Abstract
Extraction process employing Supercritical fluid carbon dioxide (SCF) yields bioactive compounds near natural forms without any artifact formation. Neem seed was subjected to SCF at different temperatures and pressure conditions. These extracts were partitioned to separate volatile fraction and were analyzed by Gas Chromatography–Mass spectroscopy along with the volatiles extracted by the hydro-distillation method. Experimental results show that there is a significant effect of pressure and temperature on isolation of a number of volatile compounds as well as retention of biologically active compounds. Twenty-five volatile compounds were isolated in the Hydro-distillate compare to the SCF extract of 100 bar, 40 °C which showed forty volatile compounds corresponds to 76.38 and 92.39% of total volatiles respectively. The majority of bioactive compounds such as Terpinen-4-ol, 1,2,4-Trithiolane, 3,5-diethyl, allyl isopropyl sulphide, Cycloisolongifolene, á-Bisabolene, (-)-α-Panasinsen, Isocaryophyllene, trans-Sesquisabinene hydrate, 1-Naphthalenol, were identified in the extract when isolated at 100 bar and 40 °C.
Keywords: Azadirachta indica, Neem seed, Kernel, Volatile, Supercritical fluid carbon dioxide extraction
Introduction
Neem (Azadirachta indica A. Juss) tree belongs to the family of Meliaceae. Neem is one of the most versatile medicinal plants having a broad spectrum of biological activity in India and its neighboring countries since immemorial. Neem seed oil and extract are proved effective against some fungi that cause infection to human body. It is used as anthelmintic, antileprotic (prevents leprosy), antibacterial, antidermatophytic activity. Neem seed spent (cake) is an active ingredient in preparation of mosquito repellent coil. A bitter compound, nimbidin is isolated from seeds which is effective in reducing fasting blood glucose (Biswas et al. 2002). The different parts of neem (seeds, leaves, flowers and bark) are used as traditional medicine for the household remedy against various human ailments. It also used as the natural pesticide, a raw material for cosmetic industry and other commodities (Ghimeray et al. 2009; Rashid et al. 2004; Olabinri et al. 2009; Aromdee and Sriubolmas 2006). Neem seed kernels constitute 50–60% of the seed and the remaining is the kernel portion. The fat content of the kernels ranges from 33 to 45%. Neem seed contains 40–45% of oil and remaining is the cellular matrix of the seeds. The oil is brownish yellow, non- drying oil with an acrid taste and unpleasant odor (Johnson and Morgan 1997). Several valuable bioactive substances from limonoids class of triterpenoids were found in neem oil. Azadirachtin (Azadirachtin A), Nimbidiol, 3-tigloylazadirachtol (Azadirachtin B), Salannol, Salannin, Nimbinin, Nimbin, Nimbidin, and 1-tigloyl-3- acetyl-11- hydroxymeliacarpin (Azadirachtin D) non volatile compounds are the major once (Mongkholkhajornsilp et al. 2005; Morgan 2009; Melwita and Ju 2010). Further, sulfur modified fatty substance like loeic acid (50–60%), palmitic acid (13–15%), stearic acid (14–19%), linoleic acid (8–16%) and arachidic acid (1–3%) are present in neem seed oil.
Volatiles isolated from neem seed oil also play a vital role in health care systems of the most traditional and modern medicines. By head space analysis insect repellent active compounds such as caryophyllene oxide, limonene, linalool, 1-hexanol and organosulphur compounds were identified from neem seed volatiles.
Hydro-distillation is a traditional technique for extraction of volatile oil. This distillation has several disadvantages, such as incomplete extraction of volatiles from plant materials, high operating temperatures with the consequent breakdown of thermally labile components, promotion of hydration reactions of chemical constituents, hydrolysis and solubilization in water of some compounds that alter the flavor and fragrance profile of many essential oils extracted by this technique. It also requires a post extraction process to remove water (Metherel et al. 2009). The other problem with this method is the production of waste water with a high biological oxygen demand (BOD). With high BOD the release of waste water is not permitted in many countries, again which requires an expensive installation of water treatment facility (O’shea et al. 2001).
In recent decades, the supercritical fluid carbon dioxide extraction (SCF-CO2) has received special attention in extraction of volatile oil from natural materials. In compressed carbon dioxide (CO2) main volatile oil constituents like hydrocarbon and oxygenated mono- and sesquiterpenes are solubilized (Stahl and Gerard 1985). In fact, CO2 is physiologically harmless, non-toxic and non-corrosive. It readily reaches critical temperature (31.1 °C) and pressure (73.8 bar). Very little work was traced about the essential oil of neem seed by Supercritical fluid extraction. The present study was undertaken to employ supercritical carbon dioxide to extract the volatiles from neem seed and characterization of the constituents.
Materials and methods
Chemicals and reagents
Analytical grade chloroform procured from Merck (Mumbai, India). Carbon dioxide (purity 99.9%) was obtained from M/S Kiran Corporation, Mysore, India. Homologous series of C8-C22 n-alkane standards were procured from Sigma (St. Louis, MO, USA). Anhydrous sodium sulfate was obtained from SLR Chemicals (Mumbai, India). All other chemicals, solvents, and reagents used were of analytical grade.
Plant material
Neem seeds were purchased from a local market at Mysore, Karnataka, India. The seeds were manually cleaned to remove the adhering soil and extraneous matter if any. These cleaned neem seeds were dried in cabinet tray drier (M/S Armstrong Smith, India) at 40 °C for 24 h. Further, neem seed was dehulled by using DIAF huller (Crompton Parkinson Ltd, Bombay). Dehulled neem seed was ground by using hammer mill (Model: CIS-100, M/s Galai Production, Israel) to get powder of 32 mesh size. The moisture content of neem seed powder is 16 ± 0.1%.
Supercritical fluid carbon dioxide extraction and partitioning of flavor volatiles
Extractions were carried out in NOVA Swiss equipment (WERKEAG, EX 1000, 1.4–1.2 V type, Switzerland) designed for working pressure of 1000 bar and temperature of 100 °C. Neem seed powder (300 g) was loaded into a steel cylinder equipped with sintered metal plate on both ends. The cylinder was then introduced into the extraction vessel, and required extractor and separator temperatures were maintained using thermostat water circulators. CO2 supplied from a gas cylinder was compressed by using two diaphragm compressors to the desired pressure by adjusting the pressure controller and heated to the specified temperature using a heat exchanger to reach the supercritical state (Zarena et al. 2012). Nine experiments were carried out in duplicates at temperatures of 40, 50 and 60 °C; the pressure of 100, 150 and 200 bar (Table 1). After completion of an experiment, the extract was collected from the separator and stored at 4 °C for further study.
