Determination of alkaloid compounds of Ricinus communis by using gas chromatography- mass spectroscopy (GC-MS)

In this study, the alkaloid compounds of Ricinus communis have been evaluated. The chemical compositions of the leaf ethanol extract of R. communis were investigated using gas chromatographymass spectroscopy (GC-MS). GC-MS analysis of R. communis alkaloid leaf ethanol extract revealed the existence of the n-haxadecanoic acid, octadecanoic acid, 1-hexadecanol. 2-Methyl, gibb-3-ene-1. 10decarboxylic acid , 2,4a. 7trihydroxy-1-methyl-8-methylene, 1.4a-lactone. 10-methyl, L-valine, ethyl ester, hexadecamethyl, tetradecamethyl, octadecamethyl, butanedioic acid, hydroxyl. diethyl ester, 1.1.3.3.5.5.7.7.9.9.11.11.13.13.15.15 hexadecamethyl, triethyl citrate, diethyl phthalate, and 3-octadecene.


INTRODUCTION
The castor oil plant Ricinus communis, also known as Palma(e) Christi or wonder tree (Figure 1), is a perennial scrub of the spurge family Euphorbiaceae.R. communis probably originates from Africa and was used in ancient Egypt and by the Romans and Greeks (Waller and Skursky, 1972).Apart from the highly toxic ricin and the less toxic R. communis agglutinin, the plant contains another toxic compound, the low molecular weight alkaloid ricinine (MW = 164.2g/mol).Ricinine or 3-cyano-4-methoxy-N-methyl-2-pyridone (CAS 524-40-3) belongs to the group of piperidine alkaloids.It was first discovered and named by Tuson in the seeds of R. communis, while searching for its medically active compounds even before ricin was known.Subsequently, its chemical structure was identified and its biosynthesis and metabolism was studied (Waller and Skursky, 1972;Mann and Byerrum, 1974).
Ricinine can be found in all parts of the plant and it is a quite strong insecticide.The castor seeds contain approximately 0.2% of the alkaloid.R. communis contains a complex cocktail of toxic substances including the type II ribosome-inactivating protein (RIP) ricin, the haemagglutinin RCA120 and the alkaloid ricinine.Furthermore, other compounds like fatty acids, flavonoids and saponins have been found to exhibit deleterious effects on bacteria, virus, fungi, invertebrates and higher animals, seemingly giving the plant some sort of protection in a hostile environment (Sitton and West, 1975; Taylor et al., 1994;Upasani et al., 2003;Bigi et al., 2004;De Assis et al., 2011) Furthermore, allergenic reactions against R. communis, in particular the seed dust, were realized.Low molecular proteins, 2S albumins, have been identified as the main allergenic compounds (Thorpe et al., 1988;Bashir et al., 1998;Deus-de-Oliveira et al., 2011).Experimental intoxication studies underline the major contribution of ricin as compared to other hazardous compounds found in the seeds (He, 2010).The oil and seed have been used as folk remedies for warts, cold tumors, indurations of the abdominal organs, whitlows, lacteal tumors, indurations of the mammary gland, corns, and moles, etc. Castor-oil is a cathartic and has labor-inducing properties.Ricinoleic acid has served in contraceptive jellies (Allardice, 1993).Ricin, a toxic protein in the seeds, acts as a blood coagulant.The oil is used externally for dermatitis and eye ailments.The seeds, which yield 45 to 50% of a fixed oil, also contain the alkaloids ricinine and toxalbumin ricin, and is considered purgative, counterirritant in scorpion-sting and fish poison.The leaves, applied to the head, is used to relieve headache and as a poultice for boil (Foster, 1990).
Phytochemical interactions of poisons lead to injury or death of living tissues.Toxicology is like science and an art like medicine.It includes observational data gathering and data utilization to predict outcome of exposure in human and animals.The ancient humans categorized some plants as harmful and some as safe.The aim of the present work is to study the toxic nature of the powder of R. communis leaves.

Collection and preparation of plant
In this research, the leaves were dried at room temperature for 10 days and when properly dried the leaves were powdered using clean pestle and mortar, and the powdered plant was size reduced with a sieve.The fine powder was then packed in airtight container to avoid the effect of humidity and then stored at room temperature.

Extraction and identification of alkaloids
The powdered leaves (2 g) were boiled in a water bath with 20 ml of 5% sulphuric acid in 50% ethanol.The mixture was cooled and filtered.A portion was reserved.Another portion of the filtrate was put in 100 ml of separating funnel and the solution was made alkaline by adding two drops of concentrated ammonia solution.Equal volume of chloroform was added and shaken gently to allow the layer to separate.The lower chloroform layer was run off into a second separating funnel.The ammoniacal layer was reserved.The chloroform layer was extracted with two quantities each of 5 ml of dilute sulphuric acid.The various extracts were then used for the following test.

Mayer's test
To the filtrate in test tube I, 1 ml of Mayer's reagent was added drop by drop.Formation of a greenish coloured or cream precipitate indicates the presence of alkaloids (Evans, 2002).

Dragendoff's test
To the filtrate in test tube II, 1 ml of Dragendoff's reagent was added drop by drop.Formation of a reddish-brown precipitate indicates the presence of alkaloids (Evans, 2002).

Wagner's test
To the filtrate in tube III, 1 ml of Wagner's reagent was added drop by drop.Formation of a reddish-brown precipitate indicates the presence of alkaloids (Evans, 2002).

