydrates, starch, and proteins. Being well-known seasoning ingredient, black pepper is 101 known to be a source of an important alkaloid piperine, which adds a strong, pungent flavor to 102 dishes [10, 20]. 103 The usage of black pepper has already been known for many centuries to treat different types of 104 health problems, including intermittent fever, influenza, muscular pain, and migraine [21] in 105 China and India. There is a growing interest from the scientific community in black pepper in 106 general and its alkaloid piperine in particular as a therapeutic agent, stimulating the appetite and 107 the production of saliva [22]. Piperine was also found to increase the orocecal transit time [23, 108 24] and to act as an anti-tumor agent in mice [25, 26], promoting the enzymatic activity of 109 pancreas and preventing diarrhea [24, 27]. Recently, some studies on the biological properties of 110 piperine have revealed its antioxidant, anticarcinogenic, anti-inflammatory, antiulcer, 111 antithyroid, and antimicrobial effects with some potential to modulate immune responses [28- 112 30]. Additionally, this compound has shown some activity to promote the absorption for some 113 drugs, diminishing their metabolism and cholesterol level in the blood [22, 31]. TE D EP AC C 114 M AN U 97 115 3. Piperine phytochemistry 116 Phytochemical analysis of black pepper had shown the presence of various chemicals, including 117 piperine as the first pharmacologically active compound isolated from the Piperaceae family 118 [32]. However, the other chemical substances were also purified subsequently from black 4 ACCEPTED MANUSCRIPT pepper, comprising phenols, flavonoids, alkaloids, amides, steroids, lignans, neolignans, 120 terpenes, chalcones, etc [24]. While some of these compounds, like piperonylamine, pipericide, 121 sarmentosine, sarmentine, chavicine (Figure 1) already identified as bioactive, the other 122 molecules (piperine) were determined to show a significantly higher pharmacological effect [32- 123 38]. In particular, piperine is believed to be the main bioactive chemical component with 124 antimicrobial activities purified from P. nigrum [39]. This chemical was first extracted from 125 Piper nigrum in 1819 by Hans Oersted [40]. In the pure form, it represents a yellow crystalline 126 powder of piperonyl-piperidine, reacting as a weak base in the solution [41, 42]. Additionally, 127 piperine has also its cis-trans isomeric structures, comprising the trans-trans (piperine), cis-trans 128 (isopiperine), cis-cis (chavicine), and trans-cis (isochavicine) isomers. Apart from piperine, none 129 of these isomers possess the pungency taste [22]. However, the piperanine, piperettine, piperylin 130 A, piperolein B, and pipericine alkaloids extracted from black paper might maintain some small 131 pungent flavor in the experiment [22, 43]. M AN U SC RI PT 119 133 EP TE D 132 Fig. 1. Some chemical substances derived from P. nigrum (Adopted from Ref. Ahmad et al., 135 2012 with modifications [26]). 136 AC C 134 137 4. Antimicrobial effects of black pepper 138 Apart from being used as a seasoning ingredient, black pepper could be applied as an 139 antimicrobial agent against various antibiotic-resistant pathogens in addition to the conventional 140 medication (Table 1). 141 5 ACCEPTED MANUSCRIPT 142 Table 1 Antimicrobial activities of piperine against different micropathogens (Adopted from 143 Ref. Aldaly et al., 2010 with modifications [39]) Erythromycin MIC 100 mg/disc 10 µg/disc 15 µg/disc (mg/ml) E. coli 20 13.5 15 6.25 Staphylococcus aureus 12 23 24 50 Klebsilla pneumonia 15 20 10 25 Proteus vulgaris 17 10 6 12.5 Pseudomonas aeruginosa 8 22 18 100 Candida albicans 23 N/A N/A 3.125 MIC: minimum inhibitory concentration RI PT Streptomycin M AN U 144 Piperine SC Tested microorganisms 145 It was determined to be most effective against the pathogenic Gram-positive strains as 147 Staphylococcus aureus, Bacillus cereus, and Streptococcus faecalis [4]. On the other hand, the 148 Gram-negative bacteria (Pseudomonas aeruginosa, Salmonella typhi, and Escherichia coli) are 149 known to be less susceptible to black pepper [44]. Moreover, the aqueous extracts of black 150 pepper might possess the permeability through lipid membranes of Gram-positive microbes at 151 the concentration of 10 µl/disc to already exhibit the antimicrobial effect [45, 46]. Some studies 152 have been conducted to investigate the antimicrobial and antifungal activity of different alkaloids 153 extracted from black pepper, including tannins, flavonoids, and glycosides [47, 48]. Furthermore, 154 the black pepper extracts can be formulated with metal-contained nanoparticles to protect the 155 agricultural crops from plant pathogens [49]. EP AC C 156 TE D 146 157 5. Piperine synthesis 158 Many synthetic strategies for the synthesis of piperine in literature have been reported, but six of 159 them will be discussed [50-55]. One of the earlier reports about piperine synthesis is Tsuboi and 160 Takeda strategy in 1979 [50]. They described this synthesis in three steps starting from cheaply 161 and commercially available aldehyde called piperonal (2) (Scheme 2). The addition of piperonal 162 (2) to acetylene suspension in the presence of base like KOH at –40 °C afforded the propargylic 163 alcohol 3 in 79% yield. The propargylic alcohol 3 was then subjected to thermal condensation 164 with N-acetylpiperidine diethyl acetal to give intermediate 4 which then undergo (3,3)6 ACCEPTED MANUSCRIPT sigmatropic rearrangement to release allene amide (5) in 74% yield. The allene amide 5 was 166 converted to a mixture of piperine (1) and isochavicine (6) with 65:35 ratio in the presence of t- 167 BuOK within overall yield of 86%. M AN U SC RI PT 165 168 169 Scheme. 