Table 1.
Yield of SCF extracts in different conditions
| No of SCF experiments | Yielda (g/100 g) | Yielda of Volatiles (mg/100 g) |
|---|---|---|
| 100 bar-40 °C | 4.61 | 24 |
| 100 bar-50 °C | 4.62 | 39 |
| 100 bar-60 °C | 1.2 | 15 |
| 150 bar-40 °C | 17.11 | 49 |
| 150 bar-50 °C | 10.72 | 35 |
| 150 bar-60 °C | 10.99 | 19 |
| 200 bar-40 °C | 31.57 | 162 |
| 200 bar-50 °C | 18.43 | 68 |
| 200 bar-60 °C | 21.39 | 67 |
aYield was given in raw material basis
All the SCF extracts were further hydro-distilled, and the distillate was partitioned with chloroform to separate the volatiles. Chloroform was removed using a rotavapor (RE111, Buchi, Switzerland) and pale yellow colored oil obtained was dried over anhydrous sodium sulfate. The flavor isolate was stored at 4 °C for further study.
Extraction of volatile oil by hydro-distillation (Conventional method)
Neem seed powder (250 g) was defatted by Soxhlet extraction using petroleum ether (60–80 °C) with 15–16 siphons in 12 h. Solvent adhering to the Marc was expelled by a screw press. Further, neem seed powder was dried in the sun for 2 h and again dried in an oven at 105 °C for 2 h. Defatted neem seed powder was subjected to hydro-distillation in Clevenger distillation until all the oil is collected (9 h). The pale brown colored oil obtained was dried over anhydrous sodium sulfate and stored at 4 °C for further analysis. All the experiments were executed in triplicate, and the result was expressed.
Chemical characterization of volatile oil and chromatographic analysis
Gas chromatography–mass spectrometry (GC–MS) analysis
The volatile oil was characterized using Perkin Elmer GC equipped with quadrupole mass spectrometer (Model: Turbomass Gold) fitted with an Elite-5 fused silica column (30.0 m × 250 µm film thickness) coated with polydimethylsiloxane (PDMS). Initially, the oven temperature was held at 35 °C for 2 min, ramp 2 °C/min to 70 °C, held for 0 min then increased at 4 °C/min to 250 °C, held for 5 min at final temperature. The carrier gas (helium) was delivered with 75 kPa inlet pressure with linear velocity (20 cm/s), injector temperature (180 °C) and detector temperature (230 °C). Samples were diluted with chloroform, and 1 µl was injected. The mixture of n-alkanes as standards (C8–C24) diluted with chloroform was also injected and analyzed at same conditions to determine the (Jennings and Shibamoto 1980; Kubra and Rao 2012) kovats’ retention indices by the following equation.
KI = 100[n + (N−n) X {log tr(unknown)−log tr(n)}/{log tr(N)−log tr(n)}]
where (n) is stand for the number of carbon atoms in the shorter chain alkane, N is the number of carbon atoms in the longer chain alkane, (tr) is the adjusted retention times for unknown compounds, tr (n), shorter-chain and tr (N), longer-chain alkanes. The total volatile production was estimated as the sum of all GC-FID peak areas in the chromatogram, and individual compounds were quantified as relative percent area. All data are means of three independent determinations expressed as the percentage of total peak area (Shivakumar et al. 2012).
Results and discussion
Supercritical fluid extraction has increased during two last decades in the extraction of plant volatile components since it is a rapid, selective and convenient method. Food grade Carbon dioxide (GRAS) which is used in SCF is an excellent alternative for chemical solvents (Pourmortazavi and Hajimirsadeghi 2007). In general, the extractability of volatile and non-volatile compounds by SCF depends on the occurrence of functional groups, molecular mass and polarity. Essential oil of coriander seeds was extracted by SCF under different conditions of temperature and pressure by Grosso et al. (2008). GC–MS analysis of volatile oil composition of SCF extract reveals that the best condition was achieved at 90 bar and 40 °C.
Extraction efficiency by evaluating SCF parameters
Neem seed (39 kg) with the moisture content of 17% was dehulled to get the kernels of 16 kg which possess the moisture content of 19.3%. The dehulled seed was ground and used for further experiments.
Supercritical fluid extraction has attracted a vast deal of attention. Indeed, it is an excellent alternative method for seed oil extraction to replace classical industrial methods. It becomes the focus of attention due to its chemical and physical properties. Furthermore, it offers recovery of solvent-free analyte from solid or semi-solid samples that are equal to or better than that of conventional extraction process (Herrero et al. 2010). CO2 is environmentally friendly and recognized as safe. It is also essential for the sample preparations of food and natural product. SCF using CO2 can be operated at low temperatures using a non-oxidant medium, which allows the extraction of thermally labile or easily oxidized compounds. In this study, the volatile composition of neem seed at three pressures (100, 150 and 200 bar) and three temperatures (40, 50 and 60 °C) in different combination was studied. And also, SCF volatile extract was compared with conventional hydro-distillation process.
In SCF, totally nine experiments conducted in duplicates and the results were analyzed. The method of extraction usually has a significant effect on the quality and quantity of the volatile oils by varying temperature and pressure in SCF extraction. The extraction yields of nine experiments were evaluated, the factor (e.g., pressure) reveal how the extraction yield changes when the level of that factor is changed. As per the earlier report (Mostafa et al. 2004), an increase in extraction pressure, at a constant temperature, leads to higher fluid density. This higher fluid density increases the solubility of the analytes.
