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Research Article - (2017) Volume 9, Issue 2

Gas Chromatography-Mass Spectrometry and Infra-Red Studies of Bioactive Phytoorganic Components of Combretum dolichopentalum Leaves

Ujowundu FN*, Ojiako AO, Nwaoguikpe RN and Ujowundu CO

Department of Biochemistry, Federal University Technology Owerri, Nigeria

*Corresponding Author:

Ujowundu FN
Department of Biochemistry, Federal University Technology Owerri, Nigeria
Tel: +2348034741650
E-mail: fnujowundu@yahoo.com

Received date: April 06, 2017; Accepted date: May 11, 2017; Published date: May 13, 2017

Citation: Ujowundu FN, Ojiako AO, Nwaoguikpe RN, Ujowundu CO (2017) Gas Chromatography-Mass Spectrometry and Infra-Red Studies of Bioactive Phytoorganic Components of Combretum dolichopentalum Leaves. Int J Drug Dev & Res 9: 10-15

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Abstract

Gas chromatography-Mass spectrometry analysis of Combretum dolichopentalum showed presence of 24 bioactive photo organic compounds which includes: Nitrocyclohexane, Phenol-2,6-bis(1,1-dimethyl)-4-methyl, methyl carbamate, 2,7 Dimethyl-1-octanol, 3,7,11,15-Tetramethyl- 2-hexadecen-1-ol, Hexadecane, Heptadecanoic acid, Octadecanoic acid, Tetratetracotane, 1-Fluorodecane, Eicosanoic acid, (E)-2,4,5- Trimethoxylpropenyl benzene, 2(4H)-Benzofuranone-5,6,7,7a-tetrahydro-4,4,7a-trimethyl-amino caproic lactam, 4-t-butyl-2-(1-methyl-2-nitroethyl) cyclohexanone, 6,10-Dimethyl-2-undecanoate, Ethyl octadecanoate, 9-Hexadecanoic acid, 2-Buthyl-1-octanol. The presence of these bioactive compounds may be the underlying scientific evidence for the many therapeutic effect of the plant extract.

Keywords

Gas chromatography-Mass spectrometry; Infra-red; Bioactive compounds; Therapeutic effect

Introduction

Plants are used as a source for many potent drugs [1], as plants synthesize lower molecular weight organic compounds which possess various biological activities [2-4]. A large number of medicinal plants and their purified constituents have shown therapeutic activities [5], and have proved as safe and effective therapeutic agents. Many plant species have been used in folklore medicine to treat various ailments [6]. The development of pharmaceuticals begins with identification of active principles, detailed biological assays and dosage formulations followed by clinical studies to establish safety, efficacy and pharmacokinetic profile of the new drug [7].

Combretum dolichopentalum is used traditionally for curing several ailments like stomach ache, gastro intestinal disorders (such as dysentery, passage of bloody stool and diarrhoea) and stomach ulcer in and around Ogwa in Imo State of Nigeria. Also in Ezinihitte Mbaise, Imo State, C. dolichopentalum is taken by women after parturition for reconditioning of the uterus [8]. Aqueous extracts of the leaves of C. dolichopentalum have antioxidant activities [9]. This study is a pioneer in identifying the organic compounds of C. dolichopentalum using GCMS analysis.

Materials and Methods

Sample collection and preparation

Combretum dolichopentalum belong to the family of Combretaceae. Fresh leaves of C. dolichopen9talum were harvested from a farm in Obinze in Owerri West Local Government Area of Imo State, Nigeria (N 5° 23’41.1’ and E 6° 57’ 14.0’). The sample specimen was deposited with voucher IMSUH12 at Imo State University herbarium. Fresh leaves of the plant were plucked from their stems, washed with distilled water and allowed to dry at room temperature (21-25°C). The dried samples were pulverized (using electric blender) and stored in an airtight container kept in a dark cupboard.

Preparation of samples for Gas Chromatography-Mass Spectrum (GC-MS) analysis

Fifty grams of the sample were soaked in absolute ethanol for 48 hours in various portions and repeatedly extracted with ethanol. The combined and concentrated ethanol extract were re-extracted using chloroform to obtain chloroform soluble portion and stored in sample bottle for GC-MS analysis.

