*Corresponding Author:
M. Arif
Faculty of Pharmacy, Integral University, Lucknow-226 026, India
E-mail: [email protected]
Date of Submission 01 February 2017
Date of Revision 10 Novemebr 2017
Date of Acceptance 08 July 2018
Indian J Pharm Sci 2018;80(5):940-946  

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Abstract

The present study was designed to convert Saudi Arabian Sukkari waste date seeds into pyrolysis liquid char oil in a slow pyrolysis reactor and determine the phytoconstituents by gas chromatography/mass spectrometry. The date seed in particle form was pyrolysed in an externally heated pan sand bath to obtain 5.2 % v/w liquid char oil. The liquid char oil (20 ml) was dissolved in 100 ml aqueous methanol (30:70) and was four times fractionated with 25 ml of n-hexane. Small portion (1 g) of both methanol and n-hexane fraction was subjected to gas chromatography/mass spectrometry analysis. Phytochemical screening of both fractions revealed the presence of steroids, alkaloids, reducing sugars, phenolics, flavonoids, terpenoids, fatty acids and amino acids. The prevailing compounds found in n-hexane fraction were cyclolanost-24-en-3-ol (11.25 %), stigmast-5-en-3 β–ol (2.02%), lupen-3-one (3.50%), isopropyl myristate (0.64%), palmitic acid, methyl ester (0.74 %), oleic acid (0.77 %), tridecane (5.66 %), and dodecane (4.42 %). Compounds found in methanol fraction were mainly stigmast-5-en-3-ol (5.77 %), propylene glycol monooleate (2.82 %), guanosine (35.92 %), DL-arabinitol (2.11 %), dodecanoic acid (8.14 %), stearic acid (4.22 %), palmitic acid (7.61 %), oleic acid (9.13 %), 1,3,5-triazine-2,4,6-triamine (1.87 %) and 2-butylbutanoic acid (1.01 %). The presence of these bioactive compounds confirmed the application of the Sukkari varieties of date seeds for various medicinal activities in future drug discovery system and could be analyzed for antiinflammatory, antioxidant, cardiovascular, anticancer and immunosuppressant activities. However, isolation of individual phytochemical constituents may proceed to find a novel drug.

Keywords

Date seed oil, pyrolysis, liquid char oil, gas chromatography/mass spectrometry (GC/MS), phytoconstituents

Date palm (Phoenix dactylifera L.) is an important agricultural commodity and has been cultivated extensively in the whole world especially in North Africa, Middle East, as well as some parts of Central and South America and Southern Europe [1]. Date fruit has always been considered an ideal food complement and a rich source of carbohydrate due to its high sugar, dietary fibre, macro and micro-nutrient contents for people of the Middle East [2]. Date palm is an important fruit crop in the Kingdom of Saudi Arabia and Sukkari variety of date palm contained the highest amount of protein and nutrients [3]. Date seeds also have potential positive health benefits and contain components with different biological actions, such as antiinflammatory, antiviral, antioxidant and some other activities but it is usually wasted [4,5]. The date seeds oil is a mixture of different types of major and minor organic compounds those belong to acids, alcohols, ketones, aldehydes, phenols, ethers, esters, sugars, furans, nitrogen compounds and multifunctional compounds [6]. The date seeds are too hard to extract out the constituents and almost unutilized. This waste seed oil can be extracted by slow pyrolysis method and used for different medicinal purpose. Pyrolysis is generally described as the thermal decomposition of the organic components into liquid products [7]. In this study, pyrolysis of Sukkari variety of date seeds cultivated in Saudi Arabia are carried out and after purification of liquid char oil products, its phytoconstituents was determined by the sophisticated analytical instruments gas chromatography/mass spectrometry (GC/MS). The underlying components in a date seeds in detail could be characterized and the valuable components would be utilized in pharmaceutical and nutraceutical industry in best possible way to minimize this waste.

Sukkari variety of date seed was directly obtained from date fruit in March 2015 in full ripe condition from Al-Madinah Al-Monwarah city markets of Saudi Arabia and was authenticated in the Department of Pharmacognosy and Phytochemistry, College of Pharmacy, Sattam Bin Abdul Aziz University, KSA (The accession no. PSA/PHAR/COG/15/04). The seeds were washed and dried at about 40° to remove the remaining moisture present and weighed.