The effect of temperature on the yield as well as the composition of extracts was evaluated. Higher temperature decreased fluid density and thus reduced extraction efficiency. When extraction was done at the different pressure of 100, 150 and 200 bar, the yield of volatile extract was more at 40 °C (Table 1). Further, it was observed that the volatiles percentage is directly related to the pressure and temperature, in turn, the density of carbon dioxide. With increasing temperature, at the pressure of 100 bar, the extract yields decreased drastically, but there is an increase in the volatiles percentage in the extract. But, at 150 and 200 bar higher pressures, there is definite increase in the volatiles percentage but not perceptible, as the total extract yields increased and the solubilization of fatty oil as well as high molecular compounds with increased density of carbon dioxide. In the extraction of pepper oil, this relationship on temperature, pressure density of volatiles correlates the earlier publications (Sankar 1989). The conventional method of hydro-distillation has been traditionally used for the extraction of volatile oils on a laboratory scale. In this study, the volatile composition of neem seed obtained by SCF extraction was compared with the conventional extraction. Further, the conventional hydro-distillation of neem seed volatile extract yielded 33 ± 0.76 mg/100 g after the removal of 53% of fat.
GC–MS analysis
Biogenetically, most of the volatile compounds are derived from mevalonic acid (MVA) pathway (i.e., triterpenoids and sesquiterpenoids). However, monoterpenoids, diterpenoids, and tetraterpenoids are biosynthesized via the plastidial methylerythritol phosphate (MEP) (Sawai and Saito 2011). In general, mono and diterpenes are more soluble than paraffin and fatty acids at low CO2 densities (T = 40–50 °C, p = 80–90 bar), whereas sesquiterpenes and oxygenated terpenes are soluble at high CO2 densities (T = 40–50 °C, p = 100–200 bar). The volatile neem isolates were subjected to GC–MS analysis. The chemical components present in the volatile oil were identified according to their order of elution on Elite-5 column.
All nine SCF experiment results were analyzed by GC–MS. At 100 bar, 40 °C forty compounds were identified, which represented 92.39% of total volatiles. Similarly, at 100 bar, 50 °C thirty-two compounds (85.9%), 100 bar, 60 °C twenty-two (56.01%), 150 bar, 40 °C thirty-three (73.34%), 150 bar, 50 °C thirty-seven (70.52%), 150 bar, 60 °C thirty-three (76.87%), 200 bar, 40 °C twenty-seven (65.71%), 200 bar, 50 °C nine (72.78%), 200 bar, 60 °C twenty three (52.47%) were identified respectively. The results of SCF volatile compounds are compiled in Table 2 (SCF 100 bar at 40, 50 and 60 °C), Table 3 (SCF 150 bar at 40, 50 and 60 °C) and Table 4 (SCF 200 bar at 40, 50 and 60 °C). Further, the hydro-distillation of neem seed volatile extract is 33 mg/100 g. Thirty-two compounds were identified which represent the 76.38% of the total volatile extract.
Table 2.
Identification of neem seed (100 bar, 40, 50 and 60 °C) volatile compounds by GC–MS
| Sl. no | RT (min) | Compound | 100 bar (ppm) | RI | ||
|---|---|---|---|---|---|---|
| 40 °C | 50 °C | 60 °C | ||||
| 1 | 16.873 | Butanal, oxime | 1.69 | – | – | 1020 |
| 2 | 25.577 | Pyrazine, 3,5-diethyl-2-methyl- | 1.21 | – | – | 1170 |
| 3 | 26.562 | Terpinen-4-ol | 1.28 | – | – | 1189 |
| 4 | 27.423 | Octanoic acid | 4.08 | 2.26 | 1207 | |
| 27.563 | 1210 | |||||
| 5 | 31.044 | Nonanoic acid | 2.94 | 2.57 | – | 1297 |
| 31.209 | 1301 | |||||
| 6 | 31.969 | 2, 4- Decadienal | 2.50 | 0.77 | – | 1323 |
| 31.969 | ||||||
| 7 | 32.2 | 1,2,4-Trithiolane, 3,5-diethyl- | 1.51 | – | – | 1330 |
| 8 | 33.72 | 2(3H)-Furanone, dihydro-5-pentyl- | 1.02 | 0.06 | – | 1373 |
| 33.72 | ||||||
| 9 | 35.146 | 1H-3a,7-Methanoazulene, 2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-, [3R-(3à,3aá,7á,8aà)]- | 1.14 | – | – | 1414 |
| 10 | 35.276 | Cyclohexane, 1-ethenyl-1-methyl-2-(1-methylethenyl)-4-(1-methylethylidene)- | 1.73 | – | – | 1418 |
| 11 | 35.421 | Cedrene | 2.06 | – | – | 1423 |
| 12 | 35.841 | (+)-epi-Bicyclosesquiphellandrene | 2.952 | – | – | 1436 |
| 13 | 35.971 | Bicyclo[3.1.1]hept-2-ene, 2,6-dimethyl-6-(4-methyl-3-pentenyl)- | 6.29 | – | – | 1440 |
| 14 | 36.066 | 1,2,4-Thiadiazole-3,5-diamine | – | – | 11.23 | 1443 |
| 15 | 36.081 | 1-Propene, 3-[(1-methylethyl)thio]- (or) Allyl isopropyl sulfide | 8.71 | 8.61 | – | 1444 |
| 36.111 | 1445 | |||||
| 16 | 36.251 | 4,7-Methanoazulene, 1,2,3,4,5,6,7,8-octahydro-1,4,9,9-tetramethyl-, [1S-(1à,4à,7à)]- | 1.23 | – | – | 1449 |
| 17 | 36.381 | γ-Muurolene | 2.53 | – | – | 1453 |
