Read Isolation of 1,2-benzenedicarboxylic acid bis(2-ethylhexyl) ester from methanol extract of the variety minor seeds of Ricinus communis Linn text version

Sani and Pateh, Nig. Journ. Pharm. Sci., October, 2009, Vol. 8 No. 2, P. 107 - 114

Nigerian Journal of Pharmaceutical Sciences Vol. 8, No. 2, October, 2009, ISSN: 0189-823X All Rights Reserved

Isolation of 1,2-benzenedicarboxylic acid bis(2-ethylhexyl) ester from methanol extract of the variety minor seeds of Ricinus communis Linn. (Euphorbiaceae)

*U.M. Sani and **U.U. Pateh *Chemistry Department, Kano State College of Arts, Science and Remedial Studies\ **Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria, Kaduna-Nigeria. Author of correspondence

Abstract The variety minor seeds of Ricinus communis plant have been chemically studied. Methanol was used in the extraction of the wet ground seeds to obtain two layers; pale yellow oil (bottom layer) and a whitish scum (upper layer). The whitish scum was dissolved in n-butanol and the solvent removed under reduced pressure to afford a whitish gummy material. The gummy material was then extracted with hot chloroform and when the chloroform extract was kept for about three days, a colourless viscous liquid (B1) was obtained. The viscous liquid was insoluble in water. Thin Layer Chromatography studies of B1, using hexane: ethyl acetate (2:1) as the solvent system, on silica-gel pre-coated plastic plate showed one single spot (Rf value = 0.65). B1 was purified by a preparative thin-layer chromatography on silica gel coated plate (20 x 20 cm and 0.05 cm thick) with hexane: ethyl acetate (2:1) as the eluent. Boiling point of B1 was found to be 385 0C. The GC/Mass spectroscopy and IR spectrum of B1 showed it to be 1, 2-benzenedicarboxylic acid bis(2-ethylhexyl) ester (a dioctyl phthalate ester). Key words: Ricinus communis, variety minor seeds, methanol extract, plasticizer, dioctyl phthalate

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Introduction

Ricinus communis L., or castor oil plant, is member of the Euphorbiaceae family that may have originated in Abyssinia, Ethiopia (Weiss, 1971). According to Trease and Evans (1989), the plant is a native of India with about 17 species. Castor seeds that are obtained in northern states of Nigeria have been classified into seven distinct varieties according to their sizes and colours (van Rheenen, 1976). However, the seeds are more commonly classified into three groups that include the larger seeds (variety major), medium seeds 107 (variety intermediate) and the smaller seeds (variety minor). The commonest variety in northern Nigeria is the variety minor. The oil extracted from the castor seeds, castor oil, makes up 35% to 55% of the weight of the seeds (Oplinger et al., 1990). Castor seeds contain a glycoprotein called ricin, which is notoriously used as an assassination weapon by the secret intelligence services (Carus, 1998). It was also reported that ricin is twice as poisonous as cobra venom (Balint, 1974). The lethal dose (LD50) of ricin in humans

Sani and Pateh, Nig. Journ. Pharm. Sci., October, 2009, Vol. 8 No. 2, P. 107 - 114