GC-MS analysis was carried out using GC-MS QP 2010 Plus Shimazu Japan equipment. An aliquot of 1 μl of the chloroform soluble portion of the sample was injected into the column with injector temperature at 230°C and carrier gas pressure of 100 kpa. The column has a length of 30 m with a diameter of 0.25 mm and a flow rate of 50 ml/min. The eluents were automatically passed into the Mass Spectrometer with a detector voltage set at 1.5 kV and sampling rate of 0.2 seconds. The Mass Spectrometer was also equipped with a computer fed Mass Spectra data bank. The Mass spectrum yielded a spectrum of compounds which was compared with the spectrum of National Institute of Standard and Technology (NIST) database with over 62,000 spectral patterns [10,11]. The identity of the spectra above 95% was used to ascertain the name, molecular weight and structure of the components in the leaves of C. dolichopentalum.

Column chromatographic separation of plant sample

Eight hundred grams of the pulverized samples were soaked in 95% ethanol for 48 hours and filtered. The filtrates were concentrated using rotary evaporator regulated at 40°C to obtain the ethanol extract. The crude ethanol extracts were partitioned between chloroform and water to afford chloroform soluble fractions. Ten grams of the chloroform soluble fractions were subjected to column chromatography over silica gel. The column was eluted as described by Abdelgadir et al. [12] with minor modifications using 100 ml petroleum ether, followed by petroleum ether/chloroform mixture and labelled as follows; (a) 100 ml petroleum ether ACD, (b) 90/10 BCD, (c) 80/20 CCD (d) 70/30 DCD, (e) 60/40 ECD, (f) 50/50 FCD, (g) 40/60 GCD, (h) 30/70 HCD (i) 20/80 ICD (j) 10/90 JCD. Further elution using chloroform and methanol mixture afforded the fraction 90/10 KCD, 80/20 LCD 70/30 MCD, 60/40 NCD, 50/50 OCD, 40/60 PCD, 30/70 QCD, 20/80 RCD 10/90 SCD.

Thin layer chromatography of column eluents

The eluents from column chromatography were subjected to thin layer chromatography following the method of Watanable et al. [13] with minor modifications using silica gel 60 G and iodine vapour for development. This was to select the pure eluents which appeared as a spot from the impure eluents that appeared as more than one spots. Fraction FCD was obtained using petroleum ether/chloroform mixture (3:1) with a Ratio of font (Rf) value of 0.82. Fraction ICD was obtained with Rf value of 0.83 which appeared as a spot. Fraction DCD had Rf value of 0.84, GCD with Rf value of 0.85, JCD with RF value of 0.86, KCD with Rf value of 0.89 and MCD with Rf value of 0.64.

Fourier Transform Infra-Red Spectroscopy (FTIR) analysis

Fourier transform infra-red spectroscopic analysis of the sample was carried out according to the method of Hashimotto et al. and Geethu et al. [14,15]. One gram of the dried plant sample was mixed with 0.5 g of potassium bromate (KBr) and 1.0 ml of nujol oil (a solvent for preparation of sample by Buck 530 IR-spectrophotometer) was added with the aid of a syringe to form a paste. The paste was introduced into the instrument sample mould and allowed to scan at a wavelength of 600-4000 nm to obtain the spectra wavelength. The eluents from the column chromatography were subjected to FTIR analysis; here 1.0 ml of the nujol oil was added to 1.0 ml of the chloroform fraction.

Results and Discussion

Identification of the chemical constituents of plants is important for the discovery of new therapeutic agents [16], and GC-MS is an important tool for this process. Gas chromatography mass spectrometer identifies the bioactive constituents of long chain hydrocarbons, alcohols, acids, ester, alkaloids, steroids, amino and nitro compounds [17]. The chemical characterization of active fraction of tested C. dolichopentalum leaves showed a mixture of fatty acids, nitrocyclohexane, furans, alkanes, organic esters, modified alkanones, alcohols, and eicosanoic acids.

A total of 24 peaks (Figure 1) were observed when the chloroform soluble portion of ethanol extract of C. dolichopentalum leaves was subjected to GC- MS (Table 1). Some of the bioactive compounds include; hexadecanoic acid, Cyclohexamine, Caprolactam, Phenol-2,6- bis(1,1-dimethylethyl)-4, methyl carbamate, 3,7,11,15-Tetramethyl-2- hexadecen-1-ol (Phytol), Heptadecanoic acid.