2. Tsuboi and Takeda strategy for synthesis of piperine (1). 170 Two years after Tsuboi and Takeda synthetic pathway, Olsen and Spessard published a two-step 172 synthesis of piperine with an efficient stereoselective control of the two double bonds (Scheme 173 3) [51]. Their two-step approach involved a vinylogous Wadsworth-Horner modified Wittig 174 condensation of piperonal with the anion derived from methyl (E)-4-diethylphosphono-2- 175 butenoate to give methyl piperate (7) with 34% yield based on peiperonal (2) and 70% based on 176 phosphonate ester. This transformation is an excellent method to yield two trans alkenes, 177 although the yield was not high based on piperonal. Piperine then was obtained with 86% yield 178 according to the methoxide-catalyzed aminolysis of methyl piperate with piperidine. EP AC C 179 TE D 171 180 181 Scheme. 3. Olsen and Spessard synthetic strategy of piperine (1). 7 ACCEPTED MANUSCRIPT 182 In 1986, Mandai et al. documented also two-step synthesis of piperine [52]. They reported a 184 highly stereoselective synthesis of piperine through a double elimination reaction of β-acetoxy 185 sulphone (Scheme 4). Their strategy involved coupling of sulphone 8, which was synthesized 186 from piperonal (2), with aldehyde 9 in the presence of a strong base n-BuLi to give acetate 10 in 187 66% yield. Double elimination of acetate 10 using t-BuOK yielded piperine in 77% yield with 188 good stereocontrol of 90% E:E. SC M AN U 189 190 RI PT 183 Scheme. 4. Mandai et al. synthetic strategy of piperine (1). 191 In 1995, Sloop reported a microscale synthesis of piperine involving transformation of methyl 193 crotonate (11) into ester 13 (Scheme 5) [53]. This access through allylic bromination of methyl 194 crotonate by N-bromosuccinimide (50% yield) followed by aldol like condensation to yield 195 methyl piperate 7 which with a moderate yield of 40%. Hydrolysis of methyl piperate 7 followed 196 by aminolysis with piperidine gave piperine with 55% yield over two steps. AC C EP 197 TE D 192 198 199 Scheme. 5. Sloop synthetic strategy of piperine (1) from methyl crotonate. 200 8 ACCEPTED MANUSCRIPT 201 In 2000, Chandrasekhar et al. reported a successful synthetic strategy through the formation of 202 dienal 16 (Scheme 6) [54]. The dienal 16 was obtained via the addition of Grignard reagent of 203 piperanol 15 to aldehyde tosylhydrazones in 80% yield. Pennick oxidation of dienal led to 204 piperic acid 13 followed by aminolysis in 73% yield overall two steps. SC 206 207 RI PT 205 Scheme. 6. Chandrasekhar et al strategy to the synthesis of piperine (1). 208 Finally, Schobert et al. in 2001 reported an intermolecular three-component reaction between 210 aldehydes, amines and ketenylidenetriphenylphosphorane (Ph3P=C=C=O) lead to a selective 211 formation of piperine (Scheme 7) [55]. Their strategy started from conversion of piperanol into 212 α,β-unsaturated aldehyde 17 in two steps; olefination with ethylidenetriphenylphosphorane to 213 give cis–trans-isomeric mixtures of 3,4-(methylenedioxy)-β-methylstyrene, and a trans-selective 214 allylic oxidation with selenium dioxide to furnish the E-aldehyde 17. The aldehyde 17 was then 215 subjected to the three-component domino reaction with ketene (Ph3P=C=C=O) and piperidine to 216 furnish piperine in 90% yield. TE D M AN U 209 219 220 Scheme. 7. Schobert et al. synthetic method piperine (1) via three-component strategy. AC C 218 EP 217 221 6. Piperine extraction 222 The piperine compound can be extracted from black pepper in the range of 6-13% by means of 223 organic solvents [56]. Several types of the volatile organic solvents have been used so far for this 224 purpose, comprising acetone, dichloromethane, ethanol, and diethyl ether under specific pressure 225 and time conditions [22]. The piperine purification process depends on various parameters, such 226 as the type of solvent used and the degree of maturation stage of black pepper [57]. Some 9 ACCEPTED MANUSCRIPT alcohol-based solvents tend to be hydrotropic and ionic chemical solutions [14], providing the 228 rapid and cheap extraction of piperine [58]. Almost 95-98% purity of the piperine extract is 229 required to be used in the pharmaceutical industry, and the additional purification might be 230 needed for this by the oleoresin extract [59]. There are some common techniques, which are used 231 for piperine extraction, such as maceration, solvent extraction, and soaking. 