| 18 | 36.656 | Cyclohexene, 3-(1,5-dimethyl-4-hexenyl)-6-methylene-, [S-(R*,S*)]- | 1.10 | – | – | 1461 |
| 19 | 36.986 | Spiro[4.5]dec-7-ene, 1,8-dimethyl-4-(1-methylethenyl)-, [1S-(1α,4β,5α)]- | 5.88 | – | – | 1472 |
| 20 | 37.527 | Cycloisolongifolene | 1.43 | – | – | 1488 |
| 21 | 37.847 | Guaia-1(10),11-diene | 10.2 | – | – | 1498 |
| 22 | 38.327 | á-Bisabolene | 9.57 | – | – | 1514 |
| 23 | 38.602 | (-)-α-Panasinsen | 1.06 | – | – | 1523 |
| 24 | 39.127 | Dodecane, 4,6-dimethyl- | – | – | 1.78 | 1541 |
| 25 | 39.332 | Isocaryophyllene | 1.22 | – | – | 1548 |
| 26 | 40.293 | Dodecanoic acid | 1.13 | – | – | 1580 |
| 27 | 40.643 | 4,6-Diethyl-1,2,3,5-tetrathiolane | 1.56 | – | – | 1591 |
| 40.753 | 1595 | |||||
| 28 | 40.833 | 2-(2-Isopropenyl-5-methyl-cyclopentyl)-acetamide | – | 0.68 | – | 1598 |
| 29 | 42.613 | trans-Sesquisabinene hydrate | 1.90 | 0.76 | – | 1661 |
| 42.614 | ||||||
| 30 | 42.899 | Cedrane or 1H-3a,7- | 1.35 | 0.82 | – | 1671 |
| 42.899 | Methanoazulene, octahydro-3,6,8,8-tetramethyl-, [3R-(3à,3aá,6à,7á,8aà)]- | |||||
| 31 | 43.349 | 1-Naphthalenol, decahydro-1,4a-dimethyl-7-(1-methylethylidene)-, [1R-(1α,4aβ,8aα)]- | 14.94 | 10.13 | – | 1687 |
| 43.424 | 1690 | |||||
| 32 | 43.684 | 4-(1,5-Dimethylhex-4- | 1.89 | 1.40 | – | 1699 |
| 43.689 | enyl)cyclohex-2-enone | |||||
| 33 | 43.794 | 2,4a-Methanonaphthalen-7(4aH)-one, 1,2,3,4,5,6-hexahydro-1,1,5,5-tetramethyl-, (2 s-cis)- (or) Isolongifolen-9-one | 2.35 | – | – | 1627 |
| 34 | 43.799 | Neoisolongifolene, 8-oxo- | – | – | 1.50 | 1703 |
| 35 | 43.804 | Isolongifolen-9-one (or) 2,4a-Methanonaphthalen-7(4aH)-one, 1,2,3,4,5,6-hexahydro-1,1,5,5-tetramethyl-, (2 s-cis)- | – | 1.04 | – | 1703 |
| 36 | 44.679 | (Z)6,(Z)9-Pentadecadien-1-ol | 1.39 | – | – | 1737 |
| 37 | 45.115 | Decane, 3-ethyl-3-methyl- | – | – | 1.75 | 1753 |
| 38 | 45.885 | Syn-Tricyclo[5.1.0.0(2,4)]oct-5-ene, 3,3,5,6,8,8-hexamethyl- | – | 1.02 | – | 1782 |
| 39 | 49.431 | Benzenamine, 4,5-dimethyl-2-(4-pyridylmethoxy)- | – | – | 1.58 | 1922 |
| 40 | 49.446 | Isobenzofuran-1(3H)-one, 3-(3-furyl)-3a,4,5,6-tetrahydro-3a,7-dimethyl- | – | 0.70 | – | 1922 |
| 41 | 49.662 | Pentadecanoic acid, 14-methyl-, methyl ester | – | – | 9.78 | 1932 |
| 42 | 49.676 | Hexadecanoic acid, methyl ester | 11.56 | 2.35 | 8.88 | 1927 |
| 49.681 | 1932 | |||||
| 51.287 | 1998 | |||||
| 43 | 50.467 | Tetradecane,1-iodo- | – | – | 1.60 | 1965 |
| 44 | 50.567 | n-Hexadecanoic acid | 69.23 | 101.8 | 233.79 | 1969 |
| 50.837 | 28.04 | 1980 | ||||
| 50.982 | 1986 | |||||
| 51.172 | 1994 | |||||
| 45 | 51.162 | (S)-(+)-6-Methyl-1-octanol | – | – | 6.68 | 1993 |
| 51.307 | Hexadecanoic acid, ethyl ester | 8.52 | 0.81 | 8.88 | 1999 | |
| 51.287 | 1998 | |||||
| 46 | 53.228 | 11,14-Eicosadienoic acid, methyl ester | – | 1.10 | 4.99 | 2083 |
| 53.638 | 2100 | |||||
| 47 | 53.378 | trans-13-Octadecenoic acid, methyl ester | – | 2.73 | – | 2089 |
| 48 | 53.658 | 9,12-Octadecadienoic acid, methyl ester | 5.04 | 0.88 | – | 2101 |
| 53.663 | ||||||
| 49 | 53.768 | 6-Octadecenoic acid, methyl | – | 2.07 | 10.85 | 2106 |
| 53.783 | ester, (Z)- | |||||
| 50 | 53.788 | 8-Octadecenoic acid, methyl ester | 11.79 | – | – | 2106 |
| 51 | 54.043 | Methyl stearate | 1.71 | 1.10 | – | 2117 |
| 54.353 | ||||||
| 52 | 54.353 | Methyl 8-methyl- nonanoate | – | – | 2.69 | 2130 |
| 53 | 54.894 | Cis-Vaccenic acid (or) 11- Octadecenoic acid, (Z)- | 8.77 | 120.74 | 145.98 | 2153 |
| 54.968 | 2156 | |||||
| 55.023 | 2158 | |||||
| 57.189 | 2246 | |||||
| 54 | 55.249 | (E)-9-Octadecenoic acid ethyl ester | 2.11 | 1.12 | 4.51 | 2167 |
| 55.264 | 2168 | |||||
| 55.278 | 2168 | |||||
| 55 | 55.389 | Octadecanoic acid (or) stearic acid | 2.76 | 35.21 | 34.28 | 2173 |
| 55.439 | 2175 | |||||
| 55.494 | 2177 | |||||
| 56 | 55.659 | Heptadecane, 9-hexyl- | – | – | 7.36 | 2184 |
| 63.562 | 5.31 | 2485 | ||||
| 57 | 55.929 | Nonadecane, 2-methyl- | – | – | 12.84 | 2195 |
| 58 | 57.844 | 2-methyltetracosane | – | 0.67 | – | 2271 |
| 59 | 61.916 | Nonadecane, 2-methyl | – | 0.72 | – | 2426 |
| 60 | 63.111 | 1,2-Benzenedicarboxylic acid, bis(6-methylheptyl) ester (or) Phthalic acid, bis(6-methylheptyl) ester | – | 9.23 | – | 2469 |
| 61 | 63.827 | Eicosane, 2-methyl- | – | 0.61 | – | 2495 |
| 62 | 64.547 | Anthracene, 9- dodecyltetradecahydro | – | – | 7.02 | 2520 |
| 63 | 65.013 | Octadecane, 3-ethyl-5-(2-ethylbutyl)- | – | – | 2.08 | 2537 |
| 64 | 65.843 | Phthalic acid, 4-methylpent-2-yl nonyl ester | – | 0.73 | – | 2565 |
| 66.288 | 1.77 | 2581 | ||||
| 66.698 | 0.63 | 2595 | ||||
| 65 | 66.858 | 1,2-Benzenedicarboxylic acid, dinonyl ester | – | 0.94 | – | 2600 |
Table 3.