Pyrolysis of date seeds was carried out according to the method of Islam et al., with some modifications [8]. At the beginning of experiment, 100 g of the seeds were milled in a heavy-duty grinder to obtain a coarse powder, which was loaded into glass funnel placed in a pan sand bath. The sand bath was heated to 400- 500° with a burner for pyrolysis and the temperature was measured by means of a mercury thermometer. Simultaneously, the glass funnel containing sample was covered with another glass funnel to prevent the excess loss of sample moisture. To release the constituents and oil, distilled water was added drop-wise to the sample. During pyrolysis, the tar and oil released were collected in a container [8,9].

The products obtained from the pyrolysis of date seed were 5.2 % v/w liquid char oil. The liquid char oil (20 ml) was dissolved in 100 ml aqueous methanol (30:70) and it was four times fractionated with 25 ml of n-hexane (Merck, for analysis) by using a separating funnel. After fractionation, layer of methanol and n-hexane was separated and the solvent was removed using a Rota-vap apparatus. In the methanol fraction chocolaty brown colour extract and in n-hexane fraction, white-coloured oily substance was obtained. The physical characteristics like viscosity, refractive index and density of the both fractions of pyrolysis liquid char oil of seeds were evaluated. Viscosity was determined as such without dilution at 27° using Brookfield DV-1 Prime viscometer (Brookfield Engineering, Inc., Middleboro, MA). The refractive index was measured by using Abbe Refractometer (BESTO) by placing one drop sample on slide and the refractometer was adjusted first with distilled water. Density of the sample was determined using Puchnometer Kit (Adam Equipment co. LTD. Bond Avenue Denbigh East Estate Milton Keynes, MK, ISV-United Kingdom) [10]. Small portion (1 g) of both fractions was subjected to GC/MS analysis [11,12].

Both methanol and n-hexane fractions of pyrolyzed seeds were subjected to preliminary phytochemical investigation for the presence of various phytoconstituents like steroids, alkaloids, reducing sugar, phenolic compounds, flavonoids, saponins, tannins, anthraquinone and amino acids [13].

The GC/MS analysis of both fractions B and C were performed using the GC/MS-QP2010 Ultra. TR 5-MS capillary standard non-polar column, with dimensions of 30 m and 0.25 mm id, and 0.25 mm film was used and flow rate of mobile phase (He as carrier gas) was set at 1.21 ml/min. The oven temperature of GC instrument was raised from 100° to 260° at 10°/min and injection volume was 5 μl. Samples which dissolved in n-hexane and methanol were run fully at a range of 10-850 m/z and the results were compared by using Wiley spectral library search program. The mass spectra detected in 30-35 min [14]. The name, molecular weight, molecular formula and structure of the component of test materials were determined while the relative percentage amount of each component was calculated by comparing its average peak area to the total areas.

The relative percent weight compared with the weight of the fresh fruits, crude fibre contents and total ash of date seeds were evaluated. The date seed was successfully converted into liquid char oil by slow pyrolysis system. The contiguous parameters like pH, density and kinematic viscosity of n-hexane and methanolic fractions of pyrolysis liquid char oil were presented in Table 1. Both fractions of pyrolysis oil are found to be slightly heavier than water with a density of 1052.4 and 1036.8 kg/m3 at 27°. Preliminary phytochemical screening of pyrolysis liquid char fractions of date seeds revealed that the n-hexane fraction contains steroids, terpenoids, amino acids and fatty compounds, whereas methanolic fraction contains steroids, terpenoids, alkaloids, reducing sugars, phenolics, flavonoids, tannins and amino acids compounds.

Sample Percentage weight (%) Crude fibre (%) Ash
(%)
Refractive index at 27° Density
(kg/m3)
Kinematic viscosity at 27° (cSt)
Seed 11.27 42.4 3.4 -- -- --
Methanol fraction 2.8 -- -- 1.442 1052.4 7.68
n-Hexane fraction 1.36 -- -- 1.468 1036.8 6.24

Table 1: Physicochemical characteristics of date seeds and methanol and n-hexane fractions of pyrolysis liquid char oil

The extraction, isolation and investigation of plant material play a vital role in the development, modernization, and quality control of herbal formulations. Hence, the present study was intended to find the bioactive compounds present in the liquid char oil obtained by slow pyrolysis of date seeds using GC-MS. Crude liquid char oil contained both polar and non-polar phytoconstituents. Sequential fractionation with n-hexane helped in separation of polar and nonpolar phytoconstituents in the respective methanol and n-hexane solvent. The results pertaining to GC/MS analysis led to the identification of a number of compounds from GC and they were identified through mass spectrometry attached with GC/MS analysis. The chromatograms obtained by n-hexane and methanol fractions of liquid char oil were shown in Figure 1A and B, respectively. The active principle, area of the peak, concentration (%), and retention time and details of the compounds were presented in Tables 2 and 3, respectively.