232 All these extraction methods require high temperature and the time-consuming with the high risk 233 of the final product degradation [60]. Some commons mistakes in the extraction technique might 234 include the improper selection of the method and the excessive usage of solvents extra usage of 235 organic solvent [60]. In fact, the microwave- or ultrasound-assisted, and supercritical fluid 236 extraction methods were developed and optimized to enhance the extraction yield of chemical 237 substances. Therefore, the modern extraction techniques summarized in Table 2 are discussed in 238 more detail in the next sections. M AN U SC RI PT 227 239 Table 2 Different extraction techniques used to extract piperine Extraction time 18 min Microwave assisted extraction (MAE) 2 min Double bypass Soxhlet apparatus (DBSA) Hydrotropic solubilization Extraction yield (w/w) 0.58% TE D Extraction technique Ultrasound assisted extraction (UAE) 94% EP AC C 240 12 ± 1 h 3.90% ± 0.10% 2h 90% to 96%* Benefits Disadvantages Reference Short running time, higher extractive yield, controllable parameters Selective, short running time, high extraction Small particle size, more filtration steps [61] More filtration steps, time consuming during cooling Long extraction time, solvent consuming [62] - [62] Easily operate, simple Minor purification steps, unlike surfactant not foaming 10 [63] ACCEPTED MANUSCRIPT 2 to 5 h 6.7% to 7.6% Efficient, selective, clean, fast High cost, less pressure resistant 30 min 3.57% - 18 min 1.96% Environment friendly, Short extraction run, high efficiency, 241 [66] SC 242 [64, 65] RI PT Supercritical fluid extraction (SFE) Ionic liquid ultrasound assisted extraction (IL-UAE) 6.1. Soxhlet extraction 244 The Soxhlet extraction technique has been used in the past for the extraction of biologically 245 active compounds [67]. However, this methodology might be considered outdated and not very 246 efficient in comparison to more advanced extraction procedures [68]. 247 The performance of this method and its modifications to extract piperine from black pepper has 248 been evaluated by Subramanian and coauthors, showing that the DBSA (Double By-Pass Soxhlet 249 Apparatus) approach outperforms the other techniques in terms of the decreased extraction time 250 due to the overall increase of extraction cycles [63]. The extraction results showed the improved 251 extraction yield in 3.9% from DBSA after 12 hours of extraction [63]. In the study of 252 Rajopadhye and coauthors, the black pepper roots were used for the Soxhlet extraction with 253 methanol, obtaining the peperine concentration of 9.56 ± 0.83 mg/g [69]. The other authors 254 applied the supercritical fluid (CO2) extraction together with the Soxhlet method to extract 255 piperine from the corn, reaching the maximal piperine concentration (56.6 mg/g) by using the 256 former methodology [69]. 257 AC C EP TE D M AN U 243 258 6.2. Hydrotropic extraction 259 Various hydrophobic molecules can be extracted from disrupted plant cells by using the 260 hydrotropic solutions [70]. The hydrotropic solutions of Piper nigrum plant extracts form 261 permeable component, allowing the adsorption of hydrophobic molecules, such as piperine, on 262 cellulose during the extraction process [71]. The whole process can be controlled by changing 263 some parameters, like particle size, temperature, and the amount of hydrotropic solvent [70]. A 264 relationship was observed between the extraction efficiency and the alkyl chain length of the 11 ACCEPTED MANUSCRIPT hydrotrope [72]. In particular, some hydrotropic solvents, including sodium p-toluene sulfonate, 266 sodium xylene sulfonate, sodium-butyl benzene sulfonate, and sodium cumene sulfonate were 267 already tested to extract piperine [71]. The latter molecule had shown the best performance in 268 this process in comparison to the other solvents due to its longest chemical chain [71]. 269 The other factor, such as the hydrophobic volume of a molecule also influences the extraction 270 process, following a similar pattern [72]. Additionally, it was determined that the extraction 271 process of piperine is optimal at 30°C, using this particular extraction technique [71]. This 272 condition was considered to be most effective for the piperine transport into the hydrotropic 273 medium and finally to on cellulose. 274 The particle size reduction of a substrate from 710 to 50 µm might also directly interfere with the 275 purity of the extracted piperine. As per reduced particle size, the cellular disintegration would be 276 increased so as the efficiency of hydrotropic solution leaching into the cellular matrix. This 277 process benefits the purity of extracted piperine from 89 to 98% [71]. SC 279 6.3. 280 The supercritical fluid extraction (SFE) method becomes a very popular technique for the 281 extraction of different drug-like molecular compounds from various sources, including plants 282 [73]. Moreover, it is mainly considered as the clean, efficient, selective and rapid extraction 283 process [73]. The SFE methodology implements different solvents with high molecular densities 284 to archive more efficient compound extraction [73]. In fact, the SFE mechanism implies the 285 effective mass transfer via fluids with much greater molecular diffusion and smaller viscosity 286 than other extraction techniques [73]. On the other hand, this method is using the temperature 287 and pressure for liquid carbon dioxide in the range of 31.1 °C and 73.8 bar, [74] with low 288 polarity level, which plays a significant role in the extraction of non-polar compounds [73]. To 289 extract the polar compounds, some chemical polar substances were used as additives to increase 290 the polarity of the mixture in a range of up to 10% of the main supercritical fluid [74]. 