Screening for secondary metabolites in Huru crepitans bark ethanol extract using GC-MS analysis: a preliminary study approach

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1 Journal of Science and Technology Advances J Sci Tech Adv. March 2017; Vol. 2 (Issue 1): Research Article Screening for secondary metabolites in Huru crepitans bark ethanol extract using GC-MS analysis: a preliminary study approach Igwe K. K. 1 *, Nwakudu.N. 1, Ijioma S.N. 1, Madubuike A.J. 1, Achi N.K. 2 1 Departmemt of Vet Physiology, Pharmacology and Biochemistry, 2 Department of Biochemistry, Michael kpara University of Agriculture, Umudike, Nigeria *For correspondence Dr. K.K. Igwe, Departmemt of Vet Physiology, Pharmacology and Biochemistry, Michael kpara University of Agriculture, Umudike, Nigeria. kkigwe191@ gmail.com Received: 12 ctober 2016 Accepted: 17 December 2016 ABSTRACT bjective: The research interest of authors was to find the phytochemicals in H. crepitans that produces the medicinal activity. Methods: Gas Chromatography-Mass Spectroscopy [GC-MS] analysis was used for screening the secondary metabolites in its bark as a preliminary study. The extract was prepared using Soxhlet extraction method with ethanol as solvent. It was concentrated at 35 o C in hot air oven. The molecular mass of the phytochemicals were established based on the molecular ion in the mass spectra. Results: The chromatogram showed eight peaks representing the presence of eight phytochemicals in the extract. The suggested compounds are 5-(hydroxymethyl)furan-2-carbaldehyde with Peak Area Percentage (PA%) of 1.65 and Retention Time (RT) 3.913; 3,5- dihydroxy-6-methyl-2,3-dihydro-4h-pyran-4-one with PA% of 2.75 and RT 5.434; Nitrocoumarin with PA% 0.91 and RT ; Methyl 14- methylpentadecanoate with PA% and RT ; Hexadecanoic acid also known as Palmitic acid with PA% 3.52 and RT ; Methyl linolelaidate with PA% and RT ; 9,12,15-ctadecatrien-1-ol with PA% and RT ; Stearic acid, methyl ester with PA% and RT Conclusions: The bioactivity studies showed that H. crepitans bark could be a urinary acidifier, inhibitor of uric acid production, antibacterial, prevents inflammation and vasodilatation. It also showed anti 5HT (Serotonin), anti HIV integrase activity and antidote activity for heavy metals poisoning. We therefore recommend the isolation and synthesis of these bioactive compounds for new drug development. Keywords: GCMS, Huru crepitans, Extraction, Preliminary study 8

2 Introduction Most cultures around the world use plants as medicine for curing various ailments and preventing diseases. These medicinal plants also play an essential role in the religion and development of human culture. 1 Analyzing medicinal plants helps us to understand the compounds in them that provide us with those medicinal activities for maximum utilization. Hura crepitans from the family of Euphorbiaceae (spurge) is an evergreen tree that is native to the tropical regions of the Amazon forest and also the North and South America. H. crepitans has its synonyms as Hura strepens Willd, Hura brasiliensis Willd and Hura senegalensis Baill. 2 This plant is also known as sandbox tree, possum wood, jabillo and dynamite tree because of the explosive sound it makes as it splits the capsule. 3 H. crepitans can grow up to 200ft (60m) 4 and the fruit it produces is in form of a large capsule that can explode, spreading its seeds as far as 160 meters per hour 5 or as far as 330ft 6 from the tree. The plant is known by possessing many dark pointed sharp spines on its smooth bark. These spines prevent animals from climbing it. Figures 1 and 2 show Huru crepitans bark and leaves respectively. The plant is usually cultivated for shade and the wood used for making furniture, while the milky sap serves as poison for arrows 7 and for catching fishes by the fishermen. 8 H. crepitans is cultivated for medicinal purposes as it is used in treating skin diseases, intestinal worms and rheumatism, while its bark is used to treat leprosy and the leaves used to treat eczema. 9 Figure 1: Shows H. crepitans bark. Phytochemicals in plant extracts have been identified using GCMS analysis by different researchers 10, 11,12,13,14 thus we use the same analysis to identify the phytochemicals in ethanol extract of H. crepitans Materials and Methods Plant materials Fresh leaves of H. crepitans were harvested from natural habitat at hafia town in Abia State, Nigeria. The plant leaves were identified at the Taxonomy section of College of Natural and Environmental Management, Michael kpara University of Agriculture, Umudike, Nigeria. Preparation of plant extract Figure 2: Shows H. crepitans bark and leaves. The plant material of H. crepitans were collected from wild, shade dried for 8 days at room temperature and pulverized to powder using electric grinder. The plant extract was prepared using Soxhlet method described by 15. 9