Identification of neem seed (150 bar, 40, 50 and 60 °C) volatile compounds by GC–MS
| Sl. no | RT (min) | Compound | 150 bar (ppm) | RI | ||
|---|---|---|---|---|---|---|
| 40 °C | 50 °C | 60 °C | ||||
| 1 | 16.358 | Amylene hydrate | 2.35 | – | 1011 | |
| 2 | 22.706 | Nonanal | – | 0.74 | – | 1110 |
| 3 | 23.331 | 2,3-Dimethyl-4-hydroxy-2-butenoic lactone | – | – | 0.49 | 1124 |
| 4 | 26.547 | 3-Cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)-, (R)- | – | 0.91 | – | 1188 |
| 5 | 27.198 | Hexan-3-yl propyl carbonate | – | – | 2.53 | 1201 |
| 6 | 30.304 | 2,6-Octadienal, 3,7-dimethyl-, (E)- | – | 0.94 | – | 1279 |
| 7 | 30.824 | Nonanoic acid | 8.38 | 2.44 | 1.89 | 1292 |
| 30.859 | 1292 | |||||
| 30.924 | 1294 | |||||
| 8 | 31.969 | 2, 4- Decadienal | 1.42 | 0.78 | 1.12 | 1323 |
| 31.974 | 1323 | |||||
| 31.979 | 1324 | |||||
| 9 | 32.204 | 1,2,4-Trithiolane, 3,5-diethyl- | 3.02 | 1.07 | 1330 | |
| 32.210 | ||||||
| 32.485 | 2.79 | |||||
| 32.495 | 1.52 | |||||
| 10 | 32.745 | 1-Propene, 1,1’-thiobis- | – | 2.06 | – | 1346 |
| 32.755 | Propanal, 2-methyl-, ethylhydrazone | – | – | 1.47 | 1346 | |
| 11 | 33.72 | 2(3H)-Furanone, dihydro-5-pentyl- | 1.58 | 0.85 | 0.48 | 1373 |
| 33.72 | 1373 | |||||
| 33.725 | 1373 | |||||
| 12 | 34.125 | n- Decanoic acid | 1.01 | – | – | 1384 |
| 13 | 34.345 | 2,6-Octadien-1-ol, 3,7-dimethyl-, acetate, (Z)- | – | 4.32 | – | 1390 |
| 14 | 34.365 | (E)-3,7-Dimethylocta-2,6-dienyl ethyl carbonate | – | – | 2.15 | 1390 |
| 15 | 35.831 | cis-muurola-4(14),5-diene | – | 0.91 | 0.515 | 1436 |
| 35.841 | ||||||
| 16 | 35.951 | Bicyclo[3.1.1]hept-2-ene, 2,6-dimethyl-6-(4-methyl-3-pentenyl)- | – | 2.34 | 1.78 | 1439 |
| 35.961 | 1440 | |||||
| 17 | 36.046 | 1-Propene, 3-[(1-methylethyl)thio]- | 26.69 | 8.97 | 7.97 | 1442 |
| 36.051 | 1443 | |||||
| 36.076 | 1443 | |||||
| 18 | 36.361 | 1-Ethyl-3-vinyl-adamantane | – | 0.75 | – | 1452 |
| 19 | 36.371 | Germacrene D (or) 1,6-Cyclodecadiene, 1-methyl-5-methylene-8-(1-methylethyl)-, [S-(E,E)]- | – | – | 0.49 | 1453 |
| 20 | 36.976 | Spiro[4.5]dec-7-ene, 1,8- | – | 2.68 | 1.47 | 1471 |
| 36.981 | dimethyl-4-(1-methylethenyl)-, [1S-(1à,4á,5à)]- | 1471 | ||||
| 21 | 37.507 | Benzene, 1-(1,5-dimethyl-4-hexenyl)-4-methyl | – | 1.25 | 0.99 | 1487 |
| 37.512 | 1488 | |||||
| 22 | 37.827 | Naphthalene, 1,2,3,5,6,7,8,8a- | – | 5.28 | 2.65 | 1497 |
| 37.837 | octahydro-1,8a-dimethyl-7-(1-methylethenyl)-, [1R-(1à,7á,8aà)]- | 1497 | ||||
| 23 | 37.832 | á-Panasinsene | 0.92 | – | – | 1497 |
| 24 | 38.297 | β-Bisabolene | 2.19 | 5.15 | 3.30 | 1513 |
| 38.302 | ||||||
| 38.312 | ||||||
| 25 | 39.132 | Undecane, 3,8-dimethyl- | 1.26 | – | – | 1541 |
| 26 | 40.22 | Dodecanoic acid | 1.14 | – | 0.70 | 1578 |
| 40.243 | ||||||
| 27 | 40.638 | 4,6-Diethyl-1,2,3,5-tetrathiolane | 2.84 | 0.99 | – | |
| 40.748 | 1.66 | |||||
| 28 | 40.873 | Heptadecane, 2,6,10,14-tetramethyl- | 1.95 | – | – | 1599 |