S No Molecular Weight Molecular Weight Molecular Formula  Molecular Structure
1 Nitrocyclohexane        129 C6H11NO2 image
2 Amino caproic lactam 113 C6H11NO image
3 Phenol-2,6-bis (1,1-dimethyl)-4 -methyl, methyl carbamate 277 C17H27NO2 image
4 2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl- 180 C11H16O2 image
5 1,2,4-trimethoxy-5-prop-1-enylbenzene 208 C12H16O3 image
6 4-t-Butyl-2-(1-methyl-2-nitroethyl) cyclohexanone cyclohexanone  241 C13H23NO3 image
7  2,7- dimethyl-1-octanol 158 C10H22O image
8  1-Tridecyne 180 C10H22O image
9  6,10-dimethyl-2-undecanone 198 C13H26O image
10 Hexadecane 226 C16H34 image
11 Methyl 14-methylpentadecanoate 270 C17H34O2 image
12 Heptedecanoic acid 270 C17H34O2 image
13  Ethyl octadecanoate  312 C20H40O2 image
14 Methyl trans-9-octadecanoate 296 C19H36O2 image
15 3,7,11,15-tetramethyl-2-hexadecen-1-ol 296 C20H40O image
16 11-Hexadecanoic acid 254 C16H30O2 image
17 Octadecanoic acid  284 C18H36O2 image
18 1-Flourodecane 160 C10H12F image
19 Eicosanoic acid 312 C20H40O2 image
20 Eicosane 282 C20H42 image
21 9-tetradecenal 210 C14H26O image
22 2-Buthyl-1-octanol 186 C12H26O image
23 Cyclododecane epoxide 182 C12H22O image
24 Tetratetracotane  618 C44H90 image

Table 1: Molecular weights, formula and structures of compounds identified from the GC-MS of ethanol extract of C. dolichopentalum.

Drug-Development-Research-chloroform-soluble-portion

Figure 1: GC-MS profile of chloroform soluble portion of leaves of C. dolichopentalum.

Hexadecanoic acid, a fatty acid with potential antimicrobial and antidiarrhoeal activities [18], is reported to cause growth inhibition and apoptosis induction in human gastric cancer cells [19]. Cyclohexamine belong to an aliphatic amine class. It is used as an intermediate in the synthesis of other organic compounds such as sulphenamide; a base reagent used as accelerators for vulcanization. Cyclohexamine is also used as a building block for pharmaceuticals, e.g., mucolytics, analgesics, and bronchodilators [20].

Phenol-2,6-bis(1,1-dimethylethyl)-4, methyl carbamate could be used to synthesize Phenol-4-[2-(aminomethyl)-4-thiazolyl]-2,6- bis(1,1-dimethylethyl) monohydrochloride which is used for the treatment of Huntington’s disease [21].

The identified hexadecanoic acid ethyl ester has antioxidant activities which are important in biological processes [22,23]. While, 3,7,11,15-Tetramethyl-2- hexadecen-1-ol (Phytol) has antimicrobial and anti-inflammatory activities [23]. Octadecanoic acid (stearic acid) is less likely to be incorporated into cholesterol esters, and thus was found to be associated with lowered LDL cholesterol when compared to other saturated fatty acids in epidemiologic and clinical studies [24]. It is also used to produce dietary supplements [25].

Heptadecanoic acid or margaric acid, is a saturated fatty acid which occurs as a trace component of fat and milk fat of ruminants [26,27].

Ethnobotanical database compounds such as furan, phenols, flavonoids and fatty acid esters possess antioxidant, antibacterial, antimicrobial, anti-inflammatory, antiproliferative, anticancer, antitumor, antidiabetic, antiarthiritic, antimalarial and automatic nerve activities [28,29]. Thus, the presence of these compounds in C. dolichopentalum implies a possibility of exhibiting afore mentioned activities as found in plants such as Litsea glutinosa [30], Suaeda maritime [31], Alpinia hainanesis and Alpinia katsumadai [32], and Macrotyloma uniflorum [33].

The spectra wavelength observed in C. dolichopentalum served as a characteristic medium to elucidate the inherent functional group and organic compounds in the plant [15,34]. The Infra-Red of the pulverized leaves (Table 2) showed different peaks, indicating transitions between vibration levels of different molecules. The peak value at 833 cm-1, was assigned C-Cl stretch of chlorine compound and 1027 cm-1 was assigned CO stretch of ether compounds (Figure 2). The medium bands at 1257 cm-1, 1632 cm-1, 3405 cm-1 were thus assigned NH stretch of amine compounds. The weak band at 1876 cm-1, and 2060 cm-1 were assigned CO stretch of unsaturated ester and carboxylic compounds. Also, 1454 cm-1, 2753 cm-1, 2856 cm-1, 2570 cm-1 and 2664 cm-1 peaks were assigned CH and SH symmetric stretch of methylene and thiol compounds. The broad band at 3065 cm-1, 3160 cm-1, and 3548 cm-1 were assigned OH stretch of primary (1°) and tertiary (3°) alcohol compounds [15,35].

Drug-Development-Research-functional-groups

Figure 2: FTIR spectra of pulverized C. dolichopentalum leaves showing absorption peaks of different functional groups.

