Comparative Evaluation of SFE and Solvent Extraction Methods on the Yield and Composition of Black Seeds ( Nigella Sativa )

LJLC253871 1 2 LJLC_030_017 Techset Composition Ltd, Salisbury, U.K. 7/12/2007 Journal of Liquid Chromatography & Related Technologiesw, 30: 1–11, 2007 Copyright # Taylor & Francis Group, LLC ISSN 1082-6076 print/1520-572X online DOI: 10.1080/10826070701540100 3 4 5 6 7 8 9 10 11 12 13 Comparative Evaluation of SFE and Solvent Extraction Methods on the Yield and Composition of Black Seeds (Nigella Sativa) 14 15 16 17 Madduri V. Rao Department of Chemistry, UAE University, Al-Ain, United Arab Emirates 18 19 Ali H. Al-Marzouqi 20 Department of Chemical and Petroleum Engineering, UAE University, Al-Ain, United Arab Emirates 21 22 23 Fatima S. Kaneez 24 Department of Biology, UAE University, Al-Ain, United Arab Emirates 25 26 S. Salman Ashraf 27 Department of Chemistry, UAE University, Al-Ain, United Arab Emirates 28 29 30 Abdu Adem 31 Department of Pharmacology and Therapeutics, UAE University, Al-Ain, United Arab Emirates 32 33 34 35 36 37 38 39 40 41 Abstract: Supercritical Fluid Extraction (SFE) conditions (temperature, pressure, and volume of CO2) were optimized to obtain high quality black seed oil rich in antioxidants. The highest extraction yield (31.7%) was obtained under the SFE condition (508C, 400 bar, and 100 mL), whereas SFE condition (508C, 100 bar, 200 mL) gave a low yield (0.84%) as lipids were not extracted. HPLC characterization of compounds in the SFE extracts indicates the presence of a large number of 42 43 44 45 Address correspondence to Ali H. Al-Marzouqi, Department of Chemical and Petroleum Engineering, UAE University, Al-Ain P. O. Box 17555, United Arab Emirates. E-mail: hassana@uaeu.ac.ae 1 LJLC253871 LJLC_030_017 2 46 47 48 49 50 51 52 53 Techset Composition Ltd, Salisbury, U.K. 7/12/2007 M. V. Rao et al. compounds in high concentrations in the extract with a low yield. The yield and composition of SFE extracts were compared with the extracts obtained by the soxhlet extraction method and the SFE extract with low yield was found to be superior. Selected SFE extracts were also subjected to GSH recovery tests, and maximum recovery (84.6%) was obtained for the extract with low yield confirming the presence of antioxidant compounds. Keywords: Black seeds, Nigella sativa, Antioxidant, SFE, HPLC characterization 54 55 56 INTRODUCTION 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 The black seed (Nigella sativa) extract, commonly known as Habbat El Baraka in the Arab world, has been in use for generations in various parts of the world, including most of the Arab population. Recent investigations of black seedSand many other herbs used for culinary, as well as medical purposes, have been shown to contain high levels of antioxidants.[1 – 2] The yield and chemical composition of black seed oil has been investigated by several researchers.[3 – 8] The antioxidant properties of black seed oil are recently reviewed.[1] Antioxidant and antimicrobial properties of black cumin are also studied.[9] It has been shown that some of the compounds isolated from black seeds have appreciable free radical scavenging properties.[10] This antioxidant property has also been reported by other investigators.[11,12] Thus, it is hypothesized that the beneficial effects of black seeds and other herbs are most likely due to their protection against cellular damage caused by oxidative stress. Supercritical fluid extraction (SFE) has received increasing attention by several authors, as this technology uses supercritical carbon dioxide (SC CO2), and, thus, no solvent residues are left behind in the product.[13 – 16] This technique has the added advantage of recovering the volatile compounds and does not alter the delicate balance of components in natural products. Very limited numbers of studies have been reported on the use of supercritical fluids for the extraction of black seeds. Fullana et al.[14] have used statistical, kinetic modeling, and simulation studies for the extraction of black seed oil using supercritical carbon dioxide. The study aimed at obtaining higher yields of oil rather than compositional quality of the extract. In another study, deacidification of black seed oil extracted by supercritical carbon dioxide was investigated.[17] These studies lack a thorough optimization of the SFE process in order to achieve black seed extract rich in antioxidant principles (with high quality and yield). Therefore, it is desirable to investigate the compositional quality of black seeds extracted by supercritical carbon dioxide. Pharmacologically active principles (thymoquinone, dithymoquinone, thymo-hydroquinone, and thymol) of black seed oil extracted using conventional solvent extraction techniques have been isolated by solid phase extraction (SPE) and HPLC separation.