3 Twenty grams (20g) of powdered sample was introduced into the extraction chamber of the Soxhlet extractor using ethanol as solvent. Temperature was maintained at 80 0 C throughout the extraction period of 48 hrs. The sample was concentrated using hot air oven at 35 0 C to obtain dried extract which was used for GCMS analysis. formula, structure and bioactivities of the phytochemicals were ascertained. Results and Discussion GCMS analysis of H. crepitans The characterization of the Phytochemicals in H. crepitans was done using GC-MS QP2010 Plus (Shimadzu, Japan). The identification of the phytochemicals in the sample was carried out using a QP2010 gas chromatography with Thermal Desorption System, TD 20 coupled with Mass Spectroscopy (Shimadzu). The ionization voltage was 70eV. Gas Chromatography was conducted in the temperature programming mode with a Restek column (0.25 mm, 60 m, XTI-5).The initial column temperature was 80 0 C for 1min, and then increased linearly at 70 0 C min-1 to C, held for 3 min followed by linear increased temperature 10 0 C min-1 to C for 10 min. The temperature of the injection port was C and the GC-MS interface was maintained at C.The sample was introduced through an all-glass injector working in the split mode, with helium carrier gas low rate of 1.2 ml min-1. The identification of compounds was accomplished by comparison of retention time and fragmentation pattern, as well as with mass spectra of the GC-MS. Figure 2: Gas Chromatogram of H. crepitans bark ethanol extract. Identification of phytochemicals in H. crepitans Gas Chromatogram of H. crepitans revealed eight peaks showing that eight different phytochemicals were present. Identity of the active phytochemicals in the extract was done by comparison of their retention indices, peak area percentage and mass spectra fragmentation pattern with those stored in the database of National Institute of Standards and Technology (NIST) and also with published literature, NIST08.LIB 16, WILEY8.LIB 17, PESTEI-3.LIB and FA-ME.LIB library sources were used for matching the identified phytochemicals from the plant material. The name, molecular weight, Figure 3: Mass Spectra of H. crepitans ethanol extract. 10

4 H H H 5-(hydroxymethyl)furan-2-carbaldehyde Hexadecanoic acid C H 3 H H H 3 C Methyl linolelaidate 3,5-dihydroxy-6-methyl-2,3-dihydro-4H-pyran- 4-one H N 9,12,15-ctadecatrien-1-ol H 3 C Nitrocoumarin H 3 C Methyl 14-methylpentadecanoate Stearic acid, methyl ester Figure 4: Shows the structural formula of phytochemicals in H. crepitans bark ethanol extract. Gas chromatogram showed eight peaks (Figure 2) representing eight phytochemicals in H. crepitans bark ethanol extract. 11