| 29 | 41.113 | Diethyl Phthalate | 1.29 | 1.18 | – | 1607 |
| 41.103 | ||||||
| 30 | 42.614 | trans-Sesquisabinene hydrate | 1.99 | – | – | 1661 |
| 31 | 42.829 | Hexasiloxane, tetradecamethyl- | – | 1.85 | 1669 | |
| 32 | 42.889 | Aromadendran | 2.33 | – | 0.64 | 1671 |
| 42.889 | ||||||
| 33 | 43.414 | 1-Naphthalenol, decahydro-1,4a-dimethyl-7-(1-methylethylidene)-, [1R-(1α,4aβ,8aα)]- (or) Eudesm-7(11)-en-4-ol; Juniper camphor | 49.15 | – | 5.81 | 1689 |
| 43.494 | 1692 | |||||
| 34 | 43.529 | Selina-6-en-4-ol | – | 7.67 | – | 1693 |
| 35 | 43.679 | 4-(1,5-Dimethylhex-4-enyl)cyclohex-2-enone | – | 3.23 | – | 1698 |
| 36 | 43.789 | 2,4a-Methanonaphthalen-7(4aH)-one, 1,2,3,4,5,6-hexahydro-1,1,5,5-tetramethyl-, (2 s-cis)- | 7.19 | 2.61 | 1.60 | 1703 |
| 43.789 | 1703 | |||||
| 43.804 | 1703 | |||||
| 37 | 44.679 | (Z)6,(Z)9-Pentadecadien-1-ol | 1.87 | 0.69 | 0.59 | 1737 |
| 44.679 | 1737 | |||||
| 44.679 | 1737 | |||||
| 38 | 45.115 | 2-Bromo dodecane | 1.19 | – | – | 1753 |
| 39 | 45.71 | Tetradecanoic acid | 1.15 | – | 0.56 | 1775 |
| 45.74 | 1776 | |||||
| 40 | 45.875 | syn-Tricyclo[5.1.0.0(2,4)]oct-5-ene, 3,3,5,6,8,8-hexamethyl- | 1.82 | – | – | 1781 |
| 41 | 46.52 | 7-Oxabicyclo[4.1.0]heptane, 1-methyl-4-(2-methyloxiranyl)- | – | 1.36 | – | 1805 |
| 42 | 47.06 | Heptasiloxane, hexadecamethyl- | – | 1.33 | – | 1827 |
| 43 | 48.386 | Phthalic acid, hex-3-yl isobutyl ester | 3.47 | – | 0.79 | 1879 |
| 48.386 | 1879 | |||||
| 44 | 49.651 | Hexadecanoic acid, methyl ester | 14.46 | 6.27 | 3.17 | 1931 |
| 49.656 | 1928 | |||||
| 49.661 | 1932 | |||||
| 45 | 50.902 | n- Hexadecanoic acid | 180.48 | 61.91 | 77.59 | 1983 |
| 50.922 | 1984 | |||||
| 51.087 | 1990 | |||||
| 46 | 51.287 | Hexadecanoic acid, ethyl ester | 16.38 | 5.67 | 10.66 | 1998 |
| 51.287 | 1998 | |||||
| 51.292 | 1999 | |||||
| 47 | 53.633 | 11,14-Eicosadienoic acid, methyl ester | – | 2.34 | – | 2100 |
| 48 | 53.643 | 9,12-Octadecadienoic acid (Z,Z)-, methyl ester (or) Linoleic acid, methyl ester | 4.33 | – | 1.14 | 2100 |
| 53.648 | ||||||
| 49 | 53.763 | 6-Octadecenoic acid, methyl ester, (Z)- | – | 5.06 | – | 2105 |
| 50 | 53.768 | trans-13-Octadecenoic acid, methyl ester | 11.05 | – | 2.84 | 2106 |
| 53.768 | ||||||
| 51 | 54.283 | Hexasiloxane, tetradecamethyl- | – | 1.34 | – | 2127 |
| 52 | 54.333 | Methyl stearate | – | 1.32 | 0.53 | 2129 |
| 54.348 | ||||||
| 53 | 54.343 | Hexadecanoic acid, 15-methyl-, methyl ester | 1.68 | – | – | 2130 |
| 54 | 54.908 | Cis-Vaccenic acid (or) 11- | 62.30 | 27.09 | 26.57 | 2153 |
| 54.913 | Octadecenoic acid, (Z)- | 2153 | ||||
| 55.009 | 2157 | |||||
| 55 | 55.239 | 9-Octadecenoic acid ethyl ester | 1.84 | 0.68 | 1.76 | 2167 |
| 55.244 | 2167 | |||||
| 55.254 | 2167 | |||||
| 56 | 55.389 | Octadecanoic acid (or) Stearic acid | 11.62 | 5.37 | 5.41 | 2173 |
| 55.394 | 2173 | |||||
| 55.459 | 2176 | |||||
Table 4.