IJPS-pyrolysis-liquid-char

Figure 1: GC/MS chromatograms
A: n-hexane and B: methanol fraction of pyrolysis liquid char oil of Sukkari date seeds

RT Peak area, % Name of compounds Molecular formula Molecular weight Compounds nature
4.151 0.59 Tetramethyl-2-hexadecen-1-ol C20H40O 296 Phytol
4.330 0.44 (14Z)-14-tricosenyl formate C24H46O2 366 Fatty ester
4.429 0.46 5-ethyl-undecane C13H28 184 Hydrocarbon
4.899 4.42 Dodecane C16H34 226 Hydrocarbon
5.045 0.75 2,5-dimethyl undecane C13H28 184 Hydrocarbon
5.228 0.38 2-ethylhexyl acrylate C11H20O2 184 Acrylic acid ester
5.390 0.39 Hexyl cyclohexane C12H24 168 Hydrocarbon
5.510 0.38 4-methyl dodecane C13H28 184 Hydrocarbon
5.558 0.70 2-methyl-6-propyl dodecane C16H34 226 Hydrocarbon
5.647 2.12 2,6,10,14-tetramethyl pentadecane C19H40 268 Hydrocarbon
5.926 5.66 Tridecane C13H28 184 Hydrocarbon
6.104 0.70 3-bromodecane C10H21Br 220 Hydrocarbon
6.190 0.74 2,4-decadienal, (E,E) C10H16O 152 Aldehyde
6.277 0.46 1-methyl naphthalene C11H10 142 Naphthalene
6.425 1.02 1-propyldecyl cyclohexane C19H38 266 Hydrocarbon
6.498 0.40 4-methyl tridecane C14H30 198 Hydrocarbon
6.617 0.56 3,8-dimethyl-decane C12H26 170 Hydrocarbon
6.673 0.96 2,6,10,14-tetramethyl-hexadecane C20H42 282 Hydrocarbon
6.821 0.63 1-tetradecanol C14H30O 214 Alcohol
7.333 0.32 1,8-dimethyl-naphthalene C12H12 156 Naphthalene
7.457 0.37 2,6,10,15-tetramethyl-heptadecane C21H44 296 Hydrocarbon
8.572 0.54 n-pentadecanol C15H32O 228 Alcohol
10.109 0.36 n-nonadecanol-1 C19H40O 284 Alcohol
10.342 0.64 Isopropyl myristate C17H34O2 270 Fatty acid ester
11.051 0.74 Palmitic acid, methyl ester C17H34O2 270 Fatty acid ester
11.488 0.29 1-Pentyl-2-propyl-cyclopentane C13H26 182 Hydrocarbon
12.157 0.23 2,6,10,14-tetramethyl-(Phytan) C20H42 282 Phytan
12.254 0.77 Oleic acid C18H34O2 282 Fatty acid
12.612 0.34 9-Octadecenoic acid (Z)-, ethyl ester C20H38O2 310 Fatty acid ester
13.349 0.84 Docosane C22H46 310 Hydrocarbon
13.906 0.45 Tetratetracontane C44H90 618 Hydrocarbon
14.346 0.54 Arachidic alcohol C20H42O 298 Fatty alcohol
14.470 1.03 Hexatriacontane C36H74 506 Hydrocarbon
14.890 0.90 Diisooctyl phthalate C24H38O4 390 Ester
15.784 1.21 n-tetratetracontane C44H90 618 Alkane
16.607 0.32 2-methylhexacosane C27H56 380 Hydrocarbon
17.016 6.71 Squalene C30H50 410 Terpenoids
17.465 0.49 Henicosyl formate C22H44O2 340 Ester
17.584 0.37 Pentatriacontane C35H72 492 Hydrocarbon
20.037 0.46 2,6,10,15,19,23-hexamethyl-1,6,10,14,18,22-tetracosahexaen-3-ol C30H50O 426 Terpenol
23.89 3.50 Lupen-3-one C30H48O 424 Lupen
25.550 2.02 Stigmast-5-en-3 β–ol C29H50O 414 Steroid
26.779 3.50 Propanoic acid, 2,2-dimethyl-, [(E,E)-3,7,11-trimethyl-2,6,10-dodecatrien-1-yl] ester C20H34O2 306 Terpene ester
27.215 4.11 9,19-cyclolanost-23-ene 3 β,25-diol C30H50O2 442 Steroid
27.645 11.25 9, 19-cyclolanost-24-en-3-ol, (3.β) C30H50O 426 Steroid
29.452 4.10 13,27-cycloursan-3-one C30H48O 424 Steroid

Table 2: Compounds present in the n-hexane fraction of pyrolysis liquid char oil of sukkari date seeds using GC/MS analysis.