[18] In addition, four novel alkaloids, namely nigellicine, nigellidine, nigellimine, and nigellimine N-oxide have LJLC253871 LJLC_030_017 Techset Composition Ltd, Salisbury, U.K. Supercritical Fluid Extraction Conditions 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 7/12/2007 3 also been isolated from black seeds.[19] Thymoquinone content fixed oil of Nigella sativa obtained from different sources has been estimated by gas chromatography.[20] Oxidative stress caused by reactive oxygen species (ROS) deplete intracellular Glutathione (GSH) levels.[21 – 23] Recently, Ashraf et al. have shown that diverse environmental pollutants including xylene, redox cycling metals, and UV radiation can cause oxidative stress in skin fibroblasts, leading to GSH depletion and causing S-thiolation of intracellular proteins.[24] GSH is recovered by the addition of extracts containing antioxidant compounds. Optimum GSH recovery indicates the maximum concentration of antioxidant compounds in the extract, which shows its protective effect against oxidative stress. In this study, intracellular GSH was measured following the method published by Coleman et al.[25] The present study focuses on optimizing SFE conditions for extraction of black seeds, aiming at obtaining extract rich in antioxidants through comparison of their composition with those obtained by traditional soxhlet extraction methods. 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 EXPERIMENTAL Supercritical Fluid Extraction The experimental apparatus consisted of a 260 mL capacity syringe pump and controller system (ISCO 260D), and an ISCO series 2000 SCF extraction system (SFX 220) consisting of a dual chamber extraction module with two 10 mL stainless steel vessels. Temperature and pressure within the vessels were measured and could be independently adjusted. The 10 mL stainless steel cell was filled with about 5 g of ground black seeds. The cell was pressurized and heated to the desired pressure (100 – 400 bar) and temperature (40 –708C) and kept for 15 minutes to reach equilibrium. A known volume of SC CO2 (50 – 400 mL) was passed through the cell at a flow rate of 1 mL/min. The extract was collected in a cold trap after depressurization of the gas. The collected sample was stored at 2188C until analysis. 127 128 129 130 Soxhlet Extraction 131 132 133 134 135 A known quantity of ground black seeds (about 5 g) was placed in a cellulose thimble and extracted with about 300 mL of either hexane or methanol for 12 hrs. Solvent was removed using a rotary evaporator operated at 458C and the final traces of solvent were removed under a stream of nitrogen. LJLC253871 LJLC_030_017 Techset Composition Ltd, Salisbury, U.K. 4 136 7/12/2007 M. V. Rao et al. Characterization of Extracts 137 138 139 140 141 142 143 144 145 146 147 148 The compounds in the extracts obtained under different SFE conditions and solvent extraction were separated by high performance liquid chromatography on Sphereclone C8 column (250 mm  4.6 mm id, 5 mm, Phenomenex) using water:methanol:2-propanol (50:45:5), at a flow rate of 1 mL/min. A 20 mL of extract solution [0.1 g of oil dissolved in methanol: 2-propanol (1:1) and made up to 10 mL] was injected and the compounds were separated. Four compounds (trans-anethole, thymoquinone, thymol, and carvacrol) were identified using commercially available standards. The relative percentage of compounds in the extract was calculated from the normalized peak areas and concentration of known compounds was calculated using the standards. 149 150 151 RESULTS AND DISCUSSION 152 153 Yields and Composition of Extracts (SFE and Soxhlet) 154 155 156 157 158 159 160 161 162 163 164 165 166 167 The extraction yield is used to evaluate the performance of the SFE process for the extraction of black seeds at different pressures, temperatures, and CO2 volumes. The extract weight is used to calculate the extraction yield, which is defined as the ratio of the extract weight to the sample weight. SFE conditions (temperature, pressure, and volume of CO2) were optimized to obtain high quality black seed oil which is rich in antioxidants. SFE yields ranged from 0.84 to 31.7% under different conditions, where as soxhlet extraction with hexane and methanol gave 28.1% and 29.2%, respectively (Table 1). The highest SFE yield was obtained at SFE condition (508C, 400 bar, and 100 mL) while the lowest SFE yield was obtained at 100 bar, 200 mL CO2 volume, and the same temperature. The higher yield obtained under high pressure condition is mainly due to the higher extraction of fats and lipids. 