291 This method was extensively applied for the peperine extraction in the first decade of the 20th 292 century [75]. The piperine yield was obtained from this extraction to be in the range from 81% 293 and 98% [76], using the pressure of 350 atm and the temperature of 60° [77]. Another SFE 294 extraction protocol developed by Kurzhals and Hubert (1980), using a mixture of propane and 295 carbon dioxide at 52°C and 78 bar, secured the piperine extraction yield up to 98% [78]. AC C EP TE D Supercritical fluid extraction M AN U 278 RI PT 265 12 ACCEPTED MANUSCRIPT 296 Furthermore, Sovova et al. (1995) have also performed the SFE extraction experiments with the 297 same parameters, resulting in the piperine extraction up to 7.6% by its weight [64]. 298 6.4. Ultrasound-assisted extraction 300 The ultrasound-assisted extraction (UAE) technique is primarily based on the principles of 301 thermal effects and cavitation, which mediate the mass transport phenomena across different 302 types of cell membranes [79]. In particular, the cavitation bubbles collapse becomes the cause of 303 micro-jets formation and disruption of cells due to the asymmetrical imploding of these bubbles 304 near to the solid surface [80], occurring at high temperature (up to 5000 K) and pressure (1000 305 bar). The thermo-physical effects produced by this process might create the cellular membrane 306 disruption and the impairment of circulating liquids in the cells [81]. All this increases the UAE 307 extraction yield through the more efficient permeation of the solvents into the plant cells [82]. 308 Overall, the advantages of this method include effective solvent permeation rate and low 309 extraction time and temperature [83]. On the other hand, the UAE extraction also depends on the 310 type of the solvent, the number of extraction cycles, temperature, ultrasound intensity and the 311 solid-solvent ratio [80]. The technique was effectively allied to the extraction of piperine from 312 Piper longum by using different organic solvents, such as ethanol, hexane, and acetone [61]. In 313 this study, acetone was found the most effective organic solvent to extract piperine as this 314 extraction is dependent on the polarity index of solvent [61]. 315 TE D M AN U SC RI PT 299 6.5. Ionic liquid extraction 317 The ionic liquid (IL) extraction is a combination of cations and anions of molten salts with the 318 melting point typically below 100°C [84]. The IL technique has some advantages, which makes 319 it the method of choice due to its more stable extraction of various chemicals, using highly polar 320 solvents and low vapor pressure [85]. 321 The physicochemical properties of ionic liquid have a significant impact on the analyte and its 322 extraction efficiencies [66]. These properties are usually correlated with ionic interactions [86]. It 323 is also worth mentioning that the hydrophobic interactions are playing an important role between 324 bio-active compounds and aqueous ILs, hydrophobic interaction in this extraction process, as it 325 was detected for the IL extraction of piperine, tannin, rutin, quercetin, and curcumin [87, 88]. 326 However, the IL approach is usually combined with other extraction methods, such as UAE, to AC C EP 316 13 ACCEPTED MANUSCRIPT achieve more efficient extraction yield [66]. For instance, the ionic liquids-ultrasound based 328 extraction (IL-UAE) was devised to enhance the extraction output and to reduce the extraction 329 time [89]. In fact, IL-UAE was utilized to extract piperine from black pepper using four different 330 anions (BF−4, BF−, H2PO−4, and PF−6) with 1-butyl-3-methylimidazolium (C4MIM) ionic liquid 331 [66]. The piperine extraction efficiency was dependent on the ionic composition in a descending 332 order in terms of their hydrophilicity as BF−4>Br−>H2PO−4>PF−6 [66]. Finally, the BF−4 ionic 333 form with C4MIM had provided the optimal extraction condition, including ultrasonic power, 334 extraction time, the solid-to-liquid ratio for the piperine purification. In particular, by using these 335 reagents at a concentration of 0.2 M with a solid-to-liquid ratio of 1:15 and an ultrasound power 336 of 500 W, the piperine extraction yield of 3.577% was obtained [66]. SC RI PT 327 338 6.6. 