Identification of neem seed (200 bar, 40, 50 and 60 °C) volatile compounds by GC–MS
| Sl. no | RT of sample | Compound | 200 bar (ppm) | KI | ||
|---|---|---|---|---|---|---|
| 40 | 50 | 60 | ||||
| 1 | 4.023 | Trichloromethane | – | – | 4.73 | 744 |
| 4.879 | 778 | |||||
| 2 | 4.078 | Methane, oxybis[dichloro- | – | 6.63 | – | 747 |
| 3 | 4.824 | Desmethyldeprenyl | – | 0.33 | – | 776 |
| 4 | 5.234 | Difluorophosphoric acid | – | 0.21 | – | 790 |
| 5 | 16.303 | Butanoic acid | – | – | 2.15 | 1010 |
| 6 | 21.795 | Pyrazine, tetramethyl- | 4.45 | – | – | 1094 |
| 7 | 23.056 | Acetic acid, 2-(thiocarboxy)hydrazide, O-methyl ester | 3.79 | – | – | 1118 |
| 8 | 23.326 | 4-Hexen-3-one, 4-methyl | 3.80 | – | – | 1124 |
| 9 | 26.192 | 3-Cyclohexen-1-ol, 4-methyl-1- | 7.74 | – | 5.26 | 1182 |
| 26.202 | (1-methylethyl)-, (R)- | 1182 | ||||
| 10 | 26.992 | Octanoic acid | 13.54 | – | 12.72 | 1197 |
| 27.143 | 1199 | |||||
| 11 | 30.274 | 2,6-Octadienal, 3,7-dimethyl-, (E)- | 6.55 | – | – | 1278 |
| 12 | 30.759 | Nonanoic acid | 9.20 | – | 13.47 | 1290 |
| 30.864 | 1292 | |||||
| 13 | 31.935 | 2,4-Decadienal | 13.99 | – | 3.37 | 1322 |
| 31.944 | 1322 | |||||
| 14 | 32.16 | 1,2,4-Trithiolane, 3,5-diethyl- | 6.39 | – | – | 1329 |
| 32.445 | 16.15 | 1337 | ||||
| 32.705 | 15.60 | 1344 | ||||
| 15 | 34.055 | n-Decanoic acid | – | – | 1.50 | 1382 |
| 16 | 35.911 | 1,3,6,10-Dodecatetraene, 3,7,11-trimethyl-, (Z,E) | 11.29 | – | – | 1438 |
| 17 | 36.931 | Benzene, 1-(1,5-dimethyl-4-hexenyl)-4-methyl- or α-Curcumene | 7.13 | – | – | 1470 |
| 18 | 37.787 | Naphthalene, 1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-methylethenyl)-, [1R-(1à,7á,8aà)]- | 15.42 | – | – | 1496 |
| 19 | 38.257 | β-Bisabolene | 23.77 | – | – | 1511 |
| 20 | 38.622 | 6-Fluoro-2-trifluoromethylbenzoic acid, 2,3-dichlorophenyl ester | – | – | 5.44 | 1524 |
| 21 | 39.087 | Tetradecane, 1-iodo- | – | – | 2.40 | 1540 |
| 22 | 40.593 | 4,6-Diethyl-1,2,3,5-tetrathiolane | 5.33 | – | – | 1590 |
| 23 | 42.834 | 1-Naphthalenol, decahydro-1,4a-dimethyl-7-(1-methylethylidene)-, [1R-(1à,4aá,8aà)]- | – | – | 141.82 | 1669 |
| 24 | 42.859 | 1H-Cycloprop[e]azulen-4-ol, decahydro-1,1,4,7-tetramethyl-, [1aR-(1aα,4β,4aβ,7α,7aβ,7bα)]- or viridiflorol | 70.59 | – | – | 1670 |
| 25 | 43.739 | Neoisolongifolene, 8-oxo- | – | – | 2.32 | 1701 |
| 26 | 45.065 | 1-Iodoundecane | – | – | 2.62 | 1751 |
| 27 | 45.82 | Benzene, 1-[1,1-dimethylethyl]-4-[2-propenyloxy]- | – | – | 1.86 | 1779 |
| 28 | 47.03 | Heptasiloxane, hexadecamethyl- | – | – | 10.25 | 1826 |
| 29 | 47.085 | Isopropyl myristate | 5.09 | – | – | 1828 |
| 30 | 49.601 | Pentadecanoic acid, 14-methyl-, | 7.54 | – | 14.70 | 1929 |
| 49.602 | methyl ester | 1929 | ||||
| 31 | 50.677 | Phthalic acid, butyl tridecyl ester | 40.16 | – | – | 1974 |
| 32 | 50.752 | n-Hexadecanoic acid | 300.13 | 30.67 | 214.16 | 1977 |
| 50.812 | 1979 | |||||
| 50.892 | 1982 | |||||
| 33 | 51.227 | Heptanoic acid, 2-ethyl- | – | – | 3.11 | 1996 |
| 34 | 53.573 | 2-Tetradecyne | 3.74 | – | 3.21 | 2097 |
| 53.588 | 2098 | |||||
| 35 | 53.708 | 9-Dodecenoic acid, methyl ester, (E)- | – | – | 9.55 | 2103 |
| 36 | 54.248 | Hexasiloxane, tetradecamethyl- | – | – | 11.40 | 2126 |
| 37 | 54.628 | 9-Decen-1-ol | – | – | 5.38 | 2142 |
| 38 | 54.854 | cis-Vaccenic acid or 11- | – | 25.40 | – | 2151 |
| 54.854 | Octadecenoic acid, (Z)- | 2151 | ||||
| 39 | 55.079 | Spiro[2.5]octane | – | 0.29 | – | 2160 |
| 40 | 55.239 | Octadecanoic acid, 2-(2-hydroxyethoxy)ethyl ester | – | – | 20.43 | 2167 |
| 41 | 55.324 | Octadecanoic acid | 47.91 | 9.50 | – | 2170 |
| 55.329 | 2171 | |||||
| 42 | 57.384 | Hexasiloxane, tetradecamethyl- | – | – | 7.91 | 2253 |
| 43 | 58.18 | Naphthalene, decahydro-1-pentadecyl- | – | 0.13 | – | 2285 |
| 44 | 60.346 | Triphenyl phosphate | – | 0.13 | – | 2368 |
| 45 | 63.607 | Oxirane, hexadecyl | 20.18 | – | – | 2487 |
| 46 | 65.663 | 1,1,1,3,5,7,7,7-Octamethyl-3,5-bis(trimethylsiloxy)tetrasiloxane | 4.54 | – | – | |
| 47 | 67.324 | Dodecane, 1-cyclopentyl-4-(3-cyclopentylpropyl)- | 3.26 | – | – | 2616 |
The major biologically active compounds of 100 bar, 40 °C of SCF extract are Terpinen-4-ol (0.44%), 2, 4-Decadienal (0.85%) (Zai-Chang et al. 2005), α-Cedrene (0.70%), Bicyclosesquiphellandrene (1%) (Aneja et al. 2005), Bicyclo [3.1.1] hept-2-ene, 2, 6-dimethyl-6-(4-methyl-3-pentenyl)- (2.1%) (Liu et al., 2011), Allyl isopropyl sulfide (2.9%) (Muhi-Eldeen et al. 2008), β-Sesquiphellandrene (0.37%) (Jayaprakasha et al. 2005), Cycloisolongifolene (0.49%) (da Silva et al. 2015), Guaia-1(10), 11-diene (3.42%), β-Bisabolene (3.27%), (−)-α-Panasinsen (0.36%), Isocaryophyllene (0.41%), trans-Sesquisabinene hydrate (0.39%), 1-Naphthalenol, decahydro-1,4a-dimethyl-7-(1-methylethylidene)-, [1R-(1α, 4aβ, 8aα)]- (5.1%), Hexadecanoic acid, methyl ester (3.95%), n- Hexadecanoic acid (23.66%), and Cis-Vaccenic acid (2.99%) (Huang et al. 2011). These above compound are biologically active as antifungal, anti-influenza, antimicrobial, and antioxidants. On the other hand, the majority of these compounds are absent in hydro-distillation of neem volatiles. Hydro-distilled neem seed volatile mainly comprises of 1,2,4-Trithiolane, 3,5-diethyl (43.91%), cycloeicosne (3.57%), n-Tetracosanol-1 (3.13%), E-15-Heptadecenal (4.07%), 1-Hexadecanol (4.51%) and others (Table 5). As shown by the results, the composition of the SCF and hydro-distillation was significantly different.
