International Journal of Drug Development and Research

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
2	Amino caproic lactam	113	C6H11NO
3	Phenol-2,6-bis (1,1-dimethyl)-4 -methyl, methyl carbamate	277	C17H27NO2
4	2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl-	180	C11H16O2
5	1,2,4-trimethoxy-5-prop-1-enylbenzene	208	C12H16O3
6	4-t-Butyl-2-(1-methyl-2-nitroethyl) cyclohexanone cyclohexanone	241	C13H23NO3
7	2,7- dimethyl-1-octanol	158	C10H22O
8	1-Tridecyne	180	C10H22O
9	6,10-dimethyl-2-undecanone	198	C13H26O
10	Hexadecane	226	C16H34
11	Methyl 14-methylpentadecanoate	270	C17H34O2
12	Heptedecanoic acid	270	C17H34O2
13	Ethyl octadecanoate	312	C20H40O2
14	Methyl trans-9-octadecanoate	296	C19H36O2
15	3,7,11,15-tetramethyl-2-hexadecen-1-ol	296	C20H40O
16	11-Hexadecanoic acid	254	C16H30O2
17	Octadecanoic acid	284	C18H36O2
18	1-Flourodecane	160	C10H12F
19	Eicosanoic acid	312	C20H40O2
20	Eicosane	282	C20H42
21	9-tetradecenal	210	C14H26O
22	2-Buthyl-1-octanol	186	C12H26O
23	Cyclododecane epoxide	182	C12H22O
24	Tetratetracotane	618	C44H90

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

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].

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

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].

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

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

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

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

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

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

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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