Gas chromatography-mass spectroscopy (GC-MS) analysis
GC-MS analysis of the methanol extract of R. communis was carried out using a Clarus 500 Perkin Elmer (Auto system XL) Gas Chromatograph equipped and coupled to a mass detector Turbo mass gold-Perkin Elmer Turbo mass 5.1 spectrometer with an Elite -1 (100% dimethyl poly siloxane), 30 m × 0.25 mm ID × 1 μm of capillary column.For GC-MS detection, an electron ionization system was operated in electron impact mode with ionization system which was operated in electron impact mode with ionization energy of 70 eV (Imad et al., 2014(Imad et al., , 2015a;;Muhanned et al., 2015).The instrument was set to an initial temperature of 110°C, and maintained at this temperature for 2 min.At the end of this period, the oven temperature was raised up to 280°C at the rate of an increase of 5°Cmin -1 , and maintained for 9 min.Helium gas (99.999%) was used as carrier gas at a constant flow rate of 1 ml/min, and an injection volume of 2 ml was employed (split ratio of 10:1).The injector temperature was maintained at 250°C, the ionsource temperature was 200°C, the oven temperature was programmed at 110°C (isothermal for 2 min), with an increase of 100°C min -1 to 200°C, then 5°C min -1 to 280°C, ending with a 9 min isothermal at 280°C.Mass spectra were taken at 70 eV; a scan interval of 0.5 s and fragments from 45 to 450 Da.The solvent delay was 0 to 2 min and the total GC-MS running time was 36 min.The samples were injected in split mode as 10:1.Mass spectral scan range was set at 45 to 450 (m/z).The mass detector used in this analysis was Turbo Mass Gold-Perkin Elmer and the software adopted to handle mass spectra and chromatograms was a Turbo Mass ver 5.2 (Imad et al., 2015b;Mohammed et al., 2015).

RESULTS
GC-MS analysis of alkaloid compound clearly showed the presence of nine compounds.The alkaloid compound, formula, molecular weight and exact mass are as shown in Table 1.The GC-MS chromatogram of the nine peaks of the compounds detected are as shown in Figure 2-4.Chromatogram GC-MS analysis of the methanol extract of R. communis showed the presence of nine major peaks and the components corresponding to the peaks were determined as follows.The first set up peaks were determined to be n-haxadecanoic acid.The second peaks were indicated to be octadecanoic acid.The next peaks were considered to be 1-hexadecanol.2-methyl, 2,ethyl ester,hexadecamethyl,tetradecamethyl,octadecamethyl,Butanedioic aciod hydroxyl. Diethyl ester,1.1.3.3.5.5.7.7.9.9.11.11.13.13.15.15 hexadecamethyl,Triethyl citrate,Diethyl Phthalate,.The identified phytocompounds have the property of antioxidant and antimicrobial activities (He, 2010;Deus-de-Oliveira, 2011).

DISCUSSION
Plant based antimicrobials have enormous therapeutic potential as they can serve the purpose with lesser side effects.Continued further exploration of plant derived antimicrobials is needed today.
Castor bean may become a weed in neglected crop land and pasture.It is not difficult to control through cultivation and mowing.Of greater concern than its weedy potential is the high toxicity of its seeds, which contain ricin, a water-soluble protein.Even a small amount of masticated seed is likely to cause death.Humans and horses are especially vulnerable.Fatal doses are from 2.5 to 6 seeds for humans and about 6 seeds for horses (CISR, 1972).The symptoms are stomach irritation, diarrhea, abdominal pain, increased heart rate, profuse sweating, collapse, and convulsions.Broken seeds can cause skin irritation.The foliage is only slightly toxic (Anonymous, 2000).It is advisable to completely eliminate castor bean from pastures, especially horse pastures, and pinch off flowers of ornamental plants to prevent possible poisoning of children.When assessing the numerous reports on intoxications with ricin, R. communis seeds or R. communis-containing feed and fertilizer, some general aspects have to be considered.The term ricin in any toxicological publication suggests a degree of homogeneity or a lack of variability that might be expected for pure chemicals (Despeyroux et al., 2000;Thullier and Griffiths, 2009).
Furthermore, other compounds like fatty acids, flavonoids and saponins have been found to exhibit deleterious effects on bacteria, virus, fungi, invertebrates and higher animals, seemingly giving the plant some sort of protection in a hostile environment (Upasani et al., 2003;De Assis et al., 2011).Furthermore, allergenic reactions against R. communis, in particular the seed dust, were realized.constitutions which may be useful for various herbal formulation as anti-inflammatory, analgesic, antipyretic, cardiac tonic and antiasthmatic.So, it may be concluded that R. communis is a very important indigenous medicinal plant which requires more exploration to utilize its medicinal property.

Figure 3 .
Figure 3. Structure of n-haxadecanoic acid present in the leaves extract of Ricinus communis using GC-MS analysis.

Figure 4 .
Figure 4. Structure of Octadecanoic acid present in the leaves extract of Ricinus communis using GC-MS analysis.

Figure 5 .
Figure 5. Structure of 1-hexadecanol.2-methyl present in the leaves extract of Ricinus communis using GC-MS analysis.

Figure 7 .
Figure 7. Structure of L-Valine, ethyl ester present in the leaves extract of Ricinus communis using GC-MS analysis.

Figure 8 .
Figure 8. Structure of Butanedioic aciod hydroxyl.Diethyl ester present in the leaves extract of Ricinus communis using GC-MS analysis.

Figure 9 .
Figure 9. Structure of Triethyl citrate present in the leaves extract of Ricinus communis using GC-MS analysis.

Figure 10 .
Figure 10.Structure of diethyl phthalate present in the leaves extract of Ricinus communis using GC-MS analysis.

Figure 11 .
Figure 11.Structure of 3-octadecene present in the leaves extract of Ricinus communis using GC-MS analysis.

Table 1 .
Compounds present in the leaves extract of Ricinus communis using GC-MS analysis.