339 The microwave-assisted extraction (MAE) has been widely implemented to extract various 340 chemical compounds [90]. This technology utilizes the microwave energy, which is absorbed by 341 chemicals in order to evaporate them from the solid raw material. Finally, the condensation of 342 these volatile compounds occurs as the recovering process [91]. MAE can be considered as 343 selective methods that favor polar molecules and solvents with high dielectric constant, 344 producing a heat during the extraction [92]. This heating process is largely generated by 345 microwaves via the ionic induction or dipole rotation [93]. The hydration or soaking phase of 346 extracted material in water plays an important role to control the extraction rate. Some other 347 factors, like the extraction temperature and microwave intensity, have also contributed to the 348 extraction process [90]. At the high microwave intensity, some cellular agglomeration occurs at 349 the beginning of the extraction phase followed by the rapid cellular disruption [94]. In particular, 350 as the power of microwaves increases the extraction rate goes high until the optimum extraction 351 yield is reached [94]. Additionally, the microwave irradiation strength was found to be directly 352 proportional to the solvent loss during the extraction [71]. For instance, when the microwave 353 intensity is increased from 300 to 450 W, the solvent consumption is also elevated from 16 to 354 20%. For instance, when microwave concentration in the range of 300 to 450 W the solvent loss 355 16% to 20%. But it decreases when the power of microwaves reduces as 150 W lost the solvent 356 up to 8%. Furthermore, the surface tension and viscosity could also contribute to the solvent loss 357 during the extraction process [95, 96]. AC C EP TE D Microwave-assisted extraction M AN U 337 14 ACCEPTED MANUSCRIPT The MAE technique was successfully used for the piperine extraction from Piper nigrum, where 359 the plant cells had experienced a dielectric heating [71]. During this extraction, the polar and 360 non-polar solvents were used, such as toluene, petroleum ether, heptane, dichloromethane, and 361 ethanol [22]. As a result, the highest extraction was achieved by applying non-polar petroleum 362 ether to intensify the piperine purity from 85 to 94% [22]. On the other hand, the other semi- 363 polar and polar solvents (dichloromethane and ethanol) provided the extraction rate from 75 to 364 80%, respectively. RI PT 358 365 7. Piperine detection 367 Several analytical techniques and quantitative methods (Table 3), including high-performance 368 liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), colorimetric 369 assays, Kjeldahl method, and ultraviolet-visible spectrophotometry (UV-Vis), are the most 370 common approaches used in the piperine detection after its extraction from white/black pepper 371 [61, 97]. 372 The Kjeldahl method or Kjeldahl digestion was applied among the first techniques to measure 373 piperine indirectly via evaluation of the total nitrogen amount in black pepper [98]. Previously, 374 this analytical technique was developed for the quantitative determination of nitrogen contained 375 in various organic substances [56]. Before that, the hydrolysis of piperine methylenedioxy group 376 by chromotropic acid was needed for some old colorimetric assays [99]. On the other hand, the 377 UV-Vis method is also a powerful technique to detect the UV absorption spectra of piperine at 378 343 nm wavelength [100] and to select this compound for its isomers [101]. Additionally, the 379 GC-MS methodology might evaluate the degradation state of piperine while identifying some 380 alkaloids (oleoresin), which are present in black pepper [102]. Presently, HPLC becomes a 381 method of choice for piperine detection with much higher precision capabilities compared to 382 UV-Vis [103]. Moreover, the high-performance thin layer chromatography (HPTLC) as a 383 modification of thin layer chromatography has also been implemented for the detection of 384 piperine from herbal products to provide the most accurate results in the experiment [104]. 385 Finally, the chemical characterization of piperine and its isomeres was also achieved by 1H 386 nuclear magnetic resonance (NMR) spectroscopy [105]. In this study, the difference in the 387 coupling constants for the olefinic protons (cis-2 J(H,H)≈11 Hz, trans-2 J(H,H)≈15 Hz) made it 388 possible to determine the configuration of the isolated compounds [105]. AC C EP TE D M AN U SC 366 15 ACCEPTED MANUSCRIPT 389 Table 3 Analytical techniques used for identification/detection of piperine Methodology Komarowsky method Phosphoric acid method Nitric acid method High performance thin liquid chromatography (HPTLC) Nuclear magnetic resonance (NMR) spectroscopy C18 Mobile Phases; Acetonitrile: water (90:10) Piperine isomers dissolved in deuterochloroform (CDCl3) to record 1H -NMR spectra Reference [99] [106] [107] [102] [97] - Mobile Phase; Acetonitrile: Water (90:10) at UV 343 nm and 1.5 ml/min flow rate Mobile Phase; Benzene: ethyl acetate: diethyl ether (60:30:10) at UV 343 nm and 01 ml/min flow rate Addition of tetramethylsilane (TMS) not required [61] [104] [105] EP 391 Piperine dissolved in organic solvents and absorbance measured at 343 nm TE D High-performance liquid chromatography (HPLC) Apolar column BP1 M AN U Gas chromatography-mass spectrometry (GC-MS) UV spectrophotometry Detection parameters Piperine heated with defined reagents, purple color develops, absorbance at 570 nm Piperine heated at 100°C for 8 min, bluish green color develops, absorbance at 635 nm Concentrated piperine, alkali and thiourea added, color changes, absorbance at 490 nm FID (Injector and Detector 300°C) RI PT Technique Colorimetric SC 390 392 8. Pharmacological effects of piperine 394 8.1. 