RT Peak area % Name of compounds Molecular formula Molecular weight Compounds nature
3.739 1.87 1,3,5-triazine-2,4,6-triamine C3H6N6 126 Aza dye
4.022 1.01 2-butylbutanoic acid C8H16O2 144 Fatty acid
4.491 2.05 2,3-dihydro-3,5-dihydroxy-6-methyl-pyran-4-one C6H8O4 144 Flavone
5.621 0.42 Pelargonic acid C9H18O2 158 Fatty acid
5.621 0.54 (3,3,4-trimethyl-4-pentenyl) benzene C14H20 188 Aromatic
7.424 35.92 Guanosine C10H13N5O5 283 Nucleoside
8.037 0.44 Methyl laurinate C13H26O2 214 Fatty ester
8.176 2.11 DL-arabinitol C5H12O5 152 Sugar
8.360 8.14 Dodecanoicacid C12H24O2 200 Fatty acid
8.593 0.70 n-pentadecanol C15H32O 228 Fatty alcohol
8.736 0.34 Phthalic acid, ethyl isoporpyl ester C13H16O4 236 Ester
8.909 0.25 1,2,4-trimethoxy-5-[(1E)-1-propenyl] benzene C12H16O3 208 Camphor
8.992 1.22 4,6-dimethyl-3-([(E)-(3-itrophenyl) methylidene] amino)-2(1H)-pyridinone C14H13N3O3 271 Alkaloids
9.113 0.10 Mandelic acid, 3,4-dihydroxy, (4-TMS) C24H34F5NO3Si3 563 Organo-silicates
9.266 0.23 Vinyl octanoate C10H18O2 170 Ester
9.644 0.56 Methyl tetradecanoate C15H30O2 242 Ester
9.917 4.22 Stearic acid C18H36O2 284 Fatty acid
10.131 1.01 n-nonadecanol-1 C19H40O 284 Wax alcohol
10.740 0.52 8-octadecanone C18H36O 268 Wax ketone
10.777 0.14 Phthalic acid, butyl tetradecyl ester C16H22O4 278 Aromatic ester
11.075 1.56 Methyl palmitate C17H34O2 270 Fatty ester
11.319 7.61 Palmitic acid C16H32O2 256 Fatty acid
11.507 0.77 n-Nonadecanol-1 C19H40O 284 Dehydag wax
12.233 5.83 Methyl 8-octadecenoate C19H36O2 296 Fatty ester
12.368 0.35 Methyl stearate C19H38O2 298 Fatty ester
12.475 9.13 Oleic acid C18H34O2 282 Fatty acid
12.758 0.76 9-tricosene, (Z) C23H46 322 Hydrocarbon
13.415 0.50 Decanoylchloride C10H19ClO 190 Acid salt
14.142 0.52 Myristaldehyde C16H32O 240 Fatty aldehyde
14.738 0.21 2-azidocholestan-3-ol C27H47O 508 Steroid
14.915 0.86 Isooctyl phthalate C24H38O4 390 Aromatic ester
15.957 2.82 Propylene glycol monooleate C21H40O3 340 Fatty ester
21.014 1.53 Stigmast-5-en-3-yl 9-octadecenoate C47H82O2 678 Steroidal ester
25.653 5.77 Stigmast-5-en-3-ol, (3.β) C29H50O 414 Steroid

Table 3: Compounds present in the methanol fraction of pyrolysis liquid char oil of sukkari date seeds using GC/MS analysis

The prevailing compounds found in the n-hexane fraction were steroidal and triterpenoids (cyclolanost- 24-en-3-ol, 11.25 %; stigmast-5-en-3 β–ol, 2.02 %; lupen-3-one, 3.50 %; squalene, 6.71 %), various fatty acids (palmitic acid, methyl ester, 0.74 %; oleic acid, 0.77 %; isopropyl myristate, 0.64 %), and various hydrocarbons tridecane (5.66 %) and dodecane (4.42 %). Compounds found in methanol fraction were mainly steroids (stigmast-5-en-3-ol, 5.77 %), glycerol (propylene glycol monooleate, 2.82 %), purine base (guanosine, 35.92 %), sugar (DL-arabinitol, 2.11 %; 2,3-dihydro-3,5-dihydroxy-6-methyl-4h-pyran-4- one, 2.05 %), fatty acids (dodecanoic acid, 8.14 %; stearic acid, 4.22 %; palmitic acid, 7.61 %; oleic acid, 9.13 %; 2-butylbutanoic acid, 1.01 %) and aza dye (1,3,5-triazine-2,4,6-triamine, 1.87 %).