5 The name retention time, peak area percentage, molecular weight, molecular formula, structural formula and bioactivity of the ethanolic phytochemical extract of H. crepitans bark are shown in (Table 1). The phytochemical 9, 12, 15 ctadecatrien-1- ol with the highest peak area percentage of sugars. 18,19,20 ligosaccharides can be glycosylated to form glycoproteins or glycolipids. 21 They can be used in cell recognition, cell binding and immune responses. The phytochemical, Methyl linolelaidate with peak area percentage of showed bioactivity of Cathecol-0-methyl transferase Table 1: Proposed phytochemicals in Hura crepitans bark Ethanol Extract. S/ No Name of Compound 1 5- (hydroxymethyl) furan-2- carbaldehyde 2 3,5-dihydroxy-6- methyl-2,3- dihydro-4hpyran-4-one Retentio n time Peak area % Molecul ar weight Molecula r formula Bioactivity C 6H 6 3 Antimicrobial, Preservative clastogenic activity, Uterotonic activity C 6H 8 4 Antimicrobial, Anti-inflammatory, Antiproliferative Antioxidant, Automatic nerve activity 3 Nitrocoumarin C 9H 5N 4 11B-HSD-Inhibitor, 17-beta-hydroxysteroid dehydrogenase-inhibitor, 5-HETE-Inhibitor, 5-HT-Inhibitor, 8-HETE-Inhibitor, Anti- HIV-Integrase, Anti-5-HT, Antidote (Heavy Metals), Antidote (Hydrazine), Aryl- Hydrocarbon-Hydroxylase-Inhibitor, Hallucinogenic, Hemangionagenic 4 Methyl 14- methylpentadeca noate 5 Hexadecanoic acid also known as Palmitic acid 6 Methyl linolelaidate 7 9,12,15- ctadecatrien-1- ol 8 Stearic acid, methyl ester C 17H 34 2 Catechol--Methyl-Transferase-Inhibitor and Methyl-Guanidine-Inhibitor C 16H 32 2 Acidifier, Acidulant, Arachidonic acid, Arachidonic-Acid-Inhibitor, ncrease Aromatic Amino Acid Decarboxylase Activity, Inhibit Production of Uric Acid C 19H 34 2 Catechol--Methyl-Transferase-Inhibitor and Methyl-Guanidine-Inhibitor C 18H 32 ligosaccharide Provider, C 19H 38 2 Urine-Acidifier, Urinary-Acidulant, Inhibit Production of Uric Acid, Increase Aromatic Amino Acid Decarboxylase Activity, Arachidonic acid-inhibitor, Methyl- Guanidine-Inhibitor, Catechol-- Methyltransferase-Inhibitor showed bioactivity as an oligosaccharide provider. An oligosaccharide is a saccharide polymer containing a small number of simple inhibitor and methyl guanidine-inhibitors. Cathecol-0-methyl-transferase (CMT) inhibitors prevent the enzymes that degrade 12

6 catecolamines such as dopamine, norepinephrine and epinephrine thus making them available.cmt is mainly located intracellularly in the CNS 22,23 but can also be found extracelullarly. Catechol-o-methyltransferase is involved in the degradation of neurotransmitters. 9, 12-ctadecadienoic acid, methyl ester, a catechol-o-methyltransferaseinhibitors opposes the degradation of neurotransmitters. Parkinson s disease is treatable with catechol-o-methyltransferaseinhibitors. 24 Methyl-guanidinase inhibitors also inhibit the hydrolase enzyme that catalyzes the hydrolysis of methyl guanidine to produce methylamine and urea. 25 ther phytochemicals with high Area peak percentage found from H. crepitans bark ethanol extract were methyl 14-methyl pentadecanoate with peak area percentage of and stearic acid methyl ester with area peak percentage of that have the same bioactivities with hexadecanoicacid with area peak percentage of 3.52 and bioactivities of cathecol-0-methyl transferase inhibitor, methylguanidine inhibitor, urine acidifiers, urinary- Acidulant, inhibit production of uric acid, increase aromatic amino acid decarboxylase activity and arachidonic acid-inhibitor. Hexadecanoic acid and hexadecanoic acid, ethyl ester have been reported to be acidifier, acidulant and arachidonic acid inhibitor 26 Acidifiers are chemicals that reduce the ph of the body. They help in food digestion in patients suffering from achlorhydria. These patients are not able to secret HCl for food digestion. These compounds may be beneficial since they increase gastric acid when ingested. Urine acidifiers are most times taken orally to change the PH level of the urine making it more acidic. Most often used in animals like dogs to treat certain types of bladder stones that are alkaline in nature, thus dissolving them 27 and with such urinary acidifier s aid in the health of the urinary tracts. Aromatic amino acid decarboxylase is a lyase enzyme that catalyzes several decarboxylation reactions that forms various neurotransmitters needed for neurotransmission, such as L-DPA decarboxylase 28,29 neurotransmitters. thereby making available Arachidonic acid is a polyunsaturated omega 6 fatty acid that is usually present in the phospholipids of cell membranes that are involved in cell signalling and also acts as an inflammatory intermediate and as vasodilator 30, therefore, Arachidonic acid inhibitors prevent inflammation and vasodilation. ther phytochemicals seen in lower percentages in the extract include 3,5-dihydroxy-6-methyl- 1,2,3-dihydro-4 H-pyran-4-one with peak area percentage 2.75, 5-(hydroxyl methyl) furan-2- carbaldehyde with peak area percentage of 1.65 and Nitrocoumarin with peak area percentage of 0.91 which have their bioactivities to include antimicrobial, anti-inflammatory, antiproliferative, antioxidant, automatic nerve activity, preservative, clastogenic activity, uterotonic activity, 11 B- HSD-inhibitor, 17- beta-hydroxysteroid dh inhibitor, 5-HETEinhibitor,, 5-HT-Inhibitor, 8-HETE-Inhibitor, Anti-HIV-Integrase, Anti-5-HT, Antidote (Heavy Metals), Antidote (Hydrazine), Aryl- Hydrocarbon - Hydroxylase-Inhibitor, Hallucinogenic, and Hemangionagenic. Clastogenic activity is the ability of an agent to induce disruption in the chromosomes, leading to deletion, addition or re-arrangement of some of the sections of the chromosomes. This activity can lead to development of cancer HETE (5-Hydroxyicosatetraenoic acid) is a metabolite of arachidonic acid produced by neutrophils which are signalling agents that amplify or dampen inflammation and allergic responses 32, thus their inhibitors prevents these responses. Anti HIV-integrase is an agent that inhibits the action of integrase enzyme which enables the integration of retroviral DNA into the host cell genome, thus it is used in the treatment of HIV as it inhibits the replication mechanism of retroviruses B- HSD-inhibitors inhibit the conversion of cortisone to the active hormone cortisol which is 13