is 1-10µg when delivered as an aerosol or by injection (Franz and Jaax, 1997). If castor seed is swallowed without chewing and there is no damage to the seed husk, it passes harmlessly through the digestive tract. However, if it is chewed and then swallowed, the intestines absorb the ricin toxin (Wesche et al., 1999). The presence of flavonoids, anthraquinones, saponins, tannins, sterols and/or triterpenes was reported in the entire plant (Tanira et al., 1989). Other studies on the aerial parts of the plant (Al-Yahya, 1986; Rizk et al., 1986) reported the presence of alkaloids, flavonoids, sterols and/or triterpenes. Rizk et al., (1986) also reported the presence of coumarines and the absence of saponins although Al-Yahya (1986) noted the presence of saponins. The presence of steroidal compounds and alkaloids in the castor seeds has also been reported by some authors (Lotti et al., 1977; Coe and Kabele-Toge et al., 1996). In most of these studies no indication made on the variety of the seeds that was studied. Fatty acid esters, derived from vegetable oils, are important plasticizers. Plasticizers are important plastics additives and when added to thermoplastics they increase their flexibility, transparency, and durability. The development of nitrocellulose led to castor oil being patented in 1856 for use as the first plasticizer. Castor oil contains 85% to 90% ricinoleic acid (Bafor et al., 1991) and derivatives of this acid find use in a variety of products including paints, printing inks, cosmetics, and pharmaceuticals (Andrew et al., 2005). Eight nitrogen-containing derivatives of ricinoleic acid were screened for their plasticizer characteristics and all except one, 4-(12-hydroxystearoyl) morpholine, were found to be satisfactory primary plasticizers for vinyl chloride-vinyl acetate copolymer (Frank et. al., 1959). These derivatives were also found to be quite similar to 1,2-benzenedicaboxylic acid bis(2-ethylhexyl) phthalate (a dioctyl phthalate) in plasticizing performance. 108

Vegetable oil-derived plasticizers are benign and not only make plastic material flexible but they also offer such benefits as its resistance to migration, evaporation and leaching, and the stability to light and heat. The most common types of synthetic plasticizers are the dialkyl or alkyl aryl esters of phthalic acid (phthalates) and the most widely-used phthalates are the dioctyl phthalate, the diisodecyl phthalate, and the diisononyl phthalate. Phthalates are not chemically bonded to the polymeric material in which they are mixed and so are easily released into the environment. Dioctyl phthalate is less volatile but it can easily be leached into food. Phthalates can therefore be introduced accidentally into solvents and lipid extracts from a variety of sources. These substances tend to cochromatograph with lipids to spread confusion and obscure compounds of interest in chromatograms. They elute with fatty acid derivatives from many of the common gas chromatography (GC) stationary phases (Shantha and Ackman, 1991).

Materials and Methods

The variety minor seeds of Ricinus communis were collected from Kano and authenticated at the herbarium of the Department of Biological Sciences, Ahmadu Bello University Zaria, where a herbarium specimen was deposited (Voucher Number: 900233). These seeds have brownish testa colour and white testa speckling. Extraction procedure The outer coating (husks) of the seeds were manually removed and then wet grounded into pulp. The wet grounded pulp (500g) was extracted with methanol in a 2-litre conical flask using a magnetic stirrer for 2 hours to afford pale yellow oil (bottom layer) and a whitish scum (upper layer). The weight and percentage of the pale yellow oily extract were recorded.

Sani and Pateh, Nig. Journ. Pharm. Sci., October, 2009, Vol. 8 No. 2, P. 107 - 114

The presence of steroidal nucleus and alkaloids in the pale yellow oil was investigated using the methods of Trease and Evans (1989). The whitish scum was dissolved in n-butanol and the solvent removed under reduced pressure to afford a whitish gummy material. The gummy The viscous liquid (B1) was purified by a preparative thin-layer chromatography on silica gel coated plate (20 x 20 cm and 0.05 cm thick); the eluting solvent also being hexane : ethyl acetate (2:1). Ethyl acetate was used to extract the viscous liquid from the silica gel. Thin layer chromatography (TLC) analysis of B1 (using hexane : ethyl acetate = 2:1 as the solvent system) on silica-gel G60 F254 precoated plastic plates (0.25 mm thickness) was carried out. The purified viscous liquid, being colourless, was tested for alkaloids using Dragendorff's spray reagent on silica-gel coated plate and its boiling point determined. Spectral analysis (GC/MS and IR) of B1 were also carried out and results recorded. The GC chromatograph was plotted between 1 to 2099 scan numbers.