The n-hexane oily fraction of liquid char oil was a semisolid at temperatures below 10° and a viscous liquid at room temperature. The semi-solid nature of the oils is an indication of the presence of major saturated and unsaturated fatty acids in sample [15]. In the GC/MS analysis of the both fractions of pyrolysis liquid char oil were found the different types of important chemical constituents, which were the saturated and unsaturated fatty acid esters, steroids and terpenoid compounds, they were previously reported in literature [10]. Fatty acids like palmitic acid, stearic acid and oleic acid (omega-9) were mainly observed in both fractions. MS spectra of these saturated and unsaturated fatty acids were readily identified by their high resolution masses 256, 284 and 282 with predicted molecular formulas of C16H32O2, C18H36O2 and C18H34O2, respectively. Five steroids were also identified along with propylene glycol monooleate (fatty acid ester) and three terpenoids like squalene, trimethyl-2,6,10-dodecatrien-1-yl (propanoic acid terpenes ester) and lupen-3-one and a number of saturated and unsaturated long chain alcohols. β-Sitosterol was found to be the most abundant of the steroids followed by stigmast-5-en-3-ol, cyclolanost- 23-ene-3,25-diol, cycloursan-3-one, stigmast-5-en-3- yl 9-octadecenoate and 2-azidocholestan-3-ol.

These terpenoids and steroidal compounds have several important medicinal activities in future drug discovery system. Such as lanostane triterpenoid and steroidal compounds have significant adaptogenic and anabolic activity. They enhance the general performance of organism during the stress condition by normalizing the physiological process and various functions of body [16]. On the other hand organic acids (octadecenoic acid, pelargonic acid and butylbutanoic acid), flavonoids and esters of aromatic phenolic compounds have been reported to exhibit a wide range of biological activities such as antioxidant, antimicrobial and mast cells stabilizing properties [17]. The fractionated oil could be used in cosmetics and other pharmaceutical care products due to the presence of fatty acids such as palmitic acid, stearic acid and oleic acid in the oil [18]. The stearic and oleic acid content in date seed oil could make this oil as effective percutaneous absorption enhancer by enhancing the diffusion of lipophilic nonsteroidal antiinflammatory drugs, which have been widely used in conditions such as chronic rheumatic disorders treatment [19]. The presence of different components in oil such as steroids, terpenoids, organic acids, flavonoids and esters of aromatic phenolic compounds can augment antiinflammatory and analgesics potency of the pharmaceutical preparations. Oleic acid as such taken in diet has augmented the high density lipoprotein content in blood and lowered the low density lipoprotein cholesterol and lipid content showing antiatherosclerotic effect [20]. This property could prevent cardiovascular diseases. Therefore, the fractionated date seed oil could be exploited by the pharmaceutical industries to develop drug formulations for the treatment of different cardiovascular and chronic rheumatic disorders.

The Sukkari variety of Saudi Arabian date seed was successfully converted into liquid char oil by slow pyrolysis system. GC/MS data of both fractions of date seeds contained major compounds. These compounds may have important medicinal activities. Phytochemical and GC/MS analysis showed several bioactive compounds including steroids, flavonoids, terpenoids, fatty acids and different types of aromatic ester compounds. These constituents might demonstrate certain pharmacological activities like antiatherosclerotic, antiinflammatory, analgesic and antirheumatic activity. Therefore, the pyrolysis liquid char oil from the date seed could be used by pharmaceutical industry for the preparation of topical formulations for the treatment of chronic rheumatic disorders and its purified fatty acids could be considered as an important candidate of potential source for the treatment of cardiovascular disorder. Moreover, GC/ MS profile could be used as biochemical markers in the pharmaceutical industries to identify the different components present in date seeds and in authentication of mother plants.

Acknowledgements

Authors wish to thank Dr. Y. T. Kamal, Assistant Professor, Department of Pharmacognosy and Phytochemistry, College of Pharmacy, Sattam Bin Abdul Aziz University, KSA for authenticating the date palm seeds.

References