7 a stress hormone, and too much of this stress hormone can lead to central obesity. 34 Hallucinogens are psychoactive agents that can cause perceptual anomalies and other substantial subjective changes in thought, emotion and consciousness which are not due to external stimulus and can manifest in variety of forms. 35 An antidote is a substance that can counteract a form of poisoning. 36 Conclusions H. crepitans bark ethanol extract showed the presence of eight phytochemicals using GCMS analysis. H. crepitans bark could be used to reduce the activity of HIV infection, reduce pain and infection, antidote to poisoning, induce hallucination, reduce weight and possibly reduce bladder stones. Thus more work could be done to elucidate the possible potentials of this H. crepitans bark as medicinal plant. Acknowledgements We are grateful to National Research Institute for Chemical Technology [NARICT], Zaria, Nigeria, for running the GC-MS analysis and Mrs Rose Sangodare for her effort to safe guard the sample for accurate result. Funding: No funding sources Conflict of interest: None declared References 1. Swain, Tony, ed. Plants in the Development of Modern Medicine. Harvard University Press. ISBN (1968) 2. The Plant List: A Working List of All Plant Species. Version 1.1, record kew Radcliffe-Smith A. A review of the family Euphorbiaceae. Nat cc Phorbol Est Swain MD, Tom Beer. Explosive Seed Dispersal in H. crepitans L. (Euphorbiaceae). New Phytologist. 1977;78: Vogel, Steven. "The Flight of the Seed of H. crepitans"(pdf). (March 2008) 6. Feldkamp, Susan Modern Biology United States: Holt, Rinehart, and Winston. p (2006). 7. Jones, David E Poison Arrows: North American Indian Hunting and Warfare. University of Texas Press Drugs and Poisons. Useful wild plants of the tropical forests. The Tropical Forests Lowie. Vol 3 pg 7 9. Tropilab Inc. Exporter and wholesale of Medical plants, herbs and tropical seeds. 10. Igwe KK, Madubuike AJ, Chika Ikenga, tuokere IE, Amaku FJ. Studies of the medicinal plant Pausinystalia yohimbe ethanol leaf extract phytocomponents by GCMS Analysis. International Journal of Science Research and management. 2016;4(5): Xu CJ, Liang YZ, Chau FT, Heyden YV. Pretreatments of chromatographic fingerprints for quality control of herbal medicines. Journal of Chromatography. A. 2006;1134: Madubuike AJ, Igwe KK, tuokere IE, Amaku FJ. Bioactivity evaluation study of Phytochemicals in Gouania Longipetala ethanol leaf extract using GC-MS analysis. International Journal of Scientific and Technical Research in Engineering (IJSTRE). 2006;1(5): Zhu H, Wang Y, Liang H, Chen Q, Zhao P, Tao J. Identification of Portulaca oleracea L. from different sources using GC-MS and FT-IR spectroscopy. Talanta. 2010;81: Igwe KK, kafor Polycarp N, Ijeh Ifeoma I. GC-MS analysis of phytocomponents in Vernonia amygdalina. Del leaves and its contractile potential in mammary tissue in female albino wister rats. ISR-JAVS. 2015;8: Jensen WB. The origin of Soxhlex Extraction. Journal Clinical Education. 2007;84(12): Stein SE. National Institute of Standards and Technology (NIST) Mass Spectral Database and Software. Version 3.02, USA

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