S No Wavelength (cm-1) Functional Group Compounds
1 833 C-Cl Chloro compound C-Cl stretch
2 1027 R-O-P Ether CO stretch
3 1257 RNH3 10 amine NH stretch
4 1454 CH3 Methyl CH stretch
5 1632 RNH2 20 amine NH stretch
6 1876 C-O-C Cyclic ester CO stretch
7 2060 RCOOH Carboxylic acid COO stretch
8 2442 R-C=N Nitriles CN anti-symmetric stretch
9 2570 CH2SH Thiol SH stretch
10 2664 CH2SH Thiol SH stretch
11 2753 CH2 Methylene CH stretch
12 2856 CH2 Methylene CH stretch
13 2935 R-S-C=N Thiocyanate SCN anti-symmetric stretch
14 3065 RCHOH 10 alcohol OH stretch
15 3160 RCHOH 10 alcohol OH stretch
16 3405 RNH2 20 amine NH stretch
17 3548 R2CHOH 30 alcohol OH stretch
18 3662 R3N 30 amine NH stretch

Table 2: FTIR spectra characteristics of crude ethanol extract of C. dolichopentalum.

The Infra-Red of the seven eluates are shown in Figures 3-9. The interpretation of the IR spectra of the eluates using the GC-MS spectra indicated Dcd eluate (Figure 3) as 4-t-Butyl-2(1-methyl-2-nitroethyl) cyclohexanone, with the Rf value of 0.84, molecular weight- 234 and an empirical formular C13H23NO3. N-H stretch occurs in the range 3500- 3300 cm-1. Eluate Fcd (Figure 4) as flourodecane an alkane which IR spectrum are usually simple with few peaks. C-H stretch occur around 3000 cm-1 and alkanes (except strained ring compounds) absorption always occurs to the right of 3000 cm-1. Gcd eluate (Figure 5) was identified as phenol-2, 6- bis (1,1-dimethyl ethyl)-4, methyl carbamate with the empirical formular C17H27NO2. Icd (Figure 6) and Kcd (Figure 7) eluates were identified as pentadecane (C15H32) and hexadecane (C16H34) respectively. Eluates Jcd (Figure 8) and Mcd (Figure 9) as 2, 7-dimethyl-1-1octanol (C10H22O) and 2-Butyl-1-octanol (C12H26O) respectively [10,11,36-38].

Drug-Development-Research-FTIR-spectra

Figure 3: FTIR spectra of eluate, Dcd (4-t-Butyl-2-(1-methyl-2-nitroethyl) cyclohexanone) of C. dolichopentalum leaves.

Drug-Development-Research-spectra-eluate-Fcd

Figure 4: FTIR spectra of eluate Fcd (1-Fluorodecane) of C. dolichopentalum leaves.

Drug-Development-Research-methyl-methyl-carbamate

Figure 5: FTIR spectra of eluate Gcd (Phenol-2,6-bis - (1,1-dimethylethyl) - 4 - methyl methyl carbamate) of C. dolichopentalum leaves.

Drug-Development-Research-dolichopentalum-leaves

Figure 6: FTIR spectra of eluate Icd (Eicosane) of C. dolichopentalum leaves.

Drug-Development-Research-Dimethyl-octanol

Figure 7: FTIR spectra of eluate Jcd (2,7-Dimethyl-1-octanol), of C. dolichopentalum.

Drug-Development-Research-Hexadecane

Figure 8: FTIR spectra of eluate Kcd (Hexadecane) of C. dolichopentalum leaves.

Drug-Development-Research-spectra-eluate

Figure 9: FTIR spectra of eluate Mcd (2-Butyl-1-octanol) of C. dolichopentalum leaves.

The present study showed that leaves of C. dolichopentalum contain a variety of biologically active phytoorganic compounds by using GC-MS and IR spectroscopy as pharmacognostic tool for the identification of plant constituents. The beneficial roles of these bioactive phytoorganic constituents can be harnessed and used in the pharmaceutical and food industries for the production of drugs and raw materials for industrial purposes.

Acknowledgements

The authors appreciate the technical support and contributions of Prof. Ukoha AI of the Department of Biochemistry and Dr. IC Iwu of the Department of Chemistry, Federal University Technology Owerri, Nigeria. The authors appreciate Mr. A Ozioko of the Bioresource Development and Conservation Program (BDCP), Research Centre, Nsukka, Enugu State and Dr. FN Mbagwu of the Department of Plant Science and Biotechnology, Imo State University, Owerri, Nigeria for their assistance in identifying and providing voucher number for the plant.

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