395 It is well-known that various spices and herbs, including a black piper, contain numerous active 396 ingredients, like flavonoids, terpenoids, phytoestrogens and minerals [26]. Among them, piperine 397 was detected to have an antioxidant potential, which might diminish oxidative stress in the cells 398 caused by the high-fat diet [108]. Moreover, piperine was also shown to decrease the level of the 399 thiobarbituric acid reactive substances via the maintenance of catalase, glutathione, glutathione 400 peroxidase, Glutathione-S-transferase, and superoxide dismutase concentrations [108]. This 401 substance could also improve the activity of biotransformation enzymes in the liver in a dose- AC C 393 Antioxidant activity 16 ACCEPTED MANUSCRIPT 402 dependent way [109]. Furthermore, several studies on the antioxidant activity of piperine have 403 been conducted to establish the reduction of lung metastatic incidence in the B16F-10 melanoma 404 cells through the alteration in lipid peroxidation and the stimulation of antioxidant enzymes [25, 405 110, 111]. RI PT 406 8.2. Anti-inflammatory activity 408 Various anti-inflammatory effects of substances extracted from plants are known for many 409 therapeutic applications in modern medicine and pharmacy to treat different disease [112]. In 410 particular, some ethanolic and hexane extracts of black pepper have exposed a significant anti- 411 inflammatory activity in mice and rats, using different dosage protocols [113]. Moreover, 412 peperine had also revealed the same activity in the interleukin (IL) 1β-activated fibroblast-like 413 synoviocytes [114], inhibiting the LPS-stimulated endotoxins [115]. Further, piperine might be 414 viewed as a potent immunomodulator, inhibiting airway inflammation a murine model of asthma 415 by the enhanced expression of TGF-beta gene in the lungs [116]. Piperine was also detected to 416 reduce the production of IL-6, MMP-13, and prostaglandin E at the concentration range of 10- 417 100 µg/ml [114]. In another study, piperine was coadministered with curcumin from Curcuma 418 longa to suppress a high fat diet-induced inflammation in the C57BL/6 mice and for the 419 prevention of metabolic syndrome [117]. Apart from that, the piperine anti-inflammatory 420 potential had been investigated at colorectal sites, inhibiting the FFA-induced TLR4 mediated 421 inflammation and acetic acid-induced ulcerative colitis in mice [118]. Finally, this compound 422 was evaluated in the carrageenan-induced inflammation assay in mice to assess the analgesic and 423 antiinflammatory activities of piperine activities at the oral dose of 6 mg/kg/day [119]. M AN U TE D EP AC C 424 SC 407 425 8.3. Anti-cancer and hepatoprotective activity 426 The anti-tumor activity of piperine has been detected after its oral administration to reduce the 427 incidence of some forms of gastrointestinal cancers [120]. An alcoholic extract of black pepper, 428 containing piperine, was found to be effective against lung cancer via altering lipid peroxidation, 429 which leads to the spread of free radical reactions and cellular damage [26]. Besides, piperine 430 might restrict the cell cycle at G1/S phase, inhibiting the HUVECs (human umbilical vein 431 endothelial cells) proliferation and migration [121]. In animal models, piperine can hinder 432 angiogenesis, suppressing the tubule formation by endothelial cells and the phosphorylation of 17 ACCEPTED MANUSCRIPT protein kinase B [121]. Some anti-cancer activity of piperine can be seen by applying it in the 434 combination with the FDA-approved antineoplastic compound docetaxel to treat castrate- 435 resistant prostate cancer [122]. By restricting the enzymatic activity of hepatic CYP3A4, piperine 436 decreases the metabolizing rate of this drug in the liver [122]. Additionally, it has also been 437 studied that the application of piperine in a nutritional supplement might also enhance the 438 docetaxel immunosuppressive effects in xenograft animal models without severe side-effects 439 [122]. Piperine was also found to be active against both androgen-dependent and independent 440 prostate cancer cell lines (LNCaP, 22RV1, PC-3, and DU-145), inducing apoptosis through the 441 activation of PARP-1 and caspase-3 proteins [115]. In the LNCaP prostate cancer cells, piperine 442 disrupts the androgen receptor expression, significantly reducing the detection of the prostate- 443 specific antigen [123]. 444 It was previously established that the methanolic extract of black pepper has the hepatoprotective 445 properties confirmed in Wistar rats with induced hepatic damage caused by ethanol- CCl4 [124]. 