168 169 170 Effect of Pressure 171 172 173 174 175 176 177 178 179 180 Figure 1 shows the influence of pressure on the yield of black seeds extracted by the SFE process at 40, 50, and 608C, when 100 mL of CO2 was passed through the sample at 1 mL/min. As it can be seen in the figure, at a constant temperature, higher pressures led to greater extraction yields. The extraction yield increased drastically (by about 150%) when the pressure was increased from 200 to 300 bar, however, the increase was only about 30% with further increases in pressure (300 to 400 bar). This was expected since an increase in pressure leads to an increase in CO2 density, resulting in a higher solubility and, hence, higher extraction yield. The increase in LJLC253871 LJLC_030_017 Techset Composition Ltd, Salisbury, U.K. 7/12/2007 Supercritical Fluid Extraction Conditions 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 5 Table 1. Experimental conditions and yield obtained during SFE & solvent extraction methods Run number Temperature (8C) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 40 40 40 40 50 50 50 50 50 50 50 50 50 60 60 60 60 70 Pressure (bar) CO2 volume (mL) 200 100 300 100 400 100 200 400 100 200 200 100 200 200 300 50 300 100 300 150 300 200 300 300 400 100 200 100 200 400 300 100 400 100 400 250 Soxhlet extraction using hexane Soxhlet extraction using methanol % Yield 9.20 24.0 30.3 27.7 0.84 9.03 15.0 12.0 24.5 29.0 29.5 29.6 31.7 8.49 14.3 22.0 30.1 30.3 28.1 29.2 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 Figure 1. Effect of pressure on the yield of black seed extract, CO2 volume ¼ 100 mL, CO2 flow rate ¼ 1 mL/min. LJLC253871 LJLC_030_017 6 Techset Composition Ltd, Salisbury, U.K. 7/12/2007 M. V. Rao et al. 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 Figure 2. Effect of temperature on the yield of black seed extract, CO2 volume ¼ 100 mL, CO2 flow rate ¼ 1 mL/min. 242 243 244 245 246 density is more significant at lower pressures since the condition is closer to the critical point of CO2. Figure 1 also shows that extraction yield was only slightly affected by the temperature. 247 248 249 Effect of Temperature 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 The influence of temperature on the yield of black seeds extracted by the SFE process is shown in Figure 2. The effect of temperature on the extraction yield was very small, especially at 200 bar. At the higher pressures (300 and 400 bar), extraction yield increased when the temperature was increased from 40 to 508C, while the yield decreased when the temperature was raised from 50 to 608C. However, at the lower pressure of 200 bar, extraction yield slightly decreased with an increase in temperature. Solubility of solutes in SC CO2 is affected by two competing factors (density of the SC CO2 and volatility of the solute), which depend on the temperature in opposite ways. Higher temperatures increase the volatility of solutes and improve their solubility and extraction. On the other hand, density of supercritical CO2 decreases with increasing temperature, reducing the solvating power of CO2 and thus reducing the solubility and extraction efficiency. This may be the reason for the varying effect of temperature on the extraction yield. 265 266 267 Effect of CO2 Density 268 269 270 Figure 3 shows the relation between the extraction yield and the density of CO2. As it can be seen in the figure, at a constant temperature the yield increases LJLC253871 LJLC_030_017 Techset Composition Ltd, Salisbury, U.K. Supercritical Fluid Extraction Conditions 7/12/2007 7 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 Figure 3. Effect of CO2 density on the yield of black seed extract, CO2 volume ¼ 100 mL, CO2 flow rate ¼ 1 mL/min. 288 289 290 291 linearly with density (r2 . 99%). The increase in yield with an increase in CO2 density is a result of the increase in the solvent power of CO2 at higher densities. 292 293 294 295 296 297 Effect of CO2 Volume Effect of CO2 volume on the yield of black seeds extracted by the SFE process at 508C, 300 bar, and flow rate of 1 mL/min is presented in Figure 4. As 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 Figure 4. Effect of CO2 volume on the yield of black seed extract, P ¼ 300 bar, T ¼ 508C, CO2 flow rate ¼ 1 mL/min. 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 Refer Table 1for SFE conditions. 161033 224251 62056 6268210 98142 104717 2856866 1159447 169233 75926 81581 258680 212806 418903 176417 63973 1697690 38282 44486 43654 47939 461706 82690 — 159236 160298 4.12 1.92 0.93 16.7 0.56 0.65 0.63 0.70 4.54 0.82 — 1.72 1.74 52856 50263 15068 114429 11987 13500 12956 39258 54351 18859 9275 22072 164036 116524 114425 62021 963125 39218 42457 43278 47079 122829 45520 29231 32186 50370 72009 62042 26241 886848 24616 26935 24518 19842 78217 17683 38094 208659 218766 333 348967 1833450 859689 12620435 714529 794133 874136 502848 328991 707695 10537172 1069196 1329217 332 843628 629543 576391 5402880 399562 479942 528813 95008 813931 529288 45604 703413 694862 331 1 2 3 5 9 10 12 12 14 17 18 19 20 330 1189016 5626146 1949514 3648451 1988219 2036113 2074734 1911104 1249078 2017271 1781142 911253 