Table 5.
Identification of neem seed (Hydro-distillation) volatile compounds by GC–MS
| Sl.no | RT (min) | Compound | RI | (ppm) |
|---|---|---|---|---|
| 1 | 7.135 | 1,3-Propanedithiol | 844 | 3.46 |
| 2 | 23.531 | Allyl mercaptan | 1128 | 2.60 |
| 3 | 26.787 | 1-Dodecene | 1193 | 1.35 |
| 4 | 28.353 | 4,6-Dimethyl-[1,2,3]trithiane | 1231 | 4.58 |
| 5 | 31.344 | 1,3-Propanedithiol | 1305 | 1.51 |
| 6 | 32.275 | 1,2,4-Trithiolane, 3,5-diethyl- | 1332 | 58.89 |
| 7 | 32.59 | 1,2,4-Trithiolane, 3,5-diethyl- | 1341 | 77.57 |
| 8 | 33.545 | 1,2,4-Trithiolane, 3,5-diethyl- | 1368 | 0.92 |
| 9 | 34.446 | 1-Hexadecanol | 1393 | 9.64 |
| 10 | 34.566 | 1,2,4-Trithiolane, 3,5-diethyl- | 1396 | 2.34 |
| 11 | 35.416 | 1H-3a,7-Methanoazulene, octahydro-3,8,8-trimethyl-6-methylene-, [3R-(3à,3aá,7á,8aà)]- | 1423 | 0.75 |
| 12 | 35.951 | Bicyclo[3.1.1]hept-2-ene, 2,6-dimethyl-6-(4-methyl-3-pentenyl)- | 1439 | 3.50 |
| 13 | 36.366 | γ-Muurolene | 1452 | 1.19 |
| 14 | 36.977 | Spiro[4.5]dec-7-ene, 1,8-dimethyl-4-(1-methylethenyl)-, [1S-(1α,4β,5α)]- | 1471 | 2.51 |
| 15 | 37.197 | 5,6-Dihydro-2,4,6-triethyl-4H-1,3,5-dithiazine | 1478 | 4.41 |
| 16 | 37.537 | Cycloisolongifolene | 1488 | 1.01 |
| 17 | 37.837 | Naphthalene, 1,2,3,5,6,7,8,8a-octahydro-1,8a-dimethyl-7-(1-methylethenyl)-, [1R-(1α,7β,8aα)]- | 1497 | 4.88 |
| 18 | 38.307 | á-Bisabolene | 1513 | 3.84 |
| 19 | 38.712 | Phenol, 2,4-bis(1,1-dimethylethyl)- | 1527 | 9.60 |
| 22 | 40.088 | 1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl-, (E)- | 1573 | 0.74 |
| 23 | 40.703 | 1-Hexadecanol | 1593 | 16.89 |
| 24 | 42.919 | 1-Naphthalenol, decahydro-1,4a-dimethyl-7-(1-methylethylidene)-, [1R-(1α,4aβ,8aα)]- | 1672 | 4.94 |
| 25 | 46.22 | E-15-Heptadecenal | 1794 | 15.23 |
| 26 | 47.056 | 1,2,4-Trithiolane, 3,5-diethyl- | 1827 | 13.75 |
| 27 | 47.646 | 1,2,4-Trithiolane, 3,5-diethyl- | 1850 | 14.72 |
| 28 | 47.821 | 1,2,4-Trithiolane, 3,5-diethyl- | 1857 | 6.15 |
| 29 | 50.162 | Oxacycloheptadec-8-en-2-one, (8Z) | 1952 | 1.06 |
| 30 | 51.192 | Cycloeicosane | 1995 | 13.36 |
| 31 | 52.563 | 4,6-Diethyl-1,2,3,5-tetrathiolane | 2054 | 1.52 |
| 32 | 55.734 | n-Tetracosanol-1 | 2187 | 8.54 |
| 33 | 59.911 | n-Tetracosanol-1 | 2351 | 3.20 |
| 34 | 63.782 | 9-Hexacosene | 2493 | 1.13 |
It is found that in all nine experiments of SCF, higher levels of compounds having carboxylic acid functional groups (25–65%). At 100 bar, 40 °C, the volatile compounds were further classified as acids (30%), alcohols (6.42%), aldehyde (0.857%), alkane (0.46%), alkene (19.48%), ester (13.93%), lactone (0.35%), n-heterocycle (0.41%), oxime (0.57%), s-heterocycle (1.05%) and α, β-unsaturated ketone (1.45%). The presence of compounds of these entire functional groups make 100 bar, 40 °C is the suitable condition for extraction of neem seed volatile in comparison with other SCF parameters and hydro-distillation process.
Conclusion
Recently, supercritical fluid carbon dioxide is used for the extraction process of bioactive compounds to get near natural forms without any artifact formation. Neem seed powder was subjected to SCF at different pressure and temperatures. Extracts were partitioned to separate volatiles and were analyzed by GC–MS along with the volatiles extracted by conventional method. There is a significant effect of pressure and temperature on isolation of number of volatile compounds as well as retention of biological active compounds by SCF. Extraction at 100 bar, 40 °C showed forty volatile compounds corresponds to 92.39% of volatiles with the major of bioactive compounds such as Terpinen-4-ol, 1,2,4-Trithiolane, 3,5-diethyl, allyl isoprophylsulphide, Cycloisolongifolene, á-Bisabolene, (−)-α-Panasinsen, Isocaryophyllene, trans-Sesquisabinene hydrate, 1-Naphthalenol.
Acknowledgements
The authors are thankful to the Director of CSIR-CFTRI for providing facilities and encouragement. The first author Swapna sonale R. acknowledges the Indian Council of Medical Research, India for providing Senior Research Fellowship.
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