446 In these experiments, ethanol-CCl4 was administered to increase the levels of triglycerides, 447 alanine transaminase, aspartate transaminase, alkaline phosphatase, and bilirubin. All these 448 parameters came to normal after the animals were treated with the methanolic extract of black 449 pepper [124]. This extract reduced the lipid peroxidation as a hepatoprotective effect at the 450 administered doses alone [125] or in combination with some antituberculosis drugs [125]. In 451 another study, the d-galactosamine-induced liver injury modeled in mice was treated with 452 piperine to normalize the concentration of glutamic oxaloacetic transaminase and pyruvic 453 transaminase levels in serum. The proposed mechanism had been found to be associated with the 454 reduced sensitivity of hepatocytes to TNF-α [126]. 455 AC C EP TE D M AN U SC RI PT 433 456 8.4. Antidiarrheal, antidepressant, and analgesic activity 457 The aqueous extract of black pepper was also assessed for its antidiarrheal via promoting the 458 antimotility and antisecretory effects in the gut at a dose of 75, 150, and 300 mg/kg due to the 459 presence of alkaloids (piperine) and carbohydrates [127]. On the other hand, in corticosterone- 460 induced mice model of depression, piperine was examined for its possible antidepressant effect 461 [128]. The depression in animals was evaluated via a decrease of sucrose utilization and an 462 increment of immobility time in the tail suspension test and forced swim test. As a result, in the 463 hippocampus of corticosterone-treated mice, levels of brain-derived neurotrophic factor protein 18 ACCEPTED MANUSCRIPT were significantly reduced in the hippocampus of corticosterone-treated mice [128]. Finally, the 465 piperine treatment of the behavioral and biochemical changes in mice induced by corticosterone 466 had reverted to normal [128]. 467 Furthermore, the acetic acid-induced twitching and tail-flick tests had shown models had shown 468 the prevention of acetic acid-induced writhing in mice after the intraperitoneal (i.p.) 469 administration of piperine at a dose of 30-70 mg/kg in comparison to indomethacin (20 mg/kg, 470 i.p.) [129]. Similarly, the i.p. injections at a dose of 30 and 50 mg/kg for piperine and at a dose of 471 5 mg/kg for morphine, had significantly increased the reaction time of mice in the tail-flick 472 assay. The analgesic effects of both substances were abolished by the pretreatment of animals 473 with naloxone (5 mg/kg i.p.), suggesting the involvement of the opioid pathway in this process 474 [129]. M AN U SC RI PT 464 475 8.5. Immuno-modulatory activity, bioavailability and cancerogenic effects 477 The immuno-modulatory activity of piperine was also been examined at a dose of 50 to 250 478 µg/ml to be cytotoxic for Dalton’s lymphoma ascites, Ehrlich ascites carcinoma and L929 cells 479 [25]. In the BALB/c mice, piperine administration caused the increment in total white blood 480 cells, bone marrow cells, and alpha-esterase positive cells [25]. 481 In a murine model of Mycobacterium tuberculosis infection, piperine was evaluated to enhance 482 the efficacy of rifampicin [130]. To examine the in-vitro immunomodulation of piperine, the 483 mouse splenocytes were used to produce cytokines together with the activation of macrophage 484 and proliferation of lymphocyte. As a result, the piperine-treated splenocytes have shown the 485 enhanced secretion of Th-1 cytokines, improved macrophage activation, and proliferation of B 486 and T cells [130]. To inhibit antigen-induced allergic reactions that control degranulation, 487 piperine can interfere with the IgE-mediated degranulation and cytokine production by RBL-2H3 488 cells [131]. 489 Some molecular mechanisms underlying piperine activities include a change in the membrane 490 dynamics accompanied by the initiation of protein synthesis linked to the cytoskeleton 491 functioning. This stimulates the passive absorption in the small intestine, thus, supporting the 492 effective drug permeation through the epithelial barriers [132]. However, piperine exhibits poor 493 bioavailability [22] that can be enhanced in situ intestinal absorption models by formulating it 494 with ethyl oleate, Tween 80, and Transcutol P as a self-emulsifying drug delivery system [22]. AC C EP TE D 476 19 ACCEPTED MANUSCRIPT Additionally, piperine amended the bioavailability of some antibiotics, like ampicillin, 496 norfloxacin [133], amoxicillin, and cefotaxime sodium [134] and herbal compounds (curcumin 497 and resveratrol) via its inhibitory effect to the liver enzymes [135]. However, some studies 498 indicated the adverse effects of piperine on cells because of the 3,4-methylenedioxybenzene 499 moiety presented in the molecule, acting as a carcinogen [136, 137]. Due to this, the piperine 500 structure resembles some other cancerogenic compounds, comprising safrole, methyl eugenol, 501 and estragole [138]. Besides, treatment of cancer cells with piperine provided diminished 502 expression of phosphorylated STAT-3 and NF-kB transcription factors together with a reduction 503 of androgen-dependent and androgen-independent tumor growth [123, 139]. Piperine could be 504 also administered as an effective antitumor agent against lung cancer via activation of caspase-3 505 and caspase-9 cascades and induction of apoptosis [140]. M AN U SC RI PT 495 506 9. 508 Piperine is a bioactive compound with a broad spectrum of therapeutic activities, which can be 509 extracted from black pepper given this plant its pungent test. Despite the various therapeutic 510 properties of piperine, its biomedical applications are still limited due to its poor bioavailability 511 and low aqueous solubility. This situation can be improved by piperine supramolecular 512 formulation with some hydrophilic substances, including unmodified cyclodextrin (CD) 513 excipients (Figure 2) [141,142]. Recent investigations on the physicochemical properties and 514 solubility of piperine complexes with α-, β-, and γ-CDs (Figure 3) has defined that the CDs 515 interact with the methylenedioxyphenyl group of piperine in a molar ratio of 1:1, influencing the 516 complex solubility [141, 142]. EP AC C 517 Future perspectives and conclusion TE D 507 20 518 TE D M AN U SC RI PT ACCEPTED MANUSCRIPT Fig. 2. Chemical structure of unmodified cyclodextrins. The high-resolution graphics were 520 prepared using the ChemDraw software [143]. AC C 521 EP 519 21 M AN U SC RI PT ACCEPTED MANUSCRIPT 522 Fig. 3. Piperine formulations with unmodified hydrophilic cyclodextrins (α-, β-, and γ-CDs) 524 shown as hypothetical scheme to improve its aqueous solubility and absorption in the gut after 525 subsequent dissociation of the inclusion complex. The high-resolution graphics were prepared 526 using the ChemDraw and AutoDock software [143, 144]. TE D 523 527 On the other hand, the quantitative structure-activity relationship (QSAR) paradigm might be 529 applied as a concept where the structural property of drug-like molecules is correlated with their 530 biological activity. It is important to quantify a biological activity in the experiments to match it 531 to the chemical characteristics of drugs, using computational modeling. In particular, this 532 technique has already been used in different biomedical applications to investigate and screen 533 various chemical substances [145-149]. Additionally, the QSAR analysis was applied to the 534 alkaloid piperine to study its pharmacokinetics with respect to the P-gp-mediated multidrug 535 resistance (Figure 4 [A]) and drug metabolism by the P450 3A4 cytochrome (Figure 4 [B]) 536 computationally [150, 151]. Recently, another computational study associated with molecular 537 docking method was conducted to discover novel piperine-derived ligands for the P-gp effective 538 inhibition in bacteria [152]. AC C EP 528 22 ACCEPTED MANUSCRIPT B A Met68 Phe724 Phe728 Met67 2.9 Trp126 RI PT Arg440 Ser975 Tyr949 SC Phe302 Phe137 Fig. 4. Piperine binding to the P-gp transporter (A) and P450 3A4 cytochrome (B) is shown 541 within the protein binding sites represented by a molecular surface with the interacting amino 542 acid residues. The piperine molecule is depicted in sticks; and the protein residues are displayed 543 as ball-and-stick models, respectively. Hydrogen bonds are visualized as dashed lines measured 544 in Å. All Hydrogen atoms are removed to enhance the overall clarity. The high-resolution 545 graphics were prepared using the AutoDock and PyMol software [144, 153]. M AN U 539 540 546 In fact, this compound has already proven to be active against different bacteria [154], so its 548 derivative forms, including piperonal, piperonylic and piperic acids have shown the similar 549 effects [155, 156]. Some inhibitory effects of piperine were confirmed in the experiment for 550 breast cancer in combination with epigallocatechin gallate, using mouse macrophages [157]. 551 Furthermore, a large library of piperine analogs, using the Autodock and Authodock Vina 552 software, was screened for the possible hit and lead compounds to bind to survinin as a member 553 of the inhibitor of apoptosis family [158]. Other results from the molecular dynamics simulations 554 using the MM-PB/GBSA (molecular mechanics Poisson-Boltzmann and generalized Born 555 surface area) approach together with the alanine scanning defined the important role of 556 hydrophobic interactions as a driving force in the piperine-protein binding [159]. 557 Moreover, the piperine cytotoxic potential and its anti-HIV activity were determined in the 558 combination with the QSAR approach [160]. Furthermore, using the computational approaches 559 to predict the peperine toxicity in vivo might be also beneficial for the animal welfare to reduce 560 the unnecessary usage of laboratory animals [161]. Some other studies used QSAR to analyze 561 the piperine analogs to inhibit the NorA efflux pump in Staphylococcus to predict the protein- AC C EP TE D 547 23 ACCEPTED MANUSCRIPT ligand binding mechanism and to measure quantitatively the ligand binding affinity to NorA 563 [145]. 564 In conclusion, phytochemical and pharmacological attributes of piperine as an active 565 pharmaceutical ingredient indicated its value for pharmaceutical chemistry and biomedicine. 566 Different synthetic strategies, extraction, and detection techniques emphasized the important role 567 of piperine for the development of novel natural remedies and future perspectives towards its 568 efficient formulation with hydrophilic excipients. In particular, some of these novel approaches 569 for optimizing delivery of piperine based on its complexation with CD and interaction with the 570 P450 3A4 cytochrome and P-gp transporter were discussed. 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