1012366 Normalized area 2.33 11.3 38.9 72.8 3.67 3.76 3.83 3.53 2.45 3.95 3.44 1.76 1.96 Conc. (mg/g) 328 Normalized area 327 Conc. (mg/g) 326 Normalized area — — — 3054848 — — — — — — — 1721 14238 Normalized area 142679 162902 49521 838478 69534 72996 68536 15980 153973 38434 68,676 10631 14589 Normalized area 0.046 0.53 0.21 2.20 0.24 0.25 0.23 0.12 0.50 0.16 0.29 0.08 0.11 Conc. (mg/g) Peak 10 (Carvacrol) 324 Normalized area 325 Peak 9 323 Run numbera 329 Peak 8 (Thymoquinone) 322 Peak 7 321 Peak 6 320 Peak 5 319 Peak 4 (t-Anethole) — — 42757 107174 50067 61076 — — — — 47466 54833 78468 Normalized area Peak 11 318 Peak 3 317 Peak 2 316 Peak 1 LJLC_030_017 a 335 HPLC characterization of active principles in the SFE and solvent extracts 334 Table 2. LJLC253871 Techset Composition Ltd, Salisbury, U.K. 8 7/12/2007 M. V. Rao et al. LJLC253871 LJLC_030_017 Techset Composition Ltd, Salisbury, U.K. Supercritical Fluid Extraction Conditions 361 362 363 364 7/12/2007 9 shown in the figure, the yield increased as more CO2 was passed through the sample. However, the extract amount approached a maximum value as the CO2 amount was increased, indicating that no more extract could be obtained by passing additional CO2 through the sample. 365 366 367 Characterization of Active Principles 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 HPLC separation of various compounds in the extracts obtained under different SFE conditions, as well as soxhlet extracts, is given in Table 2. t-Anethole, thymoquinone, and carvacrol were identified and quantitated using standards. A large number of compounds in high concentrations were present in the extract for the conditions (temperature, pressure, and CO2 volume: 508C, 100 bar, 200 mL). This may be due to the relative increase in the concentration of active principles, as lipids were not extracted under this condition. Therefore, the SFE process should be operated at the lower pressure of 100 bar to obtain extracts rich in antioxidants. Moreover, SFE extraction gave better antioxidant composition as compared to the soxhlet extraction. Since extraction of polar and non-polar compounds depends on the polarity of the extraction solvent, it is difficult to get extracts rich in antioxidants and low in fats and lipids, using common solvents such as hexane or methanol, where as optimized conditions in the SFE process provide selective extraction. Moreover, the SFE process is more flexible as compared to soxhlet extraction, since the solvent power of the CO2 can be changed by simply changing the temperature and pressure. The GSH recovery of some SFE extracts was carried out and the recovery ranged from 27.8 –84.6% (manuscript in preparation). The highest GSH recovery was found in the SFE extract obtained under the condition (508C, 100 bar, 200 mL CO2). Thymoquinone (marker compound) was found to be the highest (72.8 mg/g) in the extract obtained under this condition. The high concentration of marker compound, as well as other compounds in the extract, clearly substantiates the highest GSH recovery in the extract. 393 394 395 CONCLUSIONS 396 397 398 399 400 401 402 403 404 405 SFE conditions (temperature, pressure, and volume of CO2) were optimized to obtain quality black seed oil, which is rich in antioxidants. Extraction efficiency drastically increased with the increase in pressure and was only slightly affected by the temperature. The highest extraction yield was obtained at 508C, 400 bar, and 100 mL of CO2 volume. However, the highest antioxidants concentration was found for the condition at 508C, 100 bar, 200 mL, suggesting that the SFE process should be operated at lower pressure for obtaining higher quality extracts. Moreover, SFE extraction gave better antioxidant composition as compared to the soxhlet extraction. LJLC253871 LJLC_030_017 Techset Composition Ltd, Salisbury, U.K. 10 406 407 408 7/12/2007 M. V. Rao et al. Our current work focuses on separation of individual compounds from SFE extract to investigate their antioxidant ability (through in vitro and in vivo studies) in reducing oxidative stress caused by petrochemical pollution. 409 410 411 ACKNOWLEDGMENTS 412 413 414 415 416 The authors express their gratitude to the Research Affairs, UAE University for financially supporting this work through an Interdisciplinary Research Grant (# 01-04-2-12/04). Authors are also grateful to Ayser Solieman, Baboucarr Jobe and Shahnaz Majid for their assistance during the experimental work. 417 418 419 REFERENCES 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 1. Ali, B.H.; Blunden, G. Pharmacological and toxicological properties of Nigella sativa. Phytother. Res. 2003, 17, 299– 305. 2. Dragland, S.; Senoo, H.; Wake, K.; Holte, K.; Blomhoff, R. Several culinary and medicinal herbs are important sources of dietary antioxidants. J. 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