material was then extracted with hot chloroform and when the chloroform extract was kept for about three days, a colourless viscous liquid (B1) was obtained. Chromatographic and spectral analysis minutes, indicating that B1 is a pure compound. The total ion count was 11992 and total retention time for the elution of these ions was 34.98 minutes. The mass spectrum of B1 (Figure 2.0) indicated the [M+H]+ ion at m/z 391 and the base peak at m/z 149. The [M+H]+ ion is 95.6% of the base peak. The other prominent peaks are those at m/z 279, 167, 113, 71, and 57. The mass fragmentation pattern of B1 is given in Scheme 1.0. The major ions at m/z 167, 279, 149 (base peak) and 113 are formed by a MacLafferty rearrangement and hydrogen transfer (Scheme 1.0b, c, and d). The most abundant ion that gave m/z at 149 (base peak) can be formed through the formation of the ion having m/z at 279 in two ways (Scheme 1.0c and d). Through dealkylation of and then loss of an alkoxy group from the molecular ion two major peaks (m/z 57 and 71) are formed (Scheme 1.0a). Infra-red (IR) Spectroscopy The IR spectrum of B1 is shown in Figure 3.0. The typical diagnostic band for the ester carbonyl group, C=O (1738.59 cm-1) can be observed, indicating the presence of this functional group. The other prominent peaks are seen at 2853.46 cm-1 (­CH3 stretch), 2922.82 cm-1 (­CH2­ stretch), 1460.19 cm-1 and 723.66 cm-1 (methylene C­H bend and ­ (CH2)n­ rocking, respectively), and 1163.24 cm(C­O stretch).

Results

The weight of the methanol pale yellow extract of the seeds before it separated into two layers were found to be 240.27g representing 48.05% of the total weight of seeds used. This extract showed positive results for both steroidal compounds and alkaloids. Thin layer chromatography (TLC) studies of B1 (using hexane : ethyl acetate = 2:1 as the solvent system) on silica-gel G60 F254 pre-coated plastic plates (0.25 mm thickness) showed it to be one single spot (Rf value = 0.65). The alkaloidal test for B1 using Dragendorff's spray reagent was negative and this viscous liquid was found to boil at 385 0C. GC/Mass Spectrometry The chromatogram of B1 (given in Figure. 1.0) indicates only one major peak at 1600 scan number and retention time of 26.66 109

Sani and Pateh, Nig. Journ. Pharm. Sci., October, 2009, Vol. 8 No. 2, P. 107 - 114

Figure 1: Chromatogram of B1

Figure 2: Mass spectrum of B1

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Sani and Pateh, Nig. Journ. Pharm. Sci., October, 2009, Vol. 8 No. 2, P. 107 - 114

O

CH2CH3 C OCH2CH(CH2)3CH3 C OCH2CH(CH2)3CH3 CH2CH3 B1 e-

+H+

CH2CH3 HO C OCH2CH(CH2)3CH3 C OCH2CH(CH2)3CH3 O CH2CH3

O

(a)

+H

[M+H]+ ion, m/z 391

O C OCH=CHCH2CH3

+

O CH2CH3 C OCH2CH(CH2)3CH3 C OCH2CH(CH2)3CH3 CH2CH3 O M (molecular ion) H O CH2CH3 C (CH2)3CH3

+

C OCH=CHCH2CH3 O C O C O

+ CH3CH2CH

CH3CH2CHCH3 H+

CH3CH=CHCH3 O

m/z 57

O CH CH3CH2CH CH

m/z 71

OH C OH C OCH2CH(CH2)3CH3 CH2CH3 O CH2CH3 eCH3C CHCH2CH2CH3

(b)

CH2 O COCH2CH(CH2)3CH3 CH2CH3 O M+ (molecular ion)

C

OH C O C OCH2CH(CH2)3CH3 CH2CH3 O CH2CH3

H

m/z 279

+ CH3C=CHCH2CH2CH3

O H C OH CH2CH3 COCH2CH(CH2)3CH3 O O C OH2 COCH2CH(CH2)3CH3 O CH2CH3 O C OH C H O C O OH CH2 CH2CH3 (CH2)3CH3 O C H O

m/z 113

O H2O C C O O CH2 CH2CH3 C(CH2)3CH3 H CH2CH3 CH3C CHCH2CH2CH3

(c)

m/z 279

+ CH3C=CHCH2CH2CH3 e

CH2CH3

m/z 149

O O C C O OH OH H O C C O OH OH2 H2O

m/z 113

O C OH C O

(d)

C C OH

C OH OH

m/z 279 m/z 167

m/z 149

Scheme 1.0: Fragmentation pattern for B1. The formation of the major ions with m/z at 57, 71, and 391 {for the [M+H]+ ion} are given in (a). The other major ions with m/z at 113, 149 (base peak), 167 and 279 are formed by MacLafferty rearrangement as shown in (b), (c), and (d).

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Sani and Pateh, Nig. Journ. Pharm. Sci., October, 2009, Vol. 8 No. 2, P. 107 - 114

Figure 3.0: IR spectrum of B1

Figure 3: IR spectrum of B1

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Sani and Pateh, Nig. Journ. Pharm. Sci., October, 2009, Vol. 8 No. 2, P. 107 - 114

Discussion

The presence of steroidal compounds and alkaloids in the seeds' extract is in agreement with what has been observed by some authors (Lotti et al., 1977; KabeleToge et al., 1996). The percentage of the oil extracted (48.05%) from the seeds fell within the range reported in the literature (Oplinger et al., 1990). Eight nitrogencontaining derivatives of ricinoleic acid were screened for their plasticizer characteristics and all except one, 4-(12hydroxystearoyl) morpholine, were found to be satisfactory primary plasticizers for vinyl chloride-vinyl acetate copolymer (Frank et. al., 1959). These derivatives were also found to be quite similar to the 1,2-benzenedicarboxylic acid bis(2ethylhexyl) ester in plasticizing performance. Lipid samples may have been inadvertently contaminated with phthalates when in contact with most of the plastic materials. However, the natural occurrence of phthalates in a wide variety of plants is already in the literature (Perkins, 1967; Graham, 1973; Duc et. al., 2007) and in fact fatty foods such as milk, butter, and meats are found to be the main sources of natural bis(2-ethylhexyl) phthalate and other phthalates (Kohn et. al. 2000). Since castor oil was the first substance used and patented as a plasticizer, its plasticizing ability may not only be attributed to the amount of fatty acids it contains but the presence of bis(2ethylhexyl) phthalate will also add to that attribute. Both the IR and Mass spectra of the viscous liquid (B1) compared very well with the authentic spectra of bis(2ethylhexyl) phthalate. The fragment ion peaks at m/z 149 and 167 are considered characteristic of alkyl phthalates and ions at m/z 279 and 261 suggested that the alkyl phthalate is dioctyl phthalate (Eiichi, et. al., 1972). The dealkylation at carbon-2 to produce the C4H9+ ion, suggested that the dioctyl is 2-ethylhexyl not a normal 113

octane and carbon-2 must be a tertiary carbon. The base peak is the protonated phthalic anhydride (C8H5O3). 1,2Benzenedicarboxylic acid bis(2ethylhexyl) phthalate has been isolated from a marine alga, Sargassum weightii, and apart from its plasticizing ability it was also found to have antibacterial effect on a number of bacteria (Sastry and Rao, 1995). It was reasoned by the authors that, since other alga species collected from the same location did not show any antibacterial activity in the crude lipid extracts, the antibacterial compound extracted from S. weightii was not an impurity taken up by the alga from the environment. The viscous liquid (B1) might be natural and not synthetic compound taken in the methanol extract as impurity from the environment as there was no contact with any plastic material. This is the first report on isolation of this phthalate from the seeds of Ricinus communis.

Reference

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Isolation of 1,2-benzenedicarboxylic acid bis(2-ethylhexyl) ester from methanol extract of the variety